Calbindin-D28K, parvalbumin and calretinin in primate lower motor neurons

It has been suggested that lower motor neurons containing calcium-binding proteins (CBP) may be resistant to degeneration in motor neuron disease. The testing of this hypothesis is hampered by lack of comprehensive information regarding the presence of CBPs in motor neurons. To address this shortcoming, we investigated the distribution of the CBPs calbindin-D28K (CB), parvalbumin (PV) and calretinin (CRT) in lower motor neurons in the normal human and two non-human primates (rhesus monkey and common marmoset) using immunohistochemistry. A variable proportion of motor neurons in cranial nerve motor nuclei contained immunoreactivity for one or more CBPs. A subpopulation of spinal cord alpha-motor neurons was also CBP-positive. Comparison of staining for choline acetyltransferase (ChAT) and CBPs in the human spinal cord demonstrated that approximately 63% of ventral horn motor neurons contained PV, 53% contained CRT and 56% contained CB. CBP immunoreactivity within motor neurons was of variable staining intensity. It remains to be established whether the presence of these CBPs confers protection against the pathogenic mechanisms of motor neuron disease.     

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.     

Postnatal development of neurons containing choline acetyltransferase in rat spinal cord: an immunocytochemical study

A monoclonal antibody to choline acetyltransferase (ChAT) has been used in an immunocytochemical study of the postnatal development of ChAT-containing neurons in cervical and thoracic spinal cord. Specimens from rat pups ranging in age from 1 to 28 days postnatal (dpn) were studied and compared with adult specimens (Barber et al., '84). The development of established cholinergic neurons, the somatic motoneurons and sympathetic preganglionic cells, has been described as has that of previously unidentified ChAT-positive neurons in the dorsal, intermediate, and central gray matter. Cell bodies of somatic and visceral motoneurons contained moderate amounts of ChAT-positive reaction product at birth that gradually increased in intensity until 14-21 dpn. The most intensely stained ChAT-positive neurons in 1-5-dpn specimens were named partition cells because this cell group extended from the central gray to an area dorsal to the lateral motoneurons, and thereby divided the spinal cord into dorsal and ventral halves. Partition cells were medium to large in size with 5-7 primary dendrites, and axons that, in fortuitous sections, could be traced into the ventrolateral motoneuron pools, the ventral funiculi, or the ventral commissure. Small ChAT-positive cells clustered around the central canal and scattered in laminae III-VI of the dorsal horn were detectable at birth. These neurons were moderately immunoreactive at 11-14 dpn and intensely ChAT positive by 21 dpn. The band of ChAT-positive terminal-like structures demonstrated in lamina III of adult specimens (Barber et al., '84) was first visible in 11-14-dpn specimens. By 28 dpn, both laminae I and III contained punctate bands that approximated the density of those observed in adult spinal cord. This investigation has demonstrated ChAT within individual neurons of developing spinal cord, and has identified a group of neurons, the partition cells, that exhibit intense ChAT-positive immunoreactivity earlier than any other putative cholinergic cells in spinal cord, including motoneurons. Another important observation has been that each ChAT-positive neuronal type achieves adult levels of staining intensity at different times during development. A likely explanation for this differential staining is that various groups of neurons acquire their mature concentration of ChAT molecules at different developmental stages. In turn, this may correlate with the maturation of cholinergic synaptic activity manifest by individual cells or groups of neurons.     

Distribution of neurons expressing immunoreactivity for the 5HT3 receptor subtype in the rat brain and spinal cord

The cellular distribution of the type 3 serotonin receptor (5HT₃R) in the rat brain was established immunocytochemically by using a polyclonal antibody raised against a synthetic peptide from the deduced amino-acid sequence of the cloned 5HT₃R. The 5HT₃R-immunoreactive neurons were found in the forebrain, brainstem, and spinal cord, but within each region, the intensity of the immunoreactivity differed considerably. Within the forebrain, intensely immunoreactive cells were found in layers II–III of the neocortex, anterior olfactory nucleus, hippocampal formation, and amygdala. A few strongly immunoreactive neurons were consistently observed in the caudate putamen, and moderately or weakly labeled neurons were occasionally found in the nucleus accumbens. Within the brainstem, intensely labeled neurons were found in the trigeminal motor (V) and facial (VII) nuclei. Immunostained neurons were detected in the dorsal and the ventral horn of the spinal cord. These results reveal that the 5HT₃R-immunoreactive neurons are broadly distributed throughout the rat brain spinal cord, and suggest that this receptor can subserve significant participation in central nervous system neurotransmission. J. Comp. Neurol. 402:385–401, 1998. © 1998 Wiley-Liss, Inc.     

Distribution of high affinity choline transporter immunoreactivity in the primate central nervous system

A mouse monoclonal antibody (clone 62-2E8) raised against a human recombinant high-affinity choline transporter (CHT)-glutathione-S-transferase fusion protein was used to determine the distribution of immunoreactive profiles containing this protein in the monkey central nervous system (CNS). Within the monkey telencephalon, CHT-immunoreactive perikarya were found in the striatum, nucleus accumbens, medial septum, vertical and horizontal limb nuclei of the diagonal band, nucleus basalis complex, and the bed nucleus of the stria terminalis. Dense fiber staining was observed within the islands of Calleja, olfactory tubercle, hippocampal complex, amygdala; moderate to light fiber staining was seen in iso- and limbic cortices. CHT-containing fibers were also present in sensory and limbic thalamic nuclei, preoptic and hypothalamic areas, and the floccular lobe of the cerebellum. In the brainstem, CHT-immunoreactive profiles were observed in the pedunculopontine and dorsolateral tegmental nuclei, the Edinger-Westphal, oculomotor, trochlear, trigeminal, abducens, facial, ambiguus, dorsal vagal motor, and hypoglossal nuclei. In the spinal cord, CHT-immunoreactive ventral horn motoneurons were seen in close apposition to intensely immunoreactive C-terminals at the level of the cervical spinal cord. CHT immunostaining revealed a similar distribution of labeled profiles in the aged human brain and spinal cord. Dual fluorescent confocal microscopy revealed that the majority of CHT immunoreactive neurons contained the specific cholinergic marker, choline acetyltransferase, at all levels of the monkey CNS. The present observations indicate that the present CHT antibody labels cholinergic structures within the primate CNS and provides an additional marker for the investigation of cholinergic neuronal function in aging and disease.     

Differential effects of reserpine on brainstem catecholaminergic neurons revealed by Fos protein immunohistochemistry.

The effects of a single systemic injection of reserpine on c-fos proto-oncogene expression in catecholaminergic neurons of the rat brainstem were studied by immunohistochemistry for Fos proteins (Fos). In control rats, a few Fos immunoreactive neuronal nuclei were observed in the tectum and mesencephalic central gray. Within hours after drug injection, a substantial number of brainstem neurons stained intensely for Fos. The staining was maximal at 6 h and returned to control levels within 24 h. Double-immunohistochemical staining with antibodies to tyrosine hydroxylase revealed that in all noradrenergic (NA) neuron subgroups except the A2 group, the majority of NA neurons stained for Fos. Most adrenergic neurons were also labeled. In contrast, aside from some cells in the ventral tegmental area, reserpine did not induce Fos immunoreactivity in dopaminergic neurons. Numerous non-catecholaminergic neurons were intensely stained with Fos in the substantia nigra pars reticulata, ventral tegmental area, mesencephalic central gray, pontine nuclei and tectum. A small number of Fos immunoreactive neurons was also observed in raphe nuclei. Injection of saline (i.p.) resulted in a moderate increase in Fos immunoreactivity in the locus ceruleus, in A1/C1 neurons and in the mesencephalic central gray. The results demonstrate that acute reserpine treatment induces Fos expression in distinct populations of brainstem neurons, comprising both catecholaminergic and non-catecholaminergic neurons. Thus, induction of Fos by reserpine does not coincide with the site of action of this drug. The distribution of Fos immunoreactive NA neurons after reserpine treatment is comparable to that reported after application of stressful stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Parvalbumin and calbindin D-28k in the human motor system and in motor neuron disease

Calbindin D-28k and parvalbumin are neuronal calcium binding proteins of interest in relation to neurodegenerative diseases. Expression of calbindin and parvalbumin may be one of the determinants of selective vulnerability in these disorders. The distribution of these proteins was surveyed in the normal human motor system and in motor neuron disease (MND) using immunocytochemistry in formalin fixed post-mortem tissues. CNS tissues from 14 MND patients (mean age 61.2 years, mean post-mortem delay 24.6 h) and seven controls (mean age 62.6 years, mean post-mortem delay 25.3 h) were studied. Preliminary studies on the effects of fixation were performed. In normal cases upper and lower motor neurons showed absent expression of both proteins.
Several neuronal groups characteristically spared in MND showed varying patterns of immunoreactivity: oculomotor neurons showed parvalbumin staining of the perikaryon; the thoracic preganglionic sympathetic neurons showed calbindin staining in perikarya, Onuf's nucleus showed calbindin staining in the neuropil only. In motor neuron disease a loss of ventral horn interneurons and calbindin immunoreactive processes was observed with no other disease related changes in the spinal cord, brainstem, or motor cortex. These findings are consistent with the hypothesis that the distribution of these proteins is one determinant of selective vulnerability to the neurodegenerative processes in MND acting via disturbance of neuronal calcium homeostasis.     

HCN1 ion channel immunoreactivity in spinal cord and medulla oblongata

Hyperpolarization-activated cyclic nucleotide-gated (HCN) non-selective cation channels in neurons carry currents proposed to perform diverse functions, including the hyperpolarization activated Ih current. The 4 HCN subunits have unique but overlapping patterns of expression in the CNS. Here, we examined the distribution of HCN1 channel subunits in the brainstem and spinal cord using immunohistochemistry. At all levels of the spinal cord dorsal horn, HCN1 immunoreactivity (HCN1-IR) was predominantly absent from laminae I and II, while a dense band of punctate labeling was visible in lamina III. Labeled neurons were identified in close vicinity to the central canal, in the lateral spinal nucleus, in the ventral horn and occasionally in lamina II and III. Those in the ventral horn were identified as alpha motor neurons using retrograde tracing and/or double or triple immunostaining with neuronal markers neurofilament 200 (NF200) and choline acetyltransferase. HCN1-IR neurons in the brainstem included neurons in sensory pathways such as the dorsal column nuclei, the area postrema, the spinal trigeminal nucleus as well as identified motor neurons in motor nuclei. In the nucleus ambiguus, a mixed visceral/motor nucleus, HCN1-IR was present only in NF200-IR cells, suggesting that it is expressed in motor but not autonomic preganglionic neurons. HCN1-IR motor neurons in the nucleus ambiguus also expressed the neurokinin 1 receptor and were labeled retrogradely from the larnyx. At the light microscopic level, the NTS and inferior olive contained punctate labeling, which ultrastructural examination revealed to be present in predominantly synaptic terminals or dendrites respectively. These data therefore described the first localization of the HCN1 subunit in the spinal cord and extend previous reports from the brainstem.     

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 GABA-like immunoreactivity in the rat amygdaloid complex

The distribution of GABA-like (GABA-Li) immunoreactivity in the rat amygdaloid complex was studied by using an anti-GABA antibody. GABA-Li positive neurons and processes were present in every nucleus of the complex. Three patterns of immunoreactivity were revealed: (1) the intercalated masses and the lateral olfactory tract nucleus exhibited the most intense staining of the neuropil, and virtually every neuron was labeled, (2) the central and medial nuclei contained intensely labeled neuropil and moderately labeled neurons, and (3) in the remaining nuclei, the neuropil was weakly labeled, and relatively numerous GABA-Li neurons were present. Our results suggest that: (1) the intercalated masses and lateral olfactory tract nucleus consist of large aggregates of GABA-Li immunoreactive neurons, and (2) the lateral, basal dorsal, and the posterior cortical nuclei may constitute a significant source of GABAergic connections to other amygdaloid nuclei, in particular to the medial and central nuclei.     

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.     

Immunohistochemical localization of phospholipase D1 in rat central nervous system

Phospholipase D (PLD) is one of the intracellular signal transduction enzymes and plays an important role in a variety of cellular functions. We investigated the distribution of PLD isozyme, PLD1 in the rat brain and spinal cord using an immunological approach. Western blot analysis showed the presence of PLD1 protein in all tissues studied, with significantly higher levels in the brainstem and spinal cord, which was correlated with the results obtained from PLD activity assay. Prominent and specific signals of PLD1 were observed in many functionally diverse brain areas, including the olfactory bulb, medial septum-diagonal band complex, cerebral cortex, brainstem, cerebellum, and spinal cord. In the brainstem, the red nucleus, substantia nigra, interpeduncular nucleus, cranial motor nuclei (trigeminal motor, abducent, facial, and hypoglossal), sensory cranial nerve nuclei (spinal trigeminal, vestibular, and cochlear), as well as nuclei of the reticular formation, all showed intense immunoreactivity. Purkinje cells and deep cerebellar nuclei of the cerebellum were also labeled intensely. However, no significant labeling was found in the thalamus, epithalamus, and basal ganglia. Although many of the PLD1 immunoreactive cells were neurons, PLD1 was also expressed in glial cells such as presumed astrocytes and tanycytes. These findings suggest that PLD1 may play an important role in the central nervous system of the adult rat.     

Immunohistochemical distribution of substance P, serotonin, and methionine enkephalin in sexually dimorphic nuclei of the rat lumbar spinal cord

The purpose of the present study was to identify chemically some potential inputs to lumbar motoneurons of the rat in the spinal nucleus of the bulbocavernosus, ventral motor pool, dorsolateral nucleus, and retrodorsolateral nucleus. Substance P-like immunoreactivity and serotonin-like immunoreactivity were found in all four motor nuclei, with dense immunoreactive profiles surrounding motoneurons and their processes. Enkephalin-like immunoreactivity was restricted to the sexually dimorphic nuclei, the spinal nucleus of the bulbocavernosus, and the dorsolateral nucleus. Within the spinal nucleus of the bulbocavernosus, enkephalin-like immunoreactive profiles were apposed to the processes of motoneurons but not their somata. In contrast, enkephalin-like immunoreactivity surrounded motoneuron somata in the medial part but not the lateral part of the dorsolateral nucleus, in the location of motoneurons projecting to the ischiocavernosus muscle. Moreover, the density of serotonin-like immunoreactivity was also greater in the medial part of the dorsolateral nucleus. On the basis of the chemo-architecture and the connections of the dorsolateral nucleus, we suggest the division of this motor column into a medial part composed of ischiocavernosus motoneurons surrounded by enkephalin- and serotonin-like immunoreactivity and a lateral part that contains neurons that project to the sphincter urethrae muscle. Total spinal transection severely depleted both serotonin-like and substance P-like material in the lumbar ventral horn. No changes in the distribution of enkephalin-like immunoreactivity were observed following this lesion. It is therefore suggested that in the ventral horn, substance P- and serotonin-like material are derived from supraspinal tracts, whereas enkephalin-like material is derived from intrinsic nerve cell bodies of the spinal cord.     

Intrinsic 5HT-immunoreactive neurons in the spinal cord of the fetal non-human primate

Serotonin (5HT) immunoreactive neurons were identified in the late-term fetal spinal cord of normal non-human primates. These neurons were distributed throughout the spinal cord, being concentrated in lamina X and the subjacent ventral median fissure, while their immunoreactive fibers and terminals innervated the zone surrounding the central canal and the ventral spinal artery. Even at this late fetal stage, the dorsal and ventral spinal gray matter was virtually devoid of any positive 5HT immunoreactivity, in contrast to that seen in the adult primate. These findings suggest that the intrinsic 5HT neurons of the primate during development may modulate CSF composition or provide cues for spinal cord differentiation rather than regulate sensorimotor functions as they do in the adult.     

Expression of cholinergic phenotype by embryonic ventral horn neurons transplanted into the spinal cord in the rat

Solid grafts of E12 embryonic spinal ventral horn were transplanted into motoneuron-depleted adult lumbar spinal cord in the rat. A muscle was implanted parallel to the vertebral column with its nerve inserted into the lumbar cord at the site of transplantation so as to provide a target for innervation by the grafted neurons. Previous retrograde labelling studies have shown that modest numbers of grafted motoneuron-like cells participate in the muscle's reinnervation and these are often found outside the graft within the host spinal cord. However, Nissl stained sections show that larger numbers of neurons survive within tissue recognisable as being of graft origin. In this study we have examined the expression of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) by neurons within the graft. These enzymes are involved in cholinergic neurotransmission and are characteristic of motoneurons. Thirty-four to seventy days following transplantation the grafts contained numerous neurons with acetylcholinesterase (AChE) activity. Different patterns of AChE staining were observed which probably reflected the degree of differentiation and maturation within the graft. AChE positive neurons were found in isolation or in groups resembling developing motor pools. Most of the AChE-positive neurons appeared immature with scant cytoplasm. However, neurons could be found which appeared relatively mature with a regularly shaped nucleus, prominent nucleolus and Nissl bodies. The grafts contained few AChE-positive axons and no dense plexuses of varicose fibres around the neurons such as are found around motoneurons in the mature ventral horn. Comparisons between the size of AChE-positive neurons in the graft and the size of AChE-positive neurons in the developing ventral horn found that the size of grafted neurons to be intermediate between the sizes of spinal motoneurons at E19 and P0. Far fewer grafted neurons were found to be immunoreactive for choline acetyltransferase (ChAT) than histochemically reactive for AChE. This was consistent with our findings in the spinal cord during normal development where we found that fixation and staining procedures which labelled adult motoneurons failed to reliably demonstrate ChAT immunoreactivety in normal motoneurons prenatally, although AChE histochemical reactivity could be demonstrated as early as E16. We conclude that the grafts contain numbers of immature motoneurons which fail to proceed beyond a certain stage of development, perhaps because of a failure to form appropriate efferent and afferent connections.     

The expression of the glial glutamate transporter protein EAAT2 in motor neuron disease: an immunohistochemical study

Emerging evidence suggests that a disturbance of the glutamate neurotransmitter system may be a contributory factor to motor neuron injury in motor neuron disease. Previous autoradiographic and immunoblotting studies have suggested that there may be reduced expression of glutamate transporter proteins in pathologically affected areas of the CNS in motor neuron disease. This study further explores the possible alteration in expression of the excitatory amino acid transporter protein EAAT2 in MND, by examining the protein expression in situ, in frozen sections, using immunohistochemistry. The aim of the study was to compare the distribution and density of EAAT2 in the motor cortex and spinal cord of MND cases (n = 16) compared with neurologically normal controls (n = 12), matched for relevant parameters. A novel, previously characterized, monoclonal antibody to EAAT2 was employed. EAAT2 immunoreactivity in motor neuron disease and control cases was compared using relative optical density measurements generated by computerized image analysis. In the motor cortex, EAAT2 immunoreactivity was laminated comprising a superficial intense band (corresponding to layers 1 and 2); a paler middle band (layer 3 and part of 5) and a more intense deep layer (layers 5 and 6). In the spinal cord, the ventral horn showed strong immunoreactivity with dense perisomatic staining around motor neuron cell bodies, the substantia gelatinosa showed moderate diffuse staining and the intermediate spinal laminae showed weak staining. This general pattern of immunoreactivity was preserved in the motor neuron disease cases. However, in the motor neuron disease cases compared with controls, the optical density values for EAAT2 immunoreactivity were significantly reduced in all grey matter regions of the lumbar spinal cord (P < 0.001) and were increased in the middle laminae of the motor cortex (P < 0.05). This study indicates that glutamate transporter pathology in motor neuron disease may be a more complex phenomenon than previously recognized.     

Distribution of cholinergic neurons in the chick spinal cord during embryonic development. Comparison of ChAT immunocytochemistry with AChE histochemistry.

The location of cholinergic neurons was studied during the development of the chick embryo spinal cord. A comparison between choline acetyltransferase (ChAT) immunocytochemistry and acetylcholinesterase (AChE) histochemistry was performed. ChAT-positive neurons could be detected only from embryonic day 9 (E9) onwards by the FITC technique and from E12 onwards by the PAP technique. These neurons were located mainly in the medial and lateral motor columns in the ventral horn of the gray matter and some of them were observed in the intermediate region of the spinal cord. AChE-containing cell bodies were much more numerous than the ChAT immunoreactive ones and were distributed in the ventral horn of the gray matter, the intermediate gray region and mostly off the apical part of the dorsal horn. ChAT should provide a reliable and specific marker for cholinergic neurons.     

Spinal cord afferent systems containing the nerve terminal protein NT75

In the adult spinal cord, immunocytochemical staining for NT75 is concentrated in nerve terminals in the superficial laminae of the dorsal horn. Deeper laminae of the dorsal horn contain moderate immunocytochemical labeling, but the ventral horn is only sparsely stained. The origin of spinal nerve terminals containing NT75 was investigated with lesion techniques, colchicine treatment, and retrograde tracing in combination with immunocytochemical staining. Primary afferent neurons express NT75 immunoreactivity and account for most of the dense staining in the superficial dorsal horn and part of the labeling in the deeper laminae. It was found that corticospinal and virtually all brainstem neurons with descending projections to the spinal cord also express NT75 immunoreactivity, including those terminating in the ventral horn. Colchicine treatment of the spinal cord also resulted in NT75 staining in most, if not all, spinal neurons. It appears that neurons in all three major sources of spinal afferents (primary sensory, descending, and intrinsic systems) can express NT75 immunoreactivity, but that some neurons normally contain higher levels of the protein in their nerve terminals. Previous analysis of developing spinal cord has shown widespread, dense NT75 labeling throughout the spinal gray in the early postnatal period, which later becomes restricted to the adult pattern. These studies support the hypothesis that many spinal pathways express high levels of NT75 immunoreactivity during development, but that only certain pathways maintain high levels in the adult. © 1993 Wiley-Liss, Inc.     

Somatostatin-like immunoreactivity in the brain of the urodele amphibian Pleurodeles waltl. Colocalization with catecholamines and nitric oxide

The neuronal structures with somatostatin-like immunoreactivity have been studied in the brain of the urodele amphibian Pleurodeles waltl. Intense immunoreactivity was observed in neurons and fibers distributed throughout the brain. Within the telencephalon, the subpallial regions were densely labeled containing both cells and fibers, primarily in the striatum and amygdala. The majority of the somatostatin immunoreactive neurons were located in the preoptic area and hypothalamus, although less numerous cells were also found in the thalamus. A conspicuous innervation of the median eminence was revealed, which arises from the hypothalamic cell populations. In the brainstem, intense fiber labeling was present in the tectum and tegmentum, whereas cell bodies were located only in the tegmentum of the mesencephalon and in the interpeduncular, raphe and reticular nuclei of the rhombencephalon. Longitudinal fiber tracts throughout the brainstem were observed and they continued into the spinal cord in the laterodorsal funiculus. The localization of somatostatin in catecholaminergic and nitrergic neurons was studied by double labeling techniques with antisera against tyrosine hydroxylase and nitric oxide synthase. Catecholamines and somatostatin only colocalized in a cell population in the ventral preoptic area. In turn, the striatum and amygdala contained neurons with somatostatin and nitric oxide synthase. Our results demonstrated that the somatostatin neuronal system in the brain of Pleurodeles waltl is consistent with that observed in anuran amphibians and shares many characteristics with those of amniotes. Colocalization of somatostatin with catecholamines and nitric oxide is very restricted in the urodele brain, but in places that can be easily compared to those reported for mammals, suggesting that interactions between these neurotransmitter systems are a primitive feature shared by tetrapod vertebrates.