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Notes: Abstract: N-Acetylaspartylglutamate (NAAG), a prevalent peptide in the vertebrate nervous system, may be hydrolyzed by extracellular peptidase activity to produce glutamate and N-acetylaspartate. Hydrolysis can be viewed as both inactivating the peptide after synaptic release and increasing synaptic levels of ambient glutamate. To test the hypothesis that NAAG and the peptidase activity that hydrolyzes it coexist as a unique, two-stage system of chemical neurotransmission, 50 discrete regions of the rat CNS were microdissected for assay. In each microregion, the concentration of NAAG was determined by radioimmunoassay and the peptidase activity was assayed using tritiated peptide as substrate. The NAAG concentration ranged from 2.4 nmol/mg of soluble protein in median eminence to 64 in thoracic spinal cord. Peptidase activity against NAAG ranged from 54 pmol of glutamate produced per milligram of membrane protein per minute in median eminence to 148 in superior colliculus. A linear relationship was observed between NAAG peptidase and NAAG concentration in 46 of the 50 areas, with a slope of 2.26 and a correlation coefficient of 0.45. These data support the hypothesis that hydrolysis of NAAG to glutamate and N-acetylaspartate is a consistent aspect of the physiology and metabolism of this peptide after synaptic release. The ratio of peptide concentration to peptidase activity was 〉0.3 in the following four areas: ventrolateral medulla and reticular formation where the peptide is concentrated in axons of passage, thoracic spinal cord, where NAAG is concentrated in ascending sensory tracts as well as motoneuron cell bodies, and ventroposterior thalamic nucleus.

Notes: Abstract Concentrations of taurine have been measured in 44 microdissected rat brain nuclei or areas. Taurine is ubiquitously present and distributed unevenly in the rat brain: the ratio of the highest (pyriform cortex) to lowest (midbrain reticular formation) concentrations is 4.7:1. High taurine levels were found in cerebral cortical areas, caudate-puta-men, cerebellum, median eminence, and supraoptic nucleus. Acute pain stress reduced taurine levels in the hypothalamus and the lower brainstem nuclei but not in cortical areas. Increased locomotor and behavioral activities following a high dose of amphetamine elevated taurine concentrations significantly in the substantia nigra and locus ceruleus.

Notes: Apelin, a peptide recently isolated from bovine stomach tissue extracts, has been identified as the endogenous ligand of the human orphan APJ receptor. We established a stable Chinese hamster ovary (CHO) cell line expressing a gene encoding the rat apelin receptor fused to the enhanced green fluorescent protein, to investigate internalization and the pharmacological profile of the apelin receptor. Stimulation of this receptor by the apelin fragments K17F (Lys1-Phe-Arg-Arg-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) and pE13F (pGlu5-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) resulted in a dose-dependent inhibition of forskolin-induced cAMP production and promoted its internalization. In contrast, the apelin fragments R10F (Arg8-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) and G5F (Gly13-Pro-Met-Pro-Phe17) were inactive. The physiological role of apelin and its receptor was then investigated by showing for the first time in rodent brain: (i) detection of apelin neurons in the supraoptic and paraventricular nuclei by immunohistochemistry with a specific polyclonal anti-apelin K17F antibody; (ii) detection of apelin receptor mRNA in supraoptic vasopressinergic neurons by in situ hybridization and immunohistochemistry; and (iii) a decrease in vasopressin release following intracerebroventricular injection of K17F, or pE13F, but not R10F. Thus, apelin locally synthesized in the supraoptic nucleus could exert a direct inhibitory action on vasopressinergic neuron activity via the apelin receptors synthesized in these cells. Furthermore, central injection of pE13F significantly decreased water intake in dehydrated normotensive rats but did not affect blood pressure. Together, these results suggest that neuronal apelin plays an important role in the central control of body fluid homeostasis.

Notes: The role of afferent innervation to the hypothalamic paraventricular nucleus (PVN) on CRH mRNA and CRH receptor mRNA levels was studied in control and stressed rats. Groups of rats were subjected to unilateral transection of the stria terminalis (ST), the medial forebrain bundle at the rostral hypothalamic level (RMFB), or the lower brainstem through the medulla oblongata between the obex and the locus coeruleus (CBs). Twelve days after surgery, each group of rats was further divided into controls (basal conditions) and stressed (1 h immobilization), before collecting brains for mRNA analysis by in situ hybridization histochemistry. While ST and RMFB cuts had no effect on basal CRH mRNA levels in the PVN, CBs cut decreased CRH mRNA in the PVN ipsilaterally to the knife cut but it was without effect on the contralateral side (– 40% and –37%vs contralateral and sham-operated, respectively, P&0.01). Acute stress (rats were killed 3 h after immobilization) increased CRH mRNA levels by about 30% bilaterally, an effect which was unchanged by any of the three hemisections. Under basal conditions, CRH receptor mRNA levels in the PVN were indistinguishable from the surrounding areas in sham-operated controls, ST and RMFB operated rats. However, brainstem hemisection resulted in clear expression of CRH receptor mRNA in areas consistent with the dorsal, medial-ventral and lateral parvicellular subdivisions of the PVN, ipsilateral to the transection. CRH neurons in these subdivisions project to the lower brainstem and the spinal cord. Expression of CRH receptor mRNA in the medial-dorsal and anterior parvicellular divisions (CRH neurons with median eminence projections) was not affected by CBs cut. In these subdivisions, immobilization stress markedly increased CRH receptor mRNA levels but it did not influence CBs cut-induced CRH receptor expression. ST and RMFB hemisections were without effect on PVN CRH receptor mRNA levels under basal or stress conditions. Oxytocin (OT) and vasopressin (VP) mRNA levels in the magnocellular subdivision of the PVN were unchanged after immobilization, or following ST, RMFB or CBs cuts, whereas OT mRNA in the medial-ventral and caudal parvicellular subdivisions was decreased by 52% after CBs cut. The data demonstrate that: 1) basal CRH mRNA levels in the PVN are under tonic stimulatory influence of the lower brainstem (and/or spinal cord) afferents; 2) CRH receptor mRNA expression in PVN subdivisions (pituitary vs lower brainstem/spinal cord projecting neurons) is under different control mechanisms, and 3) immobilization-induced changes in CRH mRNA and CRH receptor mRNA levels are mediated either by neural inputs from brain areas other than those investigated here, or by humoral factors.

Notes: The concentration of cyclic AMP (cAMP) and the activity of sodium-fluoride-stimulated adenylate cyclase was measured in 29 microdissected brain areas of homozygous Brattleboro rats and their Long-Evans control rats. In ten of the investigated brain areas a decreased cAMP level was measured in Brattleboro rats. It was particularly decreased in the supraoptic nucleus, cingulate and parietal cortex, hippocampus, habenula and organum vasculosum laminae terminalis. Significantly lower cAMP levels were also found in the periventricular nucleus, bed nucleus of the stria terminalis, area postrema and locus coeruleus. An increased cAMP concentration was detected only in the subcommissural organ of Brattleboro rats. In most brain areas, where cAMP was decreased, sodium fluoride-stimulated adenylate cyclase activity was significantly increased (supraoptic nucleus, parietal cortex, periventricular nucleus, bed nucleus of the stria terminalis, locus coeruleus) or unchanged (hippocampus, habenula, organum vasculosum laminae terminalis). The coincidence of alterations in cAMP concentration and adenylate cyclase activity in brain areas of Brattleboro rats with relatively dense vasopressinergic innervation and/or vasopressin receptor population in control rats, suggests an influence of brain vasopressin on the cAMP-adenylate cyclase second messenger system.

Notes: Corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus (PVN) of the hypothalamus and in the central nucleus of the amygdala (ACE) participate in neurohumoral and behavioral responses to stress. To understand better the central regulation of CRH, the present study assessed the effects of ipsilateral surgical hemisection of the brainstem on expression of CRH mRNA in the PVN and the ACE. In situ hybridization was used to demonstrate PVN CRH mRNA expression in hemisected, sham-operated or intact rats before and after 3 h of immobilization (IMMO). In addition, hypothalamic-pituitary-adrenocortical (HPA) axis activity at baseline and during IMMO was assessed by measurements of plasma concentrations of ACTH and corticosterone. IMMO markedly increased CRH mRNA expression in the PVN in all experimental groups. Rats with brainstem hemisections had lower PVN CRH mRNA expression ipsilateral to the lesion and markedly blunted responses after IMMO, compared to values in sham-operated rats. In contrast, neither hemisection nor IMMO affected CRH mRNA expression in the ACE. Lesioned and SHAM-operated groups did not differ in baseline or IMMO-induced increases in plasma ACTH or corticosterone levels. The present results indicate that baseline levels and IMMO-induced increments in CRH mRNA expression in the PVN, but not in the ACE, depend on ipsilaterally ascending medullary tracts and that IMMO-induced HPA activation does not depend on these pathways.

Notes: We measured dopamine, norepinephrine, serotonin and epinephrine concentrations in the paraventricular nucleus and median eminence, and corticotrophin-releasing factor levels in the paraventricular nucleus. Tissue was isolated by micropunch technique from hypothalami of normal dogs, dogs treated for one week with dexamethasone (1 mg/kg/day) and dogs with spontaneous pituitary-dependent hyperadrenocorticism. Concentrations of corticotrophin-releasing factor and most of the neurotransmitters were found to be similar between our three groups of dogs. However, we found the mean dopamine concentration in the median eminence tissue to be significantly decreased in dogs with Cushing's disease and in steroid-treated dogs. Epinephrine levels were elevated in the hypothalamic paraventricular nucleus of steroid-treated dogs.

Notes: Pseudorabies virus was used to label transneuronally descending auditory projections following intracochlear injections. At different time points after injection, virus-infected cells were detected immunohistochemically in the central nervous system. Initially (25 h), virus was transported retrogradely to olivocochlear cells in the pons. At 32–72 h after injection, labelling occurred in higher order auditory brainstem nuclei as well as in the locus coeruleus and pontine dorsal raphe. At 90–108 h, virus-infected neurons were found bilaterally in the medial geniculate body and in layer V of the auditory cortex. Viral transneuronal labelling in the auditory cortex after intracochlear application confirms the existence of a continuous descending chain of neurons from the auditory cortex to the cochlea, via the medial and lateral olivocochlear systems. The transneuronal labelling of the locus coeruleus and pontine dorsal raphe suggests that noradrenergic and serotonergic inputs may substantially influence the activity of olivocochlear cells, and thus the cochlea.

Notes: 1. The influence of two angiotensin-converting enzyme inhibitors, captopril and enalapril, on the cAMP content of microdissected brain areas was examined in spontaneously hypertensive rats. Both drugs depleted systolic arterial blood pressure significantly.2. Captopril and enalapril increased the level of cAMP in catecholaminergic cell groups in the lower brain-stem. Captopril was more effective in the substantia nigra, while enalapril treatment resulted in high cAMP levels in the ventrolateral medulla oblongata (A1 catecholaminergic cell group).3. Both drugs, especially captopril, depleted cAMP content in the cingulate cortex.4. No changes in cAMP levels were measured in the primary baroreceptor centre (nucleus of the solitary tract) following either captopril or enalapril treatment.