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.     

Spinal regions involved in baroreflex control of renal sympathetic nerve activity in the rat

Spinal cord injury causes debilitating cardiovascular disturbances. The etiology of these disturbances remains obscure, partly because the locations of spinal cord pathways important for sympathetic control of cardiovascular function have not been thoroughly studied. To elucidate these pathways, we examined regions of the thoracic spinal cord important for reflex sympathetic control of arterial pressure (AP). In anesthetized rats, baroreceptor relationships between pharmacologically induced changes in AP and changes in left renal sympathetic nerve activity (RSNA) were generated in spinally intact rats and after acute surgical hemisection of either the dorsal, left, or right T8 spinal cord. None of these individual spinal lesions prevented the baroreceptor-mediated increases in RSNA caused by decreases in AP. Thus, baroreceptor-mediated increases in RSNA in rats are mediated by relatively diffuse, bilateral, descending, excitatory projections. The ability to reduce RSNA at increased AP was impaired after both dorsal and left hemisections, and baroreceptor gain was significantly decreased. Baroreceptor-induced maximum decreases in RSNA were not affected by right hemisections. However, baroreflex gain was impaired. Because both dorsal and left hemisections, but not right hemisections, attenuated the decrease in RSNA at elevated AP, we conclude that pathways involved in the tonic inhibition of spinal sources of sympathetic activity descend ipsilaterally in the dorsal spinal cord. Our results show that many lesions that do not fully transect the spinal cord spare portions of both descending excitatory pathways that may prevent orthostatic hypotension and descending inhibitory pathways that reduce the incidence of autonomic dysreflexia.     

Expression of splice variants of the NR1 subunit of the N-methyl-D-aspartate receptor in the normal and injured rat spinal cord

Quantitative western blot analysis in laminectomy control spinal cords of adult rats was used to provide the first report of the normal expression patterns of the N1, C1, C2 and C2' cassettes in the cervical, thoracic and lumbar regions of the spinal cord as a percent of total NR1 subunit protein. In all regions studied, the C1 and C2 cassettes were usually contained in less than 10% of total NR1 protein. In contrast, approximately 90% of total NR1 protein contained the C2' cassette. A significant proportion of total NR1 protein (approximately 30%) also contained the N1 cassette. These data are consistent with expression of NR1(000) (NR1-4a) and NR1(100) (NR1-4b) as the dominant splice forms in the spinal cord. Splice variant expression was also studied following incomplete, contusive spinal cord injury (SCI) to the thoracic level 8 (T8) region. This injury did not change expression of the C1 or C2 cassette in any region of the spinal cord acutely at 24 h or chronically at 1 month. There was an increase in expression of the N1 cassette in the lumbar regions 1 month after injury (p < 0.05). These data indicate that SCI induces distal changes in NR1 splice variant expression, which may play a role in the adaptive response of neurons in the chronically injured spinal cord.     

Alterations in the expression of the apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/ref-1) and DNA damage in the caudal region of acute and chronic spinal cord injured rats treated by embryonic neural stem cells

The oxidative mechanisms of injury-induced damage of neurons within the spinal cord are not very well understood. We used a model of T8-T9 spinal cord injury (SCI) in the rat to induce neuronal degeneration. In this spinal cord injury model, unilateral avulsion of the spinal cord causes oxidative stress of neurons. We tested the hypothesis that apurinic/apyrimidinic endonuclease (or redox effector factor-1, APE/Ref-1) regulates this neuronal oxidation mechanism in the spinal cord region caudal to the lesion, and that DNA damage is an early upstream signal. The embryonic neural stem cell therapy significantly decreased DNA-damage levels in both study groups - acutely (followed up to 7 days after SCI), and chronically (followed up to 28 days after SCI) injured animals. Meanwhile, mRNA levels of APE/Ref-1 significantly increased after embryonic neural stem cell therapy in acutely and chronically injured animals when compared to acute and chronic sham groups. Our data has demonstrated that an increase of APE/Ref-1 mRNA levels in the caudal region of spinal cord strongly correlated with DNA damage after traumatic spinal cord injury. We suggest that DNA damage can be observed both in lesional and caudal regions of the acutely and chronically injured groups, but DNA damage is reduced with embryonic neural stem cell therapy.     

Leptin in nucleus of the solitary tract alters the cardiovascular responses to aortic baroreceptor activation

Recent data suggests that neurons expressing the long form of the leptin receptor form at least two distinct groups within the caudal nucleus of the solitary tract (NTS): a group within the lateral NTS (Slt) and one within the medial (Sm) and gelantinosa (Sg) NTS. Discrete injections of leptin into Sm and Sg, a region that receives chemoreceptor input, elicit increases in arterial pressure (AP) and renal sympathetic nerve activity (RSNA). However, the effect of microinjections of leptin into Slt, a region that receives baroreceptor input is unknown. Experiments were done in the urethane-chloralose anesthetized, paralyzed and artificially ventilated Wistar or Zucker obese rat to determine leptin's effect in Slt on heart rate (HR), AP and RSNA during electrical stimulation of the aortic depressor nerve (ADN). Depressor sites within Slt were first identified by the microinjection of l-glutamate (Glu; 0.25 M; 10 nl) followed by leptin microinjections. In the Wistar rat leptin microinjection (50 ng; 20 nl) into depressor sites within the lateral Slt elicited increases in HR and RSNA, but no changes in AP. Additionally, leptin injections into Slt prior to Glu injections at the same site or to stimulation of the ADN were found to attenuate the decreases in HR, AP and RSNA to both the Glu injection and ADN stimulation. In Zucker obese rats, leptin injections into NTS depressor sites did not elicit cardiovascular responses, nor altered the cardiovascular responses elicited by stimulation of ADN. Those data suggest that leptin acts at the level of NTS to alter the activity of neurons that mediate the cardiovascular responses to activation of the aortic baroreceptor reflex.     

c-Fos expression in rat brain stem and spinal cord in response to activation of cardiac ischemia-sensitive afferent neurons and electrostimulatory modulation

The purpose of this study was to identify central neuronal sites activated by stimulation of cardiac ischemia-sensitive afferent neurons and determine whether electrical stimulation of left vagal afferent fibers modified the pattern of neuronal activation. Fos-like immunoreactivity (Fos-LI) was used as an index of neuronal activation in selected levels of cervical and thoracic spinal cord and brain stem. Adult Sprague-Dawley rats were anesthetized with urethane and underwent intrapericardial infusion of an "inflammatory exudate solution" (IES) containing algogenic substances that are released during ischemia (10 mM adenosine, bradykinin, prostaglandin E2, and 5-hydroxytryptamine) or occlusion of the left anterior descending coronary artery (CoAO) to activate cardiac ischemia-sensitive (nociceptive) afferent fibers. IES and CoAO increased Fos-LI above resting levels in dorsal horns in laminae I-V at C2 and T4 and in the caudal nucleus tractus solitarius. Dorsal rhizotomy virtually eliminated Fos-LI in the spinal cord as well as the brain stem. Neuromodulation of the ischemic signal by electrical stimulation of the central end of the left thoracic vagus excited neurons at the cervical and brain stem level but inhibited neurons at the thoracic spinal cord during IES or CoAO. These results suggest that stimulation of the left thoracic vagus excites descending inhibitory pathways. Inhibition at the thoracic spinal level that suppresses the ischemic (nociceptive) input signal may occur by a short-loop descending pathway via signals from cervical propriospinal circuits and/or a longer-loop descending pathway via signals from the nucleus tractus solitarius.     

Recovery of baroreflex control of renal sympathetic nerve activity after spinal lesions in the rat

Spinal cord injury (SCI) has serious long-term consequences on sympathetic cardiovascular regulation. Orthostatic intolerance results from insufficient baroreflex regulation (BR) of sympathetic outflow to maintain proper blood pressure upon postural changes. Autonomic dysreflexia occurs due to insufficient inhibition of spinal sources of sympathetic activity. Both of these conditions result from the inability to control sympathetic activity caudal to SCI. It is well established that limited motor ability recovers after incomplete SCI. Therefore, the goal of this study was to determine whether recovery of BR occurs after chronic, left thoracic spinal cord hemisection at either T(3) or T(8). Baroreflex tests were performed in rats by measuring the reflex response of left (ipsilateral) renal sympathetic nerve activity to decreases and increases in arterial pressure produced by ramped infusions of sodium nitroprusside and phenylephrine, respectively. One week after a T(3) left hemisection, BR function was modestly impaired. However, 8 wk after a T(3) left hemisection, BR function was normal. One week after a T(8) left hemisection, BR function was significantly impaired, and 8 wk after a T(8) left hemisection, BR function was significantly improved. These results indicate that BR of renal sympathetic nerve activity in rats may partially recover after spinal cord hemisections, becoming normal by 8 wk after a T(3) lesion, but not after a T(8) lesion. The nature of the spinal cord and/or brain stem reorganization that mediates this recovery remains to be determined.     

Upregulation of the transport system for TNFalpha at the blood-brain barrier

The transport system for the cytokine tumor necrosis factor-alpha (TNFalpha) at the blood-brain barrier (BBB) enables an enhanced yet saturable entry of TNFalpha from blood to the CNS. This review focuses on the selective upregulation of the transport system for TNFalpha at the BBB that is specific for type of pathology, region, and time. The upregulation is reflected by increased CNS tissue uptake of radiolabeled TNFalpha after iv injection in mice and by inhibition of this increase with excess non-radiolabeled TNFalpha. (1) Spinal cord injury (SCI): upregulation of TNFalpha uptake after thoracic transection is seen in the delayed phase of BBB disruption at the lumbar spinal cord. Thoracic SCI by compression, however, has a longer lasting impact on TNFalpha transport that involves thoracic and lumbar spinal cord, in contrast to the upregulation confined to the lumbar region in lumbar SCI by compression. Regardless, the uptake of TNFalpha by spinal cord does not parallel BBB disruption as measured by the leakage of radiolabeled albumin. (2) Experimental autoimmune encephalomyelitis (EAE): the increase in the differential permeability to TNFalpha is seen in all CNS regions (brain and cervical, thoracic, and lumbar spinal cord) and has a distinct time course and reversibility. Exogenous TNFalpha has biphasic effects in modulating functional scores. The BBB, a dynamically regulated barrier, is actively involved in disease processes.     

Blockade of the preovulatory release of gonadotropins and prolactin by spinal transections in female rats

The effect of spinal transections on the preovulatory release of gonadotropins and PRL was investigated in female rats. A preovulatory rise in serum LH, FSH and PRL and subsequent ovulation were prevented by complete spinal transections (CST) at high thoracic levels (T3-T7), but not at low thoracic and lumbar levels (T8-L5), performed at 1000-1230 h on proestrus. Norepinephrine (NE) concentrations in the preoptic-anterior hypothalamic area at 1700-1800 h on proestrus were also significantly reduced by CST at high thoracic levels, but not at lumbar levels. Either electrochemical stimulation of the suprachiasmatic part of the preoptic area or NE injection into the third ventricle at 1400-1500 h on proestrus restored ovulation in animals with CST at high thoracic levels. Animals with CST at lumbar levels exhibited relatively regular 4-day cycles, but those with CST at high thoracic levels showed prolonged periods of diestrous (8-20 days) before they resumed cyclicity. In the case of partial transections, bilateral transections of the lateral columns, but not transections of the dorsal or medial columns, of the spinal cord at T4-T5 significantly blocked the preovulatory gonadotropin release and the occurrence of ovulation. Unilateral transections of the lateral columns of the spinal cord or unilateral electrolytic lesions of the ventrolateral part of the medulla oblongata (VLMO) failed to block ovulation. When combinations of them were performed ipsilaterally, ovulation occurred, but when they were performed contralaterally, the incidence of ovulation was significantly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Degeneration of the Injured Cervical Cord Is Associated with Remote Changes in Corticospinal Tract Integrity and Upper Limb Impairment

Background

Traumatic spinal cord injury (SCI) leads to disruption of axons and macroscopic tissue loss. Using diffusion tensor imaging (DTI), we assessed degeneration of the corticospinal tract (CST) in the cervical cord above a traumatic lesion and explored its relationship with cervical atrophy, remote axonal changes within the cranial CST and upper limb function.

Methods

Nine cervical injured volunteers with bilateral motor and sensory impairment and ten controls were studied. DTI of the cervical cord and brain provided measurements of fractional anisotropy (FA), while anatomical MRI assessed cross-sectional spinal cord area (i.e. cord atrophy). Spinal and central regions of interest (ROI) included the bilateral CST in the cervical cord and brain. Regression analysis identified correlations between spinal FA and cranial FA in the CST and disability.

Results

In individuals with SCI, FA was significantly lower in both CSTs throughout the cervical cord and brain when compared with controls (p≤0.05). Reduced FA of the cervical cord in patients with SCI was associated with smaller cord area (p = 0.002) and a lower FA of the cranial CST at the internal capsule level (p = 0.001). Lower FA in the cervical CST also correlated with impaired upper limb function, independent of cord area (p = 0.03).

Conclusion

Axonal degeneration of the CST in the atrophic cervical cord, proximal to the site of injury, parallels cranial CST degeneration and is associated with disability. This DTI protocol can be used in longitudinal assessment of microstructural changes immediately following injury and may be utilised to predict progression and monitor interventions aimed at promoting spinal cord repair.     

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.     

Increased expression of spinal cord Fos protein induced by bladder stimulation after spinal cord injury

These studies examined Fos protein expression in spinal cord neurons synaptically activated by stimulation of bladder afferent pathways after spinal cord injury (SCI). In urethan-anesthetized Wistar rats after SCI for 6 wk, intravesical saline distension significantly (P 相似文献   

Respiratory resetting induced by spinal cord stimulation in the cat

Electrical stimulation (50-150 microA, 0.5-ms duration, 3-300 Hz) was performed within three different regions (lateral, ventrolateral, and ventral) of the C2-C3 spinal cord of decerebrate, vagotomized, paralyzed, and artificially ventilated cats. Spinal cord stimulation sites were located by inserting monopolar or bipolar stimulating electrodes either at the dorsolateral sulcus or at least 1 mm medial or lateral to the sulcus. With stimulation at each site, alterations in respiratory rhythm, orthodromic phrenic nerve responses, and antidromic activation of medullary respiratory-modulated neurons were examined. Phrenic nerve responses to cervical spinal cord stimulation consisted of an early excitation (2-4 ms) and/or a late excitation (4-8 ms). Stimulation of the lateral region evoked the greatest amplitude early response and stimulation of the ventrolateral region produced the greatest late excitation. All three stimulus sites elicited antidromic activation of some respiratory-modulated neurons in the dorsal (DRG) and ventral respiratory groups (VRG). The lateral region was the least effective resetting site, and it had the highest incidence of antidromic activation of both DRG and VRG neurons. The ventrolateral region of the cervical spinal cord was the most effective resetting site, but it had the lowest incidence of antidromic activation of DRG respiratory-modulated neurons. In addition, resetting responses were observed with spinal cord stimulation at similar sites in the thoracic and lumbar spinal cord regions thought to be devoid of inspiratory bulbospinal axons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of slowly conducting medullary raphe-spinal neurons, including serotonergic neurons, increases cutaneous sympathetic vasomotor discharge in rabbit

Neurons in the rostral medullary raphe/parapyramidal region regulate cutaneous sympathetic nerve discharge. Using focal electrical stimulation at different dorsoventral raphe/parapyramidal sites in anesthetized rabbits, we have now demonstrated that increases in ear pinna cutaneous sympathetic nerve discharge can be elicited only from sites within 1 mm of the ventral surface of the medulla. By comparing the latency to sympathetic discharge following stimulation at the ventral raphe site with the corresponding latency following stimulation of the spinal cord [third thoracic (T3) dorsolateral funiculus] we determined that the axonal conduction velocity of raphe-spinal neurons exciting ear pinna sympathetic vasomotor nerves is 0.8 +/- 0.1 m/s (n = 6, range 0.6-1.1 m/s). Applications of the 5-hydroxytryptamine (HT)(2A) antagonist trans-4-((3Z)3-[(2-dimethylaminoethyl)oxyimino]-3-(2-fluorophenyl)propen-1-yl)-phenol, hemifumarate (SR-46349B, 80 microg/kg in 0.8 ml) to the cerebrospinal fluid above thoracic spinal cord (T1-T7), but not the lumbar spinal cord (L2-L4), reduced raphe-evoked increases in ear pinna sympathetic vasomotor discharge from 43 +/- 9 to 16 +/- 6% (P < 0.01, n = 8). Subsequent application of the excitatory amino acid (EAA) antagonist kynurenic acid (25 micromol in 0.5 ml) substantially reduced the remaining evoked discharge (22 +/- 8 to 6 +/- 6%, P < 0.05, n = 5). Our conduction velocity data demonstrate that only slowly conducting raphe-spinal axons, in the unmyelinated range, contribute to sympathetic cutaneous vasomotor discharge evoked by electrical stimulation of the medullary raphe/parapyramidal region. Our pharmacological data provide evidence that raphe-spinal neurons using 5-HT as a neurotransmitter contribute to excitation of sympathetic preganglionic neurons regulating cutaneous vasomotor discharge. Raphe-spinal neurons using an EAA, perhaps glutamate, make a substantial contribution to the ear sympathetic nerve discharge evoked by raphe stimulation.     

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     

Distribution of alpha 1 subunit isoform of (Na,K)-ATPase in the rat spinal cord

Three isoforms of the alpha subunit of (Na,K)-ATPase have been identified in the rat central nervous system. Using a probe specific for the alpha 1 isoform, mRNA levels were measured from five sections of the rat spinal cord using slot blot techniques. Assigning a value of 1 to the slope obtained from the cervical section, the upper thoracic section was 2.6 times higher; the midthoracic section was 4.5 times higher; the lower thoracic section was 2.6 times higher; and the lumbar section was 1.7 times higher. The results suggest that alpha 1 isoform mRNA levels are not uniform throughout the spinal cord. In situ hybridization techniques showed that alpha 1 isoform mRNA was diffusely abundant in glial and central canal ependymal cells, while labeled neurons were localized exclusively in lateraily located anterior horn neurons in cervical, thoracic, and lumbar segments and in ventromedial neurons in mid-thoracic spinal cord. Also, dorsal root ganglia neurons were extensively labeled at all segments.Special issue dedicated to Dr. Bernard W. Agranoff.     

正常小儿胫神经诱发脊髓电位特征和脑瘫小儿该电位的变化

采取刺激后胫神经(PTN)诱发叠加技术,利用体表无创伤性双极记录方法观察了16例正常小儿和43例脑瘫小儿的脊髓诱发电位(SCEP)。正常小儿的SCEP自下而上潜伏时逐渐延长、电压减小。从椎体C6到T10表现为Pa-Na-Pb三相波,T10～T12为Pa-Na1-Na2-Pb波,T12～L4为多相复合波。左右侧SCEP波形相似,潜伏时、电压相同,它们之间无统计学显著差别;但不同节段之间SCEP差异显著;脊髓传导速度为57.14m/s。脑瘫小儿SCEP正常者占14％;全髓反应低下者占20％;左右侧反应不对称者占46％;节段性反应低下者占15％;其它异常约占5％。不但节段间存在显著差异,而且全脊髓左右侧电压间以及颈、腰骶髓的潜伏时间出现显著差异。脊髓传导速度减低(患侧46.22m/s,对侧53.48m/s)。结果提示:(1)正常小儿脊髓活动左右对称,不同脊髓节段对PTN刺激反应不同。(2)脑瘫小儿脊髓活动左右不对称,一侧功能下降时对侧有一定代偿力,脊髓传导速度减慢。     

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.     

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.     

Corticospinal and Reticulospinal Contacts on Cervical Commissural and Long Descending Propriospinal Neurons in the Adult Rat Spinal Cord; Evidence for Powerful Reticulospinal Connections

Descending systems have a crucial role in the selection of motor output patterns by influencing the activity of interneuronal networks in the spinal cord. Commissural interneurons that project to the contralateral grey matter are key components of such networks as they coordinate left-right motor activity of fore and hind-limbs. The aim of this study was to determine if corticospinal (CST) and reticulospinal (RST) neurons make significant numbers of axonal contacts with cervical commissural interneurons. Two classes of commissural neurons were analysed: 1) local commissural interneurons (LCINs) in segments C4-5; 2) long descending propriospinal neurons (LDPNs) projecting from C4 to the rostral lumbar cord. Commissural interneurons were labelled with Fluorogold and CST and RST axons were labelled by injecting the b subunit of cholera toxin in the forelimb area of the primary somatosensory cortex or the medial longitudinal fasciculus respectively. The results show that LCINs and LDPNs receive few contacts from CST terminals but large numbers of contacts are formed by RST terminals. Use of vesicular glutamate and vesicular GABA transporters revealed that both types of cell received about 80% excitatory and 20% inhibitory RST contacts. Therefore the CST appears to have a minimal influence on LCINs and LDPNs but the RST has a powerful influence. This suggests that left-right activity in the rat spinal cord is not influenced directly via CST systems but is strongly controlled by the RST pathway. Many RST neurons have monosynaptic input from corticobulbar pathways therefore this pathway may provide an indirect route from the cortex to commissural systems. The cortico-reticulospinal-commissural system may also contribute to functional recovery following damage to the CST as it has the capacity to deliver information from the cortex to the spinal cord in the absence of direct CST input.