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Notes: Abstract: Previous studies have provided evidence for adrenocorticotropic hormone (ACTH) effects on a wide variety of behaviors. However, the precise sites of action and the mechanisms by which these effects may be mediated have yet to be clearly elucidated. Although ACTH was shown to augment cyclic AMP levels in glial cells isolated from whole brain, other studies found little or no effect of ACTH peptides on cyclic nucleotide metabolism in slices of cerebral cortex or homogenates of whole brain. In the present study, our objective was to determine whether ACTH peptides regulate intracellular cyclic AMP levels in neurons of the cerebral cortex in primary culture. ACTH peptides stimulated cyclic AMP synthesis up to threefold in a dose-dependent manner; stimulation was complete within 5–10 min of exposure to agonists. Neurohormone efficacy was augmented by 0.1 μM forskolin (which was virtually ineffective alone); potency was unaffected. The order of potency (EC50) for increasing intracellular cyclic AMP levels was as follows: ACTH (1–24), ACTH (1–17) (10 nM) 〉 α-melanocyte stimulating hormone, β-melanocyte stimulating hormone (α-MSH, β-MSH) (100 nM) 〉 ACTH (1–10) (1 μM) 〉 ACTH (4–10) (5 μM). The hexapeptide ACTH (4–9) as well as ACTH (11–24) were inactive at concentrations as high as 10 μM. Other neuropeptides derived from proopiocortin, such as β-endorphin and Met- and Leu-enkephalin were without effect on basal or hormonally stimulated cyclic AMP synthesis. In order to determine whether distinct receptors for ACTH are present on cortical neurons, saturating concentrations of the peptide were coincubated with either vasoactive intestinal polypeptide (VIP) or the β-adrenergic agonist, isoproterenol (INE). The response to combinations of ACTH and INE were clearly additive. However, neither ACTH nor INE could further augment cyclic AMP formation at saturating concentrations of VIP. Comparison of structure-activity relationships suggest that ACTH receptors mediating the elevation of cyclic AMP formation in cortical neurons may be similar to those associated with the peptide actions on arousal rather than conditioned behavior.

Notes: The regulation of GTP-binding proteins (G proteins) was examined during the course of differentiation of neuroblastoma N1E-115 cells. N1E-115 cell membranes possess three Bordetella pertussis toxin (PTX) substrates assigned to α-subunits (Gα) of Go (a G protein of unknown function) and “Gi (a G protein inhibitory to adenylate cyclase)-like” proteins and one substrate of Vibrio cholerae toxin corresponding to an α-subunit of Gs (a G protein stimulatory to adenylate cyclase). In undifferentiated cells, only one form of Goα was found, having a pI of 5.8. Goα content increased by approximately twofold from the undifferentiated state to 96 h of cell differentiation. This is mainly due to the appearance of another Goα form having a pI of 5.55. Both Goα isoforms have similar sizes on sodium dodecyl sulfate-polyacrylamide gels, are recognized by polyclonal antibodies to bovine brain Goα, are ADP-ribosylated by PTX, and are covalently myristylated in whole N1E-115 cells. In addition, immunofluorescent staining of N1E-115 cells with Goα antibodies revealed that association of Goα with the plasma membrane appears to coincide with the expression of the most acidic isoform and morphological cell differentiation. In contrast, the levels of both Giα and Gsα did not significantly change, whereas that of the common β-subunit increased by ∼ 30% over the same period. These results demonstrate specific regulation of the expression of Goα during neuronal differentiation.

Notes: Abstract Endogenous amino acid release was measured in developing cerebellar neuronal cells in primary culture. In the presence of 25 mM K+ added to the culture medium, cerebellar cells survived more than 3 weeks and showed a high level of differentiation. These cultures are highly enriched in neurons, and electron-microscopic observation of these cells after 12 days in vitro (DIV) confirmed the presence of a very large proportion of cells with the morphological characteristics of granule cells, making synapses containing many synaptic vesicles. Synaptogenesis was also confirmed by immunostaining the cells with antisera against synapsin I and synaptophysin, two proteins associated with synaptic vesicles. From these cultures, endogenous glutamate release stimulated by 56 m M K+ was already detected after only a few days in culture, the maximal release value (1,579% increase over basal release) being reached after 10 DIV. In addition to that of glutamate, the release of aspartate, asparagine, alanine, and, particularly, γ-aminobutyric acid (GABA) was stimulated by 56 mM K+ after 14 DIV, but to a lesser extent. No increase in serine, glutamine, taurine, or tyrosine release was observed during K+ depolarization. The effect of K+ on amino acid release was strictly Ca2+-dependent. Stimulation of the cells with veratridine resulted in a qualitatively similar effect on endogenous amino acid release. In the absence of Ca2+, 30% of the veratridine effect persisted. The Ca2+-dependent release was quantitatively similar after stimulation by veratridine and K+. Treatment of cerebellar cells with tetanus toxin (5 μ/ml) for 24 h resulted in a total inhibition of the Ca2+-dependent component of the glutamate release evoked by K+ or veratridine. It is concluded that glutamate is the main amino acid neurotransmitter of cerebellar cells developed in primary culture under the present conditions and that glutamate is probably mainly released through the exocytosis of synaptic vesicles.

Notes: Abstract: Inositol-1,4,5-trisphosphate, produced in cells as a breakdown product of phosphatidylinositol-4,5-bisphosphate, induces, in many cell types, release of calcium from intracellular stores. In murine striatal neurons, differentiated in primary culture, carbachol, norepinephrine, glutamate, and neurotensin stimulate 3H-labeled inositol phosphate (3H-IP) production. The glutamate response was recently characterized as being mediated primarily by receptors of the quisqualate subtype. In the present study, we found that major differences exist between glutamate-stimulated 3H-IP formation and those stimulated by the other neuromediators. The maximal response to glutamate occurred before and during synaptogenesis and declined thereafter, whereas the maximal response to either carbachol or norepinephrine required complete neuronal differentiation. Although the glutamate response appears to be mediated exclusively by direct interaction with the neuro-transmitter receptors, responses to carbachol, norepinephrine, and neurotensin were partially or completely blocked by tetrodotoxin.

Notes: Abstract: The effects of maitotoxin (MTX) on endogenous amino acid release were tested on highly purified striatal neurons differentiated in primary culture. MTX induced a large and concentration-dependent release of γ-aminobutyric acid (GABA). This effect was abolished when experiments were performed in the absence of external Ca2+, and restored when Ca2+ ions were added after removing the MTX-containing Ca2+-free solution. MTX-induced amino acid release was not affected by 1 μM nifedipine and only slightly inhibited by 1 mM Co2+. MTX also induced a massive accumulation of 45Ca2+ in the neurons which, in contrast to the MTX-evoked GABA release, was totally blocked in the presence of 1 mM Co2+. Whereas 500 nM tetrodotoxin was without significant effect, MTX-evoked GABA release was dependent on the presence of external Na+ and sensitive to nipecotic acid, a GABA uptake inhibitor. It is concluded that, on striatal neurons, MTX induced Na+ influx only in the presence of external Ca2+. The increase in cytoplasmic Na+ ions then triggers the release of GABA.

Notes: Abstract: We tested the possibility that endogenous nitric oxide synthase activity regulated NMDA receptors in primary cultured striatal neurons. We monitored NMDA-induced increase in intra-cellular Ca2+ levels with fura-2 ratio imaging, while nitric oxide synthase activity was either increased with l-arginihe (the natural substrate of nitric oxide synthase) or inhibited using nitro-l-arginine (a specific inhibitor of nitric oxide synthase). We found that the NMDA receptor effect was slowly but strongly diminished after an l-arginine (1 mM, 15 min) treatment (l-arginine preincubation reduced the 100 μM NMDA-induced maximal effect by 30–50%). The l-arginine blockade of NMDA receptors was long-lasting but could be partially reversed by hemoglobin (100 μM, 10 min), which binds nitric oxide. This was not observed when the neurons were treated with l-arginine together with nitro-l-arginine. Our data strongly suggest that physiological nitric oxide synthase activity could regulate NMDA receptors.

Notes: The pituitary adenylate cyclase-activating polypeptide type-1 receptor (PAC1) has been involved in the survival and differentiation of neuroblasts during development. This study examined the effects of various neurotrophins on the activity of the mouse PAC1 promoter/luciferase reporter constructs in rat PC12 cells and in 8-day-old mouse cerebellar granule cells. In PC12 cells, both differentiating factors such as nerve growth factor (NGF) and mitogens such as epidermal growth factor (EGF) and insulin growth factor-1 (IGF-1) up-regulated PAC1 promoter activity by 2–4-fold in a concentration-dependent manner. Although PACAP differentiated the PC12 cells, it had no effect on the PAC1 promoter and antagonized the stimulatory effect of NGF. In cerebellar granule cells, IGF-1 and brain-derived neurotrophic factor (BDNF) also stimulated the activity of the PAC1 promoter. NGF and IGF-1 increased endogenous PAC1 mRNA levels, and the NGF-induced up-regulation is the result of an increase in transcription from PAC1 promoter instead of an increase in mRNA stability. The mitogen-activated protein kinase (MAPK) kinase inhibitor, PD98059, prevented the transcriptional effects both in PC12 and cerebellar granule cells. Moreover, expression of dominant-negative Ras protein in PC12 cells also prevented the NGF effect. Our results show that the PAC1 promoter can be up-regulated by diverse neurotrophins via an MAPK-dependent pathway and suggest a role for the Ras protein.

Notes: Abstract: If cytokines are constitutively expressed by and act on neurons in normal adult brain, then we may have to modify our current view that they are predominantly inflammatory mediators. We critically reviewed the literature to determine whether we could find experimental basis for such a modification. We focused on two “proinflammatory” cytokines, interleukin (IL)-1 and tumor necrosis factor-α (TNFα) because they have been most thoroughly investigated in shaping our current thinking. Evidence, although equivocal, indicates that the genes coding for these cytokines and their accessory proteins are expressed by neurons, in addition to glial cells, in normal brain. Their expression is region- and cell type-specific. Furthermore, bioactive cytokines have been extracted from various regions of normal brain. The cytokines’ receptors selectively are present on all neural cell types, rendering them responsive to cytokine signaling. Blocking their action modifies multiple neural “house-keeping” functions. For example, blocking IL-1 or TNFα by several independent means alters regulation of sleep. This indicates that these cytokines likely modulate in the brain behavior of a normal organism. In addition, these cytokines are likely involved in synaptic plasticity, neural transmission, and Ca2+ signaling. Thus, the evidence strongly suggests that these cytokines perform neural functions in normal brain. We therefore propose that they should be thought of as neuromodulators in addition to inflammatory mediators.

Notes: The identification of guanine nucleotide binding proteins (G proteins) in guinea-pig tissues was assessed by the adenosine diphosphate-ribosylation of the α subunit by Bordetella pertussis toxin using [α32P] nicotinamide adenine dinucleotide as the substrate followed by sodium dodecyl sulphate - polyacrylamide gel electrophoresis and autoradiography. Three tissues (inferior colliculus, neuroblastoma cells, and the organ of Corti) contained G0α (39 kD), as well as Gi2α (40 kD) and Gi1α and/or Gi3α (41 kD). The stria vascularis and the VIIIth nerve contained mainly Gi2α, Gi1α and/or Gi3α, but G0α was barely detectable. A purified preparation of outer hair cells from the organ of Corti contained all three pertussis toxin substrates including G0α, with the Gi2α (40 kD) subunit being the most prominent. The immunocytochemical localization of the G0α subunit was determined by light microscopy after incubating isolated outer hair cells, Hensen cells and the stria vascularis with affinity-purified anti-G0α antibodies. In hair cells a positive reaction was observed along the plasma membrane and around the perimeter of the cuticular plate (zona adherens). Positive reaction was also observed within the infracuticular network extending from the cuticular plate towards the nucleus in outer hair cells. Finally, the base of the outer hair cells also contained G0α. However, it is likely that the G0α that is present in this cell region is not within the hair cell itself, but rather in nerve terminals which remained attached during dissection.

Notes: The release of arachidonic acid (ArA) metabolites from mouse neurons and astrocytes in primary culture has been studied in response to ionomycin or glutamate stimulation. Cells were preincubated with [3H] ArA for 24 h and the radioactivity released was examined by HPLC. In striatal, cortical and hippocampal neurons, glutamate and ionomycin strongly stimulated the release of ArA, but neither prostaglandins (PGs) nor hydroxyeicosatetraenoic acids (HETEs) could be detected. If they were released, these latter compounds represented 〈 0.02% of the amount of ArA. In contrast, in astrocyte cultures, ionomycin (but not glutamate) strongly stimulated the release of PGs and HETEs as well as ArA. Reversed- and straight-phase HPLC analysis revealed the presence of PGD2, PGE2, PGF2α, 12-hydroxyheptadeca-5, 8,10-trienoic acid (HHT) and HETEs (15-HETE, 11-HETE and 5-HETE). Indomethacin inhibited the release of PGs and HHT, but also that of 11- and 15-HETE, indicating that these two HETEs may be produced through the cyclooxygenase pathway. Metabolism of [3H]ArA was also examined in cellular homogenates. Although 〉 50% of the [3H]ArA was metabolized to PGF2α, PGE2, PGD2, HHT, 15- and 11-HETE in cultured astrocyte homogenates, no [3H]ArA metabolism could be detected in cultured striatal neuron homogenates. Moreover, neuronal homogenates did not inhibit the metabolism of [3H]ArA observed in either astrocyte or platelet homogenates. These results indicate that central neurons in primary culture possess very low lipoxygenase and cyclooxygenase activities. They emphasize the need to identify the cellular source of ArA metabolites in the brain, particularly when considering the multiple new messenger roles proposed for these molecules, such as that of retrograde messengers involved in synaptic plasticity phenomena.