Motor neurons are rich in non-phosphorylated neurofilaments: cross-species comparison and alterations in ALS

The localization and distribution of non-phosphorylated neurofilaments (NP-NF) in the upper and lower motor neurons was investigated in the rat, the common marmoset, the rhesus monkey and man using the SMI-32 antibody. Within the spinal cord of all species studied, the most intense NP-NF immunoreactivity was observed within the ventral horn alpha-motor neurons. Concurrent staining for the cholinergic marker choline acetyltransferase (ChAT) demonstrated that virtually all of the ChAT-positive alpha-motor neurons contain NP-NF immunoreactivity. Although NP-NF staining was also observed in other neurons within the ventral and intermediate horns, these neurons were loosely scattered and contained a considerably lower staining intensity. The only other prominent NP-NF staining in the spinal cord occurred within the neurons of the dorsal nucleus of Clark and the intermediolateral cell column. Phosphorylated neurofilament (P-NF) immunoreactivity was found primarily in neuronal processes. Occasionally, a solitary motor neuron contained weak P-NF immunoreactivity. Within the brainstem, neurons in all cranial nerve motor nuclei contained intense NP-NF immunoreactivity. The distribution and apparent density of NP-NF immunoreactive neurons in these nuclei was virtually identical to that observed for neurons immunoreactive for ChAT. NP-NF immunoreactive neurons of relatively lower intensity were found in many other regions of the brainstem. All of the giant Betz cells of layer (L) V in the motor cortex contained dark NP-NF immunoreactivity. Within the spinal cord of amyotrophic lateral sclerosis (ALS) patients, both Nissl and NP-NF staining demonstrated the dramatic loss of alpha-motor neurons characteristic of this disorder. Some of the remaining motor neurons contained intense P-NF immunoreactivity. These observations suggest that NP-NF immunoreactivity is a good marker for motor neurons in health and disease and may be a useful tool for studies of motor neuron degeneration (MND).     

Organization of choline acetyltransferase-containing structures in the cranial nerve motor nuclei and spinal cord of the monkey

Cholinergic structures in the cranial nerve motor nuclei and ventral and lateral horns of the spinal cord of the monkey, Macaca fuscata, were investigated immunohistochemically with a monoclonal antibody against monkey choline acetyltransferase (ChAT). ChAT-immunoreactive perikarya and dendrites were present in the oculomotor, trochlear, abducent, trigeminal motor, facial and hypoglossal nuclei, nucleus of Edinger–Westphal, nucleus ambiguus, dorsal nucleus of the vagus, lamina IX of the cervical, thoracic and lumbar spinal cords, and intermediolateral nucleus of the thoracic spinal cord. The neuropil of the trigeminal motor, facial and hypoglossal nuclei, nucleus ambiguus and lamina IX of the cervical, thoracic and lumbar spinal cords contained many ChAT-positive bouton-like structures and they were seemingly in contact with perikarya and dendrites of motoneurons, suggesting that motoneurons in these nuclei are cholinoceptive as well as cholinergic. The oculomotor, trochlear and abducent nuclei, nucleus of Edinger–Westphal, dorsal nucleus of the vagus and intermediolateral nucleus of the thoracic spinal cord contained a small number of ChAT-immunoreactive bouton-like structures, but they did not contact with perikarya and dendrites of ChAT-positive neurons. These observations suggest that the organization of the motor nuclei is complex, at least regarding the cholinoceptivity.     

Evidence for the coexistence of acetylcholine and enkephalin in the sympathetic preganglionic neurons of rats

The localization of the cholinergic neurons in the lower thoracic segments of the spinal cord of rats was examined by a monoclonal antibody against choline acetyltransferase (ChAT). The ChAT-immunoreactive neurons were located in the intermediate as well as anterior gray matters. In the intermediate gray the highest incidence of the immunoreactive neurons was in the nucleus intermediolateralis, followed by the nucleus intercalatus pars paraependymalis and a few immunoreactive neurons were seen in the nucleus intercalatus proprius. In the sequential immunostaining of one and the same section of the spinal cord pretreated with colchicine using the ChAT antibody and a polyclonal antibody against methionine-enkephalin-argynine-glycine-leucine (Met-Enk-Arg-Gly-Leu), substantial numbers of neurons were immunostained simultaneously by the two antibodies in the intermediate gray matter. The present finding gives strong evidence for the coexistence of acetylcholine and enkephalins in, at least, some of the preganglionic neurons projecting their axons to the periphery.     

Comparisons of the immunocytochemical localization of choline acetyltransferase in the vestibular nuclei of the monkey and rat

Immunocytochemical studies of the brainstem were done in the squirrel monkey and rat using the same polyclonal antisera for choline acetyltransferase (ChAT). Cells immunoreactive for ChAT (ChATir) were evident in large numbers in visceral and motor cranial nerve nuclei in both species, but virtually no ChATir cells were seen in the vestibular nuclear complex of the rat. In the monkey ChATir cells were distributed in caudal parts of the medial (MVN) and in dorsal parts of the inferior (IVN) vestibular nuclei. Only a few immunoreactive cells were seen in the rostral MVN and none were found in cell group f of the IVN. Nearly all cells of group z and x, which do not receive primary vestibular afferents, were immunoreactive to ChAT. None of the cells in the superior and lateral vestibular nuclei, cell group y, the infracerebellar nucleus or the interstitial nucleus of the vestibular nerve were immunoreactive for ChAT. Cells immunoreactive to ChAT were present in large numbers in the rostral part of the nucleus prepositus in the monkey, but not in the rat. The relatively small number and distribution of ChATir cells in the MVN suggested they could constitute only a small fraction of the MVN neurons that contribute to a massive commissural system. Significant differences in cholinergic vestibular neurons appear to exist between the rat and the monkey.     

Type I corticosteroid receptor-like immunoreactivity in the rat CNS: distribution and regulation by corticosteroids.

Previous maps of Type I corticosteroid receptor binding in the rat central nervous system (CNS) revealed a restricted distribution of the receptor in limbic regions, hypothalamus, and circumventricular organs. More recent studies have shown a more widespread expression of the receptor, with high levels of Type I receptor mRNA in limbic, motor, and sensory systems. We have used two antisera against peptide sequences derived from the cDNA of the human Type I corticosteroid receptor to map the regional distribution and corticosteroid regulation of the intracellular location of Type I corticosteroid receptor-like immunoreactivity (Type I-ir) in the rat CNS. Neurons showing Type I-ir were observed at all levels of the CNS. Highest densities of immunoreactive neurons were observed in limbic regions, isocortex, and some thalamic nuclei. Motor, sensory, and visceral systems often showed moderate densities of immunoreactive neurons. Type I-ir glia were observed in some fiber systems, e.g., corpus callosum, medial lemniscus, cerebral peduncles, spinal trigeminal tract, and funiculi of the spinal cord. In the majority of neurons and in glia, Type I-ir showed a diffusely nuclear and cytoplasmic location. Long-term adrenalectomy reduced immunoreactivity in most neurons and glia. Neuronal Type I-ir was localized mainly in the cytoplasm after long-term adrenalectomy. Nuclear immunoreactivity was retained in some neurons in the globus pallidus, motor trigeminal nucleus, and laminae 8 and 9 of the spinal cord. Acute treatment with corticosterone or aldosterone restored neuronal and glial Type I-ir to densities below that seen in intact rats.     

Topography of choline acetyltransferase Immunoreactive Neurons and fibers in the rat spinal cord

The topography of choline acetyltransferase immunoreactivity was studied in the rat spinal cord with a monoclonal antibody. Cholinergic fibers were most prominent in lamina III of the dorsal horn and originated from cholinergic neurons within the spinal cord. Lamina X, which was rich in cholinergic neurons and fibers, provided cholinergic interconnections between the dorsal, intermediate and ventral gray. Within the ventral gray, choline acetyltransferase immunoreactive boutons were found on motor neurons. This study suggests that the cholinergic innervation of the spinal cord arises from neurons intrinsic to the spinal cord. The cholinergic neurons within the spinal cord may provide several, overlapping levels of regulation of spinal cord neurons.     

A light and electron microscopic study of GAT-1 positive cells in the monkey brainstem and spinal cord

The distribution of the GABA transporter GAT-1 was studied in the monkey brainstem and spinal cord, using an affinity purified polyclonal antibody against a synthetic peptide corresponding to the C terminus of GAT-1. Very dense staining was observed in the interpeduncular nucleus, the inferior olivary nucleus and the substantia gelatinosa of the spinal cord, whilst dense labelling was observed in the substantia nigra, cochlear nuclei, vestibular nuclei, the spinal nucleus of V, the area postrema and the ventral horn of the spinal cord. Electron microscopy showed that the labelled profiles consisted of axon terminals that formed symmetrical synapses, consistent with GABAergic terminals. Many of the nuclei that were densely labelled for GAT-1 were those that received primary auditory, vestibular, or somatosensory inputs and the high density of GAT-1 in these nuclei suggests that GAT-1 plays an important role in terminating the inhibitory effects of GABA, at these nuclei.     

Cholinergic innervation of the human thalamus: dual origin and differential nuclear distribution.

The cholinergic innervation of the human thalamus was studied with antibodies against the enzyme choline acetyltransferase (ChAT) and nerve growth factor receptor (NGFr). Acetylcholinesterase histochemistry was used to delineate nuclear boundaries. All thalamic nuclei displayed ChAT-positive axons and varicosities. Only the medial habenula contained ChAT-positive perikarya. Some intralaminar nuclei (central medial, central lateral, and paracentral), the reticular nucleus, midline nuclei (paraventricular and reuniens), some nuclei associated with the limbic system (anterodorsal nucleus and medially situated patches in the mediodorsal nucleus) and the lateral geniculate nucleus displayed the highest density of ChAT-positive axonal varicosities. The remaining sensory relay nuclei and the nuclei interconnected with the motor and association cortex displayed a lower level of innervation. Immunoreactivity for NGFr was observed in cholinergic neurons of the basal forebrain but not in cholinergic neurons of the upper brainstem. The contribution of basal forebrain afferents to the cholinergic innervation of the human thalamus was therefore studied with the aid of NGFr-immunoreactive axonal staining. The anterior intralaminar nuclei, the reticular nucleus, and medially situated patches in the mediodorsal nucleus displayed a substantial number of NGFr-positive varicose axons, presumably originating in the basal forebrain. Rare NGFr-positive axonal profiles were also seen in many of the other thalamic nuclei. These observations suggest that thalamic nuclei affiliated with limbic structures and with the ascending reticular activating system are likely to be under particularly intense cholinergic influence. While the vast majority of thalamic cholinergic input seems to come from the upper brainstem, the intralaminar and reticular nuclei, and especially medially situated patches within the mediodorsal nucleus also appear to receive substantial cholinergic innervation from the basal forebrain.     

Cholinergic innervation of the monkey amygdala: an immunohistochemical analysis with antisera to choline acetyltransferase

The organization of the cholinergic innervation of the macaque monkey amygdaloid complex was investigated by means of immunohistochemical techniques and either a polyclonal antiserum or a monoclonal antibody directed against the specific synthetic enzyme choline acetyltransferase (ChAT). Adjacent series of sections were processed histochemically for the demonstration of the degradative enzyme acetylcholinesterase (AChE) or for cell bodies with thionin. The density of ChAT immunoreactivity differed substantially among the various nuclei and cortical regions of the amygdala. In general, the distribution of ChAT immunoreactivity paralleled the pattern of AChE staining. One notable exception was the presence of AChE containing cell bodies in addition to AChE positive fibers within nearly all of the nuclear and cortical regions. In contrast, ChAT immunoreactivity was associated only with fibers and terminals. The highest density of ChAT immunoreactive fibers and terminals was consistently observed in the magnocellular subdivision of the basal nucleus. Staining was substantially less dense in the more ventrally situated parvicellular subdivision. Medially, in the adjacent accessory basal nucleus, immunoreactive fibers and terminals were densest in the magnocellular and superficial subdivisions and least prominent in the parvicellular subdivision. Of the deep nuclei, the lateral nucleus generally obtained the least ChAT immunoreactive terminals and processes. Only its more densely cellular ventrolateral portion contained appreciable fiber and terminal staining. One of the more distinctive patterns of ChAT immunoreactivity was seen in the nucleus of the lateral olfactory tract. Here, ChAT positive fibers formed pericellular basket plexuses around unstained cell bodies. This unique pattern of staining was used to delineate the boundaries of the nucleus and indicated that it is present for much of the rostrocaudal extent of the amygdala. Another region of conspicuous staining on the medial surface of the amygdala was the sulcal portion of the periamygdaloid cortex. This region, associated with the sulcus semiannularis and bordering the entorhinal cortex, consistently contained dense immunoreactivity. The central nucleus also presented a somewhat idiosyncratic pattern of ChAT staining. The lateral subdivision had a diffuse distribution of immunoreactivity in which focal patches of more densely stained terminals and occasional fine fibers were embedded. In contrast, the medial subdivision contained a larger number of thicker, stained fibers without diffuse background labeling.(ABSTRACT TRUNCATED AT 400 WORDS)     

A comparison of the distribution of central cholinergic neurons as demonstrated by acetylcholinesterase pharmacohistochemistry and choline acetyltransferase immunohistochemistry

The topographical distribution of cholinergic cell bodies has been studied in the rat brain and spinal cord by choline acetyltransferase (ChAT)-immunohistochemistry and acetylcholinesterase (AChE)-pharmacohistochemistry using diisopropylfluorophosphate (DFP). The ChAT-containing cells and the cells that stained intensely for AChE 4-8 hr after DFP were mapped in detail on an atlas of the forebrain (telencephalon, diencephalon) hindbrain (mesencephalon, rhombencephalon) and cervical cord (C2, C6). Striking similarities were observed between ChAT-positive cells and neuronal soma that stained intensely for AChE both in terms of cytoarchitectural characteristics, and with respect to the distribution of the labelled cells in many areas of the central nervous system (CNS). In the forebrain these areas include the caudatoputamen, nucleus accumbens, medial septum, nucleus of the diagonal band, magnocellular preoptic nucleus and nucleus basalis magnocellularis. In contrast, a marked discrepancy was observed in the hypothalamus and ventral thalamus where there were many neurons that stained intensely for AChE, but where there was an absence of ChAT-positive cells. No cholinergic perikarya were detected in the cerebral cortex, hippocampus, amygdala and dorsal diencephalon by either histochemical procedure. In the hindbrain, all the motoneurons constituting the well-established cranial nerve nuclei (III-VII, IX-XII) contained ChAT and exhibited intense staining for AChE. Further, a close correspondence was observed in the distribution of labeled neurons obtained by the two histochemical procedures in the midbrain and pontine tegmentum, including the laterodorsal tegmental nucleus, some areas in the caudal pontine and bulbar reticular formation, and the central gray of the closed medulla oblongata. On the other hand, AChE-intense cells were found in the nucleus raphe magnus, ventral part of gigantocellular reticular nucleus, and flocculus of the cerebellum, where ChAT-positive cells were rarely observed. According to both techniques, no positive cells were seen in the cerebellar nuclei, the pontine nuclei, or the nucleus reticularis tegmenti pontis. Large ventral horn motoneurons and, occasionally, cells in the intermediomedial zone of the cervical cord displayed ChAT-immunoreactivity and intense AChE staining. On the other hand, AChE-intense cells were detected in the dorsal portion of the lateral funiculus, but immunoreactive cells were not found in any portion of the spinal cord white matter.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of central cholinergic neurons in the baboon (Papio papio). I. General morphology

The morphological characteristics of cholinergic neurons in the central nervous system (CNS) of the baboon (Papio papio) were studied by choline acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) pharmacohistochemistry. The distributions of central cholinergic neurons as visualized by these two histochemical techniques were similar in most, but not all regions of the brain and spinal cord. Based upon these observations, central cholinergic neurons that are immunoreactive to ChAT and intensely stained for AChE by the pharmacohistochemical procedure can be divided into four major groups: (1) those in the caudate nucleus, putamen, nucleus accumbens and anterior perforated substance. These ChAT-containing and AChE-intense neurons are large and multipolar, and are scattered throughout these structures. (2) The rostral cholinergic column, which consists of a continuous mass of cholinergic perikarya situated in the medial septal nucleus, nucleus of the diagonal band, and nucleus basalis (Meynert). The ChAT-immunoreactive and AChE-intense cell bodies of the nucleus basalis are a prominent feature in the basal forebrain of the baboon. The labeled neurons are large, multipolar, and hyperchromic and show a tendency to aggregate in cell clusters. These cells are distributed within the full extent of the substantia innominata, often being associated with subcortical fiber networks such as the medullary laminae of the globus pallidus. (3) The caudal cholinergic column, which consists of a continuous group of cholinergic neurons in the caudal midbrain and pontine tegmentum. The rostral component of this group of cells is the nucleus tegmenti pedunculopontinus (subnucleus compacta) and it extends caudally to include the laterodorsal tegmental nucleus. Compared to that in other species the nucleus tegmenti pedunculopontinus in the baboon appears to occupy a relatively greater volume and is composed of a greater number of cholinergic neurons. The cells of the caudal column are large and hyperchromic. (4) Nuclei of origin of somatic and visceral efferents of the cranial nerves (III, IV, V, VI, VII, IX, X, XI, XII) and spinal nerves. In addition to these major cholinergic cell groups, a small population of ChAT-positive and AChE-intense cell bodies can be observed at the floor of the fourth ventricle and in lamina VII and X of the cervical cord. The present findings indicate that although some differences exist, the overall distribution and morphological features of cholinergic cell bodies identified in the baboon brain and spinal cord are similar to those demonstrated previously in investigations of the rhesus monkey and nonprimates.     

Ultrastructural localization of high‐affinity choline transporter in the rat anteroventral thalamus and ventral tegmental area: Differences in axon morphology and transporter distribution

The high‐affinity choline transporter (CHT) is a protein integral to the function of cholinergic neurons in the central nervous system (CNS). We examined the ultrastructural distribution of CHT in axonal arborizations of the mesopontine tegmental cholinergic neurons, a cell group in which CHT expression has yet to be characterized at the electron microscopic level. By using silver‐enhanced immunogold detection, we compared the morphological characteristics of CHT‐immunoreactive axon varicosities specifically within the anteroventral thalamus (AVN) and the ventral tegmental area (VTA). We found that CHT‐immunoreactive axon varicosities in the AVN displayed a smaller cross‐sectional area and a lower frequency of synapse formation and dense‐cored vesicle content than CHT‐labeled profiles in the VTA. We further examined the subcellular distribution of CHT and observed that immunoreactivity for this protein was predominantly localized to synaptic vesicles and minimally to the plasma membrane of axons in both regions. This pattern is consistent with the subcellular distribution of CHT displayed in other cholinergic systems. Axons in the AVN showed significantly higher levels of CHT immunoreactivity than those in the VTA and correspondingly displayed a higher level of membrane CHT labeling. These novel findings have important implications for elucidating regional differences in cholinergic signaling within the thalamic and brainstem targets of the mesopontine cholinergic system. J. Comp. Neurol. 518:1908–1924, 2010. © 2010 Wiley‐Liss, Inc.     

Co-localization of N-acetyl-aspartyl-glutamate in central cholinergic, noradrenergic, and serotonergic neurons

An immunohistochemical technique for simultaneously visualizing two different antigens has been used to investigate the presence of the acidic dipeptide, N-acetyl-aspartyl-glutamate (NAAG), in cholinergic, noradrenergic-adrenergic, and serotonergic neurons within CNS. The brain slices were processed sequentially with purified antisera against NAAG and then monoclonal antibody against choline acetyltransferase (ChAT), a marker for cholinergic neurons, or antiserum against dopamine-beta-hydroxylase (DBH), a marker of noradrenergic-adrenergic neurons, or antiserum against serotonin (5HT). Both antigens were revealed by the peroxidase reaction but with different chromogens, which are easily distinguishable. An intense double staining of NAAG-like immunoreactivity (NAAG-LI) and ChAT was observed in the motoneurons of the spinal cord as well as in the several motor components of cranial nerve nuclei including facial, ambiguus, and trigeminal nuclei. A partial colocalization of NAAG-LI and ChAT was evident in the perikarya of the basal forebrain cholinergic system, whereas cholinergic neurons of the medial septum exhibited only sporadic staining for NAAG-LI. A complete coexistence of NAAG-LI and DBH was observed in the locus coeruleus. Most of the other noradrenergic and adrenergic cell groups of the medulla region exhibited substantial co-localization with the exception of the A2 cell group, which was virtually devoid of NAAG-LI. In the dorsal raphe, only a low percentage of serotonergic neurons stained for NAAG-LI. The co-existence of NAAG-LI and serotonin was more evident in the neurons of the median raphe, although the majority of cells failed to show double staining.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of choline acetyltransferase immunoreactivity in the brain of an elasmobranch, the lesser spotted dogfish (Scyliorhinus canicula)

Although the distribution of cholinergic cells is remarkably similar across the vertebrate species, no data are available on more primitive species, such as cartilaginous fishes. To extend the evolutionary analysis of the cholinergic systems, we studied the distribution of cholinergic neurons in the brain and rostral spinal cord of Scyliorhinus canicula by immunocytochemistry using an antibody against the enzyme choline acetyltransferase (ChAT). Western blot analysis of brain extracts of dogfish, sturgeon, trout, and rat showed that this antibody recognized similar bands in the four species. Putative cholinergic neurons were observed in most brain regions, including the telencephalon, diencephalon, cerebellum, and brainstem. In the retrobulbar region and superficial dorsal pallium of the telencephalon, numerous small pallial cells were ChAT-like immunoreactive. In addition, tufted cells of the olfactory bulb and some cells in the lateral pallium showed faint immunoreactivity. In the preoptic-hypothalamic region, ChAT-immunoreactive (ChAT-ir) cells were found in the preoptic nucleus, the vascular organ of the terminal lamina, and a small population in the caudal tuber. In the epithalamus, the pineal photoreceptors were intensely positive. Many cells of the habenula were faintly ChAT-ir, but the neuropil of the interpeduncular nucleus showed intense ChAT immunoreactivity. In the pretectal region, ChAT-ir cells were observed only in the superficial pretectal nucleus. In the brainstem, the somatomotor and branchiomotor nuclei, the octavolateral efferent nucleus, and a cell group just rostral to the Edinger-Westphal (EW) nucleus contained ChAT-ir neurons. In addition, the trigeminal mesencephalic nucleus, the nucleus G of the isthmus, some locus coeruleus cells, and some cell populations of the vestibular nuclei and of the electroreceptive nucleus of the octavolateral region exhibited ChAT immunoreactivity. In the reticular areas of the brainstem, the nucleus of the medial longitudinal fascicle, many reticular neurons of the rhombencephalon, and cells of the nucleus of the lateral funiculus were immunoreactive to this antibody. In the cerebellum, Golgi cells of the granule cell layer and some cells of the cerebellar nucleus were also ChAT-ir. In the rostral spinal cord, ChAT immunoreactivity was observed in cells of the motor column, the dorsal horn, the marginal nucleus (a putative stretch-receptor organ), and in interstitial cells of the ventral funiculus. These results demonstrate for the first time that cholinergic neurons are distributed widely in the central nervous system of elasmobranchs and that their cholinergic systems have evolved several characteristics that are unique to this group.     

Distribution of hippocampal cholinergic neurostimulating peptide (HCNP) immunoreactivity in the central nervous system of the rat

Hippocampal cholinergic neurostimulating peptide (HCNP), an undecapeptide isolated from the hippocampal tissue of young rats, enhances the cholinergic development in explant cultures of medial septal nuclei. This report concerns the distribution of HCNP immunoreactivity in the central nervous system (CNS) of 11- and 28-day-old Wistar rats; two affinity-purified anti HCNP antibodies were used. Immunoblot analyses of extracts of different regions of the brain revealed a single 23 kDa band that corresponded to the presumed HCNP precursor protein. Immunostaining of the various CNS structures of the 28-day-old rats was more intense than in those of 11-day-old animals. HCNP immunoreactivity was detected in neurons as well as in glia cells, particularly oligodendroglia. The perikarya of neurons in the cerebral cortex, hippocampus, limbic cortex, caudate, putamen, arcuate nucleus of hypothalamus, trigeminal subnuclei, rostroventrolateral reticular nucleus and dorsal horn of the spinal cord were positively stained. In addition, nerve fibers and terminals in the hypothalamic subnuclei, zona incerta, thalamic subnucleus, caudate, putamen, locus coeruleus, trigeminal subnuclei, dorsal motor nucleus of the vagus, dorsal horn of the spinal cord and intermediolateral column also displayed HCNP immunoreactivity. These observations would suggest that HCNP and its related molecules may have multifunctional roles in the CNS.     

Direct spinal projections to limbic and striatal areas: Anterograde transport studies from the upper cervical spinal cord and the cervical enlargement in squirrel monkey and rat

With the anterograde tracers Phaseolus vulgaris-leucoagglutinin (PHA-L) and biotinylated dextranamine (BD), direct spinal connections from the upper cervical spinal cord (UC; C1 and C2) and the cervical enlargement (CE; C5-T1) were demonstrated in various striatal and limbic nuclei in both squirrel monkey and rat. Within each species and from each spinal level, the total number of terminals seen in the limbic and striatal areas was approximately 50–80% of the number seen within the thalamus. Labeled terminal structures were seen in the hypothalamic nuclei, ventral striatum, globus pallidus, amygdala, preoptic area, and septal nuclei. In both species, the number of labeled terminals in limbic and striatal regions was larger from UC than from CE, although the distributions to each nucleus varied with the specific lamina injected. In both species and from both UC and CE, approximately one-half of the projections to striatal and limbic areas terminated in the hypothalamus. The only region that demonstrated a topographical organization was the globus pallidus, where terminals from the CE were located dorsomedially to those from the UC. In the rat, UC and CE injections into the lateral dorsal horn and pericentral laminae resulted in the largest number of limbic and striatal terminations. The proportion of ipsilateral terminations was greatest when the medial laminae in the UC or the lateral dorsal horn in the CE received injections. Analysis of the morphology of these spinohypothalamic and spinotelencephalic terminals showed that, in the squirrel monkey, terminals from CE injections were larger than terminals from UC injections; no such size difference was evident in the rat. However, limbic and striatal terminals in the rat were generally larger than those in the squirrel monkey following injections into the UC or CE. The exact function of these direct spinal projections to various striatal and limbic areas in primates and in rodents remains to be determined. These findings, however, support recent imaging studies that suggest that the limbic system plays an important role in the mediation of chronic pain, perhaps directly through these spinolimbic and spinostriatal pathways. © 1996 Wiley-Liss, Inc.     

Cholinergic systems in the rat brain: IV. Descending projections of the pontomesencephalic tegmentum

Descending projections from cholinergic neurons in the pedunculopontine and laterodorsal tegmental nuclei, collectively referred to as the pontomesencephalotegmental (PMT) cholinergic complex, were studied by use of the fluorescent retrograde tracers fluorogold, true blue, or Evans Blue in combination with choline acetyltransferase (ChAT) immunohistochemistry of acetylcholinesterase (AChE) pharmacohistochemistry. Pedunculopontine somata positive for ChAT or staining intensely for AChE were retrogradely labeled with fluorescent tracers following infusions into the motor nuclei of cranial nerves 5, 7, and 12. ChAT-positive cells in both the pedunculopontine and laterodorsal tegmental nuclei demonstrated projections to the vestibular nuclei, the spinal nucleus of the 5th cranial nerve, deep cerebellar nuclei, pontine nuclei, locus ceruleus, raphe magnus nucleus, dorsal raphe nucleus, median raphe nucleus, the medullary reticular nuclei, and the oral and caudal pontine reticular nuclei. Fluorescent tracers used in combination with AChE pharmacohistochemistry corroborated these projections and, in addition, provided evidence for cholinergic pontomesencephalic projections to the lateral reticular nucleus and inferior olive. The majority of retrogradely labeled neurons demonstrating ChAT-like immunoreactivity were found ipsilateral to the injection site, but, in all cases, tracer-containing cholinergic cells contralateral to the infused side of the brain were detected also. More retrogradely labeled cells containing ChAT were observed in the pedunculopontine tegmental than in the laterodorsal tegmental nucleus following tracer injections at all sites with the exceptions of the locus ceruleus and dorsal raphe nucleus where the converse profile was observed. None of the pedunculopontine or laterodorsal tegmental cells immunopositive for ChAT or stained intensely for AChE contained retrogradely transported tracers following dye infusions into the cerebellar cortex or cervical spinal cord. Triple-label experiments using two tracers infused into different sites in the same animal revealed that individual ChAT-immunoreactive cells in the PMT cholinergic complex projected to more than one hindbrain site in some cases and had ascending projections as well. Certain ChAT-positive somata in the pedunculopontine and laterodorsal tegmental nuclei were found in close association with several fiber tracts, including the superior cerebellar peduncle, lateral lemniscus, dorsal tegmental tract, and medial longitudinal fasciculus.     

Expression of high affinity choline transporter during mouse development in vivo and its upregulation by NGF and BMP-4 in vitro

An important feature of cholinergic neurons is high-affinity choline transport, which allows them to reuse choline for the synthesis of ACh needed to support cholinergic neurotransmission. The choline transporter, designated CHT, was recently cloned. We applied RT/PCR to monitor the expression of CHT in the developing mouse CNS from embryonic day 14 (E14) to postnatal day 30 (P30). We found that CHT was expressed early in development, predominantly in the regions containing cholinergic neurons. In the spinal cord, CHT mRNA was present at close to adult levels at the earliest time point examined (E14) and showed almost no changes after birth. In the striatum and the septum, CHT mRNA increased steadily during embryonic stages and leveled off after birth. Surprisingly, CHT mRNA expression was also detected in other brain regions, notably in the cerebellum, where it peaked on E19, and then rapidly declined during postnatal development. CHT protein was detected by Western blotting as a band of apparent molecular weight of 70 kDa. The accumulation of this protein during development lagged behind mRNA accumulation in all tissues. We also examined the effects of NGF and BMP-4, the potent inducers of choline acetyltransferase and vesicular acetylcholine transporter genes, on CHT expression. Both factors increased CHT mRNA accumulation in primary septal cultures. The effect of NGF was dependent on the PI3K signaling, as it was abolished by the PI3K inhibitor LY294002. This result indicates that some of the signals regulating other cholinergic-specific genes also control CHT expression.     

Distribution of enkephalin and its relation to serotonin in cat and monkey spinal cord and brain stem.

The distribution of enkephalin (ENK)-like immunoreactivity (LI) in spinal cord and medulla oblongata of cat and gray monkey (Macaca fascicularis) was studied by use of immunofluorescence and peroxidase antiperoxidase (PAP) techniques. Possible coexistence between ENK- and 5-hydroxytryptamine (5-HT)-LI was also analyzed with double labeling immunofluorescence. Furthermore, in situ hybridization was used to demonstrate cell bodies in the brain stem expressing mRNA encoding for ENK. ENK-immunoreactive (IR) axonal varicosities and fibers were demonstrated throughout the spinal cord gray matter, with the highest density in the superficial dorsal horn, the area around the central canal, the intermediolateral cell column, the sacral parasympathetic nucleus, and in Onuf's nucleus. In the monkey ventral horn, ENK-IR varicose fibers could in some cases be demonstrated in very close apposition to cell bodies. A low degree of co-localization between ENK- and 5-HT-LI was seen in the spinal cord of both species. Still, fibers containing both compounds could as a rule be demonstrated in every section studied. The highest degree of coexistence was encountered in the motor nucleus of the ventral horn. Six weeks after a low thoracic spinal cord transection a decreased staining for ENK-LI was demonstrated in the ventral horn motor nucleus, whereas other parts of the spinal cord appeared unaffected. In the brain stem of cats after colchicine treatment, ENK-LI was found in a majority of the 5-HT-IR cell bodies in the raphe nuclei (nucleus raphe magnus, pallidus and obscurus) and in the lateral reticular nucleus (rostroventrolateral reticular nucleus). In cat not pretreated with colchicine, a few weakly stained ENK-IR cell bodies could be found in the midline raphe nuclei and in the lateral reticular nucleus with the PAP technique. In the monkey brain stem without colchicine treatment, using the PAP technique, heavily stained ENK-IR cell bodies could be seen in the lateral reticular nucleus whereas, as in the cat, only a few, weakly stained ENK-IR cell bodies could be seen in the midline raphe nuclei. Using in situ hybridization technique, ENK mRNA expressing cells were demonstrated in the lateral reticular nucleus while no convincing mRNA signal could be found over cell bodies in the raphe nuclei. It is concluded that part of the ENKergic innervation of the cord in both species derives from supraspinal or suprasegmental levels.(ABSTRACT TRUNCATED AT 400 WORDS)