Increased c-fos expression in spinal lumbosacral projection neurons and preganglionic neurons after irritation of the lower urinary tract in the rat

Chemical irritation of the lower urinary tract (LUT) induces c-fos expression in neurons in the lumbosacral (L₆ and S₁) spinal cord. This study used axonal tracing with fluorescent dyes to identify the types of spinal neurons expressing Fos immunoreactivity (IR) after LUT irritation in the rat. Fos-IR was detected in lateral and medial superficial dorsal horn, the sacral parasympathetic nucleus (SPN) and lamina X around the central canal. Fos-IR was detected in spinal neurons projecting to supraspinal sites (brainstem and hypothalamus), in preganglionic neurons (PGN) and in unlabeled segmental interneurons. A substantial percentage (20%) of dye labeled PGN exhibited Fos-IR after LUT irritation; and a larger percentage (36%) exhibited Fos-IR after electrical stimulation of the pelvic nerve which contains afferent pathways from all of the pelvic organs. The majority (average 55%) of Fos-positive neurons projecting to supraspinal sites were also located in the region of the SPN. A selective distribution of different types of neurons was detected in this region: PGN were located ventral to the spinal projection neurons which in turn were located ventral to the majority of unidentified Fos-positive neurons. The distribution of Fos-positive PGN and projection neurons was similar in spinal intact and spinal transected animals indicating that c-fos expression was mediated by monosynaptic afferent input or input from segmental interneurons and was not due to activation of supraspinal micturition reflex pathways.     

Increased c-fos expression in spinal neurons after irritation of the lower urinary tract in the rat.

This study utilized neuronal c-fos expression to examine the spinal pathways involved in processing nociceptive and non-nociceptive afferent input from the lower urinary tract (LUT) of the urethane-anesthetized rat. C-fos protein was detected immunocytochemically in only a small number of cells (< 2 cells/L6 section) in control animals. However, chemical irritation with 1% acetic acid or mechanical stimulation of the LUT markedly increased the number of c-fos-positive neurons (56-180 cells/L6 section) in four regions of the caudal lumbosacral (L6-S1) spinal cord: medial dorsal horn (MDH), lateral dorsal horn, dorsal commissure (DCM), and sacral parasympathetic nucleus (SPN). Only small numbers of c-fos-positive cells were detected in rostral lumbar segments, a region that is thought to receive nociceptive input from the LUT via afferent pathways in sympathetic nerves. The distribution of c-fos-positive cells in the L6 spinal cord varied according to the stimulus (i.e., urethral catheter, bladder distension, or chemical irritation). Distension of the urinary bladder increased the number of c-fos-positive cells mainly in DCM and SPN regions of the cord. In contrast, irritation of the LUT increased c-fos expression largely in DCM and MDH areas. Spinal cord transection (T8 level) did not alter the c-fos expression induced by a catheter or chemical irritation, indicating that gene expression was mediated by spinal pathways. Denervation experiments showed that c-fos expression was induced by activation of afferent pathways in the pelvic and pudendal nerves. These results suggest that neurons in several regions of the spinal cord are involved in processing afferent input from different parts of the LUT. Neurons in the DCM appear to have an important role since they respond to both nociceptive and non-nociceptive inputs and to visceral (pelvic nerve) and somatic (pudendal nerve) afferent pathways. Thus, these neurons may be involved in the mechanisms of visceral-somatic referred pain.     

The effect of glutamate antagonists on c-fos expression induced in spinal neurons by irritation of the lower urinary tract

Chemical irritation of the lower urinary tract (LUT) of the rat increases the expression of c-fos in neurons in the dorsal horn, dorsal commissure and intermediolateral region of the spinal cord. The role of glutamatergic synapses in this response was examined using two glutamate receptor antagonists, MK-801 (an NMDA antagonist) and CNQX (an AMPA antagonist). In rats with an intact spinal cord, MK-801 (3.5 mg/kg, i.v.) administered 15 min before bladder irritation decreased (50–60%) the number of c-fos-positive cells in all regions of the cord. A smaller dose of MK-801 (0.8 mg/kg, i.v.) was ineffective. In spinal transected rats (4–7 days prior to the experiment) MK-801 (3.5 mg/kg, i.v.) decreased c-fos expression only in the medial dorsal horn. CNQX (1.2 mg/kg, i.v.) was ineffective in both preparations. These results indicate that activation of NMDA receptors at glutamate synapses in the central nervous system may play a role in the processing of nociceptive input from the LUT and may also be involved in reflex pathways mediating micturition.     

Electrophysiological properties of lumbosacral preganglionic neurons in the neonatal rat spinal cord

The electrophysiological properties of parasympathetic preganglionic neurons (PGN) in L6 and S1 spinal cord slices from neonatal rats were studied using the patch clamp techniques. PGN were identified by retrograde axonal transport of a fluorescent dye (Fast Blue) injected intraperitoneally before the experiment. PGN in the intermediolateral region of the spinal cord were divided into two classes (tonic PGN and phasic PGN) on the basis of firing properties during prolonged (300 ms) depolarizing current pulses. Tonic neurons exhibited a prolonged discharge (average maximum: 5.6); whereas phasic PGN fired on average only 1.4 spikes during depolarizing pulses. PGN were usually oval in shape. The mean long axis of tonic PGN (20.7+/-0.5 microm) was significantly (P<0.05) larger than that of phasic PGN (16.7+/-0.3 microm). Tonic and phasic PGN had similar resting membrane potentials, thresholds for spike activation, input resistances and action potential durations. The duration of the after-hyperpolarization (AHP) in tonic PGN (200.5+/-11.9 ms) was longer than in phasic PGN (137.6+/-9.8 ms). 4-aminopyridine (4-AP, 0. 5 mM) reduced the threshold for spike activation in tonic and phasic PGN. 4-AP also unmasked tonic firing in phasic PGN (average maximum: 5.5 spikes during 300 ms depolarizing current pulses) and increased firing frequency by 19% in tonic PGN. These data indicate that the different discharge patterns of parasympathetic PGN are dependent in part on differences in the expression of 4-AP-sensitive K(+) channels. The two types of PGN may provide an innervation to different targets in the pelvic viscera.     

Effects of pituitary adenylate cyclase activating polypeptide on lumbosacral preganglionic neurons in the neonatal rat spinal cord

The effects of PACAP-38 on phasic and tonic preganglionic neurons (PGN) in L6 and S1 spinal cord slices from neonatal rats (5--11 days old) were studied using the whole-cell patch clamp technique. PGN were identified by retrograde axonal transport of a fluorescent dye (Fast Blue, 5 microl of 4% solution) injected into the intraperitoneal space 3--7 days prior to the study. Bath application of pituitary adenylate cyclase activating polypeptide (PACAP) (20 nM) increased the frequency of spontaneous excitatory postsynaptic potentials (EPSPs) and spontaneous firing in both types of PGN. PACAP markedly increased the number (200--800%) and frequency of action potentials elicited by depolarizing current pulses in phasic PGN, but had a smaller effect on tonic PGN. PACAP decreased the threshold for action potential generation by approximately 25% in both types of neurons (e.g. -34.0+/-1.5 to -38.4+/-1.7 mV from a holding potential of -50 mV in phasic PGN, P<0.005). PACAP did not affect the duration of the action potential. The amplitude of the spike after hyperpolarization was not changed but the duration was significantly reduced by PACAP from 204.4+/-12.2 to 106.2+/-8.1 ms in tonic but not in phasic PGN. PACAP suppressed a transient outward current that was also suppressed by 4-aminopyridine (0.5 mM). These results coupled with the immunohistochemical identification of a dense collection of PACAP fibers in the region of the PGN, raises the possibility that PACAP may function as an excitatory transmitter in lumbosacral parasympathetic reflex pathways in the neonatal rat.     

Sympathetic preganglionic neurons in the isolated spinal cord of the neonatal rat

A preparation of the isolated spinal cord of the neonatal rat was developed for the study of sympathetic preganglionic neurons (PGNs). PGNs were identified for extracellular single unit recording by their location and by antidromic activation by ventral root stimulation. PGNs could be synaptically activated by stimulation of the dorsal root and spinal pathways. Spontaneous firing was observed in 18% of the PGNs. The average firing rate was 1 Hz with a range of 0.3 to 2 Hz.PGNs (and motoneurons) were visualized by incubating vental roots in horseradish peroxidase (HRP) solutions. The location and morphology of PGNs were similar to those reported in studies using adult animals. Primary afferent fibers were visualized by incubating dorsal roots in HRP solutions. Dorsal root projections appeared mature in the neonatal rat. Primary afferents did not appear to project directly to PGNs.It is concluded that PGNs are viable in this preparation and that spinal sympathetic systems are relatively mature in the neonatal rat.     

c-fos Expression and NADPH-d reactivity in spinal neurons after fatiguing stimulation of hindlimb muscles in the rat.

The distribution of Fos-immunoreactive (Fos-ir) and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-reactive neurons in the rat lumbar spinal cord was examined following muscle fatigue caused by intermittent high-rate (100 s(-1)) electrical stimulation of the triceps surae muscle or the ventral root L5 (VRL5) for 30 min. Following both types of stimulation, the fatigue-related c-fos gene expression was more extensive in the L2-L5 segments on the stimulated side, and the majority of Fos-ir neurons were concentrated in the dorsal horn. After direct muscle stimulation, the highest number of Fos-ir neurons were detected in two regions: layer 5, and superficial layers (1 and 2(o)), although many labeled cells were also found in layers 3, 4, 6, and 7. In response to VRL5 stimulation, the maximal density of Fos-ir neurons was detected in the middle and lateral parts of layers 1 and 2(o), the zone of termination of high-threshold muscle afferents(.) Statistically significant prevalence of Fos-ir cell number was also found in layers 5 and 7 on the stimulated side. A few Fos-ir neurons were detected in the ventral horn (layer 8 and area 10) on both sides. The lamellar distribution of NADPH-d-reactive neurons was similar over all experimental groups of animals. In the L3-L6 segments, such reactive cells were arranged in two distinct regions: dorsal horn (layers 2(i), 3, and 5) and area 10; in the L1 and L2 segments, an additional cluster of NADPH-d positive cells was found in the intermediolateral cell column (IML). Double-labeled cells were not detected. We suggest that c-fos expression in response to muscle fatigue reveals activity of functionally different types of spinal neurons which could operate together with NOS-containing cells in pre-motoneuronal networks to modulate the motoneuron output.     

Suckling-induced activation of neural c-fos expression at lower thoracic rat spinal cord segments

Suckling stimulation is essential for neuroendocrine and sympathetic reflex activation during lactation. In the present study, the induction of c-fos gene expression was used to identify neuronal populations in the spinal cord activated by acute 5 min suckling or by electrical stimulation of the central stump of the first abdominal mammary nerve in lactating rats previously separated from their litters for 6 or 18 h. In addition, to investigate whether spinal sympathetic preganglionic neurons are activated by suckling, dual immunostaining (Fos and choline acetyltransferase) was performed. Fos was expressed at low levels in continuously suckled and 6 h nonsuckled mothers, but no expression was found after 18 h of nonsuckling. On the other hand, in 6 h nonsuckled rats, significant increments in Fos expression occurred in several regions after acute suckling and after electrical stimulation. Also, the pattern of Fos expression in each spinal laminae was different for the two stimuli, i.e. more intense effects of suckling in deep laminae V-X and more intense effects in laminae I-IV with electrical stimulation. Double-labeling after suckling was found only in sympathetic preganglionic neurons from the intermedio-medial cell column, whereas after electrical stimulation, double label was observed only in neurons from the intermedio-lateral cell column. On the other hand, no effect upon Fos protein expression was observed after suckling and only a minor effect after electrical stimulation of mammary nerve in 18 h nonsuckled rats. These results are consistent with previous findings on the sympathetic reflex regulation of the mammary gland, as well as on the importance of the nonsuckling interval for optimal functioning of lactation.     

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.     

Cocaine: effect on spinal projection neurons in the rat

In the anesthetized rat, systemic cocaine at an analgesic dose (25 mg/kg, IP) had no general influence on spontaneous activity in nociceptive spinoreticular tract neurons or in rostrally projecting low-threshold mechanoreceptive neurons of the spinal dorsal horn. Peripherally evoked activity was moderately (mean: 30%) reduced in 43% (6/14) of the spinoreticular tract neurons, whereas in 50% of them there was no marked change in evoked activity by cocaine. Evoked activity was slightly reduced in 25% (2/8) of the low-threshold mechanoreceptive neurons and not changed in 75% of them. The suppressive effect of a distant conditioning noxious stimulus on responses to spinoreticular tract neurons was enhanced by cocaine in 3/6 of the spinoreticular tract neurons. Primary afferent terminal excitability of A-fibers was slightly increased following cocaine as indicated by the increased amplitude (mean: 24%) of the antidromically evoked compound action potential recorded from the sural nerve. It is concluded that the previously shown marked enhancement of spontaneous activity in the bulborecticular formation is generated supraspinally. Thus, while cocaine-induced analgesia appears to be due primarily to supraspinal mechanisms, the present results suggest that spinal mechanisms are also involved possibly presynaptically.     

Parasympathetic preganglionic neurons in the sacral spinal cord

Two types of preganglionic neurons have been identified in the sacral parasympathetic nucleus (SPN) of the cat. These neurons could be differentiated by various characteristics including axonal conduction velocities, morphology, location in the nucleus, organ of innervation and central reflex mechanisms controlling their activity. Neurons having myelinated axons (B-PGNs) with conduction velocities between 3.3 and 13 m/s were located in the lateral band of the SPN and innervated the urinary bladder. Neurons with unmyelinated axons (C-PGNs) with conduction velocities of 0.5–1.4 m/s were located in the dorsal band of the nucleus and innervated the large intestine. B-PGNs were excited by distension of the bladder and inhibited by distension or mechanical stimulation of the intestine, whereas C-PGNs exhibited the opposite responses to these stimuli. C-PGNs often exhibited a low level of spontaneous discharge in absence of stimulation but exhibited marked firing (3.5–10 spikes/s) during a defecation reflex elicited by mechanical stimulation of the rectum-anal canal. The excitatory responses were elicited by C-fiber afferents via a spinal reflex pathway. B-PGNs were inactive when intravesical pressure was below the threshold for inducing micturition (5 cm H₂O) but raising the pressure above the threshold induced firing consisting of repetitive bursts of action potentials occurring at relatively high frequencies (15–60 spikes/s). These bursts coincided with rhythmic bladder contractions. The frequency of bladder contractions and associated bursts of PGN-firing and the mean PGN-firing rate (2–8 spikes/s) increased as intravesical pressure was increased in steps between 5 and 30 cm H₂O. However, as indicated by interspike interval histograms, the frequency of firing within a burst of action potentials was unchanged. It is concluded that the micturition reflex pathway is organized as a simple on-off switching circuit and that B-PGNs receive a maximal synaptic input when intravesical pressure exceeds the micturition threshold. This circuit was triggered by vesical Aδ afferents via a spinobulbospinal pathway. Transection of the spinal cord interrupted the reflex pathway and blocked micturition. However, in chronic spinal animals a spinal reflex mechanism emerged which contributed to the recovery of bladder function. This mechanism, which was weak or non-existent in animals with an intact neuraxis, exhibited a number of important differences from the normal micturiton reflex, most notably being activated by a C-fiber afferent rather than a Aδ afferent limb. The mechanism underlying the emergence of C-fiber evoked bladder reflexes in spinal animals is uncertain.     

Effect of prostaglandins on parasympathetic neurons in the rat lumbosacral spinal cord

Prostaglandin E(2)(PGE(2)) elicits a variety of effects by activating four subtypes of receptors, EP1, EP2, EP3 and EP4. We examined receptor subtypes mediating the effects of PGE(2) on parasympathetic preganglionic neurons that regulate the activity of pelvic visceral organs. In tonic parasympathetic preganglionic neurons in neonatal rat spinal slices, PGE(2) increased the firing frequency to depolarizing current pulses, induced after-discharges and inhibited spike after-hyperpolarization. PGE(2) did not affect phasic preganglionic neurons. An EP1 agonist inhibited after-hyperpolarizations and induced after-discharges, whereas EP4 agonist reduced after-hyperpolarization and increased evoked firing but did not induce after-discharges. EP2 and EP3 agonists were inactive. These results indicate that PGE(2) acting via EP1 and/or EP4 receptors modulates the excitability and/or excitatory synaptic input to tonic parasympathetic preganglionic neurons.     

Viscerovisceral convergence of urinary bladder and colorectal inputs to lumbosacral spinal neurons in rats

Extracellular potentials of single L6-S2 spinal neurons were recorded in pentobarbital anesthetized male rats. The urinary bladder was catheterized through the fundus and filled with warm saline for urinary bladder distension (UBD, 0.5-2.0 ml, 20 s). Colorectal distension (CRD) was produced by distending a latex balloon with air (20-80 mmHg, 20 s). Of 171 deeper neurons examined for responses to UBD and CRD, 49 (29%) neurons responded to both UBD and CRD; whereas 6/42 (14%) superficial neurons (depth < 0.3 mm) responded to both organs. Of 55 viscerovisceral convergent neurons, 25 (45%) neurons were excited by both UBD and CRD; the remainder exhibited multiple patterns of excitation and inhibition. In conclusion, responses of superficial and deeper lumbosacral spinal neurons to convergent inputs from urinary bladder and colon suggested that these neurons might contribute to the cross-talk that occurs between visceral organs.     

Estrogen receptor-immunoreactive neurons are present in the female rat lumbosacral spinal cord

Presence of an estrogen receptor is crucial for cells to respond to estrogen; thus, estrogen-responsive neurons should be identifiable by immunohistochemically staining for the estrogen receptor (ER). Even though spinal neurons are involved in sexual behaviors and innervation of genital organs, little information is available about ER-containing neurons in the spinal cord. Consequently, we have undertaken a study of ER-containing neurons in the female rat lumbosacral cord, an area involved in reproductive functions and predicted to contain estrogen-responsive neurons. In addition, since parasympathetic preganglionic neurons in the lumbosacral cord produce nitric oxide (NO), we also sought to determine if ER-immunoreactive (-IR) neurons contain the enzymes for NO production. Finally, we compared the distribution of ER-IR neurons to the presence of uterine cervix-related neurons. Uterine cervix-related neurons were identified by expression of FOS-immunoreactivity after vaginocervical mechanostimulation (VCS). The lumbosacral spinal cords were removed from intact, ovariectomized, and VCS-treated rats and sections stained by immunohistochemistry. ER-IR was present in the nuclei of neurons located predominately in the dorsal one-half of the spinal cord. Specific sites include the dorsal horn, lamina V, the sacral parasympathetic nucleus (SPN) (which contains preganglionic parasympathetic neurons) and extending into the lateral funiculus, and lamina X. Some ER-IR neurons were NADPH-d-positive and were localized in laminae V and VII. FOS-IR neurons had a distribution pattern similar to the distribution of neurons containing ER. The presence of ER neurons in these regions suggest that they are responsive to circulating estrogen. © 1996 Wiley-Liss, Inc.     

Ectopic expression of reelin alters migration of sympathetic preganglionic neurons in the spinal cord

Reelin, an extracellular matrix molecule, regulates neuronal positioning in the brain, brainstem, and spinal cord. Although Reelin was identified more than a decade ago, its function on neuronal migration is still poorly understood. Using a transgenic mouse that expressed reelin under the nestin promoter, we examined here the function of Reelin in control of sympathetic preganglionic neurons (SPN) migration in the spinal cord. SPN undergo primary and secondary migration to arrive at their final locations. In wildtype mice, postmitotic SPN undergo primary migration from the neuroepithelium to the ventrolateral spinal cord, and then undergo a secondary dorsal migration to their final location to form the intermediolateral column (IML). In reeler, which lacks Reelin, SPN also undergo primary migration to the ventrolateral spinal cord as in wildtype. However, during secondary migration, SPN migrate medially to cluster adjacent to the central canal. Our present study on transgenic rl/rl mutants (rl/rl ne‐reelin) shows that the initial migration of SPN (embryonic day [E]9.5–E12.5) was similar to reeler. SPN migrated from the neuroepithelium to the ventrolateral spinal cord and then back toward the central canal, despite strong reelin expression in the ventricular zone. However, SPN did not aggregate near the central canal when ectopic reelin was expressed. Only when the expression level of ectopic reelin in the ventricular zone became very weak (E18.5) were SPN found to cluster near the central canal. Postnatally, SPN in rl/rl ne‐reelin transgenic mice were located in both the IML and near the central canal. These results show that SPN position can change with location and level of reelin expression. Possible functions of Reelin on SPN migration are discussed. J. Comp. Neurol. 515:260–268, 2009. © 2009 Wiley‐Liss, Inc.     

Glutamate-immunoreactive synapses on retrogradely-labelled sympathetic preganglionic neurons in rat thoracic spinal cord

Retrograde tracing with cholera toxin B subunit (CTB) combined with post-embedding immunogold labelling was used to demonstrate the presence of glutamate-immunoreactive synapses on sympathetic preganglionic neurons that project to the adrenal medulla or to the superior cervical ganglion in rat thoracic spinal cord. At the electron microscope level, glutamate-immunoreactive synapses were found on retrogradely labelled nerve cell bodies and on dendrites of all sizes. Two-thirds of the vesicle-containing axon profiles that were directly apposed to, or synapsed on, CTB-immunoreactive sympathoadrenal neurons were glutamate positive. The proportion of glutamate-immunoreactive contacts and synapses on sympathoadrenal neurons decreased to zero when the anti-glutamate antiserum was absorbed with increasing concentrations of glutamate from 0.1 mM to 10 mM. Double immunogold labelling for glutamate and gamma-aminobutyric acid (GABA) showed that glutamate-immunoreactive profiles did not contain GABA and that GABA-immunoreactive profiles did not contain glutamate. These results suggest that glutamate is the major excitatory neurotransmitter to sympathoadrenal neurons and possibly to other sympathetic preganglionic neurons in the intermediolateral cell column of the spinal cord.     

Activation of adrenal preganglionic neurons during autonomic dysreflexia in the chronic spinal cord-injured rat

Autonomic dysreflexia (AD) occurs in a majority of high paraplegic and quadriplegic patients and is particularly characterized by a paroxysmal hypertension elicited by somatic or visceral stimuli. We have previously shown that plasma adrenaline and noradrenaline levels were significantly increased during episodes of AD in the 30-day spinal cord-injured (SCI) rats, suggesting the participation of adrenal catecholamines in the cardiovascular changes associated to AD. Thus, adrenal sympathetic preganglionic neurons (SPN) could be activated by visceral afferences leading to AD. The aim of this study was then to demonstrate whether visceral stimulation that induces AD activates adrenal SPN in chronic SCI rats. To this end, a retrograde tracer, the cholera toxin B subunit (CTB), was combined with the immunocytochemical detection of Fos protein after visceral stimulation. Chronic SCI rats received a CTB injection into the adrenal gland and, 3 days later, were stimulated by repetitive distension of the colon. Results showed that this stimulation elicited typical hypertensive episodes of AD and a significant increase in the number of double-labeled neurons (CTB/Fos immunoreactive neurons) in the thoracic spinal cord below the level of injury (T4 segment) when compared to the stimulated non-SCI rats. In conclusion, visceral stimulations in the chronic SCI rats activate adrenal SPN, which could induce release of catecholamines by the adrenal medulla. The present study brings new data on the spinal mechanisms of AD cardiovascular dysfunctions.     

大鼠Barrington核内脊髓投射神经元直接接受腰骶髓传入的电镜研究

将结合生物素的葡聚糖胺 (BDA)注射到大鼠腰骶髓后 ,在电镜下观察脑桥Barrington核内腰骶髓投射神经元与来自腰骶髓传入投射纤维间的突触联系。与先前的研究相一致 ,注射BDA到腰 6和骶 1节段后 ,光镜下可见Barrington核内出现大量顺行标记的神经末梢和一定数量的逆行标记细胞。电镜下发现标记的轴突末梢和标记的树突之间存在直接的突触连接。结果表明 ,Barrington核直接接受腰骶髓的传入投射 ,提示大鼠脑桥排尿反射的脊髓内上行投射通路中可能存在一条直接通路。     

Chemical bladder irritation provokes c-fos expression in the midbrain periaqueductal gray matter of the rat

We investigated the topographical localization of c-fos expression in the midbrain periaqueductal gray matter (PAG) to detect nociception-induced neural activity in the PAG. In conscious female Wistar rats, c-fos expression was induced by continuous intravesical infusion of saline or 0.1% acetic acid. Number of c-fos protein (Fos)-positive cells was counted at each coronal section of the PAG as well as Barrington's nucleus. Fos-positive cells were also counted at L1 and L6 of the spinal cord, where most of the hypogastric and pelvic nerve afferent terminals project, respectively. Compared with saline infusion, acetic acid infusion provoked irritative bladder responses characterized by a marked increase in the frequency of bladder contractions, and induced a significant increase in the number of Fos-positive cells in both L1 and L6 of the spinal cord. Following acetic acid infusion, there was a significant increase in the number of Fos-positive cells in all coronal sections of the PAG compared with saline infusion, especially in the caudal part of the PAG. The increase in the number of Fos-positive cells was mainly observed in the ventrolateral and lateral parts of the caudal PAG, and in the dorsal part of the rostral PAG. However, there was no difference in the number of Fos-positive cells in Barrington's nucleus between saline and acetic acid infusion. In conclusion, nociception induced by chemical bladder irritation influences neural activity in the PAG. Implication of topographical difference in Fos expression in the PAG and its relevance to changes in bladder function remain to be elucidated.