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.     

Somatostatin-like immunoreactivity in neurons, nerve terminals, and fibers of the cat spinal cord

The distribution of somatostatin-like immunoreactivity (SS) was studied in the spinal cord of untreated cats and of cats that had received colchicine at all levels of the cord. In the dorsal horn small (less than 15 microns in diameter), round neurons were found in Rexed laminae II and III at all levels. At all levels laminae IV-VI contained smaller numbers of immunoreactive neurons that were medium (between 15 and 25 microns in diameter) to large (greater than 25 microns in diameter) in size. In addition, small numbers of medium-sized neurons were observed at the dorsal and dorsomedial borders of the gray and white matter in segments C1-5. In the sacral cord (S1-3), a group of medium-sized bipolar neurons was found in the dorsolateral funiculus. In transverse sections the processes of the neurons in these two latter groups travelled in a direction parallel to the border of the gray and white matter. In the intermediate and central gray matter, in addition to the immunoreactive neurons found in the region of the intermediolateral nucleus and nucleus intercalatus of lamina VII in segments C8 to L4 (Krukoff et al., '85a), lamina VII contained immunoreactive neurons at all levels with the largest number occurring in the thoracic cord. These neurons were medium to large in size and were generally multipolar with processes travelling in all directions. Multipolar small immunoreactive neurons were also found in the central gray region (lamina X) in the thoracic and upper lumbar cord. Finally, small numbers of neurons containing SS were found in the ventral horn of the cervical and upper thoracic cord. These multipolar neurons were medium to large in size. The distribution of nerve terminals and fibers containing SS was similar to that previously described in mice, rats, guinea pigs, and primates. Although the function of somatostatin in the spinal cord is not known, its presence in neurons with short processes suggests that it may act to modify local activity in the regions where it is found, including areas involved in sensory, visceromotor, and motor functions.     

Light and electron microscopic study of an avian pretectal nucleus, the lentiform nucleus of the mesencephalon, magnocellular division

The distribution of vasoactive intestinal polypeptide (VIP) was mapped by peroxidase immunocytochemistry in the spinal cords of seven Macaca fascicularis monkeys and two cats. The animals were perfusion fixed with different chemicals. Those that were perfused with either a Zamboni fixative or 5% acrolein had significantly greater immunoreactivity outside the sacral cords; those fixed with 4% paraformaldehyde had little in nonsacral regions. VIP-like immunoreactive (VIP) axons and terminals were found in the superficial dorsal horn, reticular nucleus of lamina V, intermediomedial nucleus, and lamina X at all levels from C2 to S4; a few axons and terminals were also seen in the ventral horn. Axons were found in Lissauer's tract at all levels, and axons appeared in the dorsolateral and ventrolateral white matter at midthoracic levels; in the lumbosacral cord the number and extent of axons in the lateral and ventral white matter increased progressively in a caudal direction. VIP neurons were identified in thoracic intermediate gray lateral to the central canal and in the intercalatus (IC) and intermediolateral (IML) nuclei. Electron microscopy of the VIP terminals in laminae I and II of the cervical cord revealed they contain small round vesicles and many large granular vesicles; some are glomerular terminals and most form asymmetrical synaptic contacts onto dendrites. These results indicate VIP is much more widely distributed in the spinal cord than previously thought; VIP may be associated with both visceral thoracic and lumbosacral afferents, and with other afferents in the cervical cord; VIP neurons are present in the thoracic intermediate gray; and VIP axons in the ventral and lateral white matter indicate that the spinal cord is supplied in part by VIP sources other than primary afferents.     

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.     

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.     

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).     

Immunohistochemical Localization of Kininogen in Rat Spinal Cord and Brain

Kininogen localization has been determined by immunocytochemistry in rat spinal cord and brain using a kinin-directed kininogen monoclonal antibody. In the spinal cord, there were immunostained neurons and fibers in laminae I, II, VII, and IX, intensely stained fibers in the superficial layers of the dorsal horn, and immunoreactive glial and endothelial cells. Small neurons, satellite cells, and Schwann cells immunostained distinctly in the dorsal root ganglion. In the brain stem, there were immunoreactive neurons and fibers in the tractus solitarius and nucleus, trigeminal spinal tract and nuclei, periaqueductal gray matter, vestibular nuclei, cochlear nuclei, trapezoid body, medial geniculate nucleus, and red nucleus. Immunostained neurons and fibers were also found in cerebellum (dentate nucleus), cerebral cortex (layers III and V), hippocampus (pyramidal cell layer), and corpus callosum. Glia and endothelial cells stained in all brain regions. The widespread location of kininogen in neurons and their processes, as well as in glial and endothelial cells, indicates more than one functional role, including those proposed as a mediator, a calpain inhibitor, and a kinin precursor, in a variety of neural activities and responses.     

Localization of basic fibroblast growth factor-like immunoreactivity in the rat brain.

The immunohistochemical localization of basic fibroblast growth factor (bFGF) was studied in the adult rat brain, using a specific antibody against a synthetic bFGF fragment (the N-terminal 12 residues). Widespread but uneven regional localization of bFGF-like immunoreactive neurons and fibers was observed. Ependymal cells were also stained. The immunoreactive neurons were found in the cerebral cortex, olfactory bulb, septum, basal magnocellular nuclei, thalamus, hypothalamus, globus pallidus, hippocampus, amygdala, red nucleus, central gray of the midbrain, cerebellum, dorsal tegmental area, reticular formation, cranial motor nuclei and spinal cord. Immunoreactive fiber bundles and nerve terminals were also detected. These results indicate that bFGF is produced by or present in a specific neuronal cell population of the central nervous system.     

A cholinergic projection to the rat superior colliculus demonstrated by retrograde transport of horseradish peroxidase and choline acetyltransferase immunohistochemistry

Acetylcholinesterase (AChE) has been localized by histochemistry in the superior colliculus and in the tegmentum of the caudal midbrain and rostral pons of the rat. The pattern of AChE localization in the superior colliculus was characterized by homogeneous staining in the superficial layers and patchlike staining in the intermediate gray layer. In the tegmentum, AChE was localized in the pedunculopontine nucleus (PPN), beginning rostrally at the caudal pole of the substantia nigra and extending caudally to the level of the parabrachial nuclei, and in the lateral dorsal tegmental nucleus (LDTN) of the central gray. The localization of AChE in these nuclei overlapped the distribution of neurons stained by immunohistochemistry using an antibody to choline acetyltransferase (ChAT), the synthesizing enzyme of the neurotransmitter acetylcholine. Other neighboring areas that were stained with AChE, but that did not contain ChAT-immunoreactive neurons, included the microcellular tegmental nucleus and the ventral tegmental nucleus. Neurons in the PPN and LDTN were determined to be potential sources of the cholinergic projection to the intermediate gray layer of the rat superior colliculus by double labelling with retrograde transport of horseradish peroxidase (HRP) combined with the immunohistochemical localization of ChAT. Three populations of neurons were identified. A predominantly ipsilateral ChAT-immunoreactive population was located in the pars compacta subdivision of PPN (PPNpc). Retrograde HRP-labelled neurons in the pars dissipata subdivision of the PPN (PPNpd), located ventral to the superior cerebellar peduncle (SCP) at the level of the inferior colliculus, composed a second population that was predominantly contralateral but was not ChAT immunoreactive. A third population of retrogradely labelled neurons was predominantly ipsilateral and ChAT immunoreactive and was located in the LDTN. These findings compared favorably with the full extent of the projection from this tegmental region revealed by retrograde transport of HRP from the superior colliculus when more compatible fixation and chromogen procedures were used. The results suggest that the PPN and the LDTN are two sources of the cholinergic input to the superior colliculus. Since the PPN also has extensive efferent, and afferent, connections with basal-ganglia-related structures, this cholinergic excitatory input to the superior colliculus, like the GABA-ergic inhibitory input from the substantia nigra pars reticulata, may provide the basis for an additional influence of the basal ganglia on visuomotor behavior.     

Organization of spinal inputs to the perihypoglossal complex in the cat

First- and second-order spinal afferents to the perihypoglossal complex were sought by using axonal transport of WGA-HRP. Injections in C1, 2, and 3 dorsal root ganglia resulted in axonal labeling in the nucleus intercalatus and the external cuneate nucleus, with a number of retrogradely labeled cells seen as well in the latter. A similar pattern of axonal labeling in the nucleus intercalatus as well as several retrogradely labeled cells were found after spinal cord injections at levels C1, 2, and 3. A prominent field of labeled axons was also present in the rostral main cuneate nucleus. No labeling was seen in the perihypoglossal nuclei after injections in the spinal cord or dorsal root ganglia at levels caudal to C3. After injections of HRP into the perihypoglossal nucleus we were able to identify labeled neurons within Rexed's laminae V-VIII and the central cervical nucleus. Anterograde labeling in the main cuneate nucleus was observed after C1 to C5 ganglion and C1 to C6 cord injections. The pattern and extent of labeling in the perihypoglossal nuclei and adjacent structures seen after cerebellar injections into lobules V and VI were comparable to those previously reported and permitted evaluation of the relay from dorsal root ganglia through the intercalatus to the vermis. Topography of the cervical projections to the nucleus intercalatus is considered with respect to that of the perihypoglossal-collicular projection. A discussion is offered of the apparent importance of nucleus intercalatus as a relay of cervical and vestibular afferent information to premotor structures involved in neck motor control. The perihypoglossal complex is viewed as being organized in such a fashion as to allow the nuclei intercalatus and prepositus hypoglossi to function as key structures in the integration of inputs related to neck and ocular motor control, respectively.     

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 vegetative network in the thoracolumbar spinal cord of the guinea pig: a comparison of the distribution of AChE-enzyme activity and choline acetyltransferase-like immunoreactivity

Histochemically the acetylcholinesterase (AChE) enzyme activity and immunocytochemically the choline acetyltransferase-like immunoreactivity (ChAT-LI) were located in the components of the vegetative network of the thoracolumbar spinal cord of the guinea pig. Both reaction products showed an identical distribution among the preganglionic sympathetic cells and the processes of the vegetative network, namely: cells of the nucleus intermediolateralis pars principalis (ILp), the nucleus intermediolateralis pars funicularis (ILf), the nucleus intercalatus spinalis (IC) and the nucleus intercalatus paraependymalis (ICpe; terminology according to Petras and Cummings 1972). In longitudinal horizontal sections through the intermediate zone of the thoracolumbar spinal cord both AChE-positive- and ChAT-like immunoreactive nerve fibers were organized into two longitudinal lateral fascicles (FLL), two longitudinal medial fascicles (FLM) as well as oblique and transverse bundles that interconnect repeatedly the autonomic cell groups of this zone along the spinal cord and contribute to the ladder-like shape of the vegetative network. The ChAT-like immunostaining of the vegetative network showed that the dendrites of the ILp cells are oriented mainly in a rostrocaudal, but also in a mediolateral direction. Similar orientation of the dendrites was observed for the ICpe cell groups of the thoracolumbar intermediate zone. Thus it is evident that ILp cell bodies and dendrites are involved in the formation of the FLL, whereas the ICpe cells and their dendrites--of the FLM. The IC cells send their dendrites towards both the ILp and ICpe cells and build up together with dendrites of the ILp and ICpe cells the transverse and oblique interconnecting bundles. The vegetative network is strongly developed within the intermediate zone of T1-T4 (mostly T3) and T7-T8 segments of the spinal cord. In these segments a greater variety of interconnections between the preganglionic sympathetic cell groups which are constituents of the network are also revealed. In the remaining segments the vegetative network is more poor developed. In the first two lumbar segments the distance between the interconnecting bundles in the rostrocaudal direction diminishes to 100 microns in contrast to 300-500 microns within the upper segments. The results obtained reveal that the cholinergic preganglionic sympathetic nuclei of the intermediate zone of the thoracolumbar spinal cord together with their dendrites represent the basis (frame) of the ladder-like vegetative network to which join in addition different peptidergic fibers of supraspinal, peripheral and propriospinal origin.     

Distribution and origin of corticotropin-releasing factor-immunoreactive axons in the female rat lumbosacral spinal cord.

Corticotropin-releasing factor (CRF) is a neuropeptide traditionally known for its hormonal role in the hypothalamic/pituitary/adrenal stress axis. However, CRF has been reported in axons in sites that may be considered outside of the direct stress axis, e.g., in axons in the lumbosacral spinal cord associated with the micturition response. Whether any of these CRF-immunoreactive axons interacts with uterine-related preganglionic autonomic neurons or projection neurons in the lumbosacral spinal cord is unknown. Thus, immunohistochemistry and retrograde tracing were employed to determine the presence, distribution, and origin of CRF-immunoreactive axons in the L6/S1 spinal cord of the female rat and to ascertain whether these axons are associated with uterine-related neurons. CRF-immunoreactive axons were present in the dorsal horn, medial and lateral collateral pathways, dorsal intermediate gray, laminae VlI and X, and sacral parasympathetic nucleus of the spinal cord. Nitric oxide-synthesizing, i.e., NADPH-d-positive neurons and pseudorabies virus labeled uterine-related neurons were in the sacral parasympathetic nucleus and were closely apposed by CRF-immunoreactive axons. Injection of retrograde tracers (fluorogold or fast blue) into the L6/S1 spinal cord labeled neurons in the hypothalamic paraventricular nucleus and pontine Barrington's nucleus, and some of these neurons were immunoreactive for CRF. This study demonstrates that CRF-immunoreactive axons are present in the L6/S1 spinal cord of the female rat in areas associated with sensory and autonomic processing. Some of these axons originate from the paraventricular nucleus and Barrington's nucleus and are adjacent to uterine-related neurons. These results indicate that CRF may influence neural activity related to the female reproductive system.     

Cells of origin of the spinohypothalamic tract in the rat

We recently demonstrated that large numbers of neurons in the spinal cord of rats project directly to the hypothalamus. In the present study, we used the retrograde tracer Fluoro-Gold (FG) to examine this projection more completely. In the first series of studies, we attempted to label the entire population of spinal cord neurons that project to the hypothalamus. Injections that virtually filled the hypothalamus on one side without spreading into any other diencephalic area labeled a large number of neurons (estimated to be more than 9,000 in the case with the most neurons labeled) bilaterally at all levels of the spinal cord. Approximately 60% of the labeled neurons were contralateral to the injection. The greatest number of labeled neurons was found within the deep dorsal horn. Many were also found within the lateral spinal nucleus, the superficial dorsal horn, and the gray matter surrounding the central canal. A small number of labeled cells was located in the intermediate zone and ventral horn of the spinal gray matter. Labeled neurons were distributed bilaterally within the sacral parasympathetic nucleus and trigeminal nucleus caudalis. Injections of FG restricted to the medial hypothalamus labeled neurons within the spinal cord in a distribution similar to that produced by injections that filled the hypothalamus. However, fewer neurons were labeled in the spinal cord (estimated to be more than 6,200) and trigeminal nucleus caudalis. Injections of FG restricted to the lateral hypothalamus also labeled fewer neurons (approximately 3,300) than did injections that filled the hypothalamus. In these cases, also, the pattern of labeled neurons within the spinal cord was similar to that produced by injections within either medial or both medial and lateral hypothalamus. However, few neurons were labeled in the sacral parasympathetic nucleus following injections into the lateral hypothalamus. These findings show the distribution of a large number of spinal cord neurons that project directly to medial or lateral hypothalamic regions that are involved in autonomic, neuroendocrine, and emotional responses to somatosensory stimulation, including painful stimuli.     

Response properties of neurons of the lateral cervical nucleus in the rat.

The distribution in the spinal cord of the rat of preganglionic neurons sending fibers into the hypogastric nerve was determined with the retrograde transport of horseradish peroxidase (HRP). Labeled cells were present in the intermediate gray matter of spinal segments L₁-L₂. The majority (81%) of HRP-filled cells formed a continuous column along the midline in the dorsal gray commissure. This cell column was termed the dorsal commissural nucleus (DCN). The remainder of HRP-labeled cells were present bilaterally in the middle and lateral regions of the intermediate gray; the majority of the latter cells were located along the lateral border of the intermediate gray. The present finding of a midline preganglionic autonomic cell column in the spinal cord of a mammal is contrary to previous reports, in which sympathetic preganglionic neurons have been localized primarily in the lateral intermediate gray. The DCN may be species-specific and related to the system of short adrenergic neurons present in the pelvis.     

Distribution of glycine receptor immunoreactivity in the spinal cord of the rat: cytochemical evidence for a differential glycinergic control of lamina I and V nociceptive neurons

In this study we characterized the distribution of glycine receptor immunoreactivity in the spinal cord of the rat by using monoclonal antisera directed against the purified glycine receptor. There was dense, punctate glycine receptor immunoreactive staining in all regions of the gray matter ventral to the substantia gelatinosa. The densest staining was found in laminae III and IV of the dorsal horn. There were also distinct, tributarylike bands of punctate staining that extended well into the white matter of the lateral and ventral funiculi. The only consistent cell body staining was found in small neurons of the ventral horn. The labelled neurons were distributed among larger, unlabelled motoneurons. In general, the pattern of glycine receptor immunoreactivity was similar at all levels of the spinal cord and was comparable to that seen with binding of a tritiated glycine receptor antagonist, strychnine, to sections of rat spinal cord (Zarbin et al.: J. Neurosci. 1:532-547, '81). Two important exceptions, however, were observed. In contrast to the high levels of strychnine binding reported in the substantia gelatinosa, we found almost no glycine receptor immunoreactivity in laminae I and II of the superficial dorsal horn of the spinal cord or of the trigeminal nucleus caudalis. There was also a notable absence of antibody staining in the intermediolateral cell column of the thoracic cord. The presence of dense glycine receptor immunoreactivity in the region of lamina V and its absence in the superficial dorsal horn are discussed in terms of a possible differential glycinergic control of nociceptive neurons of laminae I and V.     

Cholinergic innervation of the superior colliculus in the cat

The superficial and intermediate gray layers of the superior colliculus are heavily innervated by fibers that utilize the neurotransmitter acetylcholine. The distribution, ultrastructure, and sources of the cholinergic innervation of these layers have been examined in the cat by using a combination of immuno-cytochemical and axonal transport methods. Putative cholinergic fibers and cells were localized by means of a monoclonal antibody to choline acetyltransferase (ChAT). ChAT immunoreactive fibers are distributed throughout the depth of the superior colliculus, with particularly dense zones of innervation in the upper part of the superficial grey layer and in the intermediate grey layer. Within the superficial grey layer, the fibers form a continuous, dense band, whereas within the intermediate grey layer the fibers are arranged in clusters or patches. Although the patches are present throughout the rostrocaudal extent of the superior colliculus, they are most prominent in middle to caudal sections. The structure of the ChAT immunoreactive terminals was examined electron microscopically. The appearance of the terminals is similar in the superficial and intermediate grey layers. They contain closely packed, mostly round vesicles, and form contacts with medium-sized dendrites that exhibit small, but prominent postsynaptic densities; a few of the terminals contact vesicle-containing profiles. To identify the sources of the cholinergic input to the superior colliculus, injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) were made in the superior colliculus and the sections were processed to demonstrate both the retrograde transport of WGA-HRP and ChAT immunoreactivity. Neurons containing both labels were found in the parabigeminal nucleus, and in the lateral dorsal and pedunculopontine tegmental nuclei of the pontomesencephalic reticular formation. Almost every cell in these nuclei that contained retrograde label was also immunoreactive for ChAT. The similarities between the laminar distributions of the ChAT terminals and the terminations of the pathway from the parabigeminal nucleus (Graybiel: Brain Res. 145:365-374, '78) support the view that the latter nucleus is a source of the cholinergic fibers in the superficial grey layer. The possibility that the pedunculopontine tegmental nucleus is a source of cholinergic fibers in the deep layers was tested by examining the distribution of labeled fibers following injections of WGA-HRP into this region of the tegmentum. Patches of labeled terminals were found in the intermediate grey layer that resemble in distribution the patches of ChAT immunoreactive fibers in this layer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of catecholaminergic projections to the spinal cord in the North American opossum, Didelphis virginiana

The intent of our study was to determine when catecholaminergic axons grow into each of their adult targets in the spinal cord of the North American opossum (Didelphis virginiana) and to identify the origin of catecholaminergic axons in the lumbosacral cord at different stages of development. Tyrosine hydroxylase-like immunoreactive axons, presumed to be catecholaminergic, were demonstrated at different stages of development by the indirect antibody peroxidase-antiperoxidase technique of Sternberger. The neurons giving rise to such axons in the lumbosacral cord were identified by using the retrograde transport of Fast Blue and immunofluorescence for tyrosine hydroxylase-like immunoreactive neurons. At birth, 12-13 days after conception, tyrosine hydroxylase-like immunoreactive axons are present in the marginal zone throughout the length of the spinal cord. Such axons are particularly numerous in the dorsolateral marginal zone, the region containing most of them in adult animals. By postnatal day 3, a few immunoreactive axons are present in the intermediate (mantle) zone of the spinal cord; and by postnatal day 8, they are most concentrated in the presumptive intermediolateral cell column. Laminae I and II of the dorsal horn are not innervated by such axons until approximately postnatal day 15. By postnatal day 44, the distribution of tyrosine hydroxylase-like immunoreactive axons in the spinal cord resembles that in adult animals, although some areas may be hyperinnervated. At birth, tyrosine hydroxylase-like immunoreactive cell bodies are present in all of the brainstem areas providing catecholaminergic projections to the spinal cord in adult animals (Pindzola et al.: Brain Behav. Evol. 32:281-292, '88); and by at least postnatal day 5, lumbosacral injections of Fast Blue retrogradely label tyrosine hydroxylase-like immunoreactive neurons in all such areas. Retrogradely labeled immunoreactive neurons were also found in areas that do not contain them in adult animals. Such areas include the dorsal part of the nucleus coeruleus and certain areas of the reticular formation. During development, spinally projecting tyrosine hydroxylase-like immunoreactive neurons are numerous medial to the nucleus ventralis lemnisci lateralis (the paralemniscal region), whereas only a few are present in the same location in adult animals. Our results suggest that catecholaminergic axons grow into the spinal cord prenatally, that they innervate their adult targets postnatally and over an extended time period, and that during some stages of development they originate from areas that do not supply them in the adult animal.