Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells

The influence of melatonin on the developmental pattern of functional nicotinic acetylcholine receptors was investigated in embryonic 8-day-old chick retinal cells in culture. The functional response to acetylcholine was measured in cultured retina cells by microphysiometry. The maximal functional response to acetylcholine increased 2.7 times between the 4th and 5th day in vitro (DIV4, DIV5), while the Bmax value for 125I-a-bungarotoxin was reduced. Despite the presence of a8-like immunoreactivity at DIV4, functional responses mediated by a-bungarotoxin-sensitive nicotinic acetylcholine receptors were observed only at DIV5. Mecamylamine (100 µM) was essentially without effect at DIV4 and DIV5, while dihydro-ß-erythroidine (10-100 µM) blocked the response to acetylcholine (3.0 nM-2.0 µM) only at DIV4, with no effect at DIV5. Inhibition of melatonin receptors with the antagonist luzindole, or melatonin synthesis by stimulation of D4 dopamine receptors blocked the appearance of the a-bungarotoxin-sensitive response at DIV5. Therefore, a-bungarotoxin-sensitive receptors were expressed in retinal cells as early as at DIV4, but they reacted to acetylcholine only after DIV5. The development of an a-bungarotoxin-sensitive response is dependent on the production of melatonin by the retinal culture. Melatonin, which is produced in a tonic manner by this culture, and is a key hormone in the temporal organization of vertebrates, also potentiates responses mediated by a-bungarotoxin-sensitive receptors in rat vas deferens and cerebellum. This common pattern of action on different cell models that express a-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.

ENDO-OLIGOPEPTIDASE-A, A PUTATIVE ENKEPHALIN-GENERATING ENZYME, IN THE VERTEBRATE RETINA

Endo-oligopeptidase A, EC 3.4.22.19, converts small enkephalin-containing peptides into the corresponding enkephalins in vitro. We investigated the presence of endooligopeptidase A in the retina and its possible colocalization with enkephalins in retinal neurons. The specific activity of endo-oligopeptidase A found in pigeon retinae (30.3 +/- 7.3 mU/mg, mean +/- standard deviation) was four times higher than in rabbit retinae (7.0 +/- 1.1 mU/mg). The enzyme activity was not modified by EDTA, but it was enhanced by dithiothreitol and inhibited by zinc and 5,5'-dithiobis(2-nitrobenzoic acid). Immunohistochemical experiments with a purified antiserum against rabbit endo-oligopeptidase A revealed labeled neurons in both the inner nuclear layer and the ganglion cell layer of pigeon and rabbit retinae. Double-labeling immunofluorescence experiments demonstrated that about 90% of neurons containing endo-oligopeptidase A-like immunoreactivity also contained [Leu5]-enkephalin-like immunoreactivity. These colocalization results may represent an important step toward the demonstration of the possible involvement of endo-oligopeptidase A in enkephalin generation in vivo.

Avaliação da teneurina-2 (Ten-2) nos astrócitos do córtex cerebral de ratos adultos após lesão mecânica. Estudo imuno-histoquímico e de expressão gênica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo da distribuição das proteínas relacionadas às teneurinas no sistema nervoso central de primatas não-humanos (Sapajus spp) e ratos (Rattus norvegicus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação da teneurina-2 (Ten-2) nos astrócitos do córtex cerebral de ratos adultos após lesão mecânica. Estudo imuno-histoquímico e de expressão gênica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo da distribuição das proteínas relacionadas às teneurinas no sistema nervoso central de primatas não-humanos (Sapajus spp) e ratos (Rattus norvegicus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evidence for glutamatergic ganglion cells in the avian retina

This dissertation presents structural, immunochemical and neurochemical evidence for glutamatergic retinotectal synaptic transmission, augmenting and extending previous physiological and anatomical studies. The evidence is especially striking when the laminar patterns of ($\sp3$H) L-glutamate receptor binding, ($\sp3$H) L-glutamate high affinity uptake (HAU) and glutamate immunoreactivity (GLIR) of the dorsal tectum are compared. All show high activity in the tectal SGFS, with a peak in the most superficial laminae of SGFS followed by dip in the b-c region, and a second broad peak in deeper SGFS. Uptake and immunoreactivity bear a stronger resemblance to one another than either does to receptor binding, consistent with the fact that HAU and GLIR are localized in the same structures: glutamatergic terminals, intrinsic cell bodies and their processes. Receptor binding, as attested by the lack of enucleation effects, is a marker of postsynaptic receptors. In summary, these results are consistent with the hypothesis that most of the retinal projection to the optic tectum is glutamatergic: (1) A glutamate/aspartate HAU system exists in the superficial laminae, and it is dependent upon an intact retinal input, as shown developmentally and by retinal ablation; (2) Glutamate-like immunoreactivity appears in retinorecipient tectal regions (partially responsive to enucleation), in cell bodies of retinal ganglion cells and displaced ganglion cells, and in a non-tectal ganglion cell projection, the ectomammilary nucleus; (3) Sodium-independent glutamate receptor binding (which remains unchanged by enucleation) is most intense in the retinorecipient regions of the tectum and the ectomammilary nucleus. This binding is pharmacologically typical of a CNS sensory structure, being dominated by the quisqualate/kainate receptor subclass. Thus, as with other sensory systems, a portion of the retinotectal projection has been shown to include glutamatergic afferents with the distribution and properties expected of the primary projection ^

Cocaine- and amphetamine-regulated transcript in the rat vagus nerve: A putative mediator of cholecystokinin-induced satiety

Cocaine- and amphetamine-regulated transcript (CART) is widely expressed in the central nervous system. Recent studies have pointed to a role for CART-derived peptides in inhibiting feeding behavior. Although these actions have generally been attributed to hypothalamic CART, it remains to be determined whether additional CART pathways exist that link signals from the gastrointestinal tract to the central control of food intake. In the present study, we have investigated the presence of CART in the rat vagus nerve and nodose ganglion. In the viscerosensory nodose ganglion, half of the neuron profiles expressed CART and its predicted peptide, as determined by in situ hybridization and immunohistochemistry. CART expression was markedly attenuated after vagotomy, but no modulation was observed after food restriction or high-fat regimes. A large proportion of CART-labeled neuron profiles also expressed cholecystokinin A receptor mRNA. CART-peptide-like immunoreactivity was transported in the vagus nerve and found in a dense fiber plexus in the nucleus tractus solitarii. Studies on CART in the spinal somatosensory system revealed strong immunostaining of the dorsal horn but only a small number of stained cell bodies in dorsal root ganglia. The present results suggest that CART-derived peptides are present in vagal afferent neurons sensitive to cholecystokinin, suggesting that the role of these peptides in feeding may be explained partly by mediating postprandial satiety effects of cholecystokinin.

Growth hormone-releasing hormone: An autocrine growth factor for small cell lung carcinoma

Antagonists of growth hormone-releasing hormone (GHRH) inhibit the growth of various cancers in vivo. This effect is thought to be exerted through suppression of the pituitary growth hormone–hepatic insulin-like growth factor I (IGF-I) axis and direct inhibition of autocrine/paracrine production of IGF-I and -II in tumors. However, other evidence points to a direct effect of GHRH antagonists on tumor growth that may not implicate IGFs, although an involvement of GHRH in the proliferation of cancer cells has not yet been established. In the present study we investigated whether GHRH can function as an autocrine/paracrine growth factor in small cell lung carcinoma (SCLC). H-69 and H-510A SCLC lines cultured in vitro express mRNA for GHRH, which apparently is translated into peptide GHRH and then secreted by the cells, as shown by the detection of GHRH-like immunoreactivity in conditioned media from the cells cultured in vitro. In addition, the levels of GHRH-like immunoreactivity in serum from nude mice bearing H-69 xenografts were higher than in tumor-free mice. GHRH(1–29)NH2 stimulated the proliferation of H-69 and H-510A SCLCs in vitro, and GHRH antagonist JV-1–36 inhibited it. JV-1–36 administered s.c. into nude mice bearing xenografts of H-69 SCLC reduced significantly (P < 0.05) tumor volume and weight, after 31 days of therapy, as compared with controls. Collectively, our results suggest that GHRH can function as an autocrine growth factor in SCLCs. Treatment with antagonistic analogs of GHRH may offer a new approach to the treatment of SCLC and other cancers.

Localization of neuropeptide Y Y1 receptors in cerebral blood vessels

The localization of neuropeptide Y (NPY) Y1 receptor (R) -like immunoreactivity (LI) has been studied in cerebral arteries and arterioles of the rat by immunohistochemistry using fluorescence, confocal, and electron microscopy. High levels of Y1-R-LI were observed in smooth muscle cells (SMCs) in the small arterioles of the pial arterial network, especially on the basal surface of the brain, and low levels in the major basal cerebral arteries. The levels of Y1-R-LI varied strongly between adjacent SMCs. Y1-R-LI was associated with small endocytosis vesicles, mainly on the outer surface of the SMCs, but also on their endothelial side and often laterally at the interface between two SMCs. NPY-immunoreactive (Ir) nerve fibers could not be detected in association with the Y1-R-rich small arterioles but only around arteries with low Y1-R levels. A dense network of central NPY-Ir nerve fibers in the superficial layers of the brain was lying close to the strongly Y1-R-Ir small arterioles. The results indicate that NPY has a profound effect on small arterioles of the brain acting on Y1-Rs, both on the peripheral and luminal side of the SMCs. However, the source of the endogenous ligand, NPY, remains unclear. NPY released from central neurons may play a role, in addition to blood-borne NPY.

Epilepsy in mice deficient in the 65-kDa isoform of glutamic acid decarboxylase

γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, is synthesized by two glutamate decarboxylase isoforms, GAD65 and GAD67. The separate role of the two isoforms is unknown, but differences in saturation with cofactor and subcellular localization suggest that GAD65 may provide reserve pools of GABA for regulation of inhibitory neurotransmission. We have disrupted the gene encoding GAD65 and backcrossed the mutation into the C57BL/6 strain of mice. In contrast to GAD67−/− animals, which are born with developmental abnormalities and die shortly after birth, GAD65−/− mice appear normal at birth. Basal GABA levels and holo-GAD activity are normal, but the pyridoxal 5′ phosphate-inducible apo-enzyme reservoir is significantly decreased. GAD65−/− mice develop spontaneous seizures that result in increased mortality. Seizures can be precipitated by fear or mild stress. Seizure susceptibility is dramatically increased in GAD65−/− mice backcrossed into a second genetic background, the nonobese diabetic (NOD/LtJ) strain of mice enabling electroencephalogram analysis of the seizures. The generally higher basal brain GABA levels in this backcross are significantly decreased by the GAD65−/− mutation, suggesting that the relative contribution of GABA synthesized by GAD65 to total brain GABA levels is genetically determined. Seizure-associated c-fos-like immunoreactivity reveals the involvement of limbic regions of the brain. These data suggest that GABA synthesized by GAD65 is important in the dynamic regulation of neural network excitability, implicate at least one modifier locus in the NOD/LtJ strain, and present GAD65−/− animals as a model of epilepsy involving GABA-ergic pathways.

Localization of choline acetyltransferase in rat peripheral sympathetic neurons and its coexistence with nitric oxide synthase and neuropeptides.

Indirect immunofluorescence methods using a mouse monoclonal antibody raised to rat choline acetyltransferase (ChAT) revealed dense networks of ChAT-immunoreactive fibers in the superior cervical ganglion, the stellate ganglion, and the celiac superior mesenteric ganglion of the rat. Numerous and single ChAT-immunoreactive cell bodies were observed in the stellate and superior cervical ganglia, respectively. The majority of ChAT-immunoreactive fibers in the stellate and superior cervical ganglia were nitric oxide synthase (NOS) positive. Some ChAT-immunoreactive fibers contained enkephalin-like immunoreactivity. Virtually all ChAT-positive cell bodies in the stellate ganglion were vasoactive intestinal polypeptide (VIP)-positive, and some were calcitonin gene-related peptide (CGRP)-positive. After transection of the cervical sympathetic trunk almost all ChAT- and NOS-positive fibers and most enkephalin- and CGRP-positive fibers disappeared in the superior cervical ganglion. The results suggest that most preganglionic fibers are cholinergic and that the majority of these in addition can release nitric oxide, some enkephalin, and a few CGRP. Acetylcholine, VIP, and CGRP are coexisting messenger molecules in some postganglionic sympathetic neurons.

Evidence for a role of endothelin 1 and protein kinase C in nitroglycerin tolerance.

We sought to examine mechanisms responsible for increased vasoconstriction that occurs during development of nitroglycerin tolerance. Rabbits were treated for 3 days with nitroglycerin patches (0.4 mg/hr), and their aortic segments were studied in organ chambers. This treatment resulted in attenuated in vitro relaxations to nitroglycerin and increased contractile sensitivity to angiotensin II, serotonin, phenylephrine, KCl, and a direct activator of protein kinase C, the phorbol ester phorbol 12,13-dibutyrate. The protein kinase C antagonists calphostin C (100 nM) and staurosporine (10 nM) corrected the hypersensitivity to constrictors in tolerant vessels, yet had minimal effects on constrictions in control vessels. Paradoxically, constrictions caused by endothelin 1 were decreased in nitrate-tolerant vessels. Immunocytochemical analysis revealed intense endothelin 1-like and big endothelin 1-like immunoreactivity in the media of nitroglycerin-tolerant but not of control aortas. The enhanced vasoconstriction to angiotensin II, serotonin, KCl, and phenylephrine could be mimicked in normal vessels by addition of subthreshold concentrations of endothelin 1, and this effect was prevented by calphostin C. We propose that increased autocrine production of endothelin 1 in nitrate tolerance sensitizes vascular smooth muscle to a variety of vasoconstrictors through a protein kinase C-mediated mechanism.

Treatment of dementia with neurotransmission modulation

The prevalence of dementia is growing in developed countries where elderly patients are increasing in numbers. Neurotransmission modulation is one approach to the treatment of dementia. Cholinergic precursors, anticholinesterases, nicotine receptor agonists and muscarinic M-2 receptor antagonists are agents that enhance cholinergic neurotransmission and that depend on having some intact cholinergic innervation to be effective in the treatment of dementia. The cholinergic precursor choline alfoscerate may be emerging as a potential useful drug in the treatment of dementia, with few adverse effects. Of the anticholinesterases, donepezil, in addition to having a similar efficacy to tacrine in mild-to-moderate Alzheimer's disease (AD), appears to have major advantages; its use is associated with lower drop-out rates in clinical trials, a lower incidence of cholinergic-like side effects and no liver toxicity. Rivastigmine is efficacious in the treatment in dementia with Lewy bodies, a condition in which the other anticholinesterases have not been tested extensively to date. Galantamine is an anticholinesterase and also acts as an allosteric potentiating modulator at nicotinic receptors to increase the release of acetylcholine. Pooled data from clinical trials of patients with mild-to-moderate AD suggest that the benefits and safety profile of galantamine are similar to those of the anticholinesterases. Selective nicotine receptor agonists are being developed that enhance cognitive performance without influencing autonomic and skeletal muscle function, but these have not yet entered clinical trial for dementia. Unlike the cholinergic enhancers, the M, receptor agonists do not depend upon intact cholinergic nerves but on intact M, receptors for their action, which are mainly preserved in AD and dementia with Lewy bodies. The M, receptor-selective agonists developed to date have shown limited efficacy in clinical trials and have a high incidence of side effects. A major recent advancement in the treatment of dementia is memantine, a non-competitive antagonist at NMDA receptors. Memantine is beneficial in the treatment of severe and moderate to-severe AD and may also be of some benefit in the treatment of mild-to-moderate vascular dementia. Drugs that modulate 5-HT, somatostatin and noradrenergic neurotransmission are also being considered for the treatment of dementia.

Type 1 nitrergic (ND1) cells of the rabbit retina: Comparison with other axon-bearing amacrine cells

NADPH diaphorase (NADPHd) histochemistry labels two types of nitrergic amacrine cells in the rabbit retina. Both the large ND1 cells and the small ND2 cells stratify in the middle of the inner plexiform layer, and their overlapping processes produce a dense plexus, which makes it difficult to trace the morphology of single cells. The complete morphology of the ND1 amacrine cells has been revealed by injecting Neurobiotin into large round somata in the inner nuclear layer, which resulted in the labelling of amacrine cells whose proximal morphology and stratification matched those of the ND1 cells stained by NADPHd histochemistry. The Neurobiotin-injected ND1 cells showed strong homologous tracer coupling to surrounding ND1 cells, and double-labelling experiments confirmed that these coupled cells showed NADPHd reactivity. The ND1 amacrine cells branch in stratum 3 of the inner plexiform layer, where they produce a sparsely branched dendritic tree of 400-600 mum diameter in ventral peripheral retina. In addition, each cell gives rise to several fine beaded processes, which arise either from a side branch of the dendritic tree or from the tapering of a distal dendrite. These axon-like processes branch successively within the vicinity of the dendritic field before extending, with little or no further branching, for 3-5 mm from the soma in ventral peripheral retina. Consequently, these cells may span one-third of the visual field of each eye, and their spatial extent appears to be greater than that of most other types of axon-bearing amacrine cells injected with Neurobiotin in this study. The morphology and tracer-coupling pattern of the ND1 cells are compared with those of confirmed type 1 catecholaminergic cells, a presumptive type 2 catecholaminergic cell, the type 1 polyaxonal. cells, the long-range amacrine cells, a novel type of axon-bearing cell that also branches in stratum 3, and a type of displaced amacrine cell that may correspond to the type 2 polyaxonal cell. (C) 2004 Wiley-Liss, Inc.