Extent of colocalization of serotonin and GABA in the neurons of the rat raphe nuclei

Previous investigations of the distribution of neurons containing both serotonin and GABA in the brainstem raphe nuclei have yielded discrepant results amongst different authors. This study attempted to clarify the distribution as well as the proportions of raphe and other brainstem neurons that contain both neurotransmitters. All the nine serotonergic cell groups known to be present in the brainstem were examined with an indirect immunofluorescence method using antibodies against serotonin and glutamic acid decarboxylase in colchicine-treated rats. Sections were incubated either simultaneously or sequentially for the two immunolabels. Brainstem neurons that were labelled for both markers were generally infrequent. Of all the serotonin cell groups in the brainstem, the nucleus raphe magnus contained the most double-labelled cells (a mean of 3.6% of a total of 625–1155 serotonin-immunoreactive cells counted in this nucleus), followed by the nucleus raphe obscurus (1.5% of a total of 220–550 serotonin-immunoreactive neurons counted). The dorsal, median and pontine raphe nuclei as well as the supralemniscal nucleus (the B9 group) contained very few double-labelled cells, which comprised a mean of 0.1–0.7% of all serotonin-immunoreactive cells in each of these nuclei. No double labelled cells were present in the caudal linear raphe nucleus or the nucleus raphe pallidus, nor in the B4 group. These results suggest that only a very small percentage of serotonergic neurons in the medullary raphe nuclei (raphe magnus and raphe obscurus) also contain GABA, whereas such cells are virtually absent in the midbrain raphe nuclei or in the non-raphe serotonergic cell groups in the brainstem.     

Coexistence of cholinergic, catecholaminergic, serotonergic, and glutamatergic neurotransmitter markers in mouse clonal hybrid neurons derived from the septal region.

Two clonal immortalized neurons designated SN6.1b and SN6.2a were isolated by limiting dilution from a mouse embryonic septal cholinergic neuronal hybrid cell line SN6 (Hammond et al., 1986). In the serum-containing medium without extra differentiating agents, one-third of SN6.1b cells stably exhibited a morphology of differentiated neurons with extensive elaborate neurites, while a majority of SN6.2a cells, along with the parent cell line SN6, were round in shape with poorly branched short processes. Neurochemical studies showed that both clones synthesized choline acetyltransferase (ChAT), dopamine, norepinephrine, serotonin, and glutamate. Immunocytochemically, they expressed a number of neuronal antigens, such as 200-kDa neurofilament protein, neuron-specific enolase, microtubule-associated protein 2, tau protein, tubulin, neural cell adhesion molecule, Thy-1.2, saxitoxin-binding sodium channel protein, ChAT, tyrosine hydroxylase, serotonin, and glutamate. The coexistence of cholinergic, catecholaminergic, serotonergic, and glutamatergic neurotransmitter markers in the clonal hybrid septal neurons that express a variety of immunocytochemical properties of differentiated neurons suggests that embryonic septal cholinergic neurons are potentially multiphenotypic with respect to neurotransmitter synthesis.     

Descending serotonergic, peptidergic and cholinergic pathways from the raphe nuclei: a multiple transmitter complex

The localization of serotonergic, various peptidergic and possibly cholinergic neurons in the medullary raphe nuclei that project to the lumbosacral spinal cord have been studied using a retrograde transport method combined with immunocytochemical and histochemical techniques. Spinally projecting neurons stained for serotonin-like, substance P-like, enkephalin-like and thyrotropin-releasing hormone-like immunoreactivity and for the histochemical marker acetylcholinesterase were all observed in each of the raphe nuclei of the medulla, as well as in the adjacent ventrolateral reticular formation. The similar distributions of the descending serotonergic and peptidergic neurons in the raphe nuclei as well as quantitative data on their relative numbers suggest that a large fraction of raphe-spinal neurons contain serotonin co-existing with one or more peptides in the same cell.     

Serotonergic dorsal raphe nucleus projections to the cholinergic and noncholinergic neurons of the pedunculopontine tegmental region: a light and electron microscopic anterograde tracing and immunohistochemical study

The serotonergic dorsal raphe nucleus is considered an important modulator of state-dependent neural activity via projections to cholinergic neurons of the pedunculopontine tegmental nucleus (PPT). Light and electron microscopic analysis of anterogradely transported biotinylated dextran, combined with choline acetyltransferase (ChAT) immunohistochemistry, were employed to describe the synaptic organization of mesopontine projections from the dorsal raphe to the PPT. In a separate set of experiments, we utilized immunohistochemistry for the serotonin transporter (SERT), combined with ChAT immunohistochemistry at the light and electron microscopic levels, to determine whether PPT neurons receive serotonergic innervation. The results of these studies indicate that: (1) anterogradely labeled and SERT-immunoreactive axons and presumptive boutons invest the PPT at the light microscopic level; (2) at the ultrastructural level, dorsal raphe terminals in the PPT pars compacta synapse mainly with dendrites and axosomatic contacts were not observed; (3) approximately 12% of dorsal raphe terminals synapse with ChAT-immunoreactive dendrites; and (4) at least 2-4% of the total synaptic input to ChAT-immunoreactive dendrites is of dorsal raphe and/or serotonergic origin. This serotonergic dorsal raphe innervation may modulate cholinergic PPT neurons during alterations in behavioral state. The role of these projections in the initiation of rapid eye movement (REM) sleep and the ponto-geniculo-occipital waves that precede and accompany REM sleep is discussed. J. Comp. Neurol. 382:302-322, 1997. © 1997 Wiley-Liss, Inc.     

Serotonergic, noradrenergic and galaninergic projections to the nucleus parafascicularis.

Immunocytochemical staining for serotonin (5-HT), tyrosine hydroxylase (TH) and galanin (GAL) was combined with horseradish peroxidase (HRP) retrograde tract-tracing technique to analyze the localizations of 5-HT-, catecholamine (CA)- and GAL-containing neurons in the brainstem which project to the nucleus parafascicularis (PF) in rats. It is demonstrated that most of the retrogradely HRP-labeled neurons (70%) in bilateral periaqueductal gray (PAG) and raphe nuclei are positively immunostained by antiserum to 5-HT, and that most of the retrogradely HRP-labeled neurons (over 80%) in bilateral locus coeruleus (LC) are positively immunostained by antisera to both TH and GAL. The possible functions of these PF-petal serotonergic, catecholaminergic (actually noradrenergic) and galaninergic projections are discussed.     

Immunohistochemical evidence for the presence of γ-aminobutyric acid and serotonin in one nerve cell. A study on the raphe nuclei of the rat using antibodies to glutamate decarboxylase and serotonin

A specific and sensitive double immunocytochemical staining for the visualization of glutamate decarboxylase (GAD) and serotonin (5-HT) on the same brain section is developed. GAD is detected with specific GAD-antibodies by means of the unlabeled antibody enzyme, peroxidase anti-peroxidase, and serotonin with an antibody against the BSA-serotonin conjugate by an indirect immunofluorescent staining. The coexistence of GAD and 5-HT in the same perikaryon is demonstrated by a peroxidase reaction superimposed on fluorescent compounds. Cell bodies containing both antigens are observed in each raphe nuclei. However, the nucleus raphe dorsalis exhibits the largest number of cells containing either GAD alone or GAD and 5-HT together. An intracellular interaction between the metabolism of GABA and serotonin could be reasonably expected. The interactions between GABAergic and serotonergic systems must be thought of in terms of intracellular and/or transynaptic controls.     

Serotonergic synaptic input to cholinergic neurons in the rat mesopontine tegmentum

Serotonergic synaptic inputs to cholinergic neurons in the laterodorsal and pedunculopontine tegmental nuclei were examined with pre-embedding dual-label immunoelectron microscopy. Numerous serotonin-immunoreactive axon terminals visualized with a silver-enhanced immunogold method were present in both of these tegmental nuclei. Serotonergic terminals occasionally made synaptic contacts with the soma and proximal dendrites of cholinergic tegmental neurons labelled with a choline acetyltransferase-immunoreactive peroxidase-anti-peroxidase diaminobenzidine reaction product. In the rostralmost region of the laterodorsal tegmental nucleus, a few serotonergic neurons of the dorsal raphe nucleus were interspersed among cholinergic neurons. Some dendrites of these serotonergic neurons appeared to contain synaptic vesicles. Both myelinated and unmyelinated serotonergic axons were present in the mesopontine tegmentum. The presence of serotonergic synapses onto tegmental cholinergic neurons is consistent with previous behavioral and electrophysiological findings suggesting an inhibitory role of serotonin in the induction of rapid eye movement sleep and its phenomenology through an action on cholinergic neurons in the mesopontine tegmentum.     

Serotonin-immunoreactive neurons and mast cells in the mouse esophagus suggest involvement of serotonin in both motility control and neuroimmune interactions

Background Serotonin is a major transmitter in the gastrointestinal tract, but little is known about the serotonergic system in the esophagus. Methods The aim of this study was to use multilabel immunofluorescence to characterize serotonin‐positive nerve cell bodies and fibers and their relationship with other neuronal and non‐neuronal elements in the mouse esophagus. Antibodies against serotonin, vesicular acetylcholine transporter (VAChT), choline acetyltransferase (ChAT), protein gene product 9.5 (PGP 9.5), and α‐bungarotoxin (α‐BT), were used. Key Results Serotonin‐containing perikarya represented ～10% of all PGP 9.5‐positive myenteric neurons. Serotonin‐positive varicose nerve fibers were found in the lamina muscularis mucosae and present on ～13% of α‐BT–labeled motor endplates in addition to VAChT‐immunoreactive motor terminals. As ChAT‐positive neurons of the compact formation of the nucleus ambiguus were negative for serotonin, serotonin‐positive varicosities on motor endplates are presumed to be of enteric origin. On the other hand, cholinergic ambiguus neurons were densely supplied with serotonin‐positive varicosities. The tela submucosa and tunica adventitia contained large numbers of serotonin‐positive mast cells, a few of which were in close association with serotonin‐positive nerve fibers. Conclusions & Inferences The mouse esophagus is endowed with a rich serotonin‐positive intrinsic innervation, including enteric co‐innervation of striated muscles. Serotonin may modulate vagal motor innervation of esophageal‐striated muscles not only at the central level via projections of the raphe nuclei to the nucleus ambiguus but also at the peripheral level via enteric co‐innervation. In addition, mast cells represent a non‐neuronal source of serotonin, being involved in neuroimmune processes.     

GABAergic neurons in the rat pontomesencephalic tegmentum: Codistribution with cholinergic and other tegmental neurons projecting to the posterior lateral hypothalamus

The present study was undertaken to determine the frequency and distribution of GABAergic neurons within the rat pontomesencephalic tegmentum and the relationship of GABAergic cells to cholinergic and other tegmental neurons projecting to the hypothalamus. In sections immunostained for glutamic acid decarboxylase (GAD), large numbers of small GAD-positive neurons (～50,000 cells) were distributed through the tegmentum and associated with a high density of GAD-positive varicosities surrounding both GAD-positive and GAD-negative cells. Through the reticular formation, ventral tegmentum, raphe nuclei, and dorsal tegineritum, GAD-positive cells were codistributed with larger cells, which included neurons immunostained on adjacent sections for glutamate, tyrosine hydroxylase (TH), serotonin, or choline acetyltransferase (ChAT). In sections dual-immunostained for GAD and ChAT, GABAergic neurons were seen to be intermingled with less numerous cholinergic cells (～2,600 GAD+ to ～ 1,400 ChAT+ cells in the laterodorsal tegmental nucleus, LDTg). Retrograde transport of cholera toxin (CT) was examined from the posterior lateral hypothalamus, where a major population of cortically projecting neurons are located. A small number of GABAergic cells were retrogradely labeled, representing a small percentage of all the GABAergic neurons (～1%) and of all the hypothalamically projecting neurons (～6%) in the tegmentum. The double-labeled GAD+/CT+ cells were commonly found ipsilaterally within (1) the deep mesencephalic reticular field, codistributed with putative glutamatergic projection neurons; (2) the ventral tegmental area, substantia nigra coinpacta, and retrorubral field, codistributed with dopaminergic projection neurons; (3) dorsal raphe, codistributed with serotonergic projection neurons; and (4) laterodorsal and pedunculopontine tegmental nuclei, codistributed with and in similar proportion to cholinergic projection cells (20–30% in LDTg). Acting as both projection and local neurons, the pontomesencephalic GABAergic cells would have the capacity to modulate the influence of the “ascending reticular activating system” and its chemically specific constituents upon cortical activation. © 1995 Wiley-Liss, Inc.     

Serotonergic connections to the ventral oral pontine reticular nucleus: implication in paradoxical sleep modulation

Cholinergic microstimulation of the ventral part of the oral pontine reticular nucleus (vRPO) in cats generates and maintains paradoxical sleep. The implication of rostral raphe nuclei in modulating the sleep-wakefulness cycle has been based on their serotonergic projections to the pontine structures responsible for the induction of paradoxical sleep. However, serotonergic neurons have also been described in brainstem structures other than the raphe nuclei. The aim of the present work is to trace the origin of the serotonergic afferents to the vRPO and to the locus coeruleus alpha and perilocus coeruleu alpha nuclei, closely related with different paradoxical sleep events. Anterograde and retrograde horseradish peroxidase conjugated with wheat germ agglutinin tracer injections in these nuclei in cats were combined with serotonin antiserum immunohistochemistry. Our results demonstrate that reciprocal connections linking the rostral raphe nuclei to those oral pontine nuclei are scarce. The percentage of double-labeled neurons after injections in the vRPO averaged 18% in rostral raphe nuclei, while a level of 82% was estimated in mesopontine tegmentum structures other than the raphe nuclei. These results showed that the main source of serotonin to the vRPO, implicated in generation and maintenance of paradoxical sleep, arises from these mesopontine tegmentum structures. This indicates that the serotonin modulation of paradoxical sleep could be the result of activation in non-raphe mesopontine tegmentum structures. The existence of a complicated network in the vRPO, which maintains a balance between different neurotransmitters responsible for the generation and alternance of paradoxical sleep episodes, is discussed.     

Effects of septal and/or raphe cell suspension grafts on hippocampal choline acetyltransferase activity, high affinity synaptosomal uptake of choline and serotonin, and behavior in rats with extensive septohippocampal lesions.

At 31 days of age, Long-Evans female rats sustained aspirative lesions of the septohippocampal pathways and, 14 days later, received intrahippocampal suspension grafts prepared from the region including the medial septum and the diagonal band of Broca (Group S, n = 11), from the region including the mesencephalic raphe (Group R, n = 11) or from both regions together (Group S+R, n = 11). Sham-operated (Group Sham, n = 9) and lesion-only (Group Les, n = 11) rats served as non-grafted controls. Seven Sham, 7 Les and 8 rats from each transplant group were tested for home cage activity (6 months after grafting) and radial maze performance (between 7.5 and 8.5 months post-grafting). One month after completion of behavioral testing, the dorsal hippocampi of these rats were prepared for measuring choline acetyltransferase (ChAT) activity and high affinity synaptosomal uptake of both [3H]choline and [3H]serotonin. The remaining rats were used for histological verifications on brain sections stained for acetylcholinesterase (AChE). The lesions increased locomotor activity, impaired radial maze learning and, in the dorsal hippocampus, reduced AChE positive staining, decreased ChAT activity (-73%) as well as high affinity uptake of both choline (-81%) and serotonin (-82%). Neither type of transplant produced any significant behavioral recovery. However, septal transplants increased hippocampal AChE positivity, restored ChAT activity and enhanced choline uptake to 116% and 70% of the values found in sham-operated rats, respectively; they had no significant effect on uptake of serotonin. Transplants from the raphe region had weak effects on hippocampal AChE positivity, increased both the ChAT activity and the choline uptake to 70% ad 38% of the sham-operated rats, respectively, and produced an (over)compensation of the serotonin uptake which reached 324% of the values found in sham-operated rats. The co-transplantation of both regions resulted in restoration of ChAT activity (113% of sham-operated rats values), choline uptake (83% of sham-operated rats) and serotonin uptake (129% of sham-operated rats). Our neurochemical data show that after extensive denervation of the hippocampus, intrahippocampal grafts of fetal neurons may foster a neurotransmitter-specific recovery which depends upon the anatomical origin of the grafted cells: a graft rich in serotonergic neurons overcompensates the serotonergic deficit, a graft rich in cholinergic neurons attenuates the cholinergic deficit, whereas a mixture of both types of grafts produces recovery from both types of deficits. Thereby, both the feasibility and the interest of the co-grafting technique are confirmed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Costorage of serotonin binding protein with serotonin in the rat CNS

Previous studies have identified two neurectoderm-specific serotonin binding proteins (SBP), one with an apparent Mr of 45 kDa, and one of 56 kDa. The current experiments were undertaken to test the hypothesis that these proteins are specific components of serotonergic neurons. Since actin has been found to bind serotonin, the relationship of the 2 forms of SBP to actin was also investigated. Antisera against purified 45 and 56 kDa SBP were raised in rabbits and shown by analyses of immunoblots and differential absorption to be monospecific and not cross-reactive. Neither antiserum reacted with purified actin and none of 3 different anti-actin sera reacted with purified 45 or 56 kDa SBP. The antisera to 45 and 56 kDa were used for immunocytochemical localization of the proteins, which was compared to that of serotonin. SBP immunoreactivity was found in rat brain and spinal cord; however, no significant differences were observed in the pattern of distribution of 45 and 56 kDa SBP-immunoreactive structures. Immunostaining of neuronal perikarya by either SBP antiserum required pretreatment of animals with colchicine. The distribution of neurons and terminals labeled by each antiserum to SBP was similar to that of neurons and terminals labeled by anti-5-HT sera. SBP-immunoreactive neuronal perikarya were present in the nuclei raphe dorsalis, raphe centralis superior, raphe medianus, raphe magnus, raphe obscurus, raphe pallidus, dorsal to the medial lemniscus in the region of the B9 cell group, near the interpeduncular nucleus, in the area postrema, the pars compacta of the substantia nigra, the dorsomedial nucleus of the hypothalamus, and the arcuate nucleus. SBP-immunoreactive fibers and terminals were present in many additional areas of the brain, as well as the spinal cord, where they paralleled those that were immunostained with antibodies to 5-HT. When double-immunostaining was used, serotonin and 45 and 56 kDa SBP immunoreactivities were found to be colocalized in both the brain and spinal cord. Cells and fibers found to be stained by one immunoreagent were also stained by the others; therefore, serotonergic neurons of the CNS probably contain both 45 and 56 kDa SBP. Moreover, it also seems likely that nonserotonergic neurons contain neither form of SBP. These data strongly suggest that SBP is an intrinsic and specific component of serotonergic neurons that can serve as a serotonergic marker.     

The brainstem projection to the lateral geniculate nucleus in the cat: identification of cholinergic and monoaminergic elements

The pontomesencephalic projection to the dorsal lateral geniculate nucleus (dLGN) of the cat was analyzed by combining retrograde transport of rhodamine-labeled latex spheres and immunohistochemistry. After injections of latex beads into the dLGN, sections of the brainstem were treated immunohistochemically for choline acetyltransferase (ChAT), serotonin (Ser), tyrosine hydroxylase (TH), and dopamine-beta-hydroxylase (DBH). Essentially, six regions in the brainstem contained retrogradely labeled cells: the superior colliculus, the parabigeminal nucleus, the dorsal raphe nuclei, the parabrachial area of the central tegmental field, the marginal nucleus of the brachium conjunctivum, and the nucleus coeruleus. Furthermore, isolated retrogradely labeled cells were present in the central nucleus of the raphe, in the cuneiform nucleus, and in the periaqueductal gray. Most serotoninergic double-labeled cells were found in the medial and lateral divisions of the dorsal raphe nuclei, but a few were also present in the central nucleus of the raphe. In the sections immunostained for ChAT, double-labeled cells were located in the central tegmental field, in the marginal nucleus of the brachium conjunctivum, and in the nucleus coeruleus. In the sections treated for TH and DBH, double-labeled cells showed a similar distribution, and like the ChAT(+) cells, they were located mainly in the central tegmental field, in the marginal nucleus of the brachium conjunctivum, and in the nucleus coeruleus. In these regions the cholinergic and noradrenergic cells that projected to the lateral geniculate nucleus were intermingled, the former predominating rostrally and the latter caudally. The majority of retrogradely labeled cells were located in the region of the central tegmental field in the vicinity of the brachium conjunctivum, and most of these cells were also ChAT-immunoreactive. We, therefore, conclude that the cholinergic projection is the most important of the central core projections ascending to the dLGN.     

Serotonergic neurons in the brainstem of the wallaby, Macropus eugenii.

The organisation and cytoarchitecture of the serotonergic neurons in a diprotodont marsupial were examined by using serial sections of the brainstem processed for serotonin immunohistochemistry and routine histology. The topographic distribution of serotonergic neurons in the brainstem of the adult wallaby (Macropus eugenii) was similar to that of eutherian mammals. Serotonergic neurons were divided into rostral and caudal groups, separated by an oblique boundary through the pontomedullary junction. Approximately 52% of the serotonergic neurons in the wallaby brainstem were located in the rostral midline nuclei (caudal linear nucleus, dorsal, median, and pontine raphe nuclei and the interpeduncular nucleus), whereas 21% were found in the caudal midline region (nuclei raphe magnus, obscurus, and pallidus). The remaining serotonergic neurons (27%) were located in more lateral regions such as the pedunculopontine tegmental nuclei, the supralemniscal nuclei (B9 group), and the ventrolateral medulla. The largest serotonergic group, the dorsal raphe, contained one-third of the brainstem serotonergic neurons and showed five subdivisions, similar to that described in other species. In contrast, the median raphe did not show clear subdivisions. The internal complexity of the raphe nuclei and the degree of lateralisation of serotonergic neurons suggest that the wallaby serotonergic system is similar in organisation to that described for the cat and rabbit. This study supports the suggestion that the serotonergic system is evolutionally well conserved and provides baseline data for a quantitative study of serotonergic innervation of the developing cortex in the wallaby.     

Distribution of N-acetylaspartylglutamate immunoreactivity in human brain and its alteration in neurodegenerative disease

The dipeptide N-acetylaspartylglutamate (NAAG) may be involved in the process of glutamatergic signaling by both acting at glutamate receptors and as a glutamate protransmitter. In the present study we determined the cellular localization and distribution of NAAG-like immunoreactivity (NAAG-LI) in normal human brain and in neurodegenerative disorders to ascertain the degree of NAAG's colocalization to putative glutamatergic pathways. Immunohistochemistry with an antibody against NAAG was performed on control, Huntington's disease (HD) and Alzheimer's disease (AD) human autopsy and biopsy brain sections from the cerebral cortex, hippocampus, amygdala, neostriatum, brainstem and spinal cord. In normal human brain, NAAG-LI was widespread localized to putative glutamatergic pyramidal neurons of the cerebral cortex and hippocampus. Punctate NAAG-LI was present in areas known to receive neuronal glutamatergic input, such as layer IV of the cerebral cortex, striatal neuropil, and the outer portion of the molecular layer of the hippocampal dentate gyrus. In the two pathologic brain regions examined, the HD neostriatum and the AD temporal cortex, we observed a widespread loss of NAAG-LI neurons. In addition NAAG-LI reactive microglia surrounding plaques were seen in AD temporal cortex but not in the HD striatum. Our results suggest that NAAG is substantially localized to putative glutamatergic pathways in human brain and that NAAG-LI neurons are vulnerable to the neurodegenerative process in HD and AD.     

Serotonergic lesion of median raphe nucleus alters nerve growth factor content and vulnerability of cholinergic septohippocampal neurons in rat.

About 45% of the serotonergic raphe neurons are reported to express nerve growth factor (NGF) receptors. We therefore investigated whether selective serotonergic lesions of the median or dorsal raphe nuclei are associated with changes in NGF protein levels of the brain and whether the loss of serotonergic function alters the vulnerability of cholinergic septohippocampal neurons. In adult rats the hippocampal NGF content changed in a biphasic way after lesion of the median raphe nucleus by 5,7-dihydroxytryptamine (5,7-DHT), with a significant increase after 2-3 weeks of up to 35%, followed by a significant reduction of 22% below control levels after 7 weeks, and a return to control levels within the following 4 weeks. By contrast, the decrease in hippocampal serotonin and 5-hydroxyindoleacetic acid remained throughout the observation period of 11 weeks, being still reduced to 15 and 30% of the control levels, respectively. In the frontal cortex the partial loss of the serotonergic innervation projecting from the median raphe was associated 5 weeks after 5,7-DHT injection with an increase in NGF protein of 39.7+/-9.6% (P<0.05), which remained elevated up to 11 weeks. At 9 weeks after 5,7-DHT, the lesion of the septohippocampal cholinergic neurons induced by the cholinotoxin ethylcholine aziridinium (AF64A) was exaggerated (P<0.05) as compared to AF64A-treated rats with intact serotonergic innervation. The present data indicate that a serotonergic lesion of the median raphe nucleus results in biphasic changes of NGF protein content and in a delayed increase in the vulnerability of septohippocampal cholinergic neurons.     

Evidence of the modulatory role of serotonin in acetylcholine release from striatal interneurons

The release of acetylcholine was studied in isolated striatal slices of the rat. The spontaneous and ouabain-stimulated release of acetylcholine was higher in those slices where serotonergic input was somehow impaired: raphe nuclei lesion orp-chlorophenylalanine pretreatment or 5,7-dihydroxytryptamine pretreatment resulted in a higher release.l-(m-chlorophenyl)-piperazine, a pure serotonin receptor stimulant andd-fenfluramine, a serotonin releaser significantly reduced the release of acetylcholine evoked by ouabain. Serotonin antagonists (cyproheptadine, mianserine and methysergide) prevented the effect of serotonin agonists. When the serotonergic neurons were destroyed either byp-chlorophenylalanine or by 5,7-dihydroxytryptamine pretreatmentd-fenfluramine had no inhibitory action; however, the effect ofl(m-chlorophenyl)-piperazine was not affected.It is suggested that there is a link between serotonergic and cholinergic neurons in the striatum: serotonin released from raphe-striatal neurons is able to inhibit the release of acetylcholine from striatal interneurons.     

Alpha 7 nicotinic receptor subunit is present on serotonin neurons projecting to hippocampus and septum

The serotonergic transmitter system regulates hippocampal activity through its raphe projection to hippocampus and medial septum/diagonal band of Broca complex (MS/DBB), and most likely also indirectly through its interaction with the cholinergic neurotransmitter system. Nicotine, e.g., enhances hippocampal serotonin release probably through presynaptic nicotinic receptors. We investigated the possible presence of the alpha 7-nicotinic subunit on serotonergic neurons projecting to hippocampus and MS/DBB. By retrograde neuronal tracing, hippocampal serotonergic neurons were identified and with double fluorescence immunostaining and Alexa-488 bound alpha-bungarotoxin the presence of active alpha 7 receptor on their soma was determined. Most of the retrogradely labeled serotonin neurons contained the alpha 7 subunit. A low degree of colocalization between alpha-bungarotoxin and serotonin-positive neurons suggest that the alpha 7 subunit may be transported anterogradely to the serotonergic axonal terminals.     

Quantitation of N-acetyl-aspartyl-glutamate in microdissected rat brain nuclei and peripheral tissues: findings with a novel liquid phase radioimmunoassay

Antibodies were raised in rabbits against the neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) coupled to bovine serum albumin via a carbodiimide linkage. One of these rabbit antisera, which preferentially recognizes coupled NAAG-like immunoreactivity (LIR), has been previously used to immunocytochemically localize NAAG-LIR. We have now employed a second of these antisera, which preferentially recognizes free NAAG, to develop a competitive liquid phase radioimmunoassay (RIA). Using this assay, we were able to detect picomole amounts of NAAG in rat tissue extracts. The specificity of the assay revealed a 60-fold greater affinity of the antibody for NAAG over N-acetyl-aspartate (NAA) and greater than one million-fold specificity for NAAG over both aspartate and glutamate. High-pressure liquid chromatographic (HPLC) separation of tissue extracts yielded only two detectable peaks of NAAG-LIR in collected fractions and these co-chromatographed with NAAG and NAA. NAAG levels determined by this liquid phase RIA and by HPLC were essentially identical after correction for the presence of NAA crossreactivity. The antibody that preferentially recognizes coupled NAAG was used to immunocytochemically localize NAAG-LIR to the red nucleus, the facial nucleus, the dorsal raphe, and the locus coeruleus. To further confirm this localization of NAAG, these and other nuclei were microdissected and levels of NAAG were determined by liquid phase RIA. Nuclei which stained intensely were found to contain high levels of NAAG by RIA and between 60 and 100% of this NAAG-LIR co-chromatographed with NAAG. These results support our previous conclusion that NAAG is co-localized in noradrenergic, serotonergic and cholinergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurotoxic lesions of the dorsolateral pontomesencephalic tegmentum-cholinergic cell area in the cat. I. Effects upon the cholinergic innervation of the brain

Kainic acid was injected bilaterally (4.8 micrograms in 1.2 microliter each side) into the dorsolateral pontomesencephalic tegmentum of cats in order to destroy cholinergic cells which are located within the pedunculopontine tegmental (PPT), laterodorsal tegmental (LDT), parabrachial (PB), and locus ceruleus (LC) nuclei in this species. The neurotoxic lesions resulted in the destruction of the majority (approximately 60%) of choline acetyltransferase (ChAT)-immunoreactive neurons and a minority (approximately 35%) of tyrosine hydroxylase (TH)-immunoreactive neurons, as well as in the destruction of other chemically unidentified neurons, in the region. The effects of these lesions upon the cholinergic innervation of the brain were investigated by comparison of brains with and without lesions which were processed for acetylcholinesterase (AChE) silver, copper thiocholine histochemistry and ChAT radio-immunohistochemistry. In the forebrain, a major and significant decrease in AChE staining, measured by microdensitometry, and associated with a decrease in ChAT immunoreactivity was found in certain thalamic nuclei, including the dorsal lateral geniculate, lateral posterior, pulvinar, intralaminar, mediodorsal and reticular nuclei. All of these nuclei receive a rich cholinergic innervation evident in both AChE histochemistry and ChAT immunohistochemistry. No significant difference in AChE staining or ChAT immunoreactivity was detected in other thalamic nuclei or in the subthalamus, hypothalamus or basal forebrain. In the brainstem, a significant decrease of AChE staining and ChAT immunoreactivity was found in the superior colliculus and the medullary reticular formation, where ChAT-immunoreactive fibers were moderately dense in the normal animal. These results indicate that the pontomesencephalic cholinergic neurons may influence the forebrain by major projections to the thalamus, involving both relay and non-specific thalamocortical projection systems, and thus act as an integral component of the ascending reticular system. They may influence the brainstem by projections onto deep tectal neurons and other reticular neurons, notably those in the medullary reticular formation, and thus also affect bulbar and bulbospinal systems.