Localization of NADPH diaphorase/nitric oxide synthase and choline acetyltransferase in the spinal cord of the frog, Rana perezi

The localization of nitrergic cells and fibers and cholinergic cells has been analyzed in the spinal cord of the anuran amphibian Rana perezi. Histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase and nitric oxide synthase immunohistochemistry revealed a concurrent pattern of labeled structures. A large population of nitrergic spinal neurons was found from the level of the obex to the filum terminale. They are abundant in the dorsal horn and intermediate gray matter, but also occur in territories of the ventral horn and, only occasionally, in somatic motoneurons. Numerous nitrergic fibers were present in the spinal white matter, particularly in the dorsal and dorsolateral funiculi. A special arrangement of nitrergic axons is present in Lissauer's tract, where a collateral system is formed. Cholinergic cells, revealed by choline acetyltransferase immunohistochemistry, were observed throughout the spinal cord. The somatic motoneurons were the most conspicuously immunoreactive cells. A large population of cholinergic cells forms a discontinuous column in the intermediate gray, from the third spinal segment to lumbar segments. These cells were organized in a medially located or intercalated cell group, and a laterally located intermediolateral group. Numerous scattered cholinergic cells were present in the central zone of the ventral horn and were absent in the dorsal horn. Double-labeling experiments revealed a high degree of codistribution of nitrergic and cholinergic cells, mainly in the intermediate gray, but colocalization of both markers in the same neurons was not found. This result contrasts with the situation found in mammals and raises the question of whether coexpression of both substances was acquired in spinal cord neurons through evolution only in amniotes or, even, only in mammals.     

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.     

Cholinergic deficits in aged rat spinal cord: restoration by GM1 ganglioside

Cholinergic neurons of spinal cord are central for the processing of motor, autonomic, and sensory modalities. Aging is associated with a variety of motor and autonomic symptoms that might be attributed, in part, to impaired spinal cord function. We found that cholinergic neurochemistry is diminished in the spinal cord of 22–24-month-old rats compared with 3-month-old rats. Choline acetyltransferase, high-affinity choline transport and hemicholinium-3 binding to the choline carrier were reduced in the aged spinal cord. The activity of the choline transporter and the hemicholinium-3 binding were decreased in all spinal segments, cervical, thoracic, lumbar and sacral. Hemicholinium-3 binding was reduced in ventral and dorsal horns along all spinal segments. The activity of choline acetyltransferase was decreased only in cervical and lumbar cord. Treatment of aged animals with GM1 induced the recovery of the presynaptic cholinergic markers in the aged spinal cord.     

Generation patterns of four groups of cholinergic neurons in rat cervical spinal cord: a combined tritiated thymidine autoradiographic and choline acetyltransferase immunocytochemical study

This report examines the generation of cholinergic neurons in the spinal cord in order to determine whether the transmitter phenotype of neurons is associated with specific patterns of neurogenesis. Previous immunocytochemical studies identified four groups of choline acetyltransferase (ChAT)-positive neurons in the cervical enlargement of the rat spinal cord. These cell groups vary in both somatic size and location along the previously described ventrodorsal neurogenic gradient of the spinal cord. Thus, large (and small) motoneurons are located in the ventral horn, medium-sized partition cells are found in the intermediate gray matter, small central canal cluster cells are situated within lamina X, and small dorsal horn neurons are scattered predominantly through laminae III-V. The relationships among the birthdays of these four subsets of cholinergic neurons have been examined by combining 3H-thymidine autoradiography and ChAT immunocytochemistry. Embryonic day 11 was the earliest time that neurons were generated within the cervical enlargement. Large and small ChAT-positive motoneurons were produced on E11 and 12, with 70% of both groups being born on E11. ChAT-positive partition cells were produced between E11 and 13, with their peak generation occurring on E12. Approximately 70% of the cholinergic central canal cluster and dorsal horn cells were born on E13, and the remainder of each of these groups was generated on E14. Other investigators have shown that all neurons within the rat cervical spinal cord are produced in a ventrodorsal sequence between E11 and E16. In contrast, ChAT-positive neurons are born only from E11 to E14 and are among the earliest cells generated in the ventral, intermediate, and dorsal subdivisions of the spinal cord. However, all cholinergic neurons are not generated simultaneously; rather their birthdays are correlated with their positions along the ventrodorsal gradient of neurogenesis. The fact that large motoneurons and medium-sized partition cells are born before small central canal cluster and dorsal horn cells would appear to support the generalization that large neurons are generated before small ones. However, the location of spinal cholinergic neurons within the neurogenic gradient seems to be more importantly associated with the time of cell generation than somal size. For example, when large and small motoneurons located at the same dorsoventral spinal level are compared, both sizes of cells are generated at the same time and in similar proportions.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Cholinergic enzymes in spinal cord infarction

Activities of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) were studied in the ventral and dorsal horns and the intermediate zone of the rabbit lumbar spinal cord (L 4–7) 24 and 96 h after ischemia caused by 20 or 40 min occlusion of the abdominal aorta. Changes of AChE and butyrylcholinesterase (BChE) activities were also detected histochemically by the direct thiocholine method. No significant changes were found immediately after ischemia. The most remarkable change after 20 min ischemia and 1 or 4 d of reperfusion was heterogenous decrease in ChAT and AChE activities in the examined parts of gray matter. The highest loss of enzyme activities was found in the ventral horns and the lowest in dorsal horns. Following 40 min ischemia and reperfusion the significant depletion in enzyme activities in all investigated zones of the gray matter was accompanied with necrotic degenerative changes. There was a relatively greater decrease in ChAT and AChE activities in the ventral horns that corresponded with a more prominent morphological damage of the cholinergic neurons in this zone of the spinal cord.     

Preferential cholinergic projections by embryonic spinal cord neurons within cocultured mouse superior cervical ganglia

The development of preferential cholinergic projections of spinal cord neurons within superior cervical ganglia (SCG) was analyzed in vitro using cocultures of SCGs (E17) with organotypic explants of fetal mouse cord (E13). The cord explants consisted of: (1) dorsal vs medioventral strips or mediodorsal vs ventral strips (dissected from levels C8-T4), or (2) transverse sections cut at various levels of the neuraxis. After 4 weeks of coculture, choline acetyltransferase (ChAT) was assayed in individual explants to quantify development of the cholinergic neurotransmitter enzyme (a) within the cord neurons, and (b) within the SCG. An index of cholinergic interaction was calculated as the relative ChAT activity in cocultured ganglion per unit ChAT activity in the ipsilateral cord strip. The highest index value (0.7) was obtained in cocultures with mediodorsal strips of cord. The index of interaction was progressively lower with medioventral (0.4), ventral (0.3) and dorsal (0.1) cord. In cocultures of transverse sections of spinal cord and SCGs, the highest indices of cholinergic interaction (expressed per hemisection of cord) were obtained with cord levels T1/T2 (1.0) and T5 (0.9). The index decreased with T9 (0.7) and was significantly lower with segments C2/C3 (0.3) and L2/L3 (0.19). Addition of a skeletal muscle target explant to the cord-SCG cocultures did not alter the preferential index of interaction between SCG and upper thoracic cord levels. Furthermore, the cholinergic cord neurons in medioventral strips did not promote increase of ChAT activity into equally accessible cocultured ganglia of inappropriate phenotype, e.g. sensory dorsal root ganglia. Decentralization of SCGs after coculture with appropriate T1/T2 cord resulted in loss of ganglionic ChAT activity. Electrical stimulation of the medial region in T1/T2 cord explants evoked compound ganglion action potentials in cocultured SCGs. The ganglion responses were blocked by hexamethonium. These data suggested that neurons located in the medial region of upper thoracic cord (presumably autonomic preganglionic) are able to develop enhanced cholinergic projections within cocultured SCGs, in comparison with neurons located in ventral cord (presumably motoneurons). In contrast, dorsal cord neurons showed no significant cholinergic interaction with SCGs. Furthermore, neurons located in upper thoracic spinal cord segments develop enhanced cholinergic projections within cocultured SCGs in comparison with neurons located in cervical and lumbar cord segments.     

Regional distribution of cholinergic neurons in human spinal cord transections in the patients with and without motor neuron disease

Regional distribution of enzymic activities in acetylcholine (ACh) metabolism was examined on thinly-sectioned transverse slices of human spinal cords obtained during autopsy of 5 motor neuron disease (MND) and 5 control patients without MND. Choline acetyltransferase (ChAT) activity was highly concentrated in the ventral horn regions (gray and white matters) of cervical, thoracic and lumbar spinal cord of non-MND patients. This enzyme activity was found to be remarkably low in the ventral gray and white matter of MND patients compared with that of the controls. Although the distribution of acetylcholinesterase (AChE) activity was found to be high in both ventral and dorsal gray matter of the spinal cord, little difference was observed between each corresponding region of MND and control patients, except relatively low enzyme activity in the cervical ventral horn region of MND patients. Muscarinic cholinergic receptors, examined as specific [3H]quinuclidinylbenzilate ([3H]QNB) binding, was also highly concentrated in the ventral and dorsal gray matter of the control spinal cord, and was strongly reduced in the ventral horn region of MND patients, indicating a quite similar distribution pattern of ChAT activity. These biochemical changes of cholinergic transmission system may be paralleled to the morphological degeneration of the spinal lower motor neurons in MND patients. Activity of 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase), a marker enzyme of central myelin structure, was evenly distributed throughout the whole spinal cord section, without regard to the gray and white matter, of both MND and control patients.     

Heterogeneous choline acetyltransferase staining in cholinergic neurons

We studied the features of choline acetyltransferase (ChAT) distribution in cholinergic structures of the central and peripheral nervous systems; specifically in the spinal cord and dorsal root ganglion (DRG) of embryo (E20), newborn, or adult rats using a goat polyclonal antibody. We found that this anti-ChAT antibody selectively labels both central and peripheral cholinergic structures. We observed cholinergic neurons with weak immunoreactivity. We demonstrated that at all stages of ontogeny that we studied, four groups of cholinergic neurons occurred in the cervical part of the spinal cord, viz., the small neurons of the dorsal horns, neurons of Rexed’s lamina X, neurons of the area of Rexed’s laminae VI–VII, and cells of the ventral horns. Interneurons of Rexed’s laminae VI–VII exhibited maximum ChAT reactivity as compared to other groups of cholinergic neurons of the spinal cord at all developmental stages we studied. We found an increase in the number of immunopositive cells of lamina X from E20 stage to the early postnatal and mature stages. In the peripheral cholinergic structures, i.e., the DRG, of the rat, all neurons expressed ChAT at all stages of ontogeny.     

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.     

Embryonic development of four different subsets of cholinergic neurons in rat cervical spinal cord

The developmental stage at which a neuron becomes committed to a neurotransmitter phenotype is an important time in its ontogenetic history. The present study examines when choline acetyltransferase (ChAT) is first detected within each of four different subsets of cholinergic neurons previously identified in the cervical enlargement of the spinal cord: namely, motor neurons, partition cells, central canal cluster cells, and dorsal horn neurons. By examining the temporal sequence of embryonic development of these cholinergic neurons, we can infer the relationships between ChAT expression and other important developmental events. ChAT was first detected reliably on embryonic day 13 (E13) by both biochemical and immunocytochemical methods, and it was localized predominantly within motor neurons. A second group of primitive-appearing ChAT-positive cells was detected adjacent to the ventricular zone on E14. These neurons seemed to disperse laterally into the intermediate zone by E15, and, on the basis of their location, were tentatively identified as partition cells. A third group of primitive ChAT-immunoreactive cells was detected on E16, both within and around the ventral half of the ventricular zone. By E17, some members of this "U"-shaped group appeared to have dispersed dorsally and laterally, probably giving rise to dorsal horn neurons as well as dorsal central canal cluster cells. Other members of this group remained near the ventral ventricular zone, most likely differentiating into ventral central canal cluster cells. Combined findings from the present study and a previous investigation of neurogenesis (Phelps et al.: J. Comp. Neurol. 273:459-472, '88), suggest that premitotic precursor cells have not yet acquired the cholinergic phenotype because ChAT is not detectable until after the onset of neuronal generation for each of the respective subsets of cholinergic neurons. However, ChAT is expressed in primitive bipolar neurons located within or adjacent to the germinal epithelium. Transitional stages of embryonic development suggest that these primitive ChAT-positive cells migrate to different locations within the intermediate zone to differentiate into the various subsets of mature cholinergic neurons. Therefore, it seems likely that spinal cholinergic neurons are committed to the cholinergic phenotype at pre- or early migratory stages of their development. Our results also hint that the subsets of cholinergic cells may follow different migration routes. For example, presumptive partition cells may use radial glial processes for guidance, whereas dorsal horn neurons may migrate along nerve fibers of the commissural pathway. Cell-cell interactions along such diverse migratory pathways could play a role in determining the different morphological, and presumably functional, phenotypes expressed by spinal cholinergic neurons.     

Immunohistochemical localization of choline acetyltransferase in rabbit spinal cord and cerebellum

Guinea pig antiserum specific for purified bovine choline acetyltransferase has been shown to cross-react with rabbit enzyme. We used the peroxidase-antiperoxidase immunohistochemical method to demonstrate the localization of choline acetyltransferase in formalin-fixed and paraffin-embedded sections of rabbit spinal cord and cerebellum. In the spinal cord, in agreement with our and others' previous results using immunofluorescent techniques, choline acetyltransferase was found in the cell bodies of the ventral horn motor neurons. In the cerebellum, choline acetyltransferase was localized exclusively in the mossy fibers and the glomeruli of the cerebellar folia. The immunohistochemical findings in the cerebellum reveal the morphological detail of cholinergic axons and their terminals. The results are consistent with published biochemical data on the cerebellar distribution of choline acetyltransferase.     

Choline acetyltransferase and substance P-like immuno-reactivity in the human spinal cord: changes in amyotrophic lateral sclerosis

The topographic location of the enzyme choline acetyltransferase (ChAT) has recently been determined within the human spinal cord². ChAT, which is regarded as a specific marker of cholinergic structures in nervous tissue, showed an area of high activity in the ventrolateral part of the ventral horn, probably related to motor neurons. In addition, an area of high ChAT activity was found in the apical part of the dorsal horn. As amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of the cortico-spinal tracts and the lower motor neurons, we considered it of value to investigate the involvement of spinal cholinergic structures in this disorder. Substance P is regarded as the transmitter of incoming pain signals to the dorsal horn of the spinal cord, a subject recently reviewed by Marx¹⁰. As disturbed sensation of pain is not a symptom of ALS, there seemed reason to correlate the spinal concentration of this peptide with the activities of ChAT in ALS.     

Regional distribution of cholinergic neurons in human spinal cord transections in the patients with and without motor neurons disease

Regional distribution of enzymic activities in acetylcholine (ACh) metabolism was examined on thinly-sectioned transverse slices of human spinal cords obtained during autopsy of 5 motor neuron disease (MND) and 5 control patients without MND. Choline acetyltransferase (ChAT) activity was highly concentrated in the ventral horn regions (gray and white matters) of cervical, thoracic and lumbar spinal cord of non-MND patients. This enzyme activity was found to be remarkably low in the ventral gray and white matter of MND patients compared with that of the controls. Although the distribution of acetylcholinesterase (AChE) activity was found to be high in both ventral and dorsal gray matter of the spinal cord, little difference was observed between each corresponding region of MND and control patients, except relatively low enzyme activity in the cervical ventral horn region of MND patients. Muscarinic cholinergic receptors, examined as specific [³H]quinuclidinylbenzilate ([³H]QNB) binding, was also highly concentrated in the ventral and dorsal gray matter of the control spinal cord, and was strongly reduced in the ventral horn region of MND patients, indicating a quite similar distribution pattern of ChAT activity. These biochemical changes of cholinergic transmission system may be paralleled to the morphological degeneration of the spinal lower motor neurons in MND patients. Activity of 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase), a marker enzyme of central myelin structure, was evenly distributed throughout the whole spinal cord section, without regard to the gray and white matter, of both MND and control patients.     

Calbindin-D28k and calretinin immunoreactivity in the spinal cord of the lizard Gekko gecko: Colocalization with choline acetyltransferase and nitric oxide synthase

The distribution of the calcium-binding proteins calbindin-D28k (CB) and calretinin (CR) was investigated in the spinal cord of the lizard Gekko gecko, by means of immunohistochemical techniques. Abundant cell bodies and fibers immunoreactive for either CB or CR were widely distributed throughout the spinal cord. Most neurons and fibers were labeled in the superficial dorsal horn, but numerous cells were also located in the intermediate gray and ventral horn. Distinct CB- and CR-containing cell populations were observed, although double immunohistochemistry revealed that 17-20% of the single-labeled cells for CB or CR in the dorsal horn contained both proteins. In addition, nitric oxide synthase was immunodetected in about 6% of the CB-positive neurons in the dorsal horn and in 10% in the ventral horn, whereas nitric oxide synthase was present in 9-13% of CR-positive cells in the dorsal horn and in 14% in the ventral horn. These doubly immunoreactive cells were restricted to areas IV, VII and VIII. Similar colocalization experiments revealed that 18-24% of the cholinergic cells in the ventral horn contained CB and 21-30% CR, with some variations throughout the length of the spinal cord. The pattern of distribution for CB and CR immunoreactivity in the spinal cord of the lizard, reported in the present study, is largely comparable to those reported for mammals, birds and anuran amphibians suggesting a high degree of conservation of the spinal systems modulated by these calcium-binding proteins.     

Proportion and location of spinal neurons receiving ventral root afferent inputs in the cat

Spinal neurons receiving ventral root afferent inputs were investigated in anesthetized and paralyzed cats. We were concerned with the afferent fibers in the ventral root that travel distally and then enter the spinal cord through the dorsal root. The questions to be answered included the proportion and distribution of spinal neurons receiving ventral root afferent inputs and their peripheral input characteristics. The 1.7 ventral root was cut near the spinal cord and the distal stump was stimulated while making a systematic search for neurons in the entire gray matter of the ipsilateral spinal cord that responded to the stimulation. The following conclusions were made: (i) the afferent fibers in the cat ventral root enter the spinal cord through the dorsal root and evoke a variety of responses (excitation, inhibition, or mixed) in a large proportion of spinal neurons (about 20%): (ii) these responses seem to be mediated largely by spinal mechanisms: (iii) spinal neurons receiving ventral root afferent inputs are situated in a wide region of the ventral spinal cord: (iv) ventral root fibers in a single root enter the spinal cord and exert their responses over a large region of the spinal cord (at least two spinal segments rostrally and caudally): (v) some of the spinal neurons that responded to ventral root stimulation were found to be ascending tract cells, suggesting that ventral root afferent inputs can reach supraspinal structures: (vi) ventral root afferent fibers converge onto spinal neurons that have a variety of peripheral receptive field characteristics: and (vii) with some exceptions, most neurons receiving ventral root inputs were excited best by mechanical and/or thermal noxious stimuli applied to the periphery.     

Effects of basic fibroblast growth factor on survival and choline acetyltransferase development of spinal cord neurons.

To investigate the biological role of basic fibroblast growth factor (bFGF) for the development of the spinal cord we studied the in vitro and in vivo effects of this protein on survival and choline acetyltransferase (ChAT)-activity of embryonic chick and rat spinal cord neurons. In vitro, bFGF (ED50 1-2.8 ng/ml) supported the survival of embryonic neurons from the ventral part of the rat spinal cord (ventral spinal cord, vsc), including motoneurons. Addition of bFGF (100 ng/ml) increased the ChAT-activity in embryonic chick vsc cultures to 150% as compared to untreated cultures (100%). The effect of bFGF was dose-dependent. In vivo-application of bFGF resulted in a similar increase of ChAT-activity in chick spinal cord. Since bFGF stimulates the ChAT-activity of spinal cord neurons in vivo and in vitro we therefore conclude that this protein may have a physiological function for the transmitter development of cholinergic spinal cord neurons.     

Immunohistochemical demonstration of some putative neurotransmitters in the lamprey spinal cord and spinal ganglia: 5-hydroxytryptamine-, tachykinin-, and neuropeptide-Y-immunoreactive neurons and fibers

The distribution of some putative neurotransmitters was investigated in the spinal cord and spinal ganglia of the lamprey, a primitive vertebrate, by using immunohistochemical methods. In the spinal cord a midline row of 5-hydroxytryptamine (5-HT)-immunoreactive neurons was present immediately ventral to the central canal over the entire length of the spinal cord. The ventral processes of these neurons formed a dense ventromedial plexus of varicosities. In the dorsal, lateral, and ventral spinal axon columns, several longitudinal 5-HT fibers were present. After chronic spinal transections the distribution of 5-HT fibers was unchanged; it is therefore concluded that there was no substantial descending 5-HT contribution and that the spinal 5-HT neurons supplied the regional 5-HT innervation. The spinal 5-HT cells sent fibers into the dorsal and ventral roots; 5-HT cell bodies and fibers were also present in the spinal dorsal root ganglia, in their dorsal, ventral, and lateral nerve branches, and in the dorsal and ventral branches of the ventral roots. Neurons and fibers containing peptides of the tachykinin (TK) family (to which, amongst others, substance P belongs) were found in the spinal cord. TK neurons in the spinal cord supplied the local TK innervation, as well as TK fibers in the dorsal and ventral roots. Fibers have been found containing either TK, or 5-HT, or both compounds. Neurons containing neuropeptide-Y (NPY)-immunoreactive material were present in a medial column just dorsal to the central canal. The NPY neurons have longitudinal, mainly descending, fibers that provide the local NPY innervation of the lamprey spinal cord. The present results provide evidence for local spinal systems containing 5-HT, TK, 5-HT and TK, or NPY, but in contrast to mammals, these compounds do not seem to arise from supraspinal neurons.     

Spinal entry route for ventral root afferent fibers in the cat

Twelve anesthetized and paralyzed cats were used to study the spinal entry routes of ventral root afferent fibers. In all animals, the spinal cord was transected at two different levels, L5 and S2. The L5 through S2 dorsal roots were cut bilaterally, making spinal cord segments L5-S2 neurally isolated from the body except for the L5-S2 ventral roots. From this preparation, a powerful excitation of the discharge rate of motor neurons and dorsal horn cells within the isolated spinal segments was observed after intraarterial injection of bradykinin (50 micrograms in 0.5 ml saline). This excitation of the spinal neurons can be considered the most convincing evidence of the potential physiologic role of the ventral root afferent fibers entering the spinal cord directly through the ventral root, because the apparent route of neuronal input from the periphery is through the ventral roots. However, additional control experiments conducted in the present study showed that the excitation persisted even after cutting all ventral roots within the isolated spinal segments, indicating that excitation was not mediated by the ventral roots. Furthermore, direct application of bradykinin on the dorsal surface of the spinal cord also increased the motoneuronal discharge rate, suggesting that excitation of spinal neurons produced by intraarterial injection of bradykinin is due to a direct action of bradykinin on the spinal cord. Thus, we provided an alternate explanation for the most convincing evidence indicating that physiologically important ventral root afferent fibers enter the spinal cord directly through the ventral root. Based on existing experimental evidence, it is likely that the majority of physiologically active ventral root afferent fibers travel distally toward the dorsal root ganglion and then enter the spinal cord through the dorsal root.