Autoradiographic localization of estrogen target cells in the spinal cord of the armadillo and baboon: a comparative study

The uptake and retention of radiolabeled estradiol by the spinal cord were examined in the baboon and the armadillo and compared to previous observations in the rat. Four females of each species were injected intracardially with 1.0-1.4 micrograms/kg body weight of 3H-estradiol and two females, one baboon and one armadillo, were injected with both labeled and 100-140 micrograms/kg body weight of unlabeled estradiol. One hour after the injections, the animals were killed and segments from the cervical, thoracic, lumbar and sacral cord were removed and processed for autoradiography. In the armadillo, labeling of neuronal nuclei were noted in laminae I & II and in alpha motor neurons. In addition, nuclei of the ependymal cells of the ventral portion of the central canal in the cervical cord concentrated radioactivity. In contrast, the baboon demonstrated only sporadic labeling of neurons in lamina II in all levels of the spinal cord. The comparison of our observations with that of the rat suggest that estrogen mediated sensations are probably coordinated at higher brain centers in the primate as opposed to the more primitive mammals.     

Distribution of Calcitonin Gene-Related Peptide in Rat and Rabbit Spinal Cords: Effect of Intrathecal 5,7-Dihydroxytryptamine

Calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) was measured in selected regions of the cervical, thoracic, and lumbar spinal cord of untreated rabbits and, following intrathecal injection of the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), in the thoracolumbar cord in rats using a sheep antiserum raised against tyrosine0 calcitonin gene-related peptide28-37. In the cervical, thoracic, and lumbar segments of the rabbit spinal cord, CGRP-LI levels were 15-50-fold higher in the dorsal than in the ventral grey region in the same segment. The only segmental variation in CGRP-LI levels was in the dorsal white region, where levels in the thoracic cord were lower than those in cervical or lumbar segments. Within individual spinal segments, the pattern of distribution of CGRP-LI in the rabbit spinal cord was analogous to that in other species previously examined, including rat, human, and cat spinal cord. Intrathecal injection of 5,7-DHT, which caused 85-91% depletion of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid from the thoracolumbar ventral spinal cord, did not affect choline acetyltransferase activity, which is colocalized with CGRP in motoneurones in this spinal cord region. In contrast, intrathecal 5,7-DHT produced a threefold increase in CGRP-LI in the ventral thoracolumbar cord, suggesting that spinal motoneurones selectively increase production of CGRP 10 days after neurotoxin-induced denervation of bulbospinal raphe neuronal input.     

Expression and ultrastructural localization of Mint2 in the spinal cord of rats

Mint protein family, as adaptor molecules, contains three members, Mint1, Mint2 and Mint3. Although Mint3 is ubiquitously expressed, Mint1 and Mint2 have been reported to express specifically in neuron. Here we demonstrated Mint1 and Mint2 expression pattern in rat spinal cord. The protein level of Mint2 was found to be higher than that of Mint1 in rat spinal by western blot. In an attempt to know Mint2 distribution in the spinal cord of rat, in situ hybridization was carried out, Mint2 mRNA was showed to be ubiquitously distributed in cervical, thoracic and lumbar sections of rat spinal cord, and high intensive signal was detected in motor neurons. These were further confirmed by fluorescent immunohistochemistry, Mint2 was also found to exist throughout gray matter especially motor neurons where Mint2 was mainly located in perikaryon, however, Mint1 was showed to be relatively lower. By electron microscope, Mint2 was found to be mainly located in vesicles in perikaryon in motor neuron of lumbar section, and at the same time Mint2 was located in axons in myelin and presynaptic terminals. These data suggest that Mint2 may play more important role in spinal cord than the other two family members.     

Changes in NMDA receptor subunit expression in response to contusive spinal cord injury

Differential assembly of N-methyl-D-aspartate (NMDA) receptor subunits determines their functional characteristics. Using in situ hybridization, we found a selective increase of the subunits NR1 and NR2A mRNA at 24 h in ventral motor neurons (VMN) caudal to a standardized spinal cord contusion injury (SCI). Other neuronal cell populations and VMN rostral to the injury site appeared unaffected. Significant up-regulation of NR2A mRNA also was seen 1 month after SCI in thoracic and lumbar VMN. The selective effects on VMN caudal to the injury site suggest that the loss of descending innervation leads to increased NMDA receptor subunit expression in these cells after SCI, which may alter their responses to glutamate. In contrast, protein levels determined by western blot analysis show decreased levels of NR2A 1 month after SCI in whole thoracic segments of spinal cord that included the injury sites. No effects of injury were seen on subunit levels in cervical or lumbar segments. Taken together with our previous study showing alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor subunit down-regulation after injury, our data suggest that glutamate receptor composition is significantly altered after SCI. These changes need to be taken into account to properly understand the function of, and potential pharmacotherapy for, the chronically injured spinal cord.     

Distribution of arginine-vasopressin in the trigeminal,dorsal root ganglia and spinal cord of the rat; depletion by capsaicin

We have measured arginine vasopressin in the neural lobe, the trigeminal ganglion (TG), dorsal root ganglia (DRG), spinal cord, trigeminal and sciatic nerves of the rat by radioimmunoassay. In control rats, the neural lobe contained 1600 pg/mg, the ganglia 52.5, 21.0, 8.5, 4.28, 3.85 pg/mg in the lumbar, sacral, cervical, thoracic, and trigeminal ganglion, respectively, the spinal cord contained 5.1, 4.3, 4.2 and 2.6 pg/mg in the lumbar, thoracic, sacral and cervical cord, respectively and the trigeminal and sciatic nerves contained 3.8 and 13 pg/mg. Neonatal capsaicin treatment depleted about 38–67% of AVP in the ganglia. Residual AVP amounted to 526.8, 30.55, 20.75, 12.88, 4.95, 2.74, 2.14, 7.94 and 2.53 pg/mg in the neural lobe, lumbar, thoracic, sacral, cervical DRG, lumbar, thoracic spinal cord, the sciatic and trigeminal nerves respectively. Capsaicin destroyed about 40.5% of total cells and 52% of AVP-immunoreactive neurons.     

Organization of bilateral spinal projections to the lateral reticular nucleus of the rat

The present study was carried out to analyze the topography of bilateral spinal projections to the lateral reticular nucleus (LRN). We used retrograde transport of fluorescent tracers Fast Blue and Diamidino Yellow to identify spinal neurons projecting to the ipsilateral and/or contralateral LRN, as well as orthograde transport of Phaseolus vulgaris leucoagglutinin to identify the LRN areas where spinoreticular axons terminate. Orthograde labeling confirmed that bilateral spinoreticular projections coming from cervical and upper-thoracic segments terminate in the magnocellular division of LRN, while those coming from the lower-thoracic, lumbar and sacral segments end in the parvocellular division of the nucleus; only a sparse spinal input has been observed in the subtrigeminal division of LRN. Retrograde labeling showed that labeled neurons were present at all spinal levels and in particular large numbers in the cervical and lumbar enlargements. Retrogradely single-labeled cells were located, with contralateral predominance, in all segments of the spinal cord, within laminae IV, V, VI, VIII, and X, whereas in laminae III and VII labeled neurons were mainly observed ipsilaterally. Furthermore, a small fraction of double-labeled cells (7.4%) was observed throughout the spinal cord, mainly in laminae III, IV, VII and VIII.     

Comparative dimensions of propriospinal neurons of different structural and functional groups in cats

A statistical comparison was made of geometric characteristics (area of cross section of the soma and proximal dendrites and d_con, the diameter of the circle of equivalent area to it) of propriospinal neurons of the cat spinal cord labeled with horseradish peroxidase. The linear dimensions of these cells differed by a factor of about seven. The mean d_con of propriospinal neurons in the cervical, thoracic, and lumbar divisions, whose axons reach level L6-7, was 39.9, 30.8, and 36.9 µm, respectively; direct correlation between the size of the neurons and the length of their axons was thus not observed. Characteristics of distribution of sizes of units in the cervical and thoracic divisions indicate the presence of two cell populations forming long propriospinal tracts; one consisting of a few, large neurons, concentrated in the cervical segments, the other consisting of small neurons, distributed among the cervical and thoracic segments. The mean d_con of neurons in the cervical division whose axons reach more caudal segments of the same cervical division was 44.2 µm (on account of a considerable number of large units in the ventral horn), evidence of the large relative size of the short-axon propriospinal neurons in this division of the spinal cord. Neurons located in the dorsal parts of the dorsal horn were the smallest in size, those located in the ventral horn were the largest. No significant differences were found in the dimensions of propriospinal neurons with uncrossed and crossed axons.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 2, pp. 238–247, March–April, 1984.     

Neurochemical and Immunocytochemical Studies on the Distribution of N-Acetyl-Aspartylglutamate and N-Acetyl-Aspartate in Rat Spinal Cord and Some Peripheral Nervous Tissues

HPLC analysis of rat spinal cord revealed a uniform distribution of N-acetyl-aspartate (NAA) across both longitudinal and dorsoventral axes. In contrast, ventral cord N-acetyl-aspartylglutamate (NAAG) levels were significantly higher than those measured in dorsal halves of cervical, thoracic, and lumbar segments. Immunocytochemical studies using an affinity-purified antiserum raised against NAAG-bovine serum albumin revealed an intense staining of motoneurons within rat spinal cord. Along with the considerable NAAG content in ventral roots, these results suggest that NAAG may be concentrated in motoneurons and play a role in motor pathways. NAAG was also present in other peripheral neural tissues, including dorsal roots, dorsal root ganglia, superior cervical ganglia, and sciatic nerve. It is interesting that NAA levels in peripheral nervous tissues were lower than those in CNS structures and that NAA levels in ventral roots and sciatic nerve were lower than NAAG levels. These findings further document a lack of correlation between NAAG and NAA levels in both central and peripheral nervous tissues. Taken together, these data demonstrate the presence of NAAG in nonglutamatergic neuronal systems and suggest a more complex role of NAAG in neuronal physiology than previously postulated.     

Spinal projections of brainstem respiratory related neurons in the cat as revealed by retrograde fluorescent markers

By means of retrograde axonal transport of fluorescent tracers, connections between brainstem respiratory related regions and the spinal cord has been studied in the cat. Neurons at the pneumotaxic center project bilaterally (90% ipsi-, 10% contra-) to cervical and lumbar spinal cord and ipsilaterally to thoracic levels. The ventrolateral nucleus of the tractus solitarius project mainly contralaterally (85%) to cervical levels and only contralaterally to thoracic levels; no efferent projections were found to lumbar levels. The ventral respiratory group showed a great number of neurons projecting to the spinal cord especially from the nucleus retroambiguus. Both nuclei, ambiguus and retroambiguus, project mainly contralaterally (70%) to the spinal cord. The B?tzinger complex showed rather scarce bilateral projections to cervical and only ipsilateral projections to lower cervical, thoracic and lumber levels.     

Autometallographic detection of mercury in rat spinal cord after treatment with organic mercury.

Autometallography was used to localize mercury in rat spinal cord after intraperitoneal administration of methylmercuric chloride (200 micrograms CH3HgCl daily). The technique permits small amounts of mercury sulfides and mercury selenides to be visualized by silver-enhancement. Mercury deposits were observed by light microscopy only in neurons. In all of the spinal cord segments selected (first cervical segment, C1; fifth cervical segment, C5; sixth thoracic segment, T6; and first lumbar segment, L1) the mercury was observed with cumulative dosages of 6000 micrograms CH3HgCl and greater. Laminae VII, VIII, and IX contained the majority of stained neurons, whereas laminae IV, V, VI, and X had a relatively lower density of mercury-containing neurons. Stained neurons were confined to specific cell groups, such as Clarke's column, nucleus intermedio-lateralis, nucleus cervicalis centralis, and nucleus dorsomedialis. At the ultrastructural level, mercury deposits were restricted to lysosomes of neurons and occasional accumulations in the lysosomes of ependymal cells.     

Spatial distribution of descending propriospinal tract neurons in the cat spinal cord

Quantitative estimates of the density of distribution of interneurons forming descending intersegmental connections in the cat spinal cord were obtained. Neurons were labeled by retrograde axonal transport of horseradish peroxidase injected unilaterally at different segmental levels. The mean number of labeled units per section 50 µ thick, in a given zone, was used as the measure of density. The density of distribution of the propriospinal neurons forming the longest tracts between the cervical and lumbosacral regions of the cord was found to be about half the density of distribution of neurons with short (not more than two segments) axons, and to be several times less than the corresponding value for neurons with axons of intermediate length. No marked local peaks of density of distribution of long-axon neurons were found at the level of the brachial enlargement. The number of neurons with crossed axons in most segments was close to half of the total number of propriospinal units. Zones of transverse section of the spinal cord with maximal concentrations of neurons forming direct and crossed propriospinal tracts of different lengths were determined at different levels. Correlation between the quantitative composition of propriospinal neuron populations with characteristics of influences transmitted by these populations is examined.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 1, pp. 96–105, January–February, 1984.     

Glial Activation and Segmental Upregulation of Interleukin-1β (IL-1β) in the Rat Spinal Cord after Surgical Incision

The present study investigated the expression patterns of glial cells and interleukin-1β (IL-1β) in the rat spinal cord after a surgical incision, which is closely related with clinical postoperative pain. Microglia and astrocytes became activated in the spinal cord following incision. Real-time polymerase chain reaction (PCR) and immunohistochemisty showed that IL-1β mRNA and protein level in the spinal cord was transiently upregulated after surgical incision. The increased IL-1β-immunoreactivity (IR) was mainly localized in neurons but not the activated microglia or astrocytes. Although obvious increase in IL-1β-IR could be observed in the lumbar segments of the spinal cord ipsilateral to a hind paw incision, significant upregulation of IL-1β was not detected in the lumbar segments following thoracic incision. The present study indicated that surgical incision could induce glial activation and segmental upregulation of IL-1β in the spinal cord. The activated glial cells and upregulated IL-1β, in turn, may be involved in the incision-induced pain hypersensitivity.     

Specific projections of sympathetic preganglionic neurons are not intrinsically determined by segmental origins of their cell bodies

Sympathetic preganglionic projections of the chick are segmentally specific. Neurons from the 16th cervical (C16) and the first thoracic (T1) spinal cord segments project almost exclusively in the rostral direction, while those from the fifth thoracic (T5) to the first lumbar (L1) spinal segments project almost exclusively in the caudal direction. Neurons from the intervening spinal cord segments (T2–4) project in rostral and caudal directions. There is also a tendency for rostrally located neurons in each segment to project rostrally and caudally located neurons to project caudally. To investigate whether specific projections of preganglionic neurons are intrinsically determined by segmental origins of their cell bodies, neural tube segments were transplanted or rotated in embryos at stages 19–26; these stages include times during and after preganglionic cell birth and just prior to axon outgrowth. When the T1 neural tube segment was replaced with the T5 or T7 neural tube segment, the transplanted T5 or T7 preganglionic neurons, now in the T1 position, projected rostrally. Conversely, when the T5 or T7 neural tube segment was replaced with the T1 neural tube, the transplanted T1 preganglionic neurons projected caudally. In addition, when individual T3 spinal cord segments were rotated 180° along the A-P axis, neurons which were originally in the caudal part of the segment projected rostrally, whereas neurons originally from the rostral part of the segment projected caudally. These results show that specific projections of preganglionic neurons are not intrinsically determined by segmental origins of their cell bodies. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 371–378, 1998     

Effects of corticospinal tract stimulation on renal sympathetic nerve activity in rats with intact and chronically lesioned spinal cords

Sympathetic preganglionic neurons and interneurons are closely apposed (presumably synapsed upon) by corticospinal tract (CST) axons. Sprouting of the thoracic CST rostral to lumbar spinal cord injuries (SCI) substantially increases the incidence of these appositions. To test our hypothesis that these additional synapses would increase CST control of sympathetic activity after SCI, we measured the effects of electrical stimulation of the CST on renal sympathetic nerve activity (RSNA) and arterial pressure (AP) in alpha-chloralose-anesthetized rats with either chronically intact or chronically lesioned spinal cords. Stimuli were delivered to the CST at intensities between 25-150 muA and frequencies between 25 and 75 Hz. Stimulation of the CST at the midcervical level decreased RSNA and AP. These decreases were not mediated by direct projections of the CST to the thoracic spinal cord because we could still elicit them by midcervical stimulation after acute lesions of the CST at caudal cervical levels. In contrast, caudal thoracic CST stimulation increased RSNA and AP. Neither the responses to cervical nor thoracic stimulation were affected by chronic lumbar SCI. These data show that the CST mediates decreases in RSNA via a cervical spinal system but excites spinal sympathetic neurons at caudal thoracic levels. Because chronic lumber spinal cord injury affected responses evoked from neither the cervical nor thoracic CST, we conclude that lesion-induced or regeneration-induced formation of new synapses between the CST and sympathetic neurons may not affect cardiovascular regulation.     

Tectal and Related Target Areas of Spinal and Dorsal Column Nuclear Projections in Hedgehog Tenrecs

The terminal distributions of spinal and dorsal column nuclear projections to tectum, pretectum, and central gray of hedgehog tenrecs (Echinops telfairi and Setifer setosus) were investigated using anterograde axonal flow and various tracer substances. In the inferior colliculus, the densest and most extensive mesencephalic projections were found within the pericentral regions. One target area, referred to as the external portion of the inferior colliculus, was represented as a semicircle of grain patches lateral and caudal to the central nucleus. This region received somesthetic afferents from the dorsal column nuclei and from spinal segments at various levels. In contrast, after high cervical injections, the pericentral portion dorsomedial to the rostral half of the central nucleus was labeled almost exclusively. This area of labeling was distinct from the labeling in the central gray and might be best compared with the intercollicular zone in other species. The superior colliculus received projections predominantly from the high cervical cord; minor projections also arose from lumbar spinal segments and the dorsal column nuclei. The terminal field covered roughly the caudal half of the colliculus and involved the stratum griseum intermediale in a patch-like fashion. Some labeling was also found in the stratum griseum profundum and in the stratum griseum superficiale. Other than in the colliculi, weak pretectal projections were observed following dorsal column nuclear injections, while the nucleus of Darkschewitsch was labeled best following lumbosacral injections. All mesencephalic target areas were labeled consistently on the contralateral side, while their ipsilateral side was involved to a varying degree: The relatively most prominent ipsilateral labeling was seen in the central gray, being roughly similar on both sides; scarcely any labeling was noted in the ipsilateral superior colliculus. Tectal injections of retrograde tracer, in addition, revealed a considerable number of labeled neurons in a relatively cell-poor region immediately ventral to the high cervial dorsal horn. This region might correspond to the lateral cervical nucleus, an aggregation of neurons that so far has only been demonstrated in higher mammals.     

The Effect of Cauda Equina Constriction on Nitric Oxide Synthase Activity

Nitric oxide synthase (NOS) activity was studied in the gray and white matter regions of the spinal cord 2 and 5 days after multiple cauda equina constrictions of the central processes of L7-Co5 dorsal root ganglia neurons. The results show considerable differences in enzyme activity in the thoracic, upper lumbar, lower lumbar, and sacral segments. Increased NOS activity was observed at 2 days after multiple cauda equina constrictions in the dorsal, lateral, and ventral columns of the lower lumbar segments and in the ventral column of the upper lumbar segments. The values returned to control levels within 5 postconstriction days. In the lateral columns of thoracic segments taken 2 and 5 days after surgery, NOS activity was enhanced by 54% and 55% and in the upper lumbar segments by 130% and 163%, respectively. Multiple cauda equina constrictions performed surgically for 2 and 5 days caused a significant increase in NOS activity predominantly in the gray matter regions of thoracic segments. A quite different response was found 5 days postconstriction in the upper lumbar segments, where the enzyme activity was significantly decreased in the dorsal horn, intermediate zone, and ventral horn. No such extreme differences could be seen in the lower lumbar segments, where NOS activity was significantly enhanced only in the ventral horn. The data correspond with a higher number of NOS immunoreactive somata, quantitatively evaluated in the ventral horn of the lower lumbar segments at 5 days after multiple cauda equina constrictions. While the great region-dependent heterogeneity in NOS activity seen 2 and 5 days after multiple cauda equina constrictions is quite apparent and suggestive of an active role played by nitric oxide in neuroprotective or neurotoxic processes occurring in the gray and white matter of the spinal cord, the extent of damage or the degree of neuroprotection caused by nitric oxide in compartmentalized gray and white matter in this experimental paradigm would be possible only using longer postconstriction periods.     

Role of Nova-1 in regulating alpha2N,a novel glycine receptor splice variant,in developing spinal cord neurons

Inhibitory glycine receptor (GlyR) subunits undergo developmental regulation, but the molecular mechanisms of GlyR regulation in developing neurons are little understood. Using RT-PCR, we investigated the regulation of GlyR alpha-subunit splice forms during the development of the spinal cord of the rat. Experiments to compare the amounts of mRNA for two known splice variants of the GlyR alpha2 subunit, alpha2A and alpha2B, in the developing rat spinal cord revealed the presence of an additional, novel variant that lacked any exon 3, herein named "alpha2N." Examination of the RNA from spinal cords of different-aged rats showed a dramatic down-regulation of alpha2N during prenatal development: alpha2N mRNA formed a significant portion of the alpha2 subunit pool at E14, but its relative level was reduced by 85% by birth and was undetectable in adults. Two proteins previously implicated in regulating the splicing of GlyR alpha2 pre-mRNA, the neurooncological ventral antigen-1 (Nova-1) and the brain isoform of the polypyrimidine tract binding protein (brPTB), underwent small changes over the same period that did not correlate directly with the changes in the level of alpha2N, calling into question their involvement in the developmental regulation of alpha2N. However, treatment of spinal cord neurons in culture with antisense oligonucleotides designed selectively to knock down one of three Nova-1 variants significantly altered the relative level of GlyR alpha2N, showing that Nova-1 isoforms can regulate GlyR alpha2 pre-mRNA splicing in developing neurons. These results provide evidence for a novel splice variant of the GlyR alpha2 subunit that undergoes dramatic developmental regulation, reveal the expression profiles of Nova-1 and brPTB in the developing spinal cord, and suggest that Nova-1 plays a role in regulating GlyR alpha2N in developing neurons.     

Cholera Toxin B Subunit Shows Transneuronal Tracing after Injection in an Injured Sciatic Nerve

Cholera toxin B subunit (CTB) has been extensively used in the past for monosynaptic mapping. For decades, it was thought to lack the ability of transneuronal tracing. In order to investigate whether biotin conjugates of CTB (b-CTB) would pass through transneurons in the rat spinal cord, it was injected into the crushed left sciatic nerve. For experimental control, the first order afferent neuronal projections were defined by retrograde transport of fluorogold (FG, a non-transneuronal labeling marker as an experimental control) injected into the crushed right sciatic nerve in the same rat. Neurons containing b-CTB or FG were observed in the dorsal root ganglia (DRG) at the L4-L6 levels ipsilateral to the tracer injection. In the spinal cord, b-CTB labeled neurons were distributed in all laminae ipsilaterally between C7 and S1 segments, but labeling of neurons at the cervical segment was abolished when the T10 segment was transected completely. The interneurons, distributed in the intermediate gray matter and identified as gamma-aminobutyric acid-ergic (GABAergic), were labeled by b-CTB. In contrast, FG labeling was confined to the ventral horn neurons at L4-L6 spinal segments ipsilateral to the injection. b-CTB immunoreactivity remained to be restricted to the soma of neurons and often appeared as irregular patches detected by light and electron microscopy. Detection of monosialoganglioside (GM1) in b-CTB labeled neurons suggests that GM1 ganglioside may specifically enhance the uptake and transneuronal passage of b-CTB, thus supporting the notion that it may be used as a novel transneuronal tracer.     

Distribution of glycine/GABA neurons in the ventromedial medulla with descending spinal projections and evidence for an ascending glycine/GABA projection

The ventromedial medulla (VM), subdivided in a rostral (RVM) and a caudal (CVM) part, has a powerful influence on the spinal cord. In this study, we have identified the distribution of glycine and GABA containing neurons in the VM with projections to the cervical spinal cord, the lumbar dorsal horn, and the lumbar ventral horn. For this purpose, we have combined retrograde tracing using fluorescent microspheres with fluorescent in situ hybridization (FISH) for glycine transporter 2 (GlyT2) and GAD67 mRNAs to identify glycinergic and/or GABAergic (Gly/GABA) neurons. Since the results obtained with FISH for GlyT2, GAD67, or GlyT2 + GAD67 mRNAs were not significantly different, we concluded that glycine and GABA coexisted in the various projection neurons. After injections in the cervical cord, we found that 29% ± 1 (SEM) of the retrogradely labeled neurons in the VM were Gly/GABA (RVM: 43%; CVM: 21%). After lumbar dorsal horn injections 31% ± 3 of the VM neurons were Gly/GABA (RVM: 45%; CVM: 12%), and after lumbar ventral horn injections 25% ± 2 were Gly/GABA (RVM: 35%; CVM: 17%). In addition, we have identified a novel ascending Gly/GABA pathway originating from neurons in the area around the central canal (CC) throughout the spinal cord and projecting to the RVM, emphasizing the interaction between the ventromedial medulla and the spinal cord. The present study has now firmly established that GABA and glycine are present in many VM neurons that project to the spinal cord. These neurons strongly influence spinal processing, most notably the inhibition of nociceptive transmission.