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Notes: Abstract Histidine decarboxylase (HDC) mRNA in various rat tissues were quantitated by using a reverse transcription-polymerase chain reaction (RT-PCR) in which a mouse mRNA was used as an internal standard. The stomach HDC mRNA level was the highest followed by the brain, skin, jejunum, spleen and liver. There was no measurable HDC mRNA in the kidney. The stomach HDC activity was also the highest followed by the brain, skin, spleen, jejunum, liver and kidney. A significant correlation (r = 0.940,p 〈 0.0001) was observed between the HDC mRNA levels and HDC activities in these tissues. We have also examined the HDC mRNA levels in fasting rats and found that HDC mRNA levels in the stomach were reduced after the 48-hr-fasting with the decrease in HDC activities. These observations indicate that there may exist a gene regulation, at least at the basal level, for the HDC activities in the rats.

Notes: Summary Previous studies of intraportal islet autotransplantation in large animals have reported graft failure after months or years. In the rat it has been reported that intraportal islet isografts eventually failed whilst islets transplanted to the renal subcapsule functioned up to a year. We made Dark Agouti (DA) rats severely diabetic with streptozotocin, then 1000 or 3000 DA islets were transplanted beneath the renal capsule or into the liver. One set of transplanted rats and untreated diabetic and normal non-diabetic littermates were monitored lifelong by measurement of plasma glucose, others were killed at 6, 12 and 18 months for measurement of haemoglobin A1c, intravenous glucose tolerance test, pancreas insulin content and histology of the kidney. Renal glomerular basement membrane thickness was measured by the orthogonal intercept method. The results showed that intraportal isografts reversed hyperglycaemia significantly faster than renal subcapsular isografts. In the renal subcapsular site, consistent reversal of diabetes was achieved with 3000 islets but not with 1000 islets. Furthermore, intraportal islet grafts with 3000 islets led to lower, normal random glucose level than renal subcapsular grafts for the first 13 months. Normoglycaemia was maintained life-long in all rats that achieved early normoglycaemia after transplantation of 3000 islets, irrespective of the site of islet transplantation. The fasting glucose, haemoglobin A1c levels, K value and glomerular basement membrane thickness of the recipients of 3000 islets to either the intraportal and subcapsular site were not significantly different from each other and the normal controls up to 18 months. We conclude that, in streptozotocin diabetic DA rats, normoglycaemia following transplantation of an adequate mass of pancreatic islet tissue (3000 islets) to the liver or beneath the renal capsule is lifelong and the development of glomerular basement membrane thickening is prevented.

Notes: Abstract A combination of either retrograde or anterograde fluorescent tracer and immunofluorescence histochemistry using the monoclonal antibody specific for the alpha isoform of calcium/calmodulin-dependent protein kinase II (CaM kinase IIα) was employed to test whether CaM kinase IIα is expressed in somata of corticospinal neurons and their axons over their whole course. After the injection of carbocyanine dye DiI into the hindlimb area of the primary motor cortex of the rat, corticospinal axons and their terminal arbors were anterogradely labeled: DiI-labeled corticospinal fibers proceeded caudally in the ipsilateral internal capsule, cerebral peduncle and medullary pyramid, crossed at the pyramidal decussation and descended in the ventralmost area of the contralateral dorsal funiculus of the spinal cord. These DiI-labeled corticospinal axons expressed strong CaM kinase IIα immunoreactivity along their course. However, their terminal arbors within the gray matter of the lumbar cord were very weakly immunostained. With the injection of Fast Blue into the lumbar enlargement of the rat, somata of corticospinal neurons in layer V of the motor cortex were retrogradely labeled. The subsequent immunofluorescent histochemistry revealed that more than 80% of Fast Blue-labeled corticospinal neurons were immunostained with CaM kinase IIα antibody. The present immunohistochemical study demonstrated that CaM kinase IIα is strongly expressed in both somata and axons of a majority of corticospinal neurons, although we could not detect this enzyme in the corticospinal terminals in the spinal target areas.

Notes: Abstract The vomeronasal organ and the olfactory bulb of the rat were cocultured from 15-day embryo siblings on collagen-coated membrane in Dulbecco's modified Eagle's medium containing fetal calf serum, horse serum, and antibiotics. At 4 days in vitro (DIV), vomeronasal axons forming two to three large fascicles were seen originating from the explants of the vomeronasal organ. Differential axonal growth was observed. Some fascicles made connections with the explants of the olfactory bulb. Twenty percent of the cocultures studied here showed the formation of connections. At 6–10 DIV many fascicles that did not connect with the olfactory bulb had degenerated, and large fascicles that were connected with the olfactory bulb survived for more than 10 DIV. The formation of connections between the vomeronasal organ and the olfactory bulb in coculture favors the survival of large nerve fascicles, but it could not be determined whether or not the presence of the olfactory bulb affects the initial orientation of the fibers and fascicles from the explants of the vomeronasal organ.

Notes: Abstract In order to determine the incidence of maternal glucocorticoids on morphological parameters in fetal development, we performed optic and electron microscopic analysis of the cerebral cortex of fetuses of 16 and 20 days of gestation, from control (C) and pregnant rats bilaterally adrenalectomized on day 1 of gestation (ADX). We also studied fetuses 20 days old from pregnant rats betamethasone-injected on days 15, 16 and 17 (BET), and adrenalectomized on day 1 and betamethasone-injected on days 15, 16 and 17 (ADX+BET). Absence of maternal glucocorticoids during gestation caused, in fetuses 16 and 20 days old, a marked increase of cellular density, laxity of tissue and lower cellular maturation in comparison with the control group. Beta-methasone injected into sham-operated animals (BET) caused a slight advance in relation to controls in developmental parameters such as cellular density, maturation and synapse formation. Betamethasone injection into adrenalectomized animals prevented the lower degree of maturation characteristic of the adrenalectomized group, although an increase of cellular density could be detected. The cerebral cortex from fetuses of 16 days of gestation from adrenalectomized mothers also showed an increase of cellular density as compared with the control group. These results show that glucocorticoids participate in prenatal rat brain in control mechanisms of cellular division and maturation.

Notes: Abstract We studied the secretory mechanism of the Harderian gland of rats. After perfusion with HEPES-buffered Ringer's solution containing NaF (10 mM) with AlCl3 (10 μM), a G-protein activator, the glandular cells of the Harderian gland showed massive exocytosis and apocrine-like protrusions on the luminal surface. Some of the secretory vacuoles aggregated within the cytoplasm, and large vacuoles were formed. Contraction of the myoepithelial cells covering the glandular endpieces caused a narrowing of the glandular lumina, which contained cytoplasmic fragments, and deformation of the basal contour of the glandular end-pieces. The basal regions of the glandular cells also bulged between the myoepithelial cells. Secretory vacuoles were also discharged to the lateral cell surface, and the intercellular spaces were dilated. The enhanced secretory activities of the glandular cells and the contraction of the myoepithelial cells were similar to those in rats stimulated with 10 μM carbachol (CCh). However, dilatation of the endoplasmic reticulum in glandular cells (type A cells), which leads to the formation of small vesicles, was observed in those glands stimulated by NaF+AlCl3, but not in those stimulated by CCh. Removal of Ca+2 from the perfusing HR or addition of EDTA (0.5 mM) diminished and inhibited NaF+AlCl3- or CCh-enhanced secretory activity of the glandular cells and also allayed the deformation of glandular cells caused by myoepithelial cell contraction. The present results demonstrate the involvement of G-proteins and Ca2+-influx in the lipid secretion of glandular cells and in the contraction of myoepithelial cells of the Harderian gland in rats.

Notes: Abstract  Consumption of certain product lots of L-tryptophan (LT) has been reported to be epidemiologically associated with an outbreak of eosinophilia-myalgia syndrome (EMS) in the United States. Since the production lots were found to contain 3-phenylamino alanine (PAA) as an impurity, its effects were studied by administering the substance orally by gavage to 5-week-old Sprague-Dawley rats. Groups of animals were given PAA for 13 consecutive weeks at dose levels of 1, 10 and 100 mg/kg per day. The animals were killed at 4 or 8 weeks. Hematological and blood biochemical tests were performed and detailed histopathological observations were made. No significant abnormalities were observed in the test animals and in particular no EMS-like conditions. A brief summary of other animal studies using several species of rats and mice performed in our laboratory since 1989 on various LT related substances is also presented. No EMS-like effects were observed in these studies.

Notes: Abstract  The effects of the herbicides paraquat, dinoseb and 2,4-D on intracellular Ca2+ levels and on vasopressin-induced Ca2+ mobilization were investigated in intact isolated hepatocytes. Incubation of rat hepatocytes with paraquat (5 mM for 60 min) and dinoseb (10 μM) resulted in a time-dependent loss of viability by approximately 25%. Viability of cells treated with 2,4-D decreased significantly, dropping to about 20% at 10 mM and 60 min incubation. Exposure of hepatocytes to paraquat (1–10 mM) for 60 min had no effect on the basal level of [Ca2+] i . Additionally, exposure to paraquat had no effect on the magnitude and on the duration of the [Ca2+] i response to vasopressin. In the presence of 2,4-D (1–10 mM), basal [Ca2+] i increases as a function of herbicide concentration. The magnitude of the Δ[Ca2+] i response decreases from 256±8 nM in control to 220±5 nM, at 10 mM 2,4-D. Exposure of hepatocytes to dinoseb (1–10 μM) had no effect on the basal level of [Ca2+] i . However, a strong concentration-dependent decrease in the magnitude of Δ[Ca2+] i in response to vasopressin was noticed at 60 min incubation. Dinoseb markedly inhibited the stimulation of the production of inositol phosphates by vasopressin stimulus. The present study demonstrates that paraquat, 2,4-D and dinoseb cause cell death in hepatocytes by mechanisms not related to an early increase in [Ca2+] i . Additionally, it has been shown for the first time that dinoseb disturbs the transduction mechanism promoted by vasopressin by inhibiting the formation of IP3.

Notes: Abstract  The uptake of methyl mercury (MeHg) by isolated rat erythrocytes was studied at 37°C using MeHg-cysteine (MeHgCySH), MeHg-glutathione (MeHgGSH), MeHg-mercaptalbumin (MeHgMASH) and the mixture of MeHgCySH with MeHgGSH, MeHgCySH with MeHgMASH, MeHgGSH with MeHgMASH at different MeHg concentrations. The measured MeHg concentrations were analyzed according to the Akaike’s information criterion in order to determine the suitable compartment model. After determining a two-compartment model, a model-independent two-compartment model was developed from the kinetics of uptake of MeHg at a concentration of 1 mmol MeHg/l packed erythrocytes using MeHgCySH, MeHgGSH and MeHgMASH, respectively. The developed two-compartment model was validated by predicting the kinetics of uptake of MeHg by rat erythrocytes at different MeHg concentrations and different mixtures of MeHg-complexes. Then, the predicted values were compared with the measured values. The results suggested: 1) MeHg uptake appeared suitable to be described by a two-compartment model, while using MeHgGSH, MeHgMASH, MeHgCySH at lower concentrations and the mixtures of MeHg-complexes; 2) MeHgCySH uptake was slowest among three kinds of MeHg-complexes, although a postulated cysteine-facilitated MeHgCySH transport system might exist in erythrocyte membrane; 3) the mixture of MeHg-complexes might facilitate MeHgCySH uptake; 4) there might be a second MeHg intracellular compartment in rat erythrocytes.

Notes: Abstract  The N-hydroxyurea derivatives 70C ((E)-N-{3-[3-(4-fluorophenoxy)phenyl]-1-(R,S)-methylprop-2-enyl}-N-hydroxyurea) and its (R) 225C and (S) 404C enantiomers, which were being developed as 5-lipoxygenase inhibitors for the treatment of certain allergic and inflammatory conditions, were found to cause severe glomerulonephropathy in the rat. The lesion appeared to be of greater severity in female rats compared with male rats. In addition, 70C and 225C treated animals appeared more severely affected than 404C treated animals. Detailed examination of the lesion in animals dosed with 225C showed that there was a clear relationship between the onset of the lesion and the dose given, i.e. the higher the dose the sooner the lesion developed. The earliest changes detected in the kidney by transmission electron microscopy were noted in the glomeruli, in which the visceral cells appeared enlarged and showed varying degrees of foot process loss. In the more advanced lesion, the degree of foot process loss became more obvious and changes in the kidney tubules were seen by light microscopy. The morphological changes were mirrored by a dose-related increase in water consumption, an increased kidney to body weight ratio and gastrointestinal oedema, suggesting impaired renal function. Shortly after the onset of foot process loss, decreases in the total plasma protein and albumin and increases in the plasma cholesterol, triglycerides, urea and creatinine were recorded. These changes, particularly the foot-process loss, together with increased proteinuria, hypoalbuminaemia, hypercholesterolaemia and lipaemia, are all characteristic of “minimal change nephrotic syndrome”. Because of the serious nature of the kidney lesion caused by these N-hydroxyureas in the rat, it was considered that it precluded their development as therapeutic agents for use in man.

Notes: Abstract  The cytotoxic effects of propyl gallate (PG), its related gallates and gallic acid have been studied in freshly isolated rat hepatocytes. Addition of PG (0.5–2.0 mM) to hepatocyte suspension elicited concentration-dependent cell death accompanied by losses of intracellular ATP, adenine nucleotide pools, glutathione (GSH) and protein thiols. The rapid loss of intracellular ATP preceded the onset of cell death caused by PG. In the comparative toxic effects of PG and related gallates at concentration of 1 mM, octyl gallate (OG), dodecyl gallate (DG) and butyl gallate (BG) elicited an abrupt depletion of ATP, followed by an acute cell death. These gallates were more toxic than PG; the toxic effects of PG were similar to those of methyl gallate (MG) and ethyl gallate (EG). In mitochondria isolated from rat liver, PG caused a concentration-dependent increase in the rate of state 4 oxygen consumption, indicating an uncoupling effect. The rate of state 3 oxygen consumption was inhibited by OG and DG. According to the respiratory control index, the order of impairment potency to mitochondria was OG〉BG, DG〉PG〉EG, MG〉gallic acid. These results indicate that PG and related gallates are toxic to hepatocytes and that the acute cytotoxicity may be due to mitochondrial dysfunction.

Notes: Abstract Transmembrane potentials from medial septal and diagonal band of Broca (MS-DBB) neurons and hippocampal field activity were recorded in curarized and urethanized rats. MS-DBB cells were studied during large amplitude irregular activity and during hippocampal θ rhythm, elicited by either sensory (i.e. stroking the fur on the animal's back) or electrical stimulation of the reticularis pontis oralis nucleus (RPO). Three types of cells were described according to their firing pattern and the characteristics of their “intracellular θ” rhythm. Type A neurons displayed continuous rhythmic oscillations in the membrane potential (Vm) of approximately 17 mV. These oscillations generated rhythmic high-frequency spike trains which were phase-locked with hippocampal θ rhythm. Type A cells revealed intracellular θ rhythm even in the absence of hippocampal θ rhythm, suggesting that the activity of this type of cell was the most important in hippocampal θ genesis. Type B cells were characterized by marked postspike afterhyperpolarization and intracellular θ oscillations of smaller amplitude than in type A cells. Type C cells revealed a post-spike afterdepolarization and a lower amplitude, intracellular θ rhythm only in the presence of hippocampal θ rhythm. Type C neurons could fire slow spikes at depolarizing (46% of cells) or hyperpolarizing (15% of cells) Vms. Type B and C cells were intracellularly stained with Lucifer yellow. Although type B and C neurons revealed dissimilar electrophysiological properties, they had comparable morphological shapes. RPO electrical stimulation generated hippocampal θ rhythm and intracellular θ rhythm in types A and B cells but not in type C cells, and increased the spike rate in type C neurons. Electrical stimulation of the fornix only evoked synaptic responses in type B and C neurons, with antidromic responses being elicited in 12% of type C cells. These results indicate that probably most of the type A rhythmic cells did not receive direct hippocampal feedback and that at least some type C cells were projecting neurons. The present findings demonstrate that θ rhythm oscillations in the Vm of MS-DBB neurons elicit different rhythmic discharge patterns.

Notes: Abstract The host response to immunologically incompatible intrastriatal neural grafts was studied using immunohistochemical techniques. Dissociated ventral mesencephalic tissue from embryonic donors of either syngeneic, allogeneic or xenogeneic (mouse) origin was stereotaxically implanted into adult rats. The brains were analysed 4 days, 2 weeks or 6 weeks after grafting with antibodies against the following antigenic structures: major histocompatibility complex (MHC) class I antigens; MHC class II antigens; complement receptor (CR) 3 (marker for microglia and macrophages); helper T-lymphocyte antigen-cluster of differentiation (CD) 4; cytotoxic T-lymphocyte antigen-CD8; tyrosine hydroxylase (TH) (marker for transplanted dopaminergic neurons). The number of surviving TH-positive cells was not different at the various time points in either the syngeneic or allogeneic groups, whereas the xenogeneic cells were all rejected by 6 weeks. The host reactions were similar in character in the syngeneic and allogeneic groups. At 4 days after implantation, there were increased levels of expression of MHC class I and II antigens. In and around the grafts, there were cellular infiltrates consisting of activated microglia, macrophages, CD4- and CD8-positive lymphocytes. At 6 weeks, MHC expression was reduced and the cellular infiltrates had subsided with only low numbers of activated microglia cells and CD8-positive lymphocytes remaining. In the xenogeneic group, at 4 days, some grafts contained cavities, possibly reflecting acute rejection. At later stages, the xenografts were heavily infiltrated by macrophages, activated microglial cells and T-lymphocytes, and at 6 weeks all the xenografts were rejected. Taken together, the results suggest that there is an inflammation caused by the implantation process which leads to an accumulation of host defence cells. This, in turn, leads to increased MHC expression in and around the grafts. In syngeneic grafts, these reactions are short lasting and weak; for allografts slightly more pronounced and longer lasting than syngeneic grafts, but not sufficient to cause rejection. For xenografts, the reactions are more intense and lead to transplant rejection. Thus, a strong sustained inflammatory response may be an important determinator for the failure of histoincompatible neural grafts. It can be speculated that a short-term anti-inflammatory treatment of graft recipients may be a sufficient immunosuppressive regimen to allow long-term graft survival.

Notes: Abstract This study describes an approach for disconnecting the septal region from the hippocampus by fimbria-fornix lesions while sparing the commissural projections. After a frontal cut through the rostral fornix, commissural fibres were labelled with the anterograde tracer Phaseolus vulgaris leucoagglutinin. The commissural fibre bundle located in the posterior-basal fornix (ventral hippocampal commissure) remained unaffected by the rostral fornix transection, whereas the absence of septal fibres in the hippocampus could be verified using AChE histochemistry. Thus, using this approach, selective studies of the septo-hippocampal projection can be performed while leaving the overwhelming portion of the commissural fibre system intact.

Notes: Abstract The object of the study was to find out how preischemic hyperglycemia (in normocapnic animals) or excessive hypercapnia (in normoglycemic animals) affect the calcium transient during ischemia, as this can be assessed by measurements of the extracellular calcium concentration ([Ca2+]e). To that extent, normocapnic-normoglycemic control animals were compared with animals with induced hyperglycemia or hypercapnia, all being subjected to 10 min of forebrain ischemia, the [Ca2+]e and d.c. potential being measured with ion-sensitive glass microelectrodes. Hyperglycemia and hypercapnia delayed the loss of ion homeostasis following induction of ischemia. Furthermore, both hyperglycemia and hypercapnia reduced the delay of Ca2+ extrusion upon recirculation. As a result, both hyperglycemia and hypercapnia significantly reduced the ischemic calcium transient, as this was assessed by calculating the duration of maximal calcium load of cells. The results make it less likely that aggravation of brain damage by hyperglycemia or excessive hypercapnia is related to a further derangement of cell calcium homeostasis.

Notes: Abstract The evoked expression of the immediate early gene (IEG)-encoded proteins c-Fos and Krox-24 was used to monitor spinal visceronociceptive processing that results from cyclophosphamide cystitis in behaving rats. Animals received a single dose of 100 mg/kg i.p. of cyclophosphamide and survived for 30 min to 5 h. Longer survival times were not considered because of ethical considerations. Cyclophosphamide-injected animals developed characteristic behavioral signs in parallel with development of bladder lesions and spinal evoked expression of IEG-encoded proteins. Histological examination of the urinary bladder was used to evaluate the degree of cystitis and as a criterion for selection of groups of animals to be quantitatively analyzed. Controls consisted of freely behaving animals including control (uninjected), sham (saline-injected) or diuretic (furosemide-injected) animals. Behavioral modifications consisted of lacrimation, piloerection, assumption of a peculiar “rounded-back” posture, which was accompanied by head immobility and various brief “crises” (tail hyperextension, abdominal retractions, licking of the lower abdomen, backward withdrawal movements). Abnormal behaviors, which first appeared (lacrimation, piloerection) at the end of postinjection hour 1, progressively increased in severity (rounded-back posture) over the following 90 min to reach a plateau at about postinjection hour 2; the rounded-back posture was maintained up to time of death. Histological modifications of bladder tissue were assessed using a 4-grade scale in a blind setting. The 1st grade consisted of control or sham animals with no bladder lesion; 2nd grade, animals with simple chorionic edema; 3rd grade, animals with chorionic edema associated with mucosal abrasion, fibrin deposit, and onset of polymorphonuclear leukocyte infiltration; 4th grade, animals with complete cystitis corresponding to an increase in severity and spread of all the signs of cystitis described above plus petechial hemorrhage. Simple chorionic edema was observed from 30 min to 3 h post-injection, but with a progressive increase in severity over time. Edema accompanied by epithelial abrasion was observed for animals that survived 3–4 h postinjection; complete inflammation was observed in animals that survived 4–5 h postinjection. The study of c-Fos- and Krox-24-encoded protein expression demonstrated that few lumbosacral spinal areas were specifically involved in the processing of visceral inputs in response to bladder stimulation. These areas were the parasympathetic column (SPN), the dorsal gray commissure (DGC as the caudal extent of lamina X), and superficial layers of the dorsal horn. Differential basal and evoked labeling between c-Fos and Krox-24 allowed a functional distinction between these spinal subregions: Krox-24 was an indicator of the first signs of inflammation (simple chorionic edema); c-Fos was an indicator of more pronounced impairments. DGC, which displayed a high level of basal Krox-24 expression and in which both Krox-24 and c-Fos were evoked in relation to disease evolution, probably codes inputs from both physiological (progressive bladder filling) and all types of pathological (inflammatory processes, overdistension) conditions. SPN, which, except for basal Krox-24 expression, responded like DGC and contains preganglionic motor cells, probably codes inputs involved in eliciting motor activity that results in bladder emptying as needed from natural filling and/or pathological situations. Laminae I and II, which responded like DGC and SPN with respect to Krox-24 expression, may code inputs from physiopathological changes of the bladder, though lamina I would be primarily involved in pain processing as it was the only region in which c-Fos expression increased during abrasion and complete inflammation. The cyclophosphamide-cystitis model has the following unique features compared with other visceral models: first, the stimulus is a “pure” visceral one, confined to one viscus (bladder; no somatic stimulation is induced by either introducing a stimulating device or from surgical wounds, and no anesthesia is required); second, it is the exact replica of a human disease, and its evolution can be efficently monitored through behavioral and histological observations; third, it can be used in behaving animals without departing from ethical rules.

Notes: Abstract After transplantation of embryonic retinal cells to injured adult retina, it is often difficult to distinguish donor from host cells. To overcome this problem, two methods were applied: labelling donor cells with the nuclear marker bromodeoxyuridine (BrdU) and use of transgenic donor tissue. BrdU was injected into timedpregnant rats on 2 or 3 consecutive days. The donor embryos were taken 1–4 days later for transplantation. The BrdU-labelled donor tissue was examined in transplants sampled up to 1 year after grafting. Labelled donor cells were specifically identified in the transplants and in the interface with the adjacent host retina. The varying intensities of cell labelling indicated differences in the initial uptake of BrdU in the S-phase, or the dilution of the label by cell divisions after BrdU injection. The best labelled cells were presumably the ones that stopped dividing shortly after injection of BrdU. As controls, the normal development of BrdU-labelled retinas from the offspring of females that had been BrdU-injected at E16 and E17 and not used for transplantation was studied. Near the time of birth, clones of labelled cells were radially distributed. In the mature retina, labelled cells were seen in all retinal layers. Embryonic retina derived from transgenic (NSE-lacZ) mice was transplanted to ‘nude’, immunodeficient rats (xenografts). These transgenic mice contain the Escherichia coli β-galactosidase gene, coupled to the promoter for neuron-specific enolase (NSE). Thus, all retinal donor cells that contain NSE could be identified by histochemistry or immunohistochemistry. The donor cells expressing the transgene could be detected several months after transplantation.

Notes: Abstract Using electrophysiological techniques in the in vitro rat auditory cortex, we have examined how spontaneous acetylcholine (ACh) release modifies synaptic potentials mediated by glutamate and γ-aminobutyric acid (GABA). Single stimulus pulses to lower layer VI elicited in layer III a four-component (A-D) extracellular field response involving synaptic potentials mediated by glutamate and GABA. The cholinesterase inhibitor eserine (10–20 μM) or the cholinergic agonist carbachol (25–50 μM) depressed by 10–50% the glutamatergic components A and C, and the GABAergic components B and D. Atropine reversed the depressive effects of eserine and carbachol. A novel finding was that the degree of depression of component A varied inversely with stimulus intensity. However, during partial pharmacological antagonism of GABAA receptors, depression of A varied directly, not inversely, with stimulus intensity. Normally, then, depression of A is offset by reduced GABAergic inhibition of A. We also tested for differential depression of responses mediated by N-methyl-d-aspartate (NMDA) versus non-NMDA glutamate receptors. Following physiological and pharmacological isolation of the responses, eserine depressed the non-NMDA, but not the NMDA, receptor-mediated potential. Since the isolated NMDA potential still could be depressed by carbachol, the data suggested that activation of NMDA receptors may reduce spontaneous ACh release. In support of this, preincubation of slices in NMDA (10–20 μM) largely prevented eserine's, but not carbachol's, depression of components A and B. These results permit three conclusions of relevance to cortical information processing: (1) spontaneous ACh release tonically depresses synaptic potentials mediated by glutamate and GABA; (2) ACh depresses responses to weak inputs to a greater degree than responses to strong inputs; (3) activation of NMDA receptors may “feed-back” to reduce ACh release, a mechanism that could place regulation of local ACh release under glutamatergic afferent control.

Notes: Abstract Slowly inactivating outward currents were examined in neurons from rat anterior cortex dissociated at postnatal day 1 and recorded after 7–48 days in vitro by the use of whole-cell patch-clamp technique, in the presence of 0.5–0.8 μM tetrodotoxin (TTX), 50 μM carbachol and 1–5 mM CsCl2. Experiments were often carried out in the additional presence of 1–5 mM CsCl2, which blocks the anomalous, inwardly rectifying I Q, the fast Ca 2 + -dependent K+ current (I C), and 50 μM carbachol, which depresses the I M current. These currents were evoked by depolarizing steps to -40+-5 mV from a conditioning hyperpolarization to -110+-10 mV. Their sensitivity to elevation from 2.5 to 12.5 mM in extracellular K+ concentration, together with their sensitivity to 5–15 mM tetraethylammonium, suggests that they are mainly carried by K+ ions. Their activation and inactivation curves show that the threshold for activation is -65 mV, that their inactivation is achieved at -75 mV and that potentials more negative than -120 mV are needed to abolish it. The time-dependence of de-inactivation gives a maximal current amplitude for conditioning hyperpolarizations of 2 s and is best described by a monoexponential function with a time constant of 0.7 s. Slow, transient K+ currents were depressed by low doses of 4-aminopyridine (30–100 μM), which indicates the occurrence of an I D-type component in the recorded K+ currents. No slowly declining K+ current was expressed when a recording solution containing 10 mM 1,2-bis(2-aminophenoxy)ethane-N, N,N′-N′-tetraacetic acid (BAPTA), instead of 1–5 mM BAPTA, was used. When recorded without Ca2+ chelator in the pipette, slowly declining K+ currents were blocked by bath-applied 40–50 μM BAPTA-aminoethoxy, revealing a large-amplitude, rapidly inactivating outward current. This residual component is insensitive to 50 μM 4-aminopyridine and may include a current more related to the I A-type. Our data provide evidence that, in cultured cortical neurons from rat, the expression of an I D-like K+ current is highly dependent on internal Ca2+ concentration.

Notes: Abstract Experiments carried out in urethane-anaesthetized rats in which single neurones were recorded extracellularly from primary somatosensory (SI) cortex employed a procedure in which one of two vibrissal inputswas temporally paired with iontophoretic applications of glutamate. Following the pairing procedure, 31% of 49 neurones studied displayed some form of synaptic plasticity, in that responses to one or both vibrissal stimuli were altered. Homosynaptic potentiation occurred in 4 neurones, and these were recorded in layers II/III only. Homosynaptic depression occurred in 6 neurones and were mainly recorded in layer IV. Heterosynaptic depression was observed in 3 neurones. Non-selective depression was observed in 2 neurones. The duration of the induced plastic changes typically exceeded 15 min, and often lasted as long as stable recordings continued. The results from experiments in which repeated glutamate applications were given alone (without synaptic input) confirmed that the non-selective changes were due to repeated glutamate applications and not the temporal pairing with synaptic responses per se. Dual recordings confirmed that plasticity was restricted to the neurone at which pairings were made, and (at the other neurone) that synaptic responses remained stable over the course of study. In some neurones homosynaptic potentiation and depression were shown to occur to the early response component (〈10 ms), suggesting that direct thalamocortical synapses are modifiable.

Notes: Abstract Synaptic depression was assessed from intracellular recordings in cortical tissue slices. Evoked postsynaptic potentials exhibited synaptic depression with an exponential or double exponential decrease (time constants: 〈1–30 s) in amplitude during repetitive afferent stimulation by short trains of suprathreshold stimuli. Depressed synaptic responses recovered with an exponential time course (time constants: 10 s-8 min) during presentation of similar short trains of stimuli every 5 or 10 s. Cortical cells recorded extracellularly in cat visual cortex show similar time constants of response decrement during adaptation to moving stripes. Postsynaptic voltage- or ion-regulated conductances and chloride conductances do not appear to be involved in synaptic depression. Input resistance changes and effects of injection of chloride indicate a lack of GABAA receptor-mediated effects. Hyperpolarizing or depolarizing neurons, and pairing polarization with afferent stimulation, also did not affect synaptic depression. This distinguishes these processes from long-term depression and long-term potentiation. Our results suggest that the most likely mechanisms of synaptic depression and adaptation in cortical cells are presynaptic decrease in transmitter release and/or receptor desensitization. Short-term postsynaptic changes may also occur after synaptic depression.

Notes: Abstract Embryonic mouse hippocampal tissue was grafted as tissue blocks to the hippocampal region of adult rats and the effect of two different immunosuppressive treatments compared. Immunosuppression with cyclosporin A, prednisolone and azathioprine or with cyclosporin A alone was compared with placebo treatment. Eight weeks' postgrafting medication with cyclosporin A, prednisolone and azathioprine had resulted in survival of 14 out of 15 grafts (93%), compared with 11 out of 14 (79%) in the group treated with cyclosporin A alone. Only 2 out of 13 grafts (15%) survived in placebo-treated animals. Transplants in the trimedication group displayed distinct cell and neuropil layers and only minimal cellular infiltration by leukocyte common antigen-expressing cells, whereas grafts in cyclosporin A- and placebo-treated groups were densely infiltrated. The results are discussed in relation to the need for extended immunosuppressive and antiinflammatory therapies after intracerebral grafting of histoincompatible tissues.

Notes: Abstract Effects of GABA and glutamate antagonists as well as dopamine agonists and antagonists on the optical responses of neostriatal (Str) slices to local electrical stimulation were examined using a voltage-sensitive dye and a high-speed image sensor. A single local stimulation applied to the Str slices evoked optical responses lasting for 40–80 ms and propagating in every direction up to about 1.5 mm. Bath application of bicuculline methiodide increased the intensity and duration of optical responses, while their spatial response patterns were unchanged. Bath application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) greatly reduced the late part of responses occurring about 4 ms after stimulation, but the early part of responses was unaffected by CNQX. The early part of the response was eliminated by application of tetrodotoxin. Bath application of N-methyl-D-aspartate antagonists, 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid and 2-amino-5-phosphonovaleric acid resulted in only small changes in the optical responses. Bath application of D1 agonist 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5,-tetrahydro-1H-3-benzazepine hydrobromide consistently increased the intensity but decreased the speed of propagation and duration of the optical reponse. Bath application of D2 agonist quinpirole had no effect on the optical response. D1 antagonist SCH 23390 and D2antagonist sulpiride also failed to change optical responses. These results indicate that the early part of the reponse is due to direct activation of the neuronal elements by electrical stimulation, while the late part of the response is due mainly to glutamatergic ex-citatory postsynaptic potentials (EPSPs) mediated by α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors. This study also suggests that dopamine may modulate AMPA/kainate responses through D1 receptors.

Notes: Abstract The aim of the present study was to characterize electrophysiologically neurones in organotypic cultures of the rat ventral mesencephalon and to compare these results with results published for the same neurones in other types of preparation. Intracellular recordings were obtained in 3- to 8-week-old organotypic slice cultures of the ventral mesencephalon prepared from newborn rats. Dopaminergic neurones were distinguished from non-dopaminergic neurones by staining with the autofluorescent serotonin analogue 5,7-dihydroxytryptamine and briefly viewing the preparation with short exposures to ultraviolet (UV) light (365 nm). Short exposures to UV light did not affect the electrophysiological properties. There were no significant differences between dopaminergic and non-dopaminergic neurones with regard to resting membrane potential or action potential threshold and amplitude, and in both types of neurone spontaneous burst activity and glutamatergic excitatory postsynaptic potentials were seen. There were differences in the following parameters, which can be used to distinguish between the two types of neurone. Dopaminergic neurones had broad action potentials (2–9 ms), high input resistance (mean 81 MΩ), were silent or fired spontaneously at a low frequency (0–9 Hz), and no spontaneous GABAA-ergic inhibitory postsynaptic potentials or inward rectification were present. In contrast, non-dopaminergic neurones had fast action potentials (0.6–3.2 ms), low input resistance (mean 32 MΩ), were silent or fired spontaneously at relatively high firing frequency (0–28 Hz), and sometimes inhibitory postsynaptic potentials and inward rectification were seen. In the presence of 1 μM tetrodotoxin and 10 mM tetraethylammonium, Ca2+ spikes could be evoked in both dopaminergic and non-dopaminergic neurones. Dopaminergic neurones in 3- to 8-week-old organotypic slice cultures have a number of distinguishing electrophysiological characteristics similar to those recorded in other types of acute or cultured preparations. However, some intrinsic regulatory mechanisms, namely the slow oscillatory potentials, inward rectification and the K+ current, I A, seem to be missing in the cultured neurones.

Notes: Abstract This longitudinal study, extending over 12 months, assessed the behavioural and biochemical effects of hippocampal sympathetic ingrowth (HSI) into the partially denervated hippocampus. Male Long-Evans rats received fimbria-fornix lesions (FIFO) or sham operations at 90 days of age. At the same time half of the rats from each group sustained bilateral ablation of the superior cervical ganglia (SCGX). A battery of behavioural tests, measuring spontaneous alternation, activity in the open field and home cage, and radial-maze performance, were employed, starting after one very short (16 days) and one extended (216 days) postoperative delay. Neurochemical analyses measuring choline acetyltransferase (ChAT) activity, high-affinity choline (HACU) and noradrenaline uptake by hippocampal synaptosomes (HANU), hippocampal noradrenaline ([NA]), serotonin ([5-HT]) and 5-hydroxyindoleacetic acid ([5-HIAA]) concentrations were carried out in a dorsal, a “middle” and a ventral region of the hippocampus. Lesion of the FIFO induced a significant and enduring deficit in radial-maze performance, in addition to a persistent locomotor hyperactivity. ChAT and HACU were significantly depleted in all three regions of the hippocampus at 12 months, and these deficits were negatively correlated with maze performance. SCGX in the presence of the FIFO lesion significantly reduced [NA] in the middle region of the hippocampus, as compared to SCGX rats, and contributed to a restoration of lesion-induced depletions in [5-HT] and [5-HIAA] in the middle and ventral hippocampal regions, whilst failing to elicit any behavioural changes at either time point. It is concluded that if lesion-induced HSI indeed occurred, as is suggested by neurochemical evidence, it had no effect upon the observed behavioural deficits elicited by transection of the FIFO in the rat.

Notes: Abstract Previous studies suggest that a population of precursor cells from the developing and adult mouse striatum can be expanded in culture using serum-free, N2-supplemented medium and mitogenic factors such as epidermal growth factor (EGF). Here we show that EGF-responsive precursor cells from embryonic rat striatum and mesencephalon can also be expanded in culture, incorporate bromodeoxy uridine (BrDU) and develop into spheres that either adhere to the surface of the culture dish or float freely in the medium. Addition of B27, a medium supplement that increases neuronal survival in primary CNS cultures, resulted in a tenfold increase in the number of proliferating cells in vitro over the first week. The effects of B27-supplemented medium on precursor cell survival were only seen when primary cultures were used, such that dividing cells grown in B27 for 1 week could then be transferred to either B27 or N2 medium and show similar survival and division rates in response to EGF. After 1, 2 or 4 weeks of growth in B27-supplemented medium, dissociated precursor cells from either striatal or mesencephalic cultures could be differentiated when exposed to a poly-1-lysine-coated substrate in serum and EGF-free medium supplemented with B27. These cells then matured into a mixed culture containing neurons (approximately 35% of cells), astrocytes (approximately 44% of cells), and oligodendrocytes (approximately 10% of cells), based on immunocytochemical staining with microtuble-associated protein (MAP2), glial fibriallary acidic protein and galactocerebrosidase. When whole spheres of precursor cells were allowed to differentiate, every one examined was found to generate neurons, astrocytes and oligodendrocytes in similar proportions. Our findings suggest that B27-supplemented medium provides an enhanced environment for dividing and differentiating multi-potential precursor cells over the first week in vitro. This culture system gives an expandable source of well-characterised, multipotential CNS precursors that can be labelled with BrdU and, as such, may prove useful for either differentiation experiments in vitro or as a source of tissue for grafting into the damaged CNS.

Notes: Abstract Status epilepticus (SE) has been related to subsequent development of epilepsy. The present work was aimed at elucidating the relationship between the duration of pilocarpine- (PILO)-induced SE and the subsequent development of epilepsy in rats. The latency for the appearance of the first spontaneous seizure, the frequency of spontaneous seizures, the cell density in the hippocampal formation and the density of supragranular neo-Timmstaining were monitored. At 30 min, 1, 2 and 6 h after the beginning of SE, animals were treated with diazepam plus pentobarbital. In non-treated rats, SE remitted spontaneously. Animals exhibiting 30 min of PILO-induced SE did not develop spontaneous seizures. Hippocampal cell counts and the density of neo-Timm staining in these animals were similar to those observed in control rats. In the other groups longer SE durations were related to: shorter latency for the appearance of the first spontaneous seizure, increased number of the spontaneous recurrent seizures, severe cell loss in the hippocampal formation, or increased supragranular neo-Timm staining. These data suggest that more than 30 min of SE is required to produce hippocampal damage with subsequent synaptic reorganization of the mossy fibre pathway that could account for SRSs observed in the PILO model of epilepsy.

Notes: Abstract The medial preoptic nucleus plays an important role in the regulation of neuroendocrine processes, vegetative functions, sexual behaviour and the modulation of the somatomotoric system. The connections of the medial preoptic nucleus to other areas of the central nervous system are very complex, and the area receives afferents using numerous transmitters and neuropeptides. Previous investigations have shown that this nucleus receives afferents from various brainstem nuclei that also contain somatostatinergic neurons. This study was carried out to investigate if somatostatin-projecting neurons of the brainstem are afferents to the medial preoptic nucleus. This was approached by combining somatostatin-mRNA in situ hybridisation with True Blue retrograde tracing. Our results demonstrate somatostinergic brain-stem projections into the medial preoptic nucleus mainly in the pedunculopontine nucleus and in the nucleus of the solitary tract (50% together). Other important somatostatinergic afferents into the medial preoptic nucleus originate in the cuneiform area, the dorsal parabrachial nucleus and in the lateral reticular nucleus (37% together). Less important are the somatostatinergic projections coming from the central grey, the laterodorsal tegmental nucleus, the locus coeruleus and the nucleus raphe magnus. Considering that these areas are involved in diverse functions such as cardiovascular regulation (nucleus of the solitary tract), transmission of visceral sensibility (dorsal parabrachial nucleus), modulation of the somatomotoric system (pedunculopontine nucleus) and in the regulation of neuroendocrine mechanisms (locus coeruleus), it seems tenable that the somatostatin projections demonstrated here also have a diverse functional quality within the medical preoptic nucleus where they terminate.

Notes: Abstract The distribution of neuronal nitric oxide synthase (NOS) immunoreactivity was examined in rat and rabbit retinas and was compared with the distribution of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase reactivity and vasoactive intestinal peptide (VIP) immunoreactivity. An antibody raised against a C-terminal fragment of a cloned rat cerebellar NOS was used to localise NOS immunoreactivity. NOS immunoreactive cells were not detected in rat retinas at postnatal day 1 or 4, but were seen from postnatal day 7 onwards. NOS immunolabelling was seen in a small population of cells in the proximal inner nuclear layer. Most of the labelled cells had the position of amacrine cells and were seen to send processes into the inner plexiform layer. A few labelled cells were at times also seen in the ganglion cell layer, which are likely to correspond to displaced amacrine cells. The same NOS-labelling pattern was seen in rat and rabbit retinas. NADPH-diaphorase staining was observed in both species, in photoreceptor inner segments, in cells with the position of horizontal cells, in a subset of amacrine and displaced amacrine cells, in large cell bodies in the ganglion cell layer, in both plexiform layers, and in endothelium. Colocalisation of NOS immunoreactivity and NADPH-diaphorase staining was only observed among amacrine cells. However, not all NADPH-diaphorase-reactive amacrine cells were found to be NOS immunoreactive. VIP immunoreactivity was also localised in rat retinas in a subpopulation of amacrine cells, but no colocalisation of NOS and VIP immunoreactivity was observed. Our observations indicate that only amacrine cells contain the NOS form recognisable by the antibody used, and suggest that different isoforms of neuronal NOS may be present in retinal cells. Further, the onset of NOS expression in rat amacrine cells appears to occur independently of neuronal activity.

Notes: Abstract We have previously demonstrated that prolonged low-protein diet leads to irreversible cell loss in the hippocampal formation of the adult rat. Because the extent of the resulting hippocampal synaptic alterations is not well characterized, we studied the contacts between mossy fibers and the dendritic excrescences of CA3 pyramidal cells (MF-CA3 synapses) using quantitative methods. Moreover, we investigated whether rehabilitation from undernutrition would influence the morphology of hippocampal synapses. To address these issues, three groups of adult rats were compared: (a) rats fed with a normal diet for 12 months (control rats); (b) rats treated during the same period with low-protein diet (undernourished rats); and (c) rats undernourished for 6 months and then switched to normal diet for 6 months (recovery rats). Timm staining and electron microscopy were employed to estimate the volume of the mossy fiber system and the number and related quantitative features of MF-CA3 synapses. The volume of the suprapyramidal bundle of the mossy fiber system and its total number of synapses were smaller in undernourished rats than in control and recovery animals. These parameters did not differ between the latter two groups. The size of mossy fiber terminals and dendritic excrescences and the surface area of synapses were smaller in undernourished than in control and recovery groups. Conversely, in recovery animals, the volume of the suprapyramidal bundle of the mossy fiber system, the size of mossy fiber terminals and dendritic excrescences, and the total number and surface area of synapses were similar to those of controls. These findings indicate that, following rehabilitation, the pre- and postsynaptic compartments of MF-CA3 synapses undergo structural alterations which compensate for the neuronal loss induced by undernutrition.

Notes: Abstract Data are presented which support the hypothesis that the pedunculopontine tegmental nucleus serves as an output station for the striatum and, in particular, has a role in the expression of behaviour stimulated from the ventrolateral caudate-putamen, a rodent homologue of the primate putamen. Rats received either bilateral ibotenate or sham lesions in the pedunculopontine tegmental nucleus and bilateral cannulation of the ventrolateral caudate-putamen. Oral motor activities were observed following microinjection of 5.0, 10.0 and 20.0 μg d-amphetamine (and vehicle-only control) into the ventrolateral caudate-putamen. As expected, orofacial behaviours such as biting and licking were observed in sham-lesioned rats following this treatment, but pedunculopontine tegmental nucleus-lesioned rats exhibited an increase in the incidence of these oral motor behaviours at all doses of amphetamine compared with the controls. This increase was the product of changes in the duration and number of times in which they engaged in oral motor behaviours, but not the latency to initiate them. There was no change in the normal oral motor activities associated with grooming. Histological analysis showed that ibotenate lesions destroyed both cholinergic and non-cholinergic neurones in the pedunculopontine tegmental nucleus. These data indicate that loss of the pedunculopontine tegmental nucleus disinhibits oral motor behaviours stimulated from the ventrolateral caudateputamen by d-amphetamine and are discussed in terms of their implications for understanding the relationships between striatal outflow and structures in the pons.

Notes: Abstract Modulation of somatosympathetic reflexes at the spinal cord and the brainstem was studied by administering opioid receptor agonists into the intrathecal space of the lumbar spinal cord and into the subarachnoid space of the cisterna magna in rats anesthetized with α-chloralose and urethane. Somatocardiac sympathetic A-and C-reflexes were elicited by electrical stimulation of myelinated (A) and unmyelinated (C) afferent fibers of the tibial nerve, respectively. Intrathecal administration of the μ-opioid receptor agonist DAMGO selectively depressed the C-reflex in a dose-dependent manner (minimum effective dose 10 ng), whereas the intrathecal injection of the δ-opioid receptor agonist DPDPE and the κ-opioid receptor agonist U-50,488H only at doses of 10 μg and 100 μg, respectively, led to a significant depression of the C-reflex. Injection of DAMGO into the cisterna magna enhanced both A-and C-reflexes in a dose-dependent manner (minimum effective dose 1 ng). The administration of neither DPDPE nor U-50,488H into the cisterna magna affected A-or C-reflexes. It is concluded that the activation of μ-opioid receptors is mainly or exclusively responsible for suppressing somatosympathetic C-reflexes at the spinal cord and for enhancing them at the brainstem.

Notes: Abstract The effect of subthalamic nucleus (STh) lesion on apomorphine-induced rotational behaviour and unit activity of substantia nigra pars reticulata (SNr) neurons was studied in normal, sham-control and unilateral 6-OHDA-lesioned rats [SN pars compacta (SNc)-lesioned]. In the latter, contraversive rotational behaviour was greatly reduced by an additional ipsilateral STh lesion. A moderate ipsiversive rotation was observed in rats with a single STh lesion. Concurrently, SN unit extracellular recordings were performed in age-matched normal rats, sham-controls for both lesions, STh-lesioned rats, SNc-lesioned rats, and SNc-lesioned rats with an ipsilateral STh lesion (SNc+STh-lesioned). Pars reticulata neurons had a higher mean firing rate in SNc-lesioned rats than in control rats. Furthermore, 68% of SNr neurons in SNc-lesioned rats had a tonic discharge pattern (against 92.3% in control rats) and 32% a mixed or bursting pattern. After STh lesion, a clear decrease in SNr firing rate was observed in SNc-lesioned rats. Moreover, STh lesion improved interspike interval regularity and decreased the occurrence of bursting patterns. In rats with a single STh lesion, the firing rate was no different from that of the sham-controls but the discharge pattern was more regular. These data show that STh lesion decreased apomorphine-induced rotational behaviour in dopamine-depleted animals. This effect could be related to the suppression of the exitatory effect of STh efferents on the SNr neurons. STh lesion both counterbalanced the increased activity of SNr neurons and regularized their discharge pattern.

Notes: Abstract A combination of retrograde cell body labeling and immunohistochemistry was employed to elucidate how oxytocinergic fibers make contact with sympathetic preganglionic neurons (SPNs) in the rat spinal cord from T1 to T4. SPNs were labeled retrogradely using cholera toxin subunit B (CTb) or horseradish peroxidase-conjugated CTb. Oxytocin-immunoreactive (ir) fibers were found in the intermediate zone, including the sympathetic preganglionic subnuclei. In the central autonomie nucleus and the intercalated nucleus, brown-stained oxytocin-ir varicosities or terminals were frequently observed to stud black-stained dendrites of SPNs. Electron microscopical observations showed that oxytocin-ir terminals form synapses with dendrites or soma of the sympathetic preganglionic neurons. The terminals contained numerous small clear round vesicles and a few large, cored vesicles. These results clearly show that a large proportion of SPNs are innervated by oxytocin-containing fibers. The origin of these fibers is discussed, and it is concluded that they are probably descending fibers from the paraventricular nucleus of the hypothalamus.

Notes: Abstract The objective of the present study was to investigate the effects of chronic activation of dopamine D2 receptors on the development of grafted fetal rat mesencephalic dopaminergic neurons. Therefore, unilaterally 6-hydroxydopamine — lesioned rats received intrastriatal mesencephalic cell suspension grafts and were subsequently chronically treated with the selective dopamine D2 receptor agonist LY 171555 (Quinpirole). After treatment for 6 consecutive weeks, the rats were processed for tyrosine-hydroxylase immunocytochemistry to assess the survival and outgrowth from grafted dopaminergic neurons. Morphological analysis revealed that, like the volume and morphology of the graft, neither the number nor the cell area of grafted dopaminergic neurons was significantly different between vehicle- and LY 171555-treated animals. To obtain a quantitative estimate of the graft-derived dopaminergic reinnervation, a computerized image analysis system was used. Using this procedure, which was based on the densitometric measurement of tyrosine hydroxylase immunoreactivity in the area adjacent to the grafted tissue, it was found that the extent of graft-derived outgrowth also appeared to be un-affected upon chronic treatment with LY 171555. It is concluded that long-term concurrent administration of a dopamine D2 receptor agonist for 6 consecutive weeks does not impair the survival and outgrowth of grafted rat fetal mesencephalic dopaminergic neurons.

Notes: Abstract The effects of microiontophoretic 5-hydroxytryptamine (5-HT) on the firing rate of red nucleus (RN) neurons were studied in urethane-anesthetized rats. The background discharge rate of almost all the neurons tested (97%) was modified by 5-HT, and generally increased (89%). Responses were dose dependent. Twenty-three percent of the excitatory responses were preceded by a short inhibitory phase. No significant difference in the effect of 5-HT was found between those RN neurons that project to the spinal cord and those that do not. The excitatory responses to 5-HT were blocked or greatly reduced by the 5-HT antagonists methysergide and ketanserin, and were even reversed in some cases. The 5-HT2/5-HT1A antagonist spiperone, in small doses, also blocked the transient inhibitory phases in addition to the excitatory effects. In RN neurons exhibiting a short-lasting inhibition in the response to 5-HT, the 5-HT1A agonist 8-hydroxy-2(di-n-propyl-amino)tetralin (8-OH-DPAT) induced inhibitory effects. These results support the hypothesis that 5-HT exerts control throughout the RN, mostly by acting on 5-HT2 receptors. Furthermore, an influence of this amine on the electrical activity of small groups of RN neurons by 5-HT1A receptors, and eventually by different mechanisms, appears probable. The functional significance of serotoninergic control of RN neuronal activity is discussed.

Notes: Abstract This study investigated astroglial responses after focal cerebral ischemia in the rat cortex induced by photothrombosis. Astrocyte activation was studied at various time points by immunocytochemistry for glial fibrillary acidic protein (GFAP) and vimentin (VIM). We found a dual astrocytic response to focal ischemia: In the border zone of the infarct, GFAP-positive astrocytes were present within 2 days and persisted for 10 weeks. These astrocytes additionally expressed VIM. Remote from the ischemic lesion, cortical astrocytes of the entire ipsilateral hemisphere transiently expressed GFAP, but not VIM, beginning on day 3 after photothrombosis. This response had disappeared on day 14. By recording DC potentials, five to seven spreading depressions (SD) could be detected on the cortical surface during the first 2 h after photothrombosis. Treatment with MK801, a non-competitive NMDA-receptor antagonist, completely abolished SD and remote ipsilateral astrocytic activation, while the reaction in the border zone of the infarct remained unchanged. Functionally, persistent astrocytosis around the infarct might be induced by leukocyte-derived cytokines, while NMDA-receptor-mediated SD might cause remote responses.

Notes: Abstract The withdrawal reflex pathways to hindlimb muscles have an elaborate spatial organization in the rat. In short, the distribution of sensitivity within the cutaneous receptive field of a single muscle has a spatial pattern that is a mirror image of the spatial pattern of the withdrawal of the skin surface ensuing on contraction in the respective muscle. In the present study, a search for neurones encoding the specific spatial input-output relationship of withdrawal reflexes to single muscles was made in the lumbosacral spinal cord in halothane/nitrous oxide-anaesthetized rats. The cutaneous receptive fields of 147 dorsal horn neurones in the L4-5 segments receiving a nociceptive input and a convergent input from A and C fibres from the hindpaw were studied. The spatial pattern of the response amplitude within the receptive fields of 118 neurones was quantitatively compared with those of withdrawal reflexes to single muscles. Response patterns exhibiting a high similarity to those of withdrawal reflexes to single muscles were found in 27 neurones located in the deep dorsal horn. Twenty-six of these belonged to class 2 (responding to tactile and nociceptive input) and one belonged to class 3 (responding only to nociceptive input). None of the neurones tested (n=20) with reflex-like response patterns could be antidromically driven from the upper cervical cord, suggesting that they were spinal interneurones. With some overlap, putative interneurones of the withdrawal reflexes to the plantar flexors of the digits, the plantar flexors of the ankle, the pronators, the dorsiflexors of the ankle, and a flexor of the knee, were found in succession in a mediolateral direction. It is concluded that neurones that are able to encode the specific spatial input-output organization of the withdrawal reflexes to single muscles do exist in the deep dorsal horn. Such reflex encoders appear to have a “musculotopic” organization. A hypothesis of the organization of the withdrawal reflex system is presented.

Notes: Abstract In brainstem-spinal cord preparations isolated from newborn rats, intrinsic burst-generating properties of preinspiratory (Pre-I) neurons in the rostral ventrolateral medulla, which have been suggested to be primary respiratory rhythm-generating neurons, were studied by “perforated” whole-cell recordings using the antibiotic nystatin. Nystatin causes small pores to be formed in the cells, through which pass small monovalent ions. For blockade of chemical synaptic transmission, perfusate Ca2+ concentration was lowered to 0.2 mM and the Mg2+ concentration was increased to 5 mM. In Iow-Ca2+, high-Mg2+ solution (referred to here as “low Ca”), 10 of 55 Pre-I neurons generated rhythmic bursts (burst type), 14 fired tonically (tonic type), and 31 were silent (silent type). Burst-type neurons showed periodic depolarization of 5–12 mV in low Ca, at a rate of 12±6.5/min. Hyperpolarization of the membrane caused decrease in or disappearance of the periodic depolarization and prolongation of the cycle period. Thus, the burst generations were voltage dependent. The firing frequency of tonictype neurons was 2.3±1.6 Hz and was decreased by hyperpolarization. In 6 of these neurons, the firing patterns changed to burst patterns during continuous hyperpolarization. Membrane depolarization by continuous outward current injection into some silent-type neurons (3 of 11 tested) induced bursting activity. Activity of C4 and Pre-I neurons was completely silent with 0.1–1 μM tetrodotoxin (TTX) added to the standard perfusate. In low Ca, burst-type neurons (n=3) were also silent with 1 μM TTX perfusion. Inspiratory neurons either became silent (n=4) or fired tonically (n=1) in low Ca. The present study by “perforated” whole-cell recordings confirmed that some Pre-I neurons possess intrinsic burst-generating properties, which were not attributable to phasic synaptic inputs.

Notes: Abstract The present study was designed to examine the effects of an intraspinal transplantation of embryonic brainstem neurons on fictive motor patterns which can develop in hindlimb nerves of adult chronic spinal rats. Seventeen adult rats were spinalized at T8-9 level and, 8 days later, a suspension of embryonic cells obtained either from the raphe region (RR, n=8) or from the locus coeruleus (LC, n=9) was injected caudally (T12–13) to the cord transection. Eight control animals (control rats) were spinalized and injected with vehicle under the same conditions. One to three months later, the animals were decorticated and fictive motor patterns were recorded in representative hindlimb nerves. The data revealed that both control and grafted spinal rats could exhibit two distinctly different fictive motor patterns, one which could be associated with stepping and the other with hindlimb paw shaking. They further showed that following transplantation of embryonic RR or LC neurons the excitability of the spinal stepping generator was increased, whereas that of the spinal neural circuits which generate hindlimb paw shaking was not significantly affected. A histological analysis performed on the spinal cord segments below the transection revealed complete absence of serotonin and noradrenaline immunoreactivity in control spinal animals and, in both types of grafted rats, an extensive monoaminergic reinnervation with synaptic contacts between monoaminergic transplanted neurons and host interneurons and/or motoneurons. The possible mechanisms by which grafted monoaminergic neurons can influence the spinal motor networks are discussed.

Notes: Abstract Calcium currents in CA1 neurons from rat hippocampus were studied with the whole-cell, patchclamp technique. Under control conditions high-voltage-activated (HVA) calcium currents activated from membrane potentials of -80 mV and -40 mV underwent “run-down”. The rate of run-down of the current activated from -40 mV was significantly attenuated by inclusion of the G-protein activator NaF (1 mM) in the pipette and also irreversibly attenuated by brief batch application of NaF (10 mM). This effect was significantly reduced by inclusion of high (10 mM) ethyleneglycoltetraacetate (EGTA) concentrations in the pipette, suggesting an involvement of calcium-dependent processes. It is suggested that activation of guanine nucleotide-binding proteins by NaF leads to a long-lasting attenuation of HVA calcium current run-down in hippocampal CA1 cells.

Notes: Abstract Adrenal medullary allografts, as well as other monoaminergic tissues, have been demonstrated in our laboratory to increase antidepressive activity when transplanted into the frontal neocortex of rats. Refinement in the optimal parameters for xenograft viability has indicated that isolated bovine chromaffin cells may be an improved source of graft donor tissue. The aim of the present study was to determine whether isolated bovine chromaffin cell grafts to the rat frontal neocortex could provide an alternative source of catecholamines for antidepressant activity. Isolated bovine chromaffin cells, isolated bovine fibroblasts, or an equal volume of vehicle were unilaterally implanted into the right or left frontal cortex or right visual cortex. All rats were assessed before and 6 weeks after transplantation using the forced swimming test, a popular measure of antidepressant activity. Bovine chromaffin cell grafts in either the right or left frontal cortex produced significant increases in antidepressant activity compared to grafts of bovine fibroblasts and sham-operated or nontransplanted rats. In contrast, bovine chromaffin cells transplanted to the visual cortex did not affect antidepressant activity. Bovine fibroblast grafts in the frontal cortex also induced slight increases in antidepressant activity, although significantly less than chromaffin cell grafts. Morphological analysis revealed robust survival of tyrosine hydroxylase-positive chromaffin cells that retained their in situ ultrastructure and occasionally formed synaptic connections with the host parenchyma. These results suggest that xenografted isolated bovine chromaffin cells can provide a viable source of catecholamines for antidepressive activity.

Notes: Abstract We investigated the expression of platelet-derived growth factor (PDGF) and its receptors in rat facial nuclei following axotomy by in situ hybridization and immunohistochemistry. Facial nuclei were examined on days 3, 6, 12, 19 and 26 postoperatively (p.o.). Strong immunoreactivity for PDGF was found in facial neurons and surrounding astrocytes on the ipsilateral side of the brainstem already after 3 days p.o. and persisted at a high level until day 26 p.o. in rats with a facial nerve cut injury. After crushing of the facial nerve, a similar increase was seen in PDGF immunoreactivity which, however, decreased after day 19 p.o., when reinnervation had occurred. Reactive gliosis appeared on the operated side and was confirmed by an increase in intensity of GFAP staining. The kinetics of PDGF A-chain mRNA expression corresponded to the PDGF immunoreactivity, whereas the B-chain mRNA was present only in the neurons. The PDGF α-receptor immunoreactivity as well as the mRNA were detected in scattered glial cells. The density of the PDGF α-receptor mRNA expressing glial cells was higher on the injured side, but the intensity of the expression per cell did not change after axotomy. An increase in PDGF β-receptor immunoreactivity was seen in the ipsilateral facial nuclei after 3–6 days p.o., however, the increase in the mRNA could not be detected. The staining persisted until day 26 p.o., when transected facial neurons showed heavier staining than those that had been crushed. Furthermore, both mRNA and protein of the β-receptor were expressed in the blood vessels after 3–6 days p.o., increasing with time. These results imply a role for PDGF in the regeneration process following nerve injury.

Notes: Abstract Development of the central olfactory system was studied in the rat with an electron microscope at three main structures: the olfactory bulb, the lateral olfactory tract, and the primary olfactory cortex (the piriform cortex). As a parameter of development, the synaptic density was examined quantitatively in the bulbar glomerulus and layer Ia (termination of bulbofugal fibers) of the piriform cortex, which are the key stations of the olfactory pathway. The synaptic densities in the glomerulus and those in layer Ia were 5.7% and 4.6% on embryonic day 19, 15.8% and 12.5% on postnatal day (P) 0, and 57.3% and 37.2% on P10, as compared with the adult (100%). As another parameter of development, the density of myelinated axons in the lateral olfactory tract was examined quantitatively. The densities of myelinated axons in the tract were 0% on P5, 15.1% on P10, and 73.5% on P21 of the adult density. Maturation in the tract was still progressing, even at P21, in terms of bundle formation and the thickness of myelin sheaths. The results show that synaptogenesis in the bulbar glomerulus is followed by synaptogenesis in layer Ia of the piriform cortex, and that myelination in the lateral olfactory tract occurs over a prolonged period, even in the stages after P21.

Notes: Abstract The colocalization of two calcium-binding proteins, parvalbumin (PV) and calbindin-D28k (CaB), which have been reported to be markers of specific subpopulations of neurons in the central nervous system, with the inhibitory amino acid neurotransmitter γ-amino-butyric acid (GABA) was investigated in neurons of laminae I–IV of the subnucleus caudalis of the rat spinal trigeminal nucleus by using post-embedding immunocytochemical methods. Cells immunoreactive for PV, CaB, and GABA were found in all four laminae of the subnucleus caudalis. A substantial proportion of PV-immunoreactive perikarya were also stained for GABA in laminae II and III (44.8% and 39.8%, respectively). However, the majority of PV-containing neurons in laminae I and IV (100% and 86%, respectively), as well as CaB-immunoreactive cells in all four laminae (98.4%), were GABA-negative. These results show that, in contrast to higher brain centers, PV-, CaB-, and GABA-immunoreactive perikarya represent significantly different populations of neurons in the subnucleus caudalis of the rat. In the light of the present findings, the differences in the neurochemical properties of the subnucleus caudalis of the spinal trigeminal nucleus and the spinal dorsal horn are also discussed.

Notes: Abstract Disruption of neuromuscular contact by nerve-crush during the early postnatal period causes increased activity and abnormal reflex responses in affected motoneurons, but such changes are not found after nerve-crush in adult animals. We found previously that neonatally lesioned cells develop an abnormal dendritic field, which may explain the functional changes. Here we have studied the dendritic morphology of the same motoneuron pool after nerve-crush at maturity in order to correlate the observed alterations in morphology with physiological findings. One to two months after sciatic nerve-crush in adult animals, motoneurons supplying the extensor hallucis longus muscles of the rat were retrogradely labelled with cholera toxin subunit-B conjugated to horseradish peroxidase. The dendritic tree of labelled cells was then analysed. Following adult nerve-crush, the dendritic tree of the motoneurons was smaller but did not display the localised increase in dendritic density seen after neonatal nerve-crush. These findings support the view that such specific morphological changes contribute to the physiological abnormalities seen only after neonatal nerve injury.

Notes: Abstract Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-β superfamily, promotes the survival, morphological differentiation, and high-affinity dopamine (DA) uptake of cultured nigral DA neurons. In order to test potential methodology for peptide delivery in vivo, GDNF-containing fibrin glue balls (8 μg/ball) were incorporated with pieces of fetal ventral mesencephalon (E15) and transplanted into the anterior chambers of sympathetically denervated adult rats. Five weeks after grafting, the numbers of TH-positive neurons and the nerve fiber density were significantly higher in the ventral mesencephalic grafts treated with GDNF-containing glue balls than in those treated with vehicle. In addition, the laminin and GFAP immunoreactivities were similar between the two groups. These data support the concept that GDNF is a potent trophic factor for DA neurons in vivo and suggest that fibrin glue may provide a unique and safe means to permit prolonged delivery of trophic molecules to CNS tissues.

Notes: Abstract By combining anterograde and retrograde axonal tracing with AChE histochemistry, we demonstrate the sources of AChE-positive afferents to embryonic neocortex, the pathways they use, their time of arrival into cortex, and their initial invasion of the cortical plate. Acetylcholinesterase (AChE) is expressed by two populations of cortical afferents: AChE is permanently present in basal forebrain fibers and has been reported to be transiently localized in axons of the principal sensory thalamic nuclei over the first few postnatal weeks beginning at the middle of the first week. We first detect AChE-positive afferents histochemically in neocortex on embryonic day seventeen (E17) and determine that they arise from the principal sensory thalamic nuclei. AChE histochemistry labels the entire length of developing thalamocortical axons, including their growth cones and branches. These AChE-positive afferents enter the neocortex by the internal capsule and take an intracortical pathway centered on the subplate layer. As soon as these axons are detected, some have already begun to extend AChE-positive collateral branches superficially toward the cortical plate. By E19, a few collaterals have entered the deep part of the cortical plate and by E21 have densely invaded all but its most superficial undifferentiated part. AChE-positive afferents from basal forebrain structues reach the neocortex by three routes: the external capsule, the internal capsule, and the cingulate bundle. Among basal forebrain components, only the substantia innominata and nucleus basalis of Meynert reach the cortex by the internal capsule. Afferents from these two sources reach neocortex on E18, but are a very minor component of the total population of AChE-positive afferents at this age. Afferents from other basal forebrain components do not reach neocortex until several days later. The spatial and temporal patterns of AChE expression in developing thalamocortical axons indicate that it is useful for delineating their innervation of the primary sensory areas of embryonic neocortex, and suggest that AChE may function in axon extension and cortical differentiation.

Notes: Abstract We examined whether the NMDA class of excitatory amino acid receptors contribute to synaptic transmission in the pathway connecting the medial geniculate body (MGB) with the lateral nucleus of the amygdala (LA) using extracellular single unit recordings and microiontophoresis. Cells were identified in LA on the basis of responsivity to electrical stimulation of the MGB. For each cell, a level of current was found for the iontophoretic ejection of the NMDA antagonist AP5 that blocked responses elicited by iontophoresis of NMDA, but had no effect on responses elicited by AMPA. Iontophoresis of AP5 with this level of current blocked the excitatory response elicited by MGB stimulation in most cells tested. Microinfusion of AP5 (25, 50, or 100 μM) also blocked the responses. Additional studies tested individual cells with both AP5 and the AMPA antagonist CNQX and showed that blockade of either NMDA or AMPA receptors interferes with synaptic transmission. Finally, iontophoretic ejection of either AP5 or CNQX blocked short-latency (〈25 ms) responses elicited in LA by peripheral auditory stimulation. Together, these results suggest that the synaptic evocation of action potentials in the thalamo-amygdala pathway depends on both NMDA and non-NMDA receptors. We hypothesize that non-NMDA receptors are most likely required to depolarize the cell sufficiently to remove the blockade of NMDA channels by magnesium and NMDA receptors are required to further depolarize the membrane to the level required for action potential generation.

Notes: Abstract Infrared images of the skull surface were obtained in urethane-anesthetized rats and gerbils before, during and after mechanical stimulation of the face and mystacial vibrissae on one side. Areas of increased temperature on the skull, localized mainly over the face area of the primary somatosensory cortex contralateral to the side of stimulation, appeared within 4–5 s after the onset of stimulation. Rarely, such temperature change was recorded bilaterally. Temperatures did not remain high on the intact skull in rats, but fell to baseline within minutes after stimulus onset regardless of stimulus duration. In rats in which the skull had been thinned and in gerbils with intact skull, temperatures remained elevated during the course of stimulation. We were unable to resolve the activation of individual vibrissae.

Notes: Abstract Electrophysiological properties of neurons as the basic cellular elements of the central nervous system and their synaptic connections are well characterized down to a molecular level. However, the behavior of complex noisy networks formed by these constituents usually cannot simply be derived from the knowledge of its microscopic parameters. As a consequence, cooperative phenomena based on the interaction of neurons were postulated. This is a report on a study of global network spike activity as a function of synaptic interaction. We performed experiments in dissociated cultured hippocampal neurons and, for comparison, simulations of a mathematical model closely related to electrophysiology. Numeric analyses revealed that at a critical level of synaptic connectivity the firing behavior undergoes a phase transition. This cooperative effect depends crucially on the interaction of numerous cells and cannot be attributed to the spike threshold of individual neurons. In the experiment a drastic increase in the firing level was observed upon increase of synaptic efficacy by lowering of the extracellular magnesium concentration, which is compatible with our theoretical predictions. This “on-off” phenomenon demonstrates that even in small neuronal ensembles collective behavior can emerge which is not explained by the characteristics of single neurons.

Notes: Abstract While sustained retinal slip is assumed to be the basic conditioning stimulus in adaptive modifications of the vestibulo-ocular reflex (VOR) gain, several observations suggest that eye motion-related signals might also be involved. We oscillated pigmented rats over periods of 20 min around the vertical axis, at 0.3 Hz and 20°/s peak velocity, in different retinal slip and/or eye motion conditions in order to modify their VOR gain. The positions of both eyes were recorded by means of a phase-detection coil system with the head restrained. The main findings came from the comparison of two basic conditions — including their respective controls — in which one or both eyes were reversibly immobilised by threads sutured to the eyes. In the first condition the animals were rotated in the light with one eye immobilised and the other eye free to move but covered. Rotation in the light in this open-loop condition immediately elicited high-gain compensatory eye movements of the non-impeded, covered eye. At the end of this training procedure, the VOR gain increased by 42.3%. In the second condition, both eyes were immobilised and one eye was covered. The result was an increase in the VOR gain of 26.3%. These two conditions were similar as to the visuo-vestibular drive during the exposure, but different as to the resulting — and allowed — eye motion, showing that the condition where the larger eye movements occurred yielded the larger VOR gain change. Our data support the idea proposed by Collewijn and Grootendorst (1979, p. 779) and Collewijn (1981, p. 146) that “[retinal] slip and eye movements seem to be relevant signals for the adaptation of the rabbit's visuo-vestibular oculomotor reflexes”. Our data also suggest that sensory information related to eye movements, more likely than efference copy, is the coding signal for eye movement which combines with the retinal slip signal to generate adaptive changes of the VOR.

Notes: Abstract The flow of information in the sensorimotor cortex may determine how somatic information modulates motor cortex neuronal activity during voluntary movement. Electrophysiological recordings and neuroanatomical tracing techniques were used to study the connections between the primary somatosensory cortex (SI) and the vibrissal representation of the primary motor cortex (MI) in rodents. Intracortical microstimulation (ICMS) was applied to the vibrissal region of the motor cortex to identify a site from which stimulation evoked movements of the vibrissae. Movements of only a single whisker were evoked by applying low-intensity stimulating current to particular locations within MI. A single injection of either horseradish peroxidase (HRP) or biocytin was made at the stimulus site in each animal, to retrogradely label cells in the somatosensory cortex. Receptive field (RF) responses were recorded from neurons in the barrel cortex to identify the sensory cortex representation of the same whisker that responded to ICMS. The site at which neurons responded predominately to manual stimulation of this particular vibrissa was marked by a small electrolytic lesion. The projection from the somatosensory cortex to the identified whisker representation in the motor cortex was determined by mapping the location of labeled neurons in tissue sections processed for either HRP or biocytin. The relationship of the labeled cells in SI to the barrel structures was determined from adjacent sections that were stained for cytochrome oxidase. In all cases, the barrel column associated with the relevant whisker contained labeled cells. Surrounding barrels also contained labeled cells, although fewer in number. Very few labeled cells were found in non-contiguous barrels. These results show that the SI to MI projection is somatotopically arranged, such that the sensory cortex representation of a whisker is morphologically connected to the motor cortex representation of the same whisker. Thus, sensory information is relayed to MI from the relevant whisker region in SI. Adjacent whisker regions also appear to relay somatic input, but presumably to a lesser degree. A second group of animals received single small injections of the anterograde tracer, Phaseolus vulgaris leucoagglutinin, to an electrophysiologically identified whisker representation in the sensory cortex. A single narrow column of labeled fibers was found in the motor cortex following such injections. Thus, the sensory cortex appears to relay somatic information from the vibrissae to restricted regions of the motor cortex in a somatotopically organized manner. Furthermore, the stimulus-evoked whisker movements suggest that certain features of the output map of the motor cortex are discretely organized. These input/output relationships suggest that complex information processing within the vibrissal sensorimotor cortex is highly organized.

Notes: Abstract The tonic discharge of 71 medial vestibular nucleus (MVN) neurones was recorded in slices of the dorsal brainstem of young adult rats. Bath application of histamine caused a dose-related excitation in 59 of the 71 cells (83%), the remaining 12 (17%) being unresponsive. Dimaprit, a selective H2 agonist, also caused excitation in all 20 cells tested. The histamine-induced excitation and the response to dimaprit were antagonised by the selective H2 antagonist ranitidine, confirming that the H2 subtype of histamine receptor is involved in mediating the effects of histamine on these cells. Triprolidine, a selective H1 antagonist, also antagonised the excitation caused by histamine, at a concentration (0.3 μM) which left the H2 receptor-mediated response to dimaprit unchanged. Thus the excitatory effects of histamine on MVN cells in the rat involve two components mediated through H1 and H2 receptor-linked mechanisms, respectively. Betahistine, a weak H1 agonist and H3 antagonist, had little excitatory action when applied on its own, but significantly reduced the excitation caused by histamine when the two drugs were applied together. The effects of betahistine were consistent with a partial-agonist action at H1 receptors on MVN cells, reducing the excitatory responses to histamine presumably by occupying these receptor sites in competition with the exogenously applied neurotransmitter. This partial-agonist action of betahistine may be an important part of its mechanism of action in the symptomatic treatment of vertigo and motion sickness, since it is likely to occur not only in the MVN but also in many brain regions, including the thalamus and cortex, which express H1 receptors and which are innervated by the hypothalamic histaminergic system. Thus the effectiveness of betahistine and other anti-H1 drugs against motion sickness may be explained by their action in reducing the effects of the excess histamine release induced in such conditions in various brain areas, including the MVN.

Notes: Abstract Stimulation of the caudal raphe nuclei alters visceral functions. The caudal raphe nuclei project to the nucleus of the solitary tract, which receives the central terminations of vagal afferents and plays an important role in the central integration of autonomic activities. The caudal raphe nuclei also project to the somatic and preganglionic autonomixc motoneurons of the spinal cord. Diamidino yellow was injected into the nucleus of the solitary tract, and fast blue was injected into either the cervical, thoracic, or lumbar spinal cord. Large numbers of double-labeled neurons were present within the caudal raphe nuclei and the adjacent reticular formation of the medial tegmental field. This observation documents that individual raphespinal and reticulospinal neurons project an axon collateral to the nucleus of the solitary tract. These data demonstrate the anatomic substrate for global modulation of the autonomic motoneuron pool by the caudal raphe nuclei.

Notes: Abstract To establish a new behavioral animal model of excitotoxicity, we injected adult rats intraocularly with a single dose of 2, 20, or 100 nmol of N-methyl-d-aspartate (NMDA). We quantified visual impairment by plotting the size of the visual field in which the rats successfully oriented towards a small, moving target. In comparison to the saline-injected (contralateral) control side, the side injected with 2 nmol of NMDA was not significantly impaired. When injected with higher doses, the rats were nearly blind immediately after surgery, with only about 20% (20 nmol NMDA) or 10% (100 nmol NMDA) of residual vision. Within about 3 weeks, however, visual performance returned to near-normal levels. Simultaneous intraocular administration of a non-competitive NMDA-antagonist, MK-801 (1 nmol), resulted in complete behavioral protection. NMDA administration led to a dose-dependent loss of cells within the ganglion cell layer, as assessed in whole-mounted retinae which were retrogradely labelled with horseradish peroxidase (HRP). Whereas 2 nmol of NMDA led to the loss of about 30% of retinal ganglion cells (RGCs), at higher NMDA doses only 13% of the RGCs survived. After the injection of 20 nmol of NMDA, large-diameter RGCs (〉22 μm) survived the lesion to a greater extent than small diameter cells (8–21 μm); at 100 nmol cells of all diameters were equally affected. The number of Nissl-stained cells with small diameters (〈11 μm), presumed to be displaced amacrine cells, was also affected by NMDA, although to a lesser degree. Analysis of behavioral performance (vision score) and the number of cells in the retina revealed a correlation of r=0.76 between visual performance and the number of HRP-filled RGCs immediately after surgery. Lower correlations were found between visual performance and cells stained with Nissl of diameters smaller than 11 μm (presumably displaced amacrine cells) or larger than 11 μm (presumed RGCs without retinofugal connections; r=0.55 and r=0.58, respectively). Because of the spontaneous recovery of vision, all correlations declined to values near 0 after 3 weeks. Thus, despite a dramatic loss of RGCs following NMDA administration, visual deficits recover significantly in adult rats within 2–3 weeks.

Notes: Abstract The present experiment assessed the locomotor response to a low dose (1 mg/kg) of systemic gemd-amphetamine in rats with cytotoxic lesions of the retrohippocampus (entorhinal and extra-subicular cortices), compared with vehicle-operated shams and unoperated controls. Under spontaneous and saline conditions, both the sham and the lesioned animals were more active than unoperated controls, and they did not differ from each other. Systemic gemd-amphetamine produced increased locomotion in all groups, but this effect was potentiated in animals with retrohippocampal lesions; two control groups did not differ from each other in their response to the drug. The present results are consistent with the suggestion that cell loss within the retrohippocampal region could affect the functional response of nucleus accumbens to amphetamine. The results are discussed in terms of the interaction between the retrohippocampus and nucleus accumbens in the control of mesolimbic dopamine release and the possible implications for schizophrenia.

Notes: Abstract Two main subclasses of ionotropic receptors for excitatory amino acids (EAAs), N-methyl-d-aspartate (NMDA) receptors and non-NMDA receptors, are involved in neurotransmission in the cortex of mammals. To examine whether EAAs are transmitters at the cortical taste area (CTA) in rats and to elucidate which types of the two ionotropic receptors operate at these synapses, we studied the effects of microiontophoretic administration of EAA antagonists on the responses of 64 taste cortical neurons to four basic taste stimuli in urethane-anesthetized rats. Both d-2-amino-5-phosphonovalerate (APV), a selective antagonist for NMDA receptors, and 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), a selective antagonist for non-NMDA receptors, suppressed most of the taste responses. The percentage of neurons suppressed by APV (70.3%) was almost the same as that suppressed by CNQX (64.1%). These suppressive effects were independent of the effects of background discharges during the prestimulus, water-rinsing period. The percentage of neurons suppressed by the antagonists did not differ between any pairs of taste stimuli. The number of neurons possessing both receptors was larger in the granular insular area (area GI), one of the two CTAs, than in the dysgranular insular area (area DI). In addition, taste responses were suppressed by CNQX or by both APV and CNQX in area GI in a significantly larger number of layer V neurons than in area DI. The present results indicate that normal excitatory transmission of taste afferents in the CTA in rats was mediated by both NMDA and non-NMDA receptors. The finding that a large fraction of neurons in the CTA in rats mediated taste information through NMDA receptors in normal transmission might be related to the higher potency of the plasticity observed in the CTA.

Notes: Abstract The effects of traumatic brain injury (TBI) on hippocampal long-term potentiation (LTP) and cellular excitability were assessed at postinjury days 2, 7, and 15. TBI was induced using a well-characterized central fluid-percussion model. LTP of the Schaffer collateral/commissural system was assessed in vivo in urethane-anesthetized rats. Significant LTP of the population excitatory postsynaptic potential (EPSP) slope was found only in controls, and no recovery to control levels was observed for any postinjury time point. Four measurement parameters reflecting pyramidal cell discharges (population spike) indicated that TBI significantly increased cellular excitability at postinjury day 2: (1) pretetanus baseline recording showed that TBI reduced population spike threshold and latency; (2) tetanic stimulation (400 Hz) increased population spike amplitudes to a greater degree in injured animals than in control animals; (3) tetanus-induced population spike latency shifts were greater in injured cases; and (4) tetanic stimulation elevated EPSP to spike ratios (E-S potentiation) to a greater degree in injured animals. These parameters returned to control levels, as measured on postinjury days 7 and 15. These results suggest that TBI-induced excitability changes persist at least through 2 days postinjury and involve a differential impairment of mechanisms subserving LTP of synaptic efficacy and mechanisms related to action potential generation

Notes: Abstract An in vitro arterially perfused medulla preparation of 3- to 8-week-old rats is described in which synchronous rhythmic activity (frequency 4.5 ± 1.7 cycles/min, burst duration 3.1 ± 1.1 s, n = 40) was recorded from hypoglossal (XII), vagal (X), or spinal (C1–2) nerves and from different classes of neurons in the region of the ventral respiratory group (VRG). Stimulation of dorsal X nerve rootlets produced a reversible blockade of rhythmic activity. Under steady-state conditions, tissue oxygen (pO2) in the VRG (depth of 600–1600 μm below the ventral surface) fell from 180 to 40 mmHg. Extracellular K+ activity (aKe) in the VRG was about 0.3 mM higher, calcium concentration ([Ca]e) did not differ, and pH (pHe) was about 0.27 units lower than in the perfusion or superfusion solution (with an aKe of 2.2 mM, a [Ca]e of 1.5 mM and a pHe of 7.4). During inspiratory XII nerve discharges, rhythmic increases of aKe by up to 0.8 mM were detected in the VRG. Perfusion of N2-gassed hypoxic solutions (5–10 min) resulted in a tissue anoxia of the VRG and a reversible cessation of rhythmic activity after 2–7 min. Such anoxia was accompanied by a rise of aKe by up to 35 mM, whereas pHe and [Ca]e fell (from mean levels of 7.17 and of 1.5 mM, respectively) by more than 0.2 pH units and 1 mM. Similar observations were made during a 2- to 5-min arrest of the perfusion pump to simulate ischaemia, whereas significantly larger changes in aKe, pHe and [Ca]e were revealed during an “ischaemia” period of 10 min. The results indicate that the rhythmic activity is generated by the functionally intact respiratory network of the VRG in which neurons are under aerobic conditions and ion homeostasis is not impaired. We conclude that the preparation is an appropriate in vitro model for the analysis of the cellular mechanisms for generation of respiratory rhythm and of metabolic perturbations like anoxia and ischaemia in the mature respiratory network.

Notes: Abstract Embryonic substantia nigra cells when transplanted into the striatum can reverse many of the defects of Parkinson's disease. The efficacy of such grafts is compromised by the poor survival of grafted dopaminergic neurones; typically, 3–10% survive transplantation. We used three tissue culture models to identify stages in the procedure for the preparation and insertion of grafts which might be responsible for this cell death and to identify environments in which survival is optimised. (1) The ventral mesencephalon was dissected from the donor brain, then placed immediately into culture contained in a collagen gel. (2) The dissected tissue fragments were enzymatically dissociated, then the cells placed into monolayer culture. (3) Enzymatically dissociated tissue was packed into 0.5-mm-diameter porous tubes, to simulate the compaction of cells into a graft deposit in the host brain. Dissociation of the tissue by itself caused the death of approximately 30% of dopaminergic neurones, as judged by the difference in cell counts between the intact embryonic day 14 (E14) mesencephalon, and cells dissociated then packed into tubes. Of the dissociated neurones approximately 60% died during the first 24 h and 87% during the first 3 days in monolayer culture, while only 7% of dopaminergic neurones in three-dimensional cultures and 11% of neurones in explant cultures died over the first 3 days. Embryonic dopaminergic neurones are clearly very vulnerable to adverse conditions during the first days after their removal from the donor brain. The excellent survival of neurones in three-dimensional and explant cultures indicates that close association with other cells, which may provide greatly improved access to trophic factors, can enable the cells to survive this period of vulnerability. In contrast to its effects in monolayer cultures, bFGF had no effect on dopaminergic neuronal survival in either explant or three-dimensional cultures.

Notes: Summary The possibility to visualize the NMDA recognition site with [3H]CGS 19755in vitro autoradiography was evaluated in rat brain and spinal cord sections and thereafter used to study the distribution of the NMDA recognition site in rat and mouse spinal cord. The [3H]CGS 19755 binding was concentrated to the dorsal horn, in particular to the substantia gelatinosa. Notable binding was also seen in the intermediate area and ventral horn, while some binding was observed in the funiculi. No major differences were seen in [3H]CGS 19755 binding at various levels of the rat or mouse spinal cord, although a more dense binding was seen in the mouse. A similar distribution of the [3H]CGS 19755 specific binding and the NMDA receptor associated ion-channel site, labeled with [3H]TCP, was found in the mouse spinal cord. Taken together, our data indicate that the NMDA recognition site can be visualized in rat and mouse spinal cord byin vitro [3H]CGS 19755 autoradiography.

Notes: Abstract Enlargement of the rat parotid salivary glands was induced by repeated administration of isoproterenol. Mean wet weights of the treated glands increased steadily to 240% of control values. Following withdrawal of the drug, quantitative histological techniques were used to investigate the balance between hypertrophy, hyperplasia and apoptosis. The volume occupied by acinar cells relative to the total gland volume together with cytoplasmic|:|nuclear area ratios as measures of hypertrophy increased during the early experimental period. Similarly, serous acinar cell mitotic counts increased, indicating that hyperplasia had occurred. Apoptosis was demonstrated at light microscopical level to be the main mechanism for cell deletion as the glands returned to normal size and weight. The results indicate that hypertrophy and hyperplasia of serous acinar cells contribute to isoproterenol-induced sialadenosis. The experimental animal model demonstrates that these proliferative changes are completed by 48 h and thereafter are balanced by apoptosis as the glands recover their normal size and weight.

Notes: Abstract Male 6-week-old Sprague Dawley rats were given a single intravenous injection of 4-hydroxyamino-quinoline 1-oxide (4HAQO) at a dose of 20 mg/kg in order to produce ultrastructural changes as possible morphological biomarkers for toxicity. Immunohistochemically demonstrated formation of 4HAQO-DNA adduct was correlated with the changes found. Nucleolar alteration, demonstrable by electron microscopy as segregation of nucleolar components into granular and fibrillar compartments, was evident in cells of the target organs, exocrine pancreas and adrenocortex, but not of the non-target liver parenchyma. Sequential observation clarified that such alteration was highest in frequency 6 h and 4 h after 4HAQO administration in pancreatic acinar cells and adrenocortical cells respectively. Electron microscopically, apoptotic changes of acinar cells were evident 2 h after injection of 4HAQO. DNA adduct formation was consistently demonstrated in the same target organs showing nucleolar segregation, the highest frequency being noted 4 h after 4HAQO treatment in both pancreatic acinar cells and adrenocortical cells. Our results thus indicate an identity of the target cells for nucleolar segregation and 4HAQO-DNA adduct formation which correlates with 4HAQO-toxicity. We suggest that nucleolar segregation occurs subsequent to the generation of DNA damage.

Notes: Abstract Measurement of parathyroid hormone (PTH) in the rat is most often performed with competitive ligand radioimmunoassays (RIA) utilizing heterologous antibodies. We report here the validation of a newly developed homologous immunoradiometric assay (IRMA) for rat PTH. Two different goat antibodies to the amino-terminal sequence of rat PTH are utilized; one is immobilized onto plastic beads to capture the PTH molecules and the other is radiolabeled for detection. To test this new IRMA, 30 Sprague-Dawley rats were randomized into three treatment groups to receive by intraperitoneal injection: (1) saline 1 ml/kg (control); (2) calcium chloride 40 mg/kg (hypercalcemic); and (3) EDTA 300 mg/kg (hypocalcemic). Blood samples were taken at 0, 30, 60, 180, and 300 minutes after administration of the assigned treatment for measurement of ionized calcium (Ca2+) and serum PTH. Most of the variance in PTH levels was found to be due to changes in Ca2+ (r2=0.780, P〈0.0001). There was also a close temporal relationship between the two, with the highest levels of PTH occurring at the same measured time points as the lowest Ca2+, and vice versa. The measured detection limit of the IRMA was 3 pg/ml with intra-and interassay coefficients of variation of 1.74% and 3.07%, respectively. Serial dilutions with pooled rat serum, synthetic rat PTH-(1–34), and synthetic human PTH-(1–34) showed good parallelism with increased specificity for the pooled and synthetic PTH, despite a degree of crossreactivity with hPTH. The assay is able to quantitate rapid changes in PTH, providing all the advantages of IRMA methodology including technical simplicity and speed of performance, and is likely to become a useful tool in investigations of bone, mineral, and renal homeostasis using the rat.

Notes: Abstract The aim of this work was to explore the reduction of fluoride concentrations in the skeleton after stopping experimental fluoride administration. Fluoride was administered to the rats at varying doses (0, 50, 100 ppm in drinking water) and for different lengths of time (4, 13, 25 weeks). A series of fluoride concentrations across the full thickness of humerus, parietal bone, and vertebra arch in rats were measured by means of an abrasive micro-sampling technique. The distribution profiles of fluoride from periosteal to endosteal surfaces, which were apparently related to the histological structure of these bones, were U shaped in the humerus, V shaped in the parietal bone, and W shaped in the vertebra arch. The average fluoride concentrations in the bones increased significantly with each increasing dose and length of fluoride administration. The relative increments were similar between the different regions or the different bones. After stopping fluoride administration, on the other hand, the relative reduction of the average fluoride concentrations in the bones were 30–100%. They were greatly related to the length after stopping fluoride administration and the dose and length of fluoride administration, but also dependent upon the type of bone and the region examined.

Notes: Abstract Long-term use of phenytoin for the treatment of epilepsy has been associated with increased thickness of craniofacial bones. The aim of the present study was to evaluate the possibility that low doses of phenytoin are osteogenic in vivo by measuring the effects of phenytoin administration on serum and bone histomorphometric parameters of bone formation in two rat experiments. In the first experiment, four groups of adult male Sprague-Dawley rats received daily I.P. injections of 0, 5, 50, or 150 mg/kg/day of phenytoin, respectively, for 47 days. Serum alkaline phosphatase (ALP) and osteocalcin were increased by 5 and 50 mg/kg/day phenytoin. The increases in osteocalcin and ALP occurred by day 7 and day 21, respectively. The tibial diaphyseal mineral apposition rate (MAR) at sacrifice (day 48) was significantly increased in rats receiving 5 mg/kg/day phenytoin. At a dose of 150 mg/kg/day, the increase in serum ALP, osteocalcin and MAR was reversed. No significant differences in serum calcium, phosphorus, or 1,25(OH)2D3 levels were seen. In a second experiment, three groups of rats received daily I.P. injection of lower doses of phenytoin (i.e., 0, 1, or 5 mg/kg/day, respectively) for 42 days. Phenytoin also did not affect the growth rate or serum calcium, phosphorus, and 25(OH)D3 levels. Daily injection of 5 mg/kg/day phenytoin significantly increased several measures of bone formation, i.e., serum ALP and osteocalcin, bone ALP, periosteal MAR, and trabecular bone volume. However, rats receiving lower doses of phenytoin (i.e., 1 mg/kg/day) did not show significant increases in the serum bone formation parameters. In contrast, metaphyseal osteoblast surface, osteoblast number, osteoid thickness, surface, and volume were all significantly increased in rats treated in 1 mg/kg/day but not with 5 mg/kg/day phenytoin, suggesting that the tibial diaphysis and metaphysis bone formation parameters might have different dose-dependent responses to phenytoin treatment. Administration of the test doses of phenytoin did not significantly affect the histomorphometric bone resorption parameters. In conclusion, these findings represent the first in vivo evidence that phenytoin at low doses (i.e., between 1 and 5 mg/kg/day) is an osteogenic agent in the rat.

Notes: Abstract Recent evidence suggests that 13-hydroxy metabolites of anthracyclines may contribute to cardiotoxicity. This study was designed to determine the pharmacokinetics of daunorubicin and the 13-hydroxy metabolite daunorubicinol in plasma and tissues, including the heart. Fisher 344 rats received 5 mg kg−1 daunorubicin i.v. by bolus injection. Rats were killed at selected intervals for up to 1 week after daunorubicin administration for determination of concentrations of daunorubicin and daunorubicinol in the plasma, heart, liver, kidney, lung, and skeletal muscle. Peak concentrations of daunorubicin were higher than those of daunorubicinol in the plasma (133±7 versus 36±2 ng ml−1;P〈0.05), heart (15.2±1.4 versus 3.4±0.4 μg g−1;P〈0.05), and other tissues. However, the apparent elimination half-life of daunorubicinol was longer than that of daunorubicin in most tissues, including the plasma (23.1 versus 14.5 h) and heart (38.5 versus 19.3 h). In addition, areas under the concentration/time curves (AUC∞) obtained for daunorubicinol exceeded those found for daunorubicin in almost all tissues, with the ratios being 1.9 in plasma and 1.7 in the heart. The ratio of daunorubicinol to daunorubicin concentrations increased dramatically with time from 〈1 at up to 1 h to 87 at 168 h in cardiac tissue. Thus, following daunorubicin injection, cumulative exposure (AUC∞) to daunorubicinol was greater than that to daunorubicin in the plasma and heart. If daunorubicinol has equivalent or greater potency than daunorubicin in causing impairment of myocardial function, it may make an important contribution to the pathogenesis of cardiotoxicity.

Notes: Abstract  We used a microdialysis technique to monitor extracellular methotrexate (MTX) levels during the steady state in a rodent model. Microdialysis probes were implanted in the muscle, liver, and kidney of anesthetized male Wistar rats. MTX (18.75–500 mg/kg) was given as a continuous infusion through a venous catheter, and blood samples were obtained through a second venous catheter. Heparinized plasma, ultrafiltered plasma, microdialysis effluent from tissues, and tissue samples (obtained at the end of experiments) were analyzed for MTX content by high-performance liquid chromatography (HPLC). Steady state was demonstrated in the blood and tissues from 2 h until the end of the experiments (6 h). Extracellular drug levels in muscle and liver displayed a linear correlation with doses, whereas kidney levels reached a plateau at an MTX dose of 150 mg/kg per 6 h. Microdialysis-fluid endpoint levels for muscle, liver, and kidney were positively correlated to the endpoint total tissue levels (r 2=0.80, 0.85,  and 0.68, respectively). In the kidneys, the maximal relative tissue MTX accumulation was measured at a total dose of 75 mg/kg per 6 h. At higher doses, the relative drug sequestration declined to less than half of the values observed at this dose. This study demonstrates that the microdialysis technique can provide reproducible data on MTX tissue exposure in an animal model and that it offers a means of serial and reproducible monitoring of extracellular-tissue MTX levels at steady state and over a wide dose range. Pending additional studies, microdialysis may be a helpful technique for elucidating the kinetics of drug delivery to both targeted and toxicity-prone tissues during chemotherapy.

Notes: Abstract  Osteonectin gene expression in relation to metallothionein mRNA expression was investigated in various tissues from Cd-treated rats. After a single 50 μmol/kg subcutaneous injection of CdCl2, Cd predominantly accumulated in the liver and metallothionein gene expression significantly increased concomitantly with Cd accumulation, but no alteration of osteonectin gene expression was observed. In the kidney and lung, both metallothionein and osteonectin mRNA increased significantly but the elevation of metallothionein mRNA levels (1 h after Cd administration) preceded that of osteonectin (3 h after administration). A significant elevation of osteonectin mRNA levels was also observed in the testis after 3 h, but that of metallothionein mRNA occurred after 6 h. Not only accumulation of Cd but also increments in both osteonectin and metallothionein mRNA were minimal in the brain, but a significant increase in gene expression was observed after 1 h for osteonectin and after 3 h for metallothionein. Since, except in the testis, metallothionein gene expression preceded osteonectin gene expression, the induced metallothionein might transpose Cd and thereby affect its levels immediately, thus reducing the levels of Cd available for accumulation in other tissues. Hence, the osteonectin-Cd interaction might be secondary to the metallothionein-Cd interaction. However, the fact that osteonectin mRNA was predominantly induced by Cd administration in the target tissues of Cd toxicity, such as the lung, kidney and testis, suggests the possible involvement of osteonectin in Cd intoxication/detoxication mechanisms.

Notes: Abstract  The results of oncogenicity studies of β-cyclodextrin in inbred Fischer 344 rats and CD-1 outbred mice are presented. Chronic feeding of β-cyclodextrin to Fischer 344 rats and CD-1 mice did not cause any treatment related carcinogenic effects. The only toxic effect was seen in mice as macroscopic distension of the large intestine with soft or fluid contents, histologically associated with the mucosa covered by mucous secretion containing exfoliated cells, and mucosal flattening and intestinal gland atrophy. Despite these observations, no differences between control and treated groups were observed concerning mortality, clinical observations or body weight and food consumption.

Notes: Abstract The pharmacokinetic profiles of amphotericin B (AmB) after administration of Amphocil®, an AmB/cholesteryl sulfate colloidal dispersion (ABCD) and the micellar AmB/deoxycholate (Fungizone®) were compared after repeated dosing in rats. After administration of ABCD and Fungizone at an equal AmB dose (1 mg/kg), AmB concentrations in plasma and most tissues were lower for the ABCD dose, especially in the kidneys where reduced drug concentration correlated with reduced nephrotoxicity. In contrast, AmB concentrations in the liver were substantially higher when ABCD was administered; however, without an accompanying increase in hepato-toxicity. Daily administration of ABCD for 14 days did not lead to AmB accumulation in plasma; while a slight accumulation was observed after multiple administration of Fungizone. AmB was eliminated more slowly from the plasma and various tissues and urinary and fecal recoveries of AmB were reduced after ABCD administration. These results suggest that ABCD may be stored in tissues in a form that is less toxic and is eliminated from the systemic circulation by a different mechanism than the free and protein-bound AmB in plasma. AmB accumulation in the spleen was observed when higher doses of ABCD (5 mg/kg) were administered, which could be due to saturation of hepatic uptake of AmB. Comparison of spleen concentrations of AmB between ABCD and Fungizone® at 5 mg/kg AmB doses was not possible because of Fungizone's toxicity in rats. In all other organs, AmB concentrations reached or approached a steady state within two weeks of dosing with ABCD. Urinary and fecal clearances of AmB were not different between ABCD and Fungizone administration. In summary, the distribution and elimination characteristics of AmB in rats were substantially altered when it was administered as ABCD in comparison to Fungizone. Nephrotoxicity of AmB in rats was reduced after administration of ABCD apparently because of the altered tissue distribution pattern. Thus, ABCD (Amphocil®) may be a clinically beneficial formulation of AmB in patients with systemic fungal infections.

Notes: Abstract Changes in plasma activated partial thromboplastin times (APTT) and prothrombin times (PT) in mice, rats, rabbits, dogs, monkeys and human were examined for up to 96 h at storage temperatures of 4 and 25°C. Prolongation of APTT in rats was rapid and marked, with times doubling within 24 h post-sampling. Plasma APTT of human and monkey were also affected, but to a lesser extent. No effect was observed in mice, rabbits and dogs. On the other hand, the magnitude of PT changes was much smaller than that observed with APTT in all species. No significant differences were noted between the results from samples stored at 4°C or 25°C for either test. The false prolongation of APTT is clearly undesirable in a toxicity study, especially in rats. It is important therefore to minimise these changes by performing this test under strict time-controlled conditions.

Notes: Abstract  The inhibition of neointima formation by drugs is a major goal to prevent restenosis following angioplasty. In the present study, the effect of etofibrate on blood lipids and vessel wall was investigated using a balloon injury rat model. Two weeks after ballooning the common carotid artery neointima formation was quantified by morphometric measurement of the neointimal area and cellularity in vessel cross sections, and by fluorometric evaluation of the DNA content. Etofibrate (160 mg/kg/day) had no effect on plasma triglyceride levels, but reduced serum cholesterol by about 25%. The injury-induced increase of both the neointimal area and the DNA-content was significantly inhibited by 47% (P〈0.005) and 34% (P〈0.05), respectively, in the drug-treated animals in comparison to the untreated control rats. The ratio of neointima and media was significantly (P〈0.001) reduced from 152.9±11.6% (controls) to 82.84±12.59% in the etofibrate-treated group. The cellularity (numerical profile and volume density of nuclei) in the neointima was similar in both groups. In conclusion, injury-induced neointima formation is reduced in etofibrate-treated animals, which could be due to an inhibition of smooth muscle cell proliferation.

Notes: Abstract The inhibition of neointima formation by drugs is a major goal to prevent restenosis following angioplasty. In the present study, the effect of etofibrate on blood lipids and vessel wall was investigated using a balloon injury rat model. Two weeks after ballooning the common carotid artery neointima formation was quantified by morphometric measurement of the neointimal area and cellularity in vessel cross sections, and by fluorometric evaluation of the DNA content. Etofibrate (160 mg/kg/day) had no effect on plasma triglyceride levels, but reduced serum cholesterol by about 25%. The injury-induced increase of both the neointimal area and the DNA-content was significantly inhibited by 47% (P 〈0.005) and 34% (P 〈0.05), respectively, in the drug-treated animals in comparison to the untreated control rats. The ratio of neointima and media was significantly (P 〈 0.001) reduced from 152.9 ± 11.6% (controls) to 82.84 ± 12.59% in the etofibrate-treated group. The cellularity (numerical profile and volume density of nuclei) in the neointima was similar in both groups. In conclusion, injury-induced neointima formation is reduced in etofibrate-treated animals, which could be due to an inhibition of smooth muscle cell proliferation.

Notes: Abstract The aim of this study was to examine the potencies of several recently identified selective somatostatin (SRIF)-receptor ligands as inhibitors of electrogenic ion transport in the rat distal colonic mucosa with the view to identifying the SRIF receptor type involved. Under basal conditions, cumulative administration of SRIF and SRIF2g decreased short circuit current (SCC), a measure of electrogenic ion transport, with EC50 values of 4 nM and 9 nM respectively. The peptidase inhibitors, phosphoramidon (1 μM) and amastatin (10 μM), had no effect on the potencies of either SRIF or SRIF28. The inhibitory action of SRIF on basal SCC was suppressed by piretanide and diphenylamine-2-carboxylate, compatible with the assumption that the Na+K+2Cl− co-transporter and Cl− channels, respectively, may be involved in this antisecretory action of SRIF. Tetrodotoxin (1 μM) had no effect on the antisecretory action of SRIF, suggesting that the process was not neuronally mediated. All of the SRIF analogues examined, with the exception of BIM-23056, maximally inhibited basal SCC to a similar extent as SRIF. Seglitide and octreotide were both more potent antisecretory agents than SRIF (respective EC50 values, 0.4 nM and 1.5 nM) suggesting that this effect was mediated by a receptor belonging to the SRIF1 receptor group. The most distinguishing feature of the rank order of agonist potencies was the high potency of the selective sst2 receptor ligand, BIM-23027 (EC50, value 0.32 nM), the weaker potency exhibited by the selective sst5 receptor ligand, L-362855 (EC50 value 21 nM), and the lack of agonist activity displayed by the selective sst3 receptor ligand, BIM-23056 (EC50 value 〉 1000 nM). This profile is comparable with that observed in binding studies on the recombinant sst2 receptor. Forskolin-stimulated secretion was suppressed by SRIF analogues with the rank order of agonist potencies BIM-23027 〉 SRIF 〉 L-362855 〉 BIM-23056 which resembled that exibited under basal conditions. However, the absolute potencies of these agonists were lower (respective EC50 values 2 nM, 14 nM, 38 nM and 〉 1000 nM) whilst the magnitude of inhibition was about three fold greater. BIM-23027 and SRIF (both 30 nM) also inhibited carbachol-stimulated increases in basal SCC by 60–70%, while a similar concentration of L-362855 inhibited these responses by 11 %. BIM-23056 (1 μM) had no effect on carbachol-simulated secretion. Radioligand binding studies on rat colonic mucosal membranes using [125I]-Tyr11-SRIF suggested heterogeneity of SRIF binding sites. Thus, SRIF and SRIF28 competed for binding (IC50 values, 0.32 and 0.63 nM, respectively) with Hill slopes less than unity; while seglitide and BIM-23027 both maximally displaced only 30–40% of specific binding with apparent high affinity (respective pIC50 values, 10.1 nM and 10.0). In conclusion, SRIF decreases basal as well as both cAMP and Ca2+-dependent Cl− secretion in rat colonic mucosa. The rank order of agonist potencies suggests that receptors resembling the recombinant sst2 receptor mediate inhibition of basal and forskolin-stimulated secretion. Radioligand binding studies suggest that BIM-23027 interacts with a sub-population of [125I]Tyr11-SRIF binding sites in rat colonic mucosal membranes which probably correspond to the receptors mediating the antisecretory effects described here.

Notes: Abstract We observed incidentally that rat sciatic nerve in contact with oxidized cellulose (OC), an absorbable hemostatic agent, underwent focal fiber degeneration, and we undertook studies to determine the mechanism of its production. Topically applied OC generated acute nerve damage within the adjacent nerve fascicle of rat sciatic nerve in a dose-dependent fashion (r = 0.99, P 〈 0.01, threshold amount: 9.9 mg). In single teased fibers, the predominant type of myelinated fiber damage was axonal degeneration. The subperineurial blood flow of the rat sciatic nerve was serially measured by microelectrode hydrogen polarography, and the reduction at 90 min after application of OC was not greater than that of controls. A thin polyethylene membrane interposed between OC and the sciatic nerve almost completely prevented the nerve damage. These data suggest that the chief mechanism of nerve damage by OC was neither compression nor ischemia, but was a diffusible chemical mechanism. Care should be taken to avoid direct OC application around peripheral nerves.

Notes: Abstract We observed incidentally that rat sciatic nerve in contact with oxidized cellulose (OC), an absorbable hemostatic agent, underwent focal fiber degeneration, and we undertook studies to determine the mechanism of its production. Topically applied OC generated acute nerve damage within the adjacent nerve fascicle of rat sciatic nerve in a dose-dependent fashion (r=0.99, P〈0.01, threshold amount: 9.9 mg). In signle teased fibers, the predominant type of myelinated fiber damage was axonal degeneration. The subperineurial blood flow of the rat sciatic nerve was serially measured by microelectrode hydrogen polarography, and the reduction at 90 min ather application of OC was not greater than that of controls. A thin polyethylene membrane interposed between OC and the sciatic nerve almost completely prevented the nerve damage. These data suggest that the chief mechanism of nerve damage by OC was neither compression nor ischemia, but was a diffusible chemical mechanism. Care should be taken to avoid direct OC application around preripheral nerves.

Notes: Abstract Paraneoplastic cerebellar degeneration accompanying gynecological or breast malignancies is frequently associated with an autoantibody response, termed "type I" or "anti-Yo" directed against cytoplasmic antigens of cerebellar Purkinje cells. The role of this antibody response in the pathogenesis of paraneoplastic cerebellar degeneration is unknown; however, it is also not known whether anti-Purkinje cell antibodies from the systemic circulation bind to targe t Purkinje cell antigens under the conditions of brain inflammation and blood-brain barrier disruption, which are frequently present at the onset of cerebellar symptoms. Inbred Lewis rats received intraperitoneal injections of type I or normal IgG in the setting of blood-brain barrier disruption induced by adoptive transfer of experimental allergic encephalomyelitis (EAE) and were killed after 24, 48, and 96 h. Brains of these animals were studied histologically for evidence of EAE and immunohistochemically for binding of human or endogenous rat IgG to target neurons. Rat IgG was detected around vessels and in Purkinje cells of all animals studied. Human IgG was detected around vessels of all animals. In animals examined 96 h after receiving type I human IgG, human IgG was identified within processes of Purkinje cells and within occasional Purkinje cell bodies. Uptake of type I IgG by other cell types was not observed, and neuronal uptake of IgG was not seen in brains of animals receiving normal human IgG. Ou r data demonstrate that circulating type I IgG is internalized by cerebellar Purkinje cells in the setting of blood-brain barrier disruption and suggest a mechanism by which an antibody response directed against cytoplasmic antigens of Purkinje cells may reach target antigens at the onset of paraneoplastic cerebellar degeneration.

Notes: Abstract Histochemical fibre-type composition and myosin heavy chain isoform component in the soleus muscle were studied in normoxic rats at postnatal ages of 5, 10, 15, and 20 weeks and in rats exposed to hypobaric hypoxia (460 torr) for 5 weeks from postnatal ages of 5, 10, and 15 weeks. The increase in the percentage of type I fibres and the concomitant decrease in that of type IIa fibres in the soleus muscle of normoxic rats were observed until 15 weeks of age. On the other hand, no change in the fibre-type composition of the muscle during postnatal development was observed in hypoxic rats, irrespective of the age at which they were exposed to hypoxia. The changes in the myosin heavy chain isoform component (MHC I and MHC IIa) of the muscle during postnatal development and by hypoxia corresponded well with those in the muscle fibre-type composition. It is concluded that hypobaric hypoxia inhibits the growth-related shift of muscle fibre-types from type IIa to type I and of myosin heavy chain isoforms from MHC IIa to MHC I in the rat soleus muscle, and that there are no changes in the muscle fibre-type composition or the myosin heavy chain isoform component caused by hypoxia after the shifts in these parameters which occur during postnatal development are completed.

Notes: Abstract In rat hippocampal slices epileptiform activity was induced by superfusion with Mg2+-free artificial cerebrospinal fluid (ACSF). Paroxysmal depolarization shifts (PDS) were evoked by electrical stimulation of Schaffer collaterals. To investigate the afterpotentials that follow PDS, intracellular recordings were made from CA1 pyramidal cells. The experiments revealed that several components are engaged in the generation of PDS afterpotentials in Mg2+-free ACSF. A long lasting component which determined the overall duration of the PDS afterhyperpolarization was blocked by intracellular application of ethylenebis(oxonitrilo)-tetraacetate (EGTA); concomitantly, the afterhyperpolarizations following depolarizing current injections were blocked. This indicated that the long lasting component was due to a slow Ca2+-activated K+ current. The block of Ca2+-activated K+ current uncovered a depolarizing PDS afterpotential with an N-shaped voltage dependence, suggesting that this depolarizing afterpotential component may be due to an N-methyl d-aspartate (NMDA) conductance. Intracellular injection of Cl− revealed that the PDS were followed by Cl− currents lasting about 500 ms. This component could be blocked by application of bicuculline suggesting that it is due to a synaptically GABA-mediated (i.e. γ-aminobutyric acid) Cl− current. A comparison of PDS afterpotentials in Mg2+-free ACSF and those in other models of epileptiform activity suggests that similar sequences of inhibitory components are activated in spite of different pharmacological alterations of membrane conductances which induce the epileptiform discharges.

Notes: Abstract Glial cells in the hilus of the dentate gyrus of the rat were investigated using the patch-clamp technique in acute slices of the hippocampal formation. According to their voltage-gated current patterns, two classes of glial cells — putative astrocytes and presumed glial precursor cells — were apparent. The glutamate receptor agonists kainate, glutamate, and α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) evoked inward currents at a holding potential of −70 mV in astrocytes and presumed glial precursor cells. Inward currents could also be induced in nucleated patches, indicating a direct action on glial receptors. In presumed hilar glial precursor cells, 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 μM) blocked the kainate-induced current, while it was partially inhibited by Zn2+ (2 mM) and Evans Blue (10 μM). Cyclothiazide (100 μM), in contrast, potentiated this current, indicating the presence of AMPA receptors. In 90% of the presumed glial precursor cells the excitatory amino-acid-evoked current voltage (I/V) relations were linear or outwardly rectifying and reversed close to 0 mV, which is characteristic for non-specific cation channels. To determine the permeability to Ca2+, I/V relations were determined in a Na+-free solution containing 40 mM Ca2+ and showed reversal potentials of a wide variation ranging from −63 mV to + 1 mV with corresponding P Ca/ P Cs permeability ratios of between 0.09 and 2.10. Statistical analysis revealed that the permeability to Ca2+ significantly decreased with an advance in age (r=−0.596; n=21; P〈0.01). These data suggest that the Ca2+ influx mediated by the activation of AMPA receptors expressed in presumed hilar glial precursor cells is dependent on the developmental stage.

Notes: Abstract The aim of the experiments was to study the effects of a moderate heat load on sleep in young (26-day-old) rats and to determine whether the sleep-promoting effect of heat results from stimulation of the homeostatic sleep process. The changes in sleep-wake activity, electroencephalogram slow wave activity (SWA) during non-rapid eye movement sleep (NREMS) and cortical temperature (T crt) were determined during and after long (24-h) and short (2.5-h) heat loads (elevation of ambient temperature from 26° C to 32° C), and after total sleep deprivation (SD) combined with a short-term heat load. The heat exposures elicited increases in T crt and rectal temperature (2 and 1.7° C respectively). The long-term heat load induced persistent, albeit slight enhancements in NREMS. Rapid eye movement sleep (REMS) increased with a 12-h delay during the 24-h heat load. Heat elicited an immediate large increase in SWA. After this initial increase, SWA declined and tended to fall below the baseline level during the last 12 h of the 24-h heat load. SWA and REMS were significantly suppressed after termination of 24-h heat loading. The increased SWA during the short-term heat load was not followed by subsequent alterations in sleep when the ambient temperature had returned to normal. However, after the combination of SD with the shortterm heat load the durations of NREMS and SWA were significantly enhanced compared with those found after SD at 26° C. The results are interpreted as suggesting that heat increases NREMS in the young rat by the same mechanism as is involved in the enhancement of NREMS after SD: a stimulation of sleep drive.

Notes: Abstract Recordings of isometric force were obtained for twitches and (sub)maximal tetani of gastrocnemius medialis (MG) and tibialis anterior (TA) muscle units in female Wistar rats. We assessed the relationships between unit properties that have all been associated with “speed”: (1) the relative degree of peak force attained during repetitive activation at 40 Hz (P 40/P max), (2) the relative degree of final twitch fusion during the same test burst (Fus-end), and (3) various measures of the time-course of single twitches, including twitch time-to-peak and a parameter referred to as “initial fusion ratio” (Fus-in; relative decline from peak force at 25 ms from twitch onset). The various measures of twitch time-course were significantly correlated to each other with correlation coefficients varying over a fairly wide range (0.35–0.64 for MG; 0.50–0.80 for TA). Twitch time-course was also significantly correlated with Fus-end during the 40-Hz repetitive activation; the highest correlation coefficient (0.69 for MG, 0.80 for TA) was obtained for Fus-in, which was also numerically similar to Fus-end. Thus, the degree of fusion indeed seemed to be largely dependent upon aspects of twitch time-course. However, the relative degree of force mobilization obtained in the same contractions elicited by stimulation at 40 Hz was not consistently better correlated with Fus-end than with measures of single twitch time-course. Furthermore, in fast-twitch units having the same twitch time-to-peak, the force mobilization elicited by stimulation at 40 Hz (P 40 P max) was the same for MG and TA, while the degree of fusion was significantly smaller for TA than for MG units. The results demonstrate the complexity of the concept of isometric “speed” and underline the need for using several speed indicators in parallel in studies concerning the differentiation of muscle (unit) properties.

Notes: Abstract The present study was designed to examine possible interactions between exogenous CCK and the 5-HT1A receptor subtype mediated serotonergic effects on feeding in rats. The somatodendritic 5-HT1A receptor agonist 8-OH-DPAT (0.32 mg/kg sc) evoked feeding in freely feeding rats. This effect was attenuated by treatment with CCK-8 (1, 5 and 25 μg/kg ip). In food deprived rats, CCK-8 (40 μg/kg ip) significantly reduced the size of a test meal. Treatment with the 5-HT1A receptor antagonist WAY-100135 (10 mg/kg ip) antagonized this anorectic effect of CCK-8. WAY-100135 on its own did not affect food intake. These results suggest the involvement of the 5-HT1A receptor subtype in mediating 5-HT-CCK interactions in the control of food intake in rats.

Notes: Abstract  The present study was designed to examine possible interactions between exogenous CCK and the 5-HT1A receptor subtype mediated serotonergic effects on feeding in rats. The somatodendritic 5-HT1A receptor agonist 8-OH-DPAT (0.32 mg/kg sc) evoked feeding in freely feeding rats. This effect was attenuated by treatment with CCK-8 (1, 5 and 25 μg/kg ip). In food deprived rats, CCK-8 (40 μg/kg ip) significantly reduced the size of a test meal. Treatment with the 5-HT1A receptor antagonist WAY-100135 (10 mg/kg ip) antagonized this anorectic effect of CCK-8. WAY-100135 on its own did not affect food intake. These results suggest the involvement of the 5-HT1A receptor subtype in mediating 5-HT-CCK interactions in the control of food intake in rats.

Notes: Abstract We measured the diffusion coefficient of myoglobin (D Mb) inside mammalian skeletal muscle cells with a microinjection technique. A small bolus of horse Mb was injected into a single muscle fibre and the subsequent time-dependent changes of the Mb profiles along the fibre axis were measured with a microscope-photometer. For fibres of the rat soleus muscle at 22° C, a D Mb of 1.3·10−7 cm2/s was found, confirming a result obtained previously by us for rat diaphragm muscle with a photo-oxidation technique. In the extensor digitorum longus muscle of the rat, a higher value of 1.9 · 10−7 cm2/s was measured. Auxotonic muscle contractions did not change the apparent D Mb. For the temperature range between 22 ° C and 37 ° C, a temperature coefficient, Q 10, of 1.5 was calculated. The implication of this result for the role of Mb in the facilitation of oxygen transport was examined. Model calculations show that with this relatively low D Mb value, the intracellular oxygen supply can be improved only slightly.

Notes: Abstract Effects of cholinergic and adrenergic agonists on the secretion of the fluorescent dye calcein were examined to clarify the involvement of calcium ions in the secretion of calcein from acinar cells dispersed from the rat parotid gland. Addition of carbachol (CCh) and noradrenalin (NA), but not isoproterenol (IPR), enhanced the net release of calcein from acinar cells during the subsequent 10 min in a dose range from 10−8 M to 10−6 M. The net release of calcein reached a maximum 7 min after the addition of CCh. The release of calcein was suppressed by the simultaneous additions of atropine with CCh, or phenoxybenzamine with NA. Addition of CCh induced a sustained dosedependent increase in the intracellular levels of calcium ions, ([Ca2+]i). Addition of NA at 10−6 M increased [Ca2+]i. Phenoxybenzamine completely inhibited the NA-induced increase, but propranolol did not. The removal of extracellular calcium ions did not influence the release of calcein induced by 10−6 M CCh, but it abolished the sustained increase in [Ca2+]i. The transient increase in [Ca2+]i induced by CCh was observed in the absence of extracellular calcium ions. A calcium ion chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) inhibited the CCh-induced release of calcein. The calcium ionophore, A23187 (2.5×10−6 M), but not 10−3 M dibutyryl cAMP, evoked the release of calcein. It also increased [Ca2+]i. Removal of extracellular calcium ions suppressed the A23187-induced release of calcein. These results suggest that the release of calcein from parotid acinar cells is transiently induced through an increase in [Ca2+]i by muscarinic and α-adrenergic agonists and may represent the initial process of salivary secretion.

Notes: Abstract The angiotensin I converting enzyme inhibitor (ACEI) perindopril (2 mg/kg body weight), the peripheral vasodilator dihydralazine (DHL) (25 mg/kg body weight) or distilled water was given daily from birth to day 14 to neonatal rats. Blood pressure, plasma creatinine, plasma renin activity (PRA), substrate (PRS) and concentration (PRC) and renin content of kidney tissue sections were evaluated on days 14 and 28. By day 14, a high mortality in the ACEI group was observed. ACEI, but not DHL, led to a significant fall (P 〈 0.01) in blood pressure, 57 ± 11 versus 89 ± 25 in the DHL group and 103 ± 24 mmHg in controls, and to a dramatic increase in plasma creatinine. PRA and PRS were undetectable in ACEI-treated rats; in contrast, PRC and renal staining with anti-renin antibody were significantly increased in ACEI rats. On day 28, the blood pressure was normal in all groups and plasma creatinine returned to the normal range in ACEI rats. PRA, PRS and PRC were not significantly different in the ACEI group and controls. These results suggest that the renin-angiotensin system (RAS) plays a major postnatal role in the neonatal rat. Inhibition of the RAS during the first 2 weeks of life leads to high mortality, severe hypotension, reversible renal failure and a defect in circulating angiotensinogen.

Notes: Abstract In the neonate, chronic unilateral ureteral obstruction (UUO) reduces renal blood flow (RBF) of the ipsilateral kidney and increases RBF of the opposite kidney. To determine whether renal nerves mediate or modulate these responses complete left UUO in the neonatal rat was used as a model of severe obstructive uropathy, and was compared with sham-operated controls. At 24–28 days of age, animals underwent left or right mechanical renal denervation or left sham renal denervation. One week after denervation, animals were anesthetized and blood pressure and heart reate were measured. Cardiac output and RBF were determined by the radioactive microsphere technique. UUO increased blood pressure and heart rate, and decreased RBF in the obstructed kidney, regardless of denervation. While left UUO increased RBF to the intact opposite kidney in rats with left renal denervation, this was attenuated by right renal denervation. Thus, in the neonatal rat, UUO modulates systemic renal hemodynamics, possibly through activation of the renin-angiotensin system. While renal nerves do not mediate the vasoconstriction of the obstructed kidney, renal nerves modulate vascular tone of the kidney contralateral to UUO.

Notes: Abstract The effects of three antidepressants, desipramine (2.5–20 mg/kg) tranylcypromine (0.63–2.5 mg/kg) mianserin (1.25–10 mg/kg) and three non-antidepressants, chlordiazepoxide (CDP; 1.25–10 mg/kg) haloperidol (0.02–0.16 mg/kg)d-amphetamine (0.31–1.25 mg/kg) were evaluated in rats responding for water reinforcement under a DRL 72s schedule. The antidepressants all produced dose-related decreases in overall response rates, but no significant changes in reinforcement frequency. In contrast, the anxiolytic CDP did increase the number of reinforcers obtained. Haloperidol decreased both reinforcers and responses whilstd-amphetamine stimulated responding, thereby decreasing reinforcement frequency. An analysis of the modes of inter-response times (IRTs) revealed no significant shifts in the peaks of the IRT distributions for most of the drugs tested. Amphetamine, however, (0.31 and 0.63 mg/kg) decreased the modal values in correspondence with the shift to the left of the peak of responding caused by this compound. These results are discussed in the context of the use of the DRL 72s procedure as a screening test for antidepressant drugs.

Notes: Abstract An experiment was performed to determine the relationship between saccharin preference and the self-administration of morphine via the oral and intravenous routes. On the basis of voluntary intake of a saccharin solution by male rats, low and high preference groups were formed. Rats selected for high saccharin preference self-administered more morphine intravenously than rats selected for low preference. The two groups did not differ in oral morphine intake. The positive relationship between the intake of saccharin and intravenous morphine self-administration may be due to their mediation by a common mechanism. Measures of taste sensitivity or preference may be useful in identifying individuals at risk for drug abuse.

Notes: Abstract Studies examining differential sensitivity to psychoactive drugs in mice suggest that genotype may play a critical role. Furthermore, an involvement of genotype in mediating individual differences in sensitivity to the rewarding effects of several drugs of abuse has also been postulated. The aim of this study was to examine the conditioned rewarding and dopamine-releasing effects of morphine in two outbred rat strains commonly used in addiction research. Additionally, the behavioural and neuroendocrine responses of these strains to the stress of novelty were also examined. Basal locomotor activity was higher in Wistar rats than Sprague-Dawley following exposure to a novel environment. In contrast, elevations in plasma corticosteroid levels following novelty exposure did not differ between the two strains. In a counterbalanced place preference conditioning procedure, increasing doses of morphine (1.0–10.0 mg/kg SC) produced significant conditioned place preferences (CPP) in both Wistar and Sprague-Dawley strains. However, Wistar rats required a significantly larger dose of morphine (5.0 mg/kg) to produce a significant CPP than the Sprague-Dawley rats. In the latter strain, CPP occurred with doses of 3.0 mg/kg and greater. In parallel microdialysis experiments, both strains showed significant dose-related increases in dopamine release in the nucleus accumbens following acute morphine challenge (1.0–10.0 mg/kg SC). Again in Wistar rats, a larger dose of morphine was necessary to produce a significant increase in comparison to Sprague-Dawley rats. These results show that genetically distinct rat strains can show differential sensitivity to opioids, more specifically to drug-seeking responses.

Notes: Abstract In rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle, pretreatment with the D1 DA antagonists, SCH 23390 (7-chloro-8-hydroxy-2,3,4,5-tetrahydro-3-methyl-1-phenyl-1H-3-benzazepine) and A66359 (1-[2-bromo-4, 5-dimethoxybenzyl]-7-hydroxy-6-methoxy-2-methyl-1,2,3,4 tetrahydroisoquinoline), but not the D2 DA antagonist raclopride inhibited the contralateral circling induced by the benzazepine D1 DA agonists SKF 38393 (7-H, 3-H analogue of SCH 23390), SKF 80723 (7-H, 3-H, 6-Br analogue) and SKF 83959 (7-H, 6-Cl, 3′-CH3 analogue). In MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) treated common marmosets, administration of SKF 80723 and SKF 83959 increased locomotor activity and reversed the motor disability. Grooming and oral activities were also increased. Pretreatment with SCH 23390 and A66359 inhibited all the behavioural changes induced by both D1 DA agonists. In general, higher doses of A66359 and more especially SCH 23390 were needed to inhibit SKF 83959 and SKF 80723 induced increases in oral activity and grooming than locomotor activity. Raclopride pretreatment did not affect SKF 83959 and SKF 80723 induced oral activity and grooming, though it reduced the duration of the locomotor changes induced by the D1 DA agonists. These findings demonstrate that the behavioural effects of benzazepine D1 DA agonists in the 6-OHDA lesioned rat and MPTP-treated marmoset are mediated by D1 DA receptor sites, although in the primate, stimulation of D2 DA receptors by endogenous DA may be necessary in facilitating the antiparkinsonian effects of D1 DA agonists. The differential sensitivities of locomotor/motor disability and oral/grooming behaviours to antagonism by D1 DA antagonists may indicate the involvement of multiple D1 DA receptor subtypes in mediating benzazepine D1 DA agonist induced behaviours in the MPTP-treated marmoset.

Notes: Abstract Two experiments were designed to assess the effect of a “novel” environment on the development of sensitization to the psychomotor activating effects ofd-amphetamine. In the first experiment, rats with a unilateral 6-hydroxydopamine lesion of the mesostriatal dopamine system received ten daily injections of amphetamine (2 mg/kg), either in their home cages or in novel test cages. The home and novel cages were physically identical (cylindrical transparent buckets), but one group lived and were tested in these cages, whereas the other group was transported from the stainless steel hanging cages where they lived to these novel test cages, for each test session. The first injection of amphetamine produced significantly more rotational behavior in animals tested in a novel environment than in animals tested at home. In addition, animals tested in a novel environment showed greater sensitization than animals tested at home, so the difference between the two groups was even more pronounced following the last injection. In a second experiment, locomotor activity was quantified in rats that received ten injections of either saline or 1.5 mg/kg amphetamine, in their home cages or in a physically identical novel environment. Again, there was a significantly greater locomotor response to the first injection of amphetamine, and greater sensitization, in animals tested in a novel environment than in animals tested at home. These data indicate that environmental factors can exert a large effect on the susceptibility to sensitization, and mechanisms by which this may occur are discussed.

Notes: Abstract To clarify the influence of opioids on social play, the effects of morphine on playful and non-playful social behavior in juvenile rats was investigated under different conditions. Environmental variables employed were different (dim and intense) levels of illumination during testing, familiarity to the test cage, and different periods of social isolation prior to testing. Under dim light conditions, morphine markedly increased playful social behavior, such as pinning, boxing/wrestling and following/chasing, whereas non-playful social behavior such as social exploration and contact behavior was hardly affected. This effect of morphine was independent of duration of previous isolation and dose-dependent, with a maximal effect at 1.0 mg/kg. The mechanism of this effect is interpreted as an action on the rewarding aspects of play. A dose of 0.1 mg/kg of morphine abolished the initial suppression of play induced by unfamiliarity to the test cage, without influencing total levels of play. This may be an effect of morphine on the integration of sensory stimuli. Under intense light conditions, where playful behavior was completely suppressed, morphine itself hardly affected such behavior, but decreased some aspects of non-playful social behavior. These results suggest that in juvenile rats playful and non-playful forms of social behavior are differentially regulated. In addition, opioid systems may be involved at different levels in the regulation of social play.

Notes: Abstract This study investigated the alterations of the 5-HT1A and 5-HT1B autoreceptor function following chronic treatment with fluvoxamine using osmotic minipumps. The 5-HT1A and 5-HT1B autoreceptor function were studied using microdialysis in the dorsal hippocampus. The effect of the 5-HT1A receptor agonist 8-OH-DPAT (0.3 mg/kg, SC) and the 5-HT1B receptor agonist RU-24969 (100 nM through the dialysis probe for 30 min) on 5-HT release was compared with rats chronically treated with saline. 8-OH-DPAT decreased 5-HT release to 55% and 60% of baseline, while RU-24969 decreased 5-HT release to 66% and 70% of baseline value in the saline and fluvoxamine group, respectively. In both cases, differences between the saline and fluvoxamine groups were not statistically significant. Plasma levels of fluvoxamine after 21 days of treatment ranged from 3 to 5 ng/ml. Fluvoxamine concentration in rat brain during treatment was estimated between 100 and 200 nM, which approximates to the IC50 value of fluvoxamine on the 5-HT transporter in synaptosomes and is 50 times higher than the Kd value for the 5-HT reuptake site. In conclusion, no evidence was found for changes in 5-HT1A,B receptor function using 8-OH-DPAT and RU-24969 as probes after continuous treatment with fluvoxamine by means of osmotic minipumps.

Notes: Abstract A multiple choice serial reaction time task was used to investigate visual attention in rats. The intelligence panel consisted of a transparent touch-sensitive device, placed directly in front of a video monitor. Amphetamine (0.2–1.6 mg/kg IP) increased errors of omission and decreased latency to respond, but had no effect on accuracy. The 5-HT agonist quipazine (0.6–2.4 mg/kg IP) increased errors of omission, but did not affect other parameters. ICV administration of hemicholinium-3 (1–4 µg) had no effect upon performance. Thus, psychopharmacological manipulations can reliably alter performance in the touch window box, suggesting potential new avenues for rat cognitive testing.

Notes: Abstract Since the hippocampus is likely to be a major site of phencyclidine (PCP) action, the effects of various doses of PCP (1.8, 18 or 36 nM) as well as 3.6 nM MK-801 or saline injected directly into the dentate gyrus of the hippocampus was tested for acquisition of a spatial navigation task (dry land version of a water maze) using a paradigm that assesses short term memory based on learning within a day and long term memory based on learning between days. Results indicated that relative to saline or 1.8 nM PCP injected rats, rats with 18 or 36 nM PCP or 3.6 nM MK-801 injections were impaired in acquisition of the task as measured by increased distances traveled to find the food location between days but not within days. In additional experiments 36 nM PCP or 3.6 nM MK-801 did not produce any deficits in the acquisition of an object discrimination task. It is suggested that PCP through its blocking action of the NMDA receptor in the dentate gyrus or CA1 region of the dorsal hippocampus mediates the consolidation of new spatial location information.

Notes: Abstract The present series of experiments examined the effects of five benzodiazepine receptor (BZR) partial inverse agonists on the behaviour of rats on an elevated plus maze. The drugs were tested in a standard plus maze with 3-cm walls added to the open arms, as this has been shown to increase the sensitivity of the plus maze to anxiogenic-like drug effects (Jones and Cole 1995). The drugs tested were FG 7142 (0–100 mg/kg),β-CCE (0–30 mg/kg), ZK 132 556 (0–100 mg/kg), ZK 90 886 (0–30 mg/kg) and Ro 15–4513 (0–30 mg/kg). In addition, to allow a comparison with previous studies, the effects of three reference substances, DMCM (0–2.5 mg/kg), pentylenetetrazol (PTZ; 0–30 mg/kg) and yohimbine (0–5 mg/kg), were also examined. These three reference compounds produced a dose-dependent reduction in the duration of open arm exploration and the total number of open arm entries, indicative of anxiogenic-like effects. DMCM produced significant effects at the doses of 1.25 and 2.5 mg/kg, PTZ at 30 mg/kg, and yohimbine at 5 mg/kg. The BZR partial inverse agonist FG 7142 (10, 30 and 100 mg/kg) also reduced the duration of open arm exploration and the total number of arm entries. The minimally effective dose resulted in a receptor occupancy of approximately 80%. Ro 15–4513 also produced anxiogenic-like effects, but only at a dose (30 mg/kg) that resulted in a receptor occupancy of approximately 95%. In contrast, the other BZR partial inverse agonists, ZK 132 553 and ZK 90 886, did not significantly reduce the duration of open arm exploration, even at doses that produced greater than 95% receptor occupancies.β-CCE also did not reduce open arm exploration at any dose tested (0–30 mg/kg). The GABA shift, a biochemical index of intrinsic activity, indicates that these latter three compounds are more inverse agonistic than Ro 15–4513. In summary, these results demonstrate that not all BZR receptor partial inverse agonists have anxiogenic-like activity in the rat plus maze, and that the GABA shift, a biochemical index of intrinsic efficacy, does not predict which BZR partial inverse agonists are anxiogenic.