Sexual dimorphism in sympathetic preganglionic neurons of the rat hypogastric nerve

Horseradish peroxidase (HRP) applied to one hypogastric nerve labelled sensory neurons in T11-L3 dorsal root ganglia (DRG) bilaterally and preganglionic neurons (PGN) in the spinal cord segments T13-L3. An average of 130 small DRG neurons were labelled per animal (male or female). These were concentrated in the L1 + L2 DRGs (92%). About 75% were located ipsilateral to the site of HRP application. Central projections from DRG neurons were noted throughout Lissauer's tract and in the marginal zones (medial and lateral) near the borders of Lissauer's tract. A short projection was also seen extending to the dorsolateral funiculus. More than 90% of the preganglionic neurons were located in segments L1 + L2. Most of these were found in the dorsal commissural nucleus (75%) and most of the remainder were located bilaterally in the intermediolateral columns. Somewhat more intermediolateral neurons were labelled on the ipsilateral side than on the contralateral side. There were a few intercalating neurons and a very few funicular cells. An average of 415 PGNs were labelled in the male animals and 110 in the females, demonstrating a strong sexual dimorphism. No dimorphism was found in the sensory components.     

Neuropathic pain is associated with alterations of nitric oxide synthase immunoreactivity and catalytic activity in dorsal root ganglia and spinal dorsal horn

Previous experiments have suggested that nitric oxide may play an important role in nociceptive transmission in the spinal cord. To assess the possible roles of neuronal nitric oxide synthase (nNOS) in spinal sensitization after nerve injury, we examined the distribution of nNOS immunoreactivity in dorsal root ganglia (DRGs) and dorsal horn of the corresponding spinal segments. NOS catalytic activity was also determined by monitoring the conversion of [3H]arginine to [3H]citrulline in the lumbar (L4-L6) spinal cord segments and DRGs in rats 21 days after unilateral loose ligation of the sciatic nerve. Behavioral signs of tactile and cold allodynia developed in the nerve-ligated rats within 1 week after surgery and lasted up to 21 days. Immunocytochemical staining revealed a significant increase (approximately 6.7-fold) of nNOS-immunoreactive neurons and fibers in the DRGs L4-L6. No significant changes were detected in the number of nNOS-positive neurons in laminae I-II of the spinal segments L4-L6 ipsilateral to nerve ligation. However, an increased number of large stellate or elongated somata in deep laminae III-V of the L5 segment expressed high nNOS immunoreactivity. The alterations of NOS catalytic activity in the spinal segments L4-L6 and corresponding DRGs closely correlated with nNOS distribution detected by immunocytochemistry. No such changes were detected in the contralateral DRGs or spinal cord of sham-operated rats. The results indicate that marked alterations of nNOS in the DRG cells and in the spinal cord may contribute to spinal sensory processing as well as to the development of neuronal plasticity phenomena in the dorsal horn.     

Changes in galanin immunoreactivity in rat lumbosacral spinal cord and dorsal root ganglia after spinal cord injury

Alterations in the expression of the neuropeptide galanin were examined in micturition reflex pathways 6 weeks after complete spinal cord transection (T8). In control animals, galanin expression was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the superficial dorsal horn; (3) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (4) the lateral collateral pathway in lumbosacral spinal segments. Densitometry analysis demonstrated significant increases (P < or = 0.001) in galanin immunoreactivity (IR) in these regions of the S1 spinal cord after spinal cord injury (SCI). Changes in galanin-IR were not observed at the L4-L6 segments except for an increase in galanin-IR in the dorsal commissure in the L4 segment. In contrast, decreases in galanin-IR were observed in the L1 segment. The number of galanin-IR cells increased (P < or = 0.001) in the L1 and S1 dorsal root ganglia (DRG) after SCI. In all DRG examined (L1, L2, L6, and S1), the percentage of bladder afferent cells expressing galanin-IR significantly increased (4-19-fold) after chronic SCI. In contrast, galanin expression in nerve fibers in the urinary bladder detrusor and urothelium was decreased or eliminated after SCI. Expression of the neurotrophic factors nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) was altered in the spinal cord after SCI. A significant increase in BDNF expression was present in spinal cord segments after SCI. In contrast, NGF expression was only increased in the spinal segments adjacent and rostral to the transection site (T7-T8), whereas spinal segments (T13-L1; L6-S1), distal to the transection site exhibited decreased NGF expression. Changes in galanin expression in micturition pathways after SCI may be mediated by changing neurotrophic factor expression, particularly BDNF. These changes may contribute to urinary bladder dysfunction after SCI.     

Sympathetic preganglionic efferent and afferent neurons mediated by the greater splanchnic nerve in rabbit

Motion sickness, a multisymptom disorder characterized by abnormal gastrointestinal motility and emesis, can be induced by vestibular effects on the sympathetic portion of the autonomic nervous system. However, the vestibular-autonomic pathways are unknown. As a first step in the analysis, we identified the locus of preganglionic sympathetic neurons (PSNs) and dorsal root afferent ganglionic neurons (DRGs) which supply sympathetic innervation to major portions of the gastrointestinal tract in the rabbit. Retrograde labeling of neurons was obtained by application of horseradish peroxidase (HRP) to the cut end of the greater splanchnic nerve. Labeled PSNs were found, ipsilaterally, within the T1 to T11 spinal cord segments, with the highest density of neurons in T6. Most PSNs were located within the intermediolateral column (IML), but a significant portion also occurred within the lateral funiculus (LF), the intercalated region (IC) and the central autonomic area (CA). The proportion of labeling between the four regions depended on the spinal cord segment. In the midthoracic levels, the distribution of labeled neurons was denser in the IML and LF, and in the caudal thoracic segments, the majority were localized in the IC and CA. Labeled cells in these four areas varied morphologically from large fusiform neurons in the IC to small fusiform neurons in the LF, small stellate neurons in the CA, and medium-size stellate neurons in the IML. The DRGs were labeled in thoracic segments T1 to T12, with the majority between T5 and T11. These labeled DRG somata of the greater splanchnic nerve were smaller in comparison with unlabeled ones.     

Central nervous system neurons infected by pseudorabies virus injected into the rat urinary bladder following unilateral transection of the pelvic nerve

Following the transection of one pelvic nerve and both hypogastric nerves, the urinary bladder of male Sprague-Dawley rats was injected with pseudorabies virus (PRV; Bartha strain). The central stump of the transected pelvic nerve was labelled with fast blue (FB), and rats were maintained for 2, 2. 5, and 3 days following viral infection. Tissue was processed with antisera against PRV and choline acetyltransferase (CAT). In the L6-S1 spinal cord, neurons in the ipsilateral intermediolateral area (IML) were labelled after 2 days. After 2. 5 days, labelled neurons were also found in the dorsal gray commissure (DGC), the ipsilateral superficial dorsal horn, and the contralateral IML area. After 3 days, many labelled neurons appeared in the superficiai dorsal horns and, bilaterally, in the L6-S1 dorsal root ganglia. In both IMLs, two groups of PRV-labelled neurons were found: (1) CAT-positive preganglionic cells and (2) smaller, CAT-negative cells located slightly dorsal to the preganglionic neurons. No other doubly stained neurons were found in the spinal cord. Contralateral DRG neurons stained for either PRV or FB or both. Ipsilateral DRG neurons stained only for PRV. PRV-immunoreactive (IR) neurons appeared in the brainstem only after 3 days. These were located primarily in the pontine micturition centers (equal numbers), the ventral locus coeruleus, and the raphe and lateral reticular areas. © 1995 Wiley-Liss, Inc.     

Upregulation of brain-derived neurotrophic factor in the sensory pathway by selective motor nerve injury in adult rats

Selective motor nerve injury by lumbar 5 ventral root transection (L5 VRT) induces neuropathic pain, but the underlying mechanisms remain unknown. Previously, increased expression and secretion of brain-derived neurotrophic factor (BDNF) had been implicated in injury-induced neuropathic pain in the sensory system. In this study, as a step to examine potential roles of BDNF in L5 VRT-induced neuropathic pain, we investigated BDNF gene and protein expression in adult rats with L5 VRT. L5 VRT induced a dramatic upregulation of BDNF mRNA in intact sensory neurons in the ipsilateral L5 dorsal root ganglia (DRG), in non-neuronal cells in the ipsilateral sciatic nerve, and in motoneurons in the ipsilateral spinal cord. L5 VRT also induced de novo synthesis of BDNF mRNA in spinal dorsal horn neurons and in glial cells in the white matter of the ipsilateral spinal cord. Consistent with the mRNA expression pattern, BDNF protein was also mainly upregulated in all populations of sensory neurons in the ipsilateral L5 DRG and in spinal neurons and glia. Quantitative analysis by ELISA showed that the BDNF content in the DRG and sciatic nerve peaked on day 1 and remained elevated 14 days after L5 VRT. These results suggest that increased BDNF expression in intact primary sensory neurons and spinal cord may be an important factor in the induction of neuropathic pain without axotomy of sensory neurons.     

Spinal cord microglial cells and DRG satellite cells rapidly respond to transection of the rat sciatic nerve

Transection of the rat sciatic nerve induces retrograde changes in the dorsal root ganglia (DRG) neurons and in the motoneurons in the ventral grey matter of the lumbar L4-L6 spinal cord segments. In the ipsilateral dorsal grey matter and in the ipsilateral nucleus gracilis, transganglionic changes occur in the terminal fields of the centrally projecting axons of injured DRG neurons. As revealed by immunocytochemistry, the neuronal reactions were associated with a rapid proliferation and activation of microglial cells in the lumbar spinal cord as well as in the nucleus gracilis. Reactive microglial cells were detected as early as 24 h after sciatic axotomy. The microglial reaction had a maximum around day 7 postlesion and disappeared around 6 weeks after axotomy. In addition to light microscopy, activated, perineuronal microglia were identified by immuno-electron microscopy in the ventral grey matter. In the DRG, satellite cells constitutively expressed major histocompatibility complex (MHC) class II antigens. Sciatic axotomy led to a proliferation of satellite cells and an increased expression of MHC class II molecules in particular. This satellite cell reaction started 24 h after axotomy and continued to increase gradually until about 6 weeks after the lesion. Resident macrophages, detected in the DRG interstitial tissue by their expression of monocyte/macrophage markers, also reacted to sciatic axotomy. Our data suggest that (1) sciatic axotomy leads to a rapid microglial reaction in both the ventral and dorsal grey matter of the lumbar spinal cord and in the ipsilateral nucleus gracilis; (2) the immunophenotype of activated microglia following sciatic axotomy is comparable with that observed after axotomy of cranial nerves, e.g. the facial nerve; (3) satellite cells in DRG constitutively express MHC class II molecules; and (4) sciatic axotomy leads to a rapid activation of satellite cells and interstitial macrophages in the axotomized DRG.     

Peripheral axotomy affects nicotinamide adenine dinucleotide phosphate diaphorase and nitric oxide synthases in the spinal cord of the rabbit

Using nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and nitric oxide synthase (NOS) immunocytochemistry combined with radioassay of calcium-dependent NOS activity, we examined the occurrence of NADPHd staining and NOS immunoreactivity (NOS-IR) in the dorsal root ganglia (DRG) neurons, dorsal root afferents, and axons projecting via gracile fascicle to gracile nucleus 14 days after unilateral sciatic nerve transection in the rabbit. Mild to moderate NADPHd staining and NOS-IR appeared in a large number of small and medium-sized to large neurons in the ipsilateral L4-L6 DRG, accompanied by enhanced NOS-IR of thick myelinated fibers in the ipsilateral L4-L6 dorsal roots. A noticeable increase in the density of punctate NADPHd staining occurred throughout laminae I-IV in the ipsilateral medial part of the dorsal horn in L4-L6 segments. Concurrently, a statistically significant decrease in the number of small NADPHd-exhibiting neurons in laminae I-II and, in contrast to this, a statistically significant increase of medium-sized to large NADPHd-stained somata in the ipsilateral laminae III-VI of L4-L6 segments were found. A detailed compartmentalization of L4-L6 segments into gray and white matter regions disclosed substantially increased catalytic NOS activity and inducible NOS mRNA levels in the dorsal horn and dorsal column ipsilaterally to the peripheral injury. A noticeable increase in the number of thick myelinated NADPHd-exhibiting and NOS-IR axons was noted in the ipsilateral gracile fascicle, terminating in dense, punctate NADPHd staining in the neuropil of the gracile nucleus. These observations indicate that the de novo-synthesized NOS in the lesioned primary afferent neurons resulting after sciatic nerve transection may be involved in an increase in NADPHd staining and NOS-IR in the ipsilateral dorsal roots and dorsal horn of L4-L6 segments, whence NOS could be supplied to ascending axons of the gracile fascicle.     

Distribution of sympathetic preganglionic neurons innervating the kidney in the rat: PRV transneuronal tracing and serial reconstruction.

The organization of spinal motor circuitry to the kidney is not well-characterized and changes in renal innervation have been associated with disease states such as hypertension found in the spontaneously hypertensive rat or renal hypertension. Here, we describe the segmental and intra-segmental organization of the spinal motor circuitry that was resolved after neurotropic viral injection into the kidney and retrograde transneuronal transport to the spinal cord. In the first experiment, the serial reconstruction of infected neurons in the thoracolumbar spinal cord from T8-L1 was performed following injection of pseudorabies virus (PRV, Bartha strain) into either the cranial pole, the caudal pole or both the cranial and caudal poles of the left kidney in male rats. In the second experiment, rats received injections of two different PRV strains that were genetically engineered to express unique reporter molecules; one of the engineered strains was injected into the cranial pole and the other was injected into the caudal pole. Either 3- or 4-day post-infection, the animals were anesthetized and sacrificed by transcardial perfusion. PRV-infected neurons were located by immunocytochemistry against either PRV itself (experiment 1) or the unique marker proteins (experiment 2). After injection of both poles of the kidney, the majority of the infected neurons were found in the ipsilateral intermediolateral cell column (IML) from T10 to T12 with the mode at T11. Infected neurons were found in discrete neuron clusters in the intermediolateral cell column along the longitudinal axis in a repeating pattern of high and low density that has been called "beading". Three observations indicated a topographic distribution of renal sympathetic preganglionic neurons (SPN). First, after injection into either the cranial or caudal poles of the kidney, the mode of infected cells was located in segments T11 and T12, respectively. The one spinal segment shift in the mode suggested a topographic distribution. Second, in spinal segments T8-L1, comparison of the distributions of the neurons innervating each pole of the left kidney revealed an overlap in the distribution, except in the T11 segment. In the T11 segment, the neurons projecting to each pole tended to segregate into separate populations. Third, in rats that received injections of two PRV strains that were genetically engineered to express unique markers into opposite poles of the kidney, a segregation of neurons projecting to the cranial and caudal poles of the kidney was noted again in the T11 spinal segment and the segregation at adjacent spinal levels was obvious. The analysis of the distribution of infected neurons within each spinal cord segment (intra-segmental distribution) revealed three different patterns along the cranial-caudal dimension. In segments T8-T10, >60% of the infected neurons were located in the caudal half of the spinal segment. In segments T12-L1, >60% of the infected neurons were located in the cranial half of the spinal segment. In segment T11, the neurons were more evenly distributed throughout the segment. These intra-segmental distribution patterns were found after both 3- or 4-day survival periods post-infection and were found in most animals. The distribution of clusters of neurons revealed a similar intra-segmental pattern. Thus, as was described previously for the sympathetic postganglionic neurons that innervate the kidney, the present work indicates a topographic organization in the second-order neurons in the renal sympathetic efferent pathway. The physiological significance of this anatomical organization remains to be determined.     

Spinal neurons activated with the urethrogenital reflex in the male rat

The urethrogenital (UG) reflex is a spinal ejaculatory-like reflex. The location of spinal neurons activated by the UG reflex was examined in the male rat using the immediate early gene, c-fos. In addition, co localization of neurons containing galanin and choline acetyl transferase (ChAT) and serotonin fibers with fos-immunoreactive (fos-I) nuclei was examined. Activation of the UG reflex resulted in a significant increase in fos positive nuclei in segments T13-S1, compared to controls in which the UG reflex was not activated. Spinal circuits involved in the UG reflex include neurons relaying afferent information from the pudendal sensory nerve, in the dorsal horn and medial cord of L5-S1. Interneurons specifically activated with the UG reflex were identified in the medial, intermediate and lateral gray. A small proportion of parasympathetic and sympathetic preganglionic neurons in the intermediolateral cell column (IML) of L5-S1 and IML and medial gray of T13-L2, respectively, was activated with the UG reflex. A significant increase in the number of galanin containing neurons expressing c-fos in the medial gray of L3-L4 was also observed with the UG reflex. Serotonin fibers and varicosities were found throughout the spinal cord and were especially dense in the ventral horn, IML and medial gray. Fos activated neurons were found in close apposition to serotonin fibers in the IML and medial gray. These studies demonstrate the multisegmental intraspinal circuitry responsible for ejaculatory-like responses and demonstrate the potential involvement of galanin, acetylcholine and serotonin in mediation of the UG reflex.     

The difference of osteocalcin-immunoreactive neurons in the rat dorsal root and trigeminal ganglia: co-expression with nociceptive transducers and central projection

The co-expression of osteocalcin (OC) with the capsaicin receptor (VR1) and vanilloid receptor 1-like receptor (VRL-1) was examined in the dorsal root (DRG) and trigeminal ganglia (TG). Virtually all OC-immunoreactive (ir) DRG neurons were devoid of VR1- and VRL-1-immunoreactivity (ir). In the TG, 14.1% of OC-ir neurons were also immunoreactive for VR1. Only 1.7% of OC-ir TG neurons co-expressed VRL-1-ir. The distribution of OC-ir was also examined in the spinal cord and trigeminal sensory nuclei. In the spinal cord, the superficial laminae of the dorsal horn were devoid of OC-ir. The neuropil was weakly stained in other regions of the spinal horns. The medullary dorsal horn (MDH) contained numerous OC-ir varicose fibers in laminae I and II. These fibers were occasionally observed originating from the spinal trigeminal tract. The neuropil was weakly stained in deeper laminae of the MDH, and the rostral parts of the trigeminal sensory nuclei. The present study suggests that OC-ir TG nociceptors send their unmyelinated axons to the superficial laminae of the MDH.     

Tracing of afferent and efferent pathways in the left inferior cardiac nerve of the cat using retrograde and transganglionic transport of horseradish peroxidase

Retrograde and transganglionic transport of horseradish peroxidase (HRP) was used to trace afferent and efferent pathways in the left inferior cardiac nerve of the cat. Cardiac efferent and afferent neurons were located, respectively, in the stellate ganglion (average cell count per experiment: 2679) and in the ipsilateral dorsal root ganglia (DRG) from C8 to T9 (average cell count per experiment: 213). Labeled cardiac afferent projections to the spinal cord were most dense in segments T2–T6 where they were located in Lissauer's tract and in lamina 1 on the lateral border of the dorsal horn. Labeled affrent axons extended ventrally through lamina 1 into lamina 5 and the dorsolateral region of lamina 7 in proximity to the intermediolateral nucleus. A weak projection was noted on the medial side of the dorsal horn. These sites of termination are similar to projections by other sympathetic afferent pathways (i.e. renal, hypogastric and splanchnic nerves) to the lower thoracic and lumbar spinal cord, indicating that visceral afferents may have a uniform pattern of termination at various segmental levels. This pattern of termination in regions of the gray matter containing spinothalamic tract neurons and neurons involved in autonomic mechanisms is consistent with the known functions of sympathetic afferent pathways in nociception and in the initiation of autonomic reflexes.     

Transneuronal labeling of spinal interneurons and sympathetic preganglionic neurons after pseudorabies virus injections in the rat medial gastrocnemius muscle.

The distribution of retrogradely and transneuronally labeled neurons was studied in CNS of rats 4 days after injections of the Bartha strain of pseudorabies virus (PRV) into the medial gastrocnemius (MG) muscle. Tissue sections were processed for immunohistochemical detection of PRV. Retrogradely labeled cells were identified in the ipsilateral MG motor column in the caudal L4 and the L5 spinal segments. In order to evaluate the efficacy of PRV retrograde cell body labeling, the number of PRV retrogradely labeled neurons in the MG motor column was compared to the number labeled with two conventional retrograde cell body markers--Fluoro-Gold and cholera toxin-HRP. A ratio of 1:3 representing medium-sized (less than 30 microns) versus large neurons (greater than 30 microns) was found in the Fluoro-Gold dye experiments; a 1:2 ratio was seen in the PRV experiments. In contrast, when cholera toxin-HRP was used as a retrograde marker, mainly large neurons were labeled; the medium-to-large cell body ratio was 1:10 suggesting cholera toxin-HRP may have a greater affinity for the terminals of alpha-motoneurons as opposed to gamma-motoneurons. Transneuronally labeled cells were identified in the L1-L6 spinal gray matter, intermediolateral cell column (T11-L2), lateral spinal nucleus and medial part of lamina VII in C4 and C5 spinal segments, brainstem (caudal raphe nuclei, rostral ventrolateral medulla, A5 cell group, paralemniscal nucleus, locus coeruleus, subcoeruleus nucleus, red nucleus) and paraventricular hypothalamic nucleus. In the L5 spinal cord, transneuronally labeled neurons were seen in the ipsilateral spinal laminae I and II and bilaterally in spinal laminae IV-VIII, and X. Similar results were obtained in rats that had chronic unilateral L3-L6 dorsal rhizotomies indicating most of the labeling was due to retrograde transneuronal cell body labeling. In order to determine whether PRV was transported into the spinal cord by the dorsal root axons, the ipsilateral dorsal root ganglia (DRGs) were examined for PRV immunoreactivity; none was found. However, using the polymerase chain reaction, viral DNA was shown to be present in the ipsilateral DRGs indicating that some of spinal cord cell body labeling may have resulted from anterograde transneuronal labeling, as well.     

Pseudorabies virus tracing of neural pathways between the uterine cervix and CNS: effects of survival time, estrogen treatment, rhizotomy, and pelvic nerve transection

The transneuronal tracer, pseudorabies virus (PRV), was used to identify pathways from the uterine cervix which may be involved in induction of analgesia and abbreviation of estrus by vaginocervical stimulation. In Experiment I, PRV immunoreactivity (PRV-IR) in brain and spinal cord was examined 3-5 days after injection into the cervix of ovariectomized (OVX) female rats given estrogen (E) or control treatments. No differences in viral labeling were observed between OVX and OVX+E females at any time. PRV-infected cells were observed to increase as a function of time and at progressively higher CNS levels. PRV-IR neurons were first observed on day 3 post-infection at L6 in the SPN. Increased labeling was observed at day 4 in the SPN and the DGC at L6 and S1 spinal segments. Dorsal horn neurons showed PRV-IR by 4.5 days. Five days post-infection, labeling was seen in the IML and lamina X in T12-L1 segments, and in medullary raphe, A5, nPGi, nGi, DMV, lateral reticular, Barrington's nuclei, and in the midbrain PAG. In Experiment II, the effects of bilateral L6 dorsal root rhizotomy (RH) combined with unilateral (UPx) or bilateral (BPx) pelvic nerve transection on PRV infectivity were examined 5 days after infection. Despite reductions in substance P labeling in the dorsal horn following RH, PRV-IR neurons persisted in this area. In RH+UPx females, labeling persisted bilaterally in the SPN and DGC at L6. RH+BPx almost completely eliminated the PRV labeling in L6 and S1. Horizontal sections showed distinct patterns of infectivity within the IML of thoracolumbar and SPN of lumbosacral segments consistent with infection in the hypogastric and pelvic nerves, respectively. Our data indicate that retrograde transport of PRV occurs via the hypogastric and pelvic nerves after injection of the virus into the uterine cervix. Furthermore, significant intraspinal processing is likely to occur between thoracolumbar and lumbosacral levels in the modulation of reproductive tract function.     

Dermatomes and the central organization of dermatomes and body surface regions in the spinal cord dorsal horn in rats

Dermatomes and the associated central projection fields were studied with the application of fluorescent neurotracer, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), to 21 reference points on rat trunk and hindlimb skin. Segmental distribution and rostrocaudal central level of dorsal root ganglion (DRG) neurons innervating reference points were examined and DiI-induced fluorescent areas were mapped in the horizontal plane through lamina II of the dorsal horn. Segmental levels of DRG neurons innervating reference points were generally identical to the level determined using dye-extravasation methods. However, innervation of the first digit was situated in the L4 dermatome, not the L3 reported previously using those methods. Generally, afferents from a reference point projected to a single field in the ipsilateral dorsal horn. Reference points on ventral and dorsal median lines of the trunk were represented bilaterally. Afferents from reference points located on the ventral median line of the hindlimb projected to two separate fields: one on the medial margin of spinal cord segments L2-L5 and the other on the medial half of spinal cord segment L5. From the distribution of central projection fields of reference points, central projection fields of dermatomes were revealed as even in shape and located within corresponding spinal cord segments. The arrangement of peripheral and central fields of dermatomes and body surface regions suggests that peripheral and central projection fields of cutaneous afferent fibers are reshaped from the common prototypical pattern that exhibits an orderly and evenly sequenced arrangement.     

Segmental distribution and central projectionsof renal afferent fibers in the cat studied by transganglionic transport of horseradish peroxidase

The segmental and central distributions of renal nerve afferents in adult cats and kittens were studied by using retrograde and transganglionic transport of horseradish peroxidase (HRP). Transport of HRP from the central cut ends of the left renal nerves labeled afferent axons in the ipsilateral minor splanchnic nerves and sensory perikarya in the dorsal root ganglia from T12 to L4. The majority of labeled cells (85%) were located between L1 and L3. A few neurons in the contralateral dorsal root ganglia were also labeled. Labeled cells were not confined to any particular region within a dorsal root ganglion. Some examples of bifurcation of the peripheral and central processes within the ganglion were noted. A small number of preganglionic neurons, concentrated in the intermediolateral nucleus, were also identified in some experiments. In addition, many sympathetic postganglionic neurons were labeled in the renal nerve ganglia, the superior mesenteric ganglion, and the ipsilateral paravertebral ganglia from T12 to L3 Transganglionic transport of HRP labeled renal afferent projections to the spinal cord of kittens from T1 1 to L6, with the greatest concentrations between Ll and L3. These afferents extended rostrocaudally in Lissauer's tract and sent collaterals into lamina I. In the transverse plane, a major lateral projection and a minor medial projection were observed along the outer and inner margins of the dorsal horn, respectively. From the lateral projection many fibers extended medially in laminae V and VI forming dorsal and ventral bundles around Clarke's nucleus. The dorsal bundle was joined by collaterals from the medial afferent projection and crossed to the contralateral side. The ventral bundle extended into lamina VII along the lateroventral border of Clarke's nucleus. Some afferents in the lateral projection could be followed ventrally into the dorsolateral portion of lamina VII in the vicinity of the intermediolateral nucleus. In the contralateral spinal cord, labeled afferent fibers were mainly seen in laminae V and VI These results provide the first anatomical evidence for sites of central termination of renal afferent axons. Renal inputs to regions (laminae I, V, and VI) containing spinoreticular and spinothajamic tract neurons may be important in the mediation of supraspinal cardiovascular reflexes as well as in the transmission of activity from nociceptors in the kidney. In addition, the identification of a bilateral renal afferent projection in close proximity to the thoracolumbar autonomic nuclei is consistent with the demonstration in physiological experiments of a spinal pathway for the renorenal sympathetic reflexes.     

Immunohistochemical localization of calretinin in the dorsal root ganglion and spinal cord of the rat

Calretinin (CR). a recently identified calcium-binding protein, is present in nervous tissue, including sensory pathways, where it may play an important role in regulation of cellular activity. Using immunocytochemistry, we examined the cellular localization of CR in dorsal root ganglia (DRG) and spinal cord of normal rats and after multiple unilateral dorsal root ganglionectomies. In DRG, CR-immunoreactive cell bodies and axons were a small subpopulation (10%) of medium- to large-sized neurons. In the spinal cord, CR-like immunoreactivity (LI) in neurons and fibers was found in all laminae except motoneurons. Dense fiber networks were also found in Clarke's column. The densest staining of both cell bodies and fibers was in the superficial laminae, especially lamina II, and in the lateral spinal and lateral cervical nuclei. CR-immunoreactive fibers were also observed in the fasciculi cuneatus and gracilis. Fasciculus gracilis exhibited the greatest number of labeled axons at the lumbosacral levels, but few labeled axons were found at the rostral thoracic and cervical levels. In contrast, the corticospinal tract at the base of the dorsal column was devoid of CR-immunoreactive fibers. Unilateral multiple lumbar ganglionectomies resulted in a loss of CR-LI in the dorsal columns ipsilateral to the surgery. In the spinal gray matter ipsilateral to the ganglionectomies, CR-LI was reduced in Clarke's column and slightly enhanced in the medial third of lamina II. Our observations demonstrate a unique distribution pattern of CR-LI compared to other calcium-binding proteins in the spinal cord, and suggest a role for CR in nociceptive and proprioceptive pathways.     

Segmental distribution of peptide-like immunoreactivity in cell bodies of the thoracolumbar sympathetic nuclei of the cat

The distribution of leucine-enkephalin, methionine-enkephalin, neurotensin, somatostatin, substance P, oxytocin, vasopressin, and neurophysin II in cell bodies of sympathetic autonomic nuclei of the thoracolumbar (T-L) spinal cord was studied immunohistochemically in cats after intrathecal administration of colchicine. Neurons containing only enkephalin-, neurotensin-, somatostatin-, and substance P-like immunoreactivity (ENK, NT, SS, SP, respectively) were found in the intermediolateral nucleus pars principalis (IMLp) and pars funicularis (IMLf), the nucleus intercalatus (IC), and the central autonomic area (CA). The size, shape, location, and numbers of the peptide-positive neurons in the IMLp, IMLf, and IC suggested that they were sympathetic preganglionic neurons (SPN). This was confirmed by a combined retrograde tracing/immunohistochemical study showing that most of these neurons at the levels of the T-L cord known to provide preganglionic fibers to the stellate ganglion were SPN. On the other hand, the functional identification of the neurons in the CA is uncertain as neurons were not observed which were both retrogradely labelled and contained ENK, NT, SS, or SP. Immunoreactive neurons in each area were counted in ten sections from each segment from C8 to L4. In the IMLp, the SPN with ENK were greatest in number (up to 25) in segments T4-T7 and L2-L3. The maximum number of SPN containing NT was found in segments T4-T7 (45 neurons). Of the four peptides, neurons containing SS were found in the greatest number (up to 48 in segments T2-T6); neurons containing SP were found in the smallest number (15 or fewer per segment). Few SPN containing each of the four peptides were found in the IC; CA neurons with ENK and NT were also few in number. A comparison of the numbers of immunoreactive neurons in the IML with earlier estimates for the total numbers of SPN in the IML at each level showed that the proportions of IML neurons containing each of the four peptides were fairly consistent throughout the T-L cord, with some exceptions. These results suggest that the innervation of visceral organs is not obviously peptide-specific, although some organs may be innervated by a greater proportion of SPN containing one of these peptides. Finally, the presence of ENK, NT, SS, and SP in SPN suggests that these four peptides act as neurotransmitters in preganglionic pathways to sympathetic ganglia.     

Hindlimb movement in the cat induced by amplitude-modulated stimulation using extra-spinal electrodes

Hindlimb movement in the cat induced by electrical stimulation with an amplitude-modulated waveform of the dorsal surface of the L5-S1 spinal cord or the L5-S1 dorsal/ventral roots was investigated before and after acute spinal cord transection at the T13-L1 level. Stimulation of the spinal cord or dorsal/ventral root at the same spinal segment induced similar movements including coordinated multi-joint flexion or extension. The induced movements changed from flexion to extension when the stimulation was moved from rostral (L5) to caudal (S1) spinal segments. Stimulation of a dorsal or ventral root on one side induced only ipsilateral hindlimb movement. However, stimulation on the dorsal surface of the spinal cord along the midline or across the spinal cord induced bilateral movements. The extension induced by stimulation of L7 dorsal root produced the largest ground reaction force that was strong enough to support body weight. Dorsal root stimulation induced a larger ground reaction force than ventral root stimulation and produced a more graded recruitment curve. Stepping at different speeds could be generated by combined stimulation of the rostral (L5) and the caudal (L6/L7) spinal segments with an appropriate timing between the different stimulation channels. Acute transection of the spinal cord did not change the responses indicating that the induced movements did not require the involvement of the supraspinal locomotor centers. The methods and the stimulation strategy developed in this study might be utilized to restore locomotor function after spinal cord injury.