Cholinergic elements in the zebrafish central nervous system: Histochemical and immunohistochemical analysis

Recently, the zebrafish has been extensively used for studying the development of the central nervous system (CNS). However, the zebrafish CNS has been poorly analyzed in the adult. The cholinergic/cholinoceptive system of the zebrafish CNS was analyzed by using choline acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) histochemistry in the brain, retina, and spinal cord. AChE labeling was more abundant and more widely distributed than ChAT immunoreactivity. In the telencephalon, ChAT-immunoreactive (ChAT-ir) cells were absent, whereas AChE-positive neurons were observed in both the olfactory bulb and the telencephalic hemispheres. The diencephalon was the region with the lowest density of AChE-positive cells, mainly located in the pretectum, whereas ChAT-ir cells were exclusively located in the preoptic region. ChAT-ir cells were restricted to the periventricular stratum of the optic tectum, but AChE-positive neurons were observed throughout the whole extension of the lamination except in the marginal stratum. Although ChAT immunoreactivity was restricted to the rostral tegmental, oculomotor, and trochlear nuclei within the mesencephalic tegmentum, a widespread distribution of AChE reactivity was observed in this region. The isthmic region showed abundant AChE-positive and ChAT-ir cells in the isthmic, secondary gustatory and superior reticular nucleus and in the nucleus lateralis valvulae. ChAT immunoreactivity was absent in the cerebellum, although AChE staining was observed in Purkinje and granule cells. The medulla oblongata showed a widespread distribution of AChE-positive cells in all main subdivisions, including the octavolateral area, reticular formation, and motor nuclei of the cranial nerves. ChAT-ir elements in this area were restricted to the descending octaval nucleus, the octaval efferent nucleus and the motor nuclei of the cranial nerves. Additionally, spinal cord motoneurons appeared positive to both markers. Substantial differences in the ChAT and AChE distribution between zebrafish and other fish species were observed, which could be important because zebrafish is widely used as a genetic or developmental animal model.     

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the central nervous system of a chondrostean, the siberian sturgeon (Acipenser baeri)

All studies to date of cholinergic systems of bony fishes have been done in teleosts. To gain further insight into the evolution of the cholinergic systems of bony fishes, we have studied the brain of a chondrostean fish, the Siberian sturgeon (Acipenser baeri, Brandt), by using an antibody against choline acetyltransferase (ChAT). This study showed the presence of ChAT-immunoreactive (ChAT-ir) neurons in the preoptic region (parvocellular and magnocellular preoptic nuclei and suprachiasmatic nucleus), the periventricular and tuberal hypothalamus, the saccus vasculosus, the dorsal thalamus, and the habenula. The mesencephalic tegmentum contained ChAT-ir cells in the torus semicircularis and torus lateralis. The isthmus contained several cholinergic populations: the nucleus isthmi, the lateral nucleus of the valvula, the secondary visceral nucleus, and the dorsal tegmental nucleus. The motor neurons of the cranial nerves and the spinal motor column were strongly immunoreactive. The medial (sensory) trigeminal nucleus also contained a ChAT-ir neuronal population. The distribution of ChAT-ir neurons in the sturgeon brain showed some notable differences with that observed in teleosts, such as the absence of cholinergic cells in the telencephalon and the optic tectum. Several brain regions were richly innervated by ChAT-ir fibers, particularly the telencephalon, optic tectum, thalamus, posterior tubercle, and interpeduncular nucleus. The hypothalamo-hypophyseal tract, the tract of the saccus vasculosus, the fasciculus retroflexus, and an isthmo-mesencephalo-thalamic tract were the most conspicuous cholinergic bundles. Comparative analysis of these results suggests that teleosts have conserved most traits of the cholinergic system of the sturgeon, having acquired new cholinergic populations during evolution.     

The adult central nervous cholinergic system of a neurogenetic model animal, the zebrafish Danio rerio

The central nervous cholinergic system of the zebrafish (Danio rerio), a model animal for neurogenetics, is documented here using immunohistochemical methods for visualizing choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme. Neuronal cell bodies containing ChAT are present in the telencephalon (lateral nucleus of ventral telencephalic area), preoptic region (anterior/posterior parvocellular and magnocellular preoptic nuclei), diencephalon (habenula, dorsal thalamus, posterior tuberculum), mesencephalon (Edinger-Westphal (EW) nucleus, oculomotor nerve nucleus, rostral tegmental nucleus, tectal type XIV neurons), isthmic region (nucleus lateralis valvulae, secondary gustatory-viscerosensory nucleus, nucleus isthmi (NI), perilemniscal nucleus, superior reticular nucleus (SRN)) and rhombencephalon (trochlear, trigeminal, abducens, facial, glossopharyngeal-vagal motor nerve nuclei, rostral and caudal populations of octavolateralis efferent neurons). In addition, some ChAT positive neurons are present in the rhombencephalic reticular formation, the central gray, and in cells accompanying the descending trigeminal tract. Obvious ChAT positive terminal fields are present in the supracommissural nucleus of area ventralis telencephali and the medial zone of area dorsalis telencephali, parvocellular superficial pretectal nucleus, torus semicircularis, medial octavolateralis nucleus, facial, glossopharyngeal, and vagal lobes, and in the inferior lobe (around the periventricular nucleus of the lateral recess and in the diffuse nucleus). The identification of all central nervous cholinergic systems provided here in this model system is pivotal for future detailed studies of their development and maintenance, e.g., with regard to the zebrafish ventral telencephalic and isthmic superior reticular neuronal populations, likely representing the homologues of at least part of the cholinergic basal forebrain and pedunculopontine/laterodorsal tegmental ascending activating systems of mammals, respectively.     

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the brain of the adult trout and tract-tracing observations on the connections of the nuclei of the isthmus

The distribution of cholinergic neurons and fibers was studied in the brain and rostral spinal cord of the brown trout and rainbow trout by using an antiserum against the enzyme choline acetyltransferase (ChAT). Cholinergic neurons were observed in the ventral telencephalon, preoptic region, habenula, thalamus, hypothalamus, magnocellular superficial pretectal nucleus, optic tectum, isthmus, cranial nerve motor nuclei, and spinal cord. In addition, new cholinergic groups were detected in the vascular organ of the lamina terminalis, the parvocellular and magnocellular parts of the preoptic nucleus, the anterior tuberal nucleus, and a mesencephalic tegmental nucleus. The presence of ChAT in the magnocellular neurosecretory system of trout suggests that acetylcholine is involved in control of hormone release by neurosecretory terminals. In order to characterize the several cholinergic nuclei observed in the isthmus of trout, their projections were studied by application of 1,1;-dioctadecyl-3,3,3;, 3;-tetramethylindocarbocyanine perchlorate (DiI) to selected structures of the brain. The secondary gustatory nucleus projected mainly to the lateral hypothalamic lobes, whereas the nucleus isthmi projected to the optic tectum and parvocellular superficial pretectal nucleus, as previously described in other teleost groups. In addition, other isthmic cholinergic nuclei of trout may be homologs of the mesopontine system of mammals. We conclude that the cholinergic systems of teleosts show many primitive features that have been preserved during evolution, together with characteristics exclusive to the group.     

Afferent connections of the optic tectum in lampreys: an experimental study

Tectal afferents were studied in adult lampreys of three species (Ichthyomyzon unicuspis, Lampetra fluviatilis, and Petromyzon marinus) following unilateral BDA injections into the optic tectum (OT). In the secondary prosencephalon, neurons projecting to the OT were observed in the pallium, the subhipoccampal lobe, the striatum, the preoptic area and the hypothalamus. Following tectal injections, backfilled diencephalic cells were found bilaterally in: prethalamic eminence, ventral geniculate nucleus, periventricular prethalamic nucleus, periventricular pretectal nucleus, precommissural nucleus, magnocellular and parvocellular nuclei of the posterior commissure and pretectal nucleus; and ipsilaterally in: nucleus of Bellonci, periventricular thalamic nucleus, nucleus of the tuberculum posterior, and the subpretectal tegmentum, as well as in the pineal organ. At midbrain levels, retrogradely labeled cells were seen in the ipsilateral torus semicircularis, the contralateral OT, and bilaterally in the mesencephalic reticular formation and inside the limits of the retinopetal nuclei. In the hindbrain, tectal projecting cells were also bilaterally labeled in the dorsal and lateral isthmic nuclei, the octavolateral area, the sensory nucleus of the descending trigeminal tract, the dorsal column nucleus and the reticular formation. The rostral spinal cord also exhibited a few labeled cells. These results demonstrate a complex pattern of connections in the lamprey OT, most of which have been reported in other vertebrates. Hence, the lamprey OT receives a large number of nonvisual afferents from all major brain areas, and so is involved in information processing from different somatic sensory modalities.     

Ontogeny of choline acetyltransferase (ChAT) immunoreactivity in the brain of the urodele amphibian Pleurodeles waltl

The ontogeny of cholinergic neurons has been studied in the brain of the urodele amphibian Pleurodeles waltl by means of choline acetyltransferase (ChAT) immunohistochemistry. Embryonic and larval stages were studied. The earliest ChAT immunoreactive (ChATi) cells were the primary motoneurons in the upper spinal cord, at embryonic stage 29. Slightly later, before hatching, the cranial nerve motor nuclei were immunopositive as well as non-motor populations in the developing inferior reticular nucleus, the nucleus of the solitary tract, the dorsal column nucleus and the retina. At initial larval stages, ChATi cells were located for the first time in the suprachiasmatic nucleus and the isthmic tegmentum. In addition, moderate immunoreactivity appeared in the Mauthner cells. During the period of active larval life the cholinergic systems maturated progressively and new ChATi cell groups were found in the caudal telencephalon and the habenula. Also extensive fiber labeling occurred at active larval stages. No transient ChAT expression was observed and even the Mauthner cells maintained their immunoreactivity after metamorphosis. A general caudorostral spatio-temporal sequence of appearance of cholinergic structures was found in the brain. Comparison of the results observed in the urodele with previous data available only in amniotes shows numerous similarities and suggests a conservative developmental pattern of cholinergic systems in vertebrates.     

Immunohistochemical localization of ryanodine binding proteins in the central nervous system of gymnotiform fish.

The ryanodine receptor, an integral membrane protein of the sarcoplasmic reticulum in muscle, embodies a high conductance channel permeable to calcium ions. Recent studies have identified ryanodine-binding proteins in avian and mammalian central nervous systems. These neuronal ryanodine receptors appear to function as Ca2+ channels which may gate the release of Ca2+ from caffeine-sensitive intracellular pools in neurons. In the present investigation, we employed monoclonal antibodies against ryanodine-binding proteins of avian muscle cells to the brain of weakly electric gymnotiform fish. Immunoprecipitation and Western blot analysis revealed two isoforms in the fish brain, with molecular weights comparable to those of avian and fish muscle ryanodine-binding proteins. By employing immunohistochemical techniques, we mapped these proteins in fish brain. Ryanodine receptor-like immunoreactivity was found in nerve cell bodies as well as dendrites and axonal processes. The ryanodine-binding protein is distributed throughout the neuraxis in specific cell types of the gymnotiform brain. In the telencephalon, immunoreactive cells were found in the glomerular layer of the olfactory bulb, in the supracommissural subdivision of the ventral telencephalon, and in the intermediate rostral subdivision of the ventral telencephalon. In the diencephalon, immunoreactive cells or fibers were observed in the nucleus prethalamicus and the habenula, within the nucleus at the base of the optic tract and the adjacent dorsal tegmental nucleus, the pretectal nuclei A and B, and the nucleus electrosensorius. In addition, immunopositive cells were seen in several nuclei of the hypothalamus, with the inferior and lateral subdivision of the nucleus recessus lateralis displaying the highest concentration of neurons. In the mesencephalon, the optic tectum contained the greatest number of immunopositive cells. In the rhombencephalon, labelling was seen in the nucleus of the lateral valvula, central gray, lateral tegmental nucleus, in boundary cells of the nucleus praeminentialis, efferent octavolateral nucleus, an area adjacent to the medial edge of the lateral reticular nucleus, nucleus medialis, and electrosensory lateral line lobe. As in avian brain, cerebellar Purkinje cells were positive for ryanodine-binding protein, although only subsets of Purkinje cells were labelled.     

Cholinergic systems in the rat brain: I. Projections to the limbic telencephalon

The cholinergic projections to the limbic telecephalon in the rat were investigated by use of fluorescent tracer histology in combination with choline-O-acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) histochemistry (pharmacohistochemical regimen). Propidium iodide or Evans Blue was infused into the olfactory bulb, hippocampus, dorsal retrohippocampal region, amygdala, and the entorhinal, perirhinal, pyriform, insular, and cingular cortices. Retrogradely transported fluorescent labels and ChAT and/or AChE were microscopically analyzed on the same brain section. Virtually all of the cholinergic projections to the limbic telencephalon derived from the basal forebrain cholinergic system composed of neurons associated with the medial septal nucleus, nuclei of the vertical and horizontal limbs of the diagonal band, the magnocellular preoptic area, the subpallidal substantia innominata and its rostral extension into the regions of the ventral pallidum laterally and the lateral preoptic area medially, and the nucleus basalis. The cingulate cortex received a small cholinergic projection from the dorsolateral tegmental nucleus in the brainstem. All of the presumed cholinergic innervation of the olfactory bulb, hippocampus, and dorsal retrohippocampal area and the majority of cholinergic afferents to posterior cingulate and entorhinal cortices derived from the medial septal nucleus, vertical and horizontal limbs of the diagonal band, magnocellular preoptic area, and rostral substantia innominata. Putative cholinergic afferents to the amygdala and to pyriform, insular, perirhinal, and anterior cingulate cortices orginated from ChAT-positive cells concentrated more caudally in the basal forebrain cholinergic system. Within the basal forebrain, no simple topographic pattern emerged to explain the cholinergic innervation of the limbic telencephalon, although an essentially reverse rostrocaudal organization was observed for afferents to the cingular region. It was noted, however, that most regions of the limbic telencephalon received cholinergic input from rostral portions of the basal forebrain cholinergic system, an observation inviting speculation that anterior aspects of the basal forebrain provide cholinergic afferents primarily to limbic structures in the telencephalon whereas more caudal portions are the source of cholinergic fibers preferentially innervating non-limbic regions. Of the total number of projection neurons innervating a given region of the limbic telencephalon, a greater proportion was ChAT-positive if phylogenetically newer target structures were innervated.(ABSTRACT TRUNCATED AT 400 WORDS)     

The immunohistochemical localization of choline acetyltransferase in the cat brain

The distribution of neurons displaying choline acetyltransferase (ChAT) immunoreactivity was examined in the feline brain using a monoclonal antibody. Groups of ChAT-immunoreactive neurons were detected that have not been identified previously in the cat or in any other species. These included small, weakly stained cells found in the lateral hypothalamus, distinct from the magnocellular rostral column cholinergic neurons. Other small, lightly stained cells were also detected in the parabrachial nuclei, distinct from the caudal cholinergic column. Many small ChAT-positive cells were also found in the superficial layers of the superior colliculus. Other ChAT-immunoreactive neurons previously detected in rodent and primate, but not in cat, were observed in the present study. These included a dense cluster of cells in the medial habenula, together with outlying cells in the lateral habenula. Essentially all of the cells in the parabigeminal nucleus were found to be ChAT-positive. Additional ChAT-positive neurons were detected in the periolivary portion of the superior olivary complex, and scattered in the medullary reticular formation. In addition to these new observations, many of the cholinergic cell groups that have been previously identified in the cat as well as in rodent and primate brain such as motoneurons, striatal interneurons, the magnocellular rostral cholinergic column in the basal forebrain and the caudal cholinergic column in the midbrain and pontine tegmentum were confirmed. Together, these observations suggest that the feline central cholinergic system may be much more extensive than previous studies have indicated.     

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.     

Distribution of choline acetyltransferase immunopositive structures in the rat brainstem

The distribution of neurons, fibers and terminal fields in rat brainstem displaying positive immunoreactivity to a polyclonal antiserum to human placental choline acetyltransferase (ChAT) is described. The antiserum was used at the high dilution of 1:10,000 and was coupled with a sensitive detection system using the nickel ammonium sulfate intensification method. In addition to previously described ChAT immunopositive groups of large cells in the cranial motor nuclei, and the parabrachial and reticular complexes, many small or medium size, weakly immunopositive neurons were identified. Some of these appeared in structures in the region of the fourth ventricle, including the area postrema. Others were in structures associated with the superior olivary complex, including the lateral superior olive, and the medioventral, lateroventral and superior periolivary nuclei. Scattered, weakly positive cells were seen in numerous other structures, including the ventral tegmental area of Tsai, central gray, superior colliculus, spinal nucleus of nerve 5, dorsal cochlear nucleus and non-motor regions of the spinal cord. The prominent ascending fiber tract of the laterodorsal tegmental pathway was traceable from the parabrachial area to the subgeniculate region of the thalamus. Prominent terminal fields were seen in a number of brainstem structures, including the superior colliculus, pontine nuclei, anterior pretectal nucleus, interpeduncular nucleus and spinal nucleus of nerve 5. The association of small ChAT positive cells and terminal fields with many sensory structures suggests a significant cholinergic participation in the physiology of sensory function.     

Cholinergic neurons in the brain of a teleost fish (Porichthys notatus) located with a monoclonal antibody to choline acetyltransferase

A monoclonal antibody (Ab8) to choline acetyltransferase (ChAT) was used to locate structures showing ChAT-like immunoreactivity (ChAT-IR) in the brain of a teleost fish, the midshipman (Porichthys notatus). ChAT is the synthetic enzyme for acetylcholine found in neurons using that neurotransmitter; thus ChAT-IR may be interpreted as indicating putative cholinergic activity. Robust staining is seen in all cranial nerve motor nuclei. In addition, the brainstem of Porichthys is distinguished by two other expansive ChAT-IR zones: a sonic motor nucleus, which innervates swimbladder “drum” muscles, and an octavolateralis efferent nucleus, which innervates acoustic, vestibular, and lateral line end organs. Scattered labeled cells are found in several cranial sensory nuclei–the vagal lobe, and the main and descending trigeminal nuclei. ChAT-IR cells form restricted subpopulations in other noncranial nerve nuclei, including the granule cell layer of the cerebellum; superior, medial, and inferior divisions of the reticular formation; the stratum periventriculare of the midbrain's optic tectum; and the nucleus isthmi in the midbrain tegmentum. In the telencephalon, a dense population of ChAT-IR cells is found in the ventral nucleus of area ventralis; terminals and fine fibers are found in the dorsal, medial, and central nuclei of area dorsalis. Together, the data represent the first complete report of ChAT-IR cell bodies in the brain of any nonmammal with the monoclonal antibody Ab8, which has already been extensively used on a variety of vertebrate brains. The results are thus discussed from a comparative viewpoint, considering reports of ChAT-IR in different taxa.     

Distribution of calcitonin gene-related peptide-like immunoreactivity in the brain of the small-spotted dogfish,scyliorhinus canicula L

The distribution of neuropeptides has been useful in comparing neuronal aggregates of elasmobranchs with those in other vertebrates. The distribution of calcitonin gene-related peptide (CGRP)-like immunoreactivity in the brain of the dogfish was examined with an antiserum to rat α-CGRP. Western blot analysis confirms that our antiserum recognizes a single peptide in the dogfish brain very similar to mammalian CGRP. CGRP-like immunoreactivity was located in discrete neuronal groups. CGRP-like-immunoreactive (CGRP-ir) neurons were found in the motor nuclei III, IV, V, VI, VII, IX, and X of the brainstem motor column and in the octavolateral efferent neurons. In the isthmal region, two groups of CGRP-ir neurons appeared in the parabrachial region and reticular substance. Three other CGRP-ir cell groups were observed in the mesencephalon: in the ventral tegmental area, in the substantia nigra, and one widely scattered but numerous population in superficial layers of the optic tectum. In the diencephalon, CGRP-ir cells were observed in the magnocellular preoptic nucleus and the organon vasculosum hypothalami. A population of CGRP-ir cells was also observed in the entopeduncular nucleus in the impar telencephalon. CGRP-ir fibers of central origin were widely distributed in the brain, but the most conspicuous areas were found in the ventral telencephalon, the hypothalamus, the mesencephalic lateral reticular area, and the dorsolateral isthmal region. The neurointermediate lobe of the hypophysis was also richly innervated by CGRP-ir fibers. CGRP-ir sensory fibers of cranial nerves IX and X and of dorsal spinal roots formed very conspicuous terminal fields in the lobus vagi and Cajal's nucleus commissuralis and in the dorsal region of the substantia gelatinosa, respectively. Comparison of the distribution of fibers and perikarya in dogfish and other vertebrates suggests that this CGRP-ir system has been well conserved during evolution. © 1995 Wiley-Liss, Inc.     

Mesencephalic cholinergic nuclei in progressive supranuclear palsy

Using an antibody against choline acetyltransferase (ChAT), mesencephalic cholinergic cell nuclei were studied in autopsy material from 3 cases of progressive supranuclear palsy (PSP) and 4 controls. ChAT-immunoreactive neurons were quantified in sections that spanned the rostrocaudal extent of each nucleus. In PSP, there was a significant decrease in the number of neurons with detectable immunoreactivity for ChAT in and adjacent to the central gray substance in the following nuclei: the nucleus of Edinger-Westphal (69%); the rostral interstitial nucleus of the medial longitudinal fasciculus (97%); the interstitial nucleus of Cajal (78%). A cell loss was also evident in a group of neurons found in the deep layers of the superior colliculus (93%). In contrast, the estimated number of ChAT-immunoreactive cell bodies in cranial nerves III and IV, in the mesencephalic reticular formation, and in the parabigeminal nucleus was not different from that of controls. The results are compatible with the notion that, in PSP, there is a regionally selective destruction of cholinergic neurons.     

Preganglionic parasympathetic neurons projecting to the sphenopalatine ganglion contain nitric oxide synthase in the rabbit

We have investigated the possible presence of nitric oxide synthase (NOS) and choline acetyltransferase (ChAT) in brainstem preganglionic parasympathetic neurons projecting to the sphenopalatine ganglion in rabbits, using combined retrograde axonal tracing and immunohistochemistry. Retrogradely labeled neurons were observed in the ipsilateral rostral medulla and caudal pons, in a region laterodorsal to the facial motor nucleus. Double-labeling experiments demonstrated that 75±5% of retrogradely labeled neurons contained NOS immunoreactivity, while all of retrogradely labeled neurons contained ChAT immunoreactivity. These observations suggest that nitric oxide could influence cholinergic transmission from preganglionic endings in the sphenopalatine ganglion.     

Cholinergic systems in the rat brain: III. Projections from the pontomesencephalic tegmentum to the thalamus, tectum, basal ganglia, and basal forebrain

The ascending cholinergic projections of the pedunculopontine and dorsolateral tegmental nuclei, referred to collectively as the pontomesencephalotegmental (PMT) cholinergic complex, were investigated by use of fluorescent tracer histology in combination with choline-O-acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) pharmacohistochemistry. Propidium iodide, true blue, or Evans blue was infused into the anterior, reticular, mediodorsal, central medial, and posterior nuclear areas of the thalamus; the habenula; lateral geniculate; superior colliculus; pretectal/parafascicular area; subthalamic nucleus; caudate-putamen complex; globus pallidus; entopeduncular nucleus; substantia nigra; medial septal nucleus/vertical limb of the diagonal band area; magnocellular preoptic/ventral pallidal area; and lateral hypothalamus. In some animals, separate injections of propidium iodide and true blue were made into two different regions in the same rat brain, usually a dorsal and a ventral target, in order to assess collateralization patterns. Retrogradely transported fluorescent labels and ChAT and/or AChE were analyzed microscopically on the same brain section. All of the above-delimited targets were found to receive cholinergic input from the PMT cholinergic complex, but some regions were preferentially innervated by either the pedunculopontine or dorsolateral tegmental nucleus. The former subdivision of the PMT cholinergic complex projected selectively to extrapyramidal structures and the superior colliculus, whereas the dorsolateral tegmental nucleus was observed to provide cholinergic input preferentially to anterior thalamic regions and rostral portions of the basal forebrain. The PMT cholinergic neurons showed a tendency to collateralize extensively.     

Autoradiographic localization of dihydrotestosterone and testosterone concentrating neurons in the brain of the oyster toadfish

Vertebrate species with male mating calls or songs tend to have sexually dimorphic sonic neurons that concentrate gonadal steroids. The distribution of [³H]dihydrotestosterone- and testosterone-concentrating neurons was examined in oyster toadfish (Opsanus tau), males of which produce a courtship boatwhistle call. Labeled cells in the forebrain were found in the posterior nucleus of the dorsal telencephalon (Dp), a pallial structure, the supracommissural nucleus of the ventral telencephalon (Vs), nucleus preopticus parvocellularis anterior (PPa) and other preoptic nuclei, the ventral, dorsal and caudal hypothalamus. Positive brainstem areas included the optic tectum, torus semicircularis, nucleus lateralis valvula, a periventricular nucleus of the rostral medulla and the inferior reticular formation. Compared to estrogen, androgens labeled fewer sites in the forebrain and more in the brainstem. Two of the positive sites, Vs and PPa, have been implicated in boatwhistle production. Many sites that connect to these areas in teleosts likewise concentrate steroids. Unlike the situation in frogs, birds, and one other teleost, the toadfish sonic motor nucleus did not concentrate androgens. Androgen labeling in the posterior nucleus of the dorsal telencephalon represents the first autoradiographic demonstration of steroid concentration in the pallium of a teleost forebrain.     

Nitric oxide synthase and NADPH diaphorase distribution in the bullfrog (Rana catesbeiana) CNS: pathways and functional implications

The gas nitric oxide (NO) is emerging as an important regulator of normal physiology and pathophysiology in the central nervous system (CNS). The distribution of cells releasing NO is poorly understood in non-mammalian vertebrates. Nitric oxide synthase immunocytochemistry (NOS ICC) was thus used to identify neuronal cells that contain the enzyme required for NO production in the amphibian brain and spinal cord. NADPH-diaphorase (NADPHd) histochemistry was also used because the presence of NADPHd serves as a reliable indicator of nitrergic cells. Both techniques revealed stained cells in all major structures and pathways in the bullfrog brain. Staining was identified in the olfactory glomeruli, pallium and subpallium of the telencephalon; epithalamus, thalamus, preoptic area, and hypothalamus of the diencephalon; pretectal area, optic tectum, torus semicircularis, and tegmentum of the mesencephalon; all layers of the cerebellum; reticular formation; nucleus of the solitary tract, octaval nuclei, and dorsal column nuclei of the medulla; and dorsal and motor fields of the spinal cord. In general, NADPHd histochemistry provided better staining quality, especially in subpallial regions, although NOS ICC tended to detect more cells in the olfactory bulb, pallium, ventromedial thalamus, and cerebellar Purkinje cell layer. NOS ICC was also more sensitive for motor neurons and consistently labeled them in the vagus nucleus and along the length of the rostral spinal cord. Thus, nitrergic cells were ubiquitously distributed throughout the bullfrog brain and likely serve an essential regulatory function.     

Basal forebrain cholinergic and noncholinergic projections to the thalamus and brainstem in cats and monkeys

The projections of basal forebrain neurons to the thalamus and the brainstem were investigated in cats and primates by using retrograde transport techniques and choline acetyltransferase (ChAT) immunohistochemistry. In a first series of experiments, the lectin wheat germ-agglutinin conjugated with horseradish peroxidase (WGA-HRP) was injected into all major sensory, motor, intralaminar, and reticular (RE) thalamic nuclei of cats and into the mediodorsal (MD) and pulvinar-lateroposterior thalamic nuclei of macaque monkeys. In cats numerous neurons of the vertical and horizontal limbs of the diagonal band nucleus and the substantia innominata (SI), including its rostromedial portion termed the ventral pallidum (VP), were retrogradely labeled after WGA-HRP injections in the rostral pole of the RE complex, the MD, and anteroventral/anteromedial (AV/AM) thalamic nuclei. Fewer retrogradely labeled cells were observed in the same areas after injections in the ventromedial (VM) thalamic nucleus, and none or very few after other thalamic injections. After RE, MD, and AV/AM injections, 7-20% of all retrogradely labeled cells in the basal forebrain were also ChAT positive, while none of the retrogradely labeled neurons following VM injections displayed ChAT immunoreactivity. The basal forebrain projection to the MD nucleus was shown to arise principally from VP in both cats and macaque monkeys. In a second series of experiments performed in cats, injections of WGA-HRP in the brainstem peribrachial (PB) area comprising the pedunculopontine nucleus led to retrograde labeling of a moderate number of neurons in the lateral part of the VP, SI, and preoptic area (POA), only a few of which displayed ChAT immunoreactivity. In addition, a large number of retrogradely labeled cells were observed in the bed nuclei of the anterior commissure and stria terminalis after PB injections. In a third series of experiments, the use of the retrograde double-labeling method with fluorescent tracers in squirrel monkeys allowed us to identify a significant number of basal forebrain neurons sending axon collaterals to both the RE thalamic nucleus and PB brainstem area, while no double-labeled neurons were disclosed after injections confined to the ventral anterior/ventral lateral (VA/VL) thalamic nuclei and PB area or following injections in the cerebral cortex and PB area. Our findings reveal the existence of cholinergic and noncholinergic basal forebrain projections to the thalamus and the brainstem in both cats and macaque monkeys. We suggest that these projections may play a crucial role in the control of thalamic functions in mammals.     

Afferent and efferent connections of the torus semicircularis in the sea lamprey: an experimental study

The afferent and efferent connections of the torus semicircularis (TS) of larval sea lampreys were studied with horseradish peroxidase, carbocyanine dye (DiI) and fluorescein-coupled or Texas-Red-coupled dextran amine tract-tracing methods. Application of tracers to the TS or to the octavolateral area revealed the presence of bilateral projections from the octavolateral area to the torus semicircularis, mainly from the mechanoreceptive regions (medial and ventral octavolateral nuclei) though also from the electroreceptive (dorsal octavolateral nucleus) region. The nucleus of the descending root of the trigeminal nerve projects to the contralateral TS, mostly from neurons located rostral to the obex. Fairly numerous reticular cells of the rhombencephalon project to the torus semicircularis. In the mesencephalon, scattered cells in the tegmentum, and some in the tectum, have toral projections, mostly ipsilateral. Numerous thalamic neurons, as well as fairly numerous neurons of the posterior tubercle, hypothalamus and preoptic region, and a few neurons in the ventral telencephalon (striatum, septum), were labeled after tracer application to the TS. The torus semicircularis mainly projects to the thalamus, the hypothalamus and the reticular rhombencephalic nuclei. Our results reveal for the first time a complex pattern of connections of the lamprey TS, which suggests that it is a multisensory center integrating head cutaneous sensitivity with mechano- and electrosensory information from the octavolateral area and with visual information. A number of afferents from the forebrain also appear to contribute to TS function.