Synapses on axons of sympathetic preganglionic neurons in rat and rabbit thoracic spinal cord

Axosomatic and axodendritic synapses occur on sympathetic preganglionic neurons, but it is not yet known whether their axons receive synaptic input, which could be particularly effective at regulating sympathetic outflow. Here, we examined retrogradely labelled sympathetic preganglionic axons to see if they received synapses. Cholera toxin B subunit (CTB) or CTB conjugated to horseradish peroxidase (CTB-HRP) was used to label neurons projecting to the rat or rabbit superior cervical ganglion, the rat adrenal medulla, or the rabbit stellate ganglion. At the light microscopic level, small groups of CTB-immunoreactive axons travelled through the ventral horn near its lateral boundary, with occasional axons taking a more medial course. The axons passed through the ventrolateral funiculus to exit at the ventral roots. In parasagittal section, a few axons branched within the ventral horn, sending processes rostrally and caudally for short distances before they turned ventrally to exit the spinal cord. At the ultrastructural level, CTB-immunoreactive rat and rabbit sympathetic preganglionic axons were almost exclusively unmyelinated. In contrast, labelling with CTB-HRP revealed both myelinated and unmyelinated axons in the ventral horn, the ventrolateral white matter, and the ventral roots. CTB-HRP also allowed the detection of the initial segment of a sympathetic preganglionic axon. Synapses, with vesicles clustered presynaptically and membrane specializations postsynaptically, were found on some unmyelinated CTB-immunoreactive axons. Occasional axons received several synapses. Synapses were most common on CTB-containing axons just ventral to the intermediolateral cell column. One synapse was found on an axon within 2 μm of its origin from a proximal dendrite. Rare synapses were found several hundred micrometers ventral to the intermediolateral cell column. One branching axon had synapses just below the branch point on both the main axon and the axonal branch. These findings indicate an extensive synaptic input to the axons of at least some sympathetic preganglionic neurons. These axoaxonic synapses could have a profound effect on sympathetic activity. © 1995 Wiley-Liss, Inc.     

Specificity of spinal projections from hypothalamic and brainstem areas which innervate sympathetic preganglionic neurons

The specificity and topographic organization of afferent projections to the intermediolateral column (IML) were examined using retrograde transport of fluorescent tracers injected into pairs of thoracic spinal segments. Neurons within the hypothalamus (parvocellular paraventricular nucleus, dorsomedial nucleus and lateral hypothalamus), pons (Kolliker-Fuse and A5 nuclei) and medulla (ventrolateral nucleus of the solitary tract and rostral ventrolateral medulla) each appeared to innervate only a single spinal segment. Neurons in each cell group projecting to different spinal segments were intermixed and showed no evidence of topographic organization. These results provide a potential anatomical substrate for organ-specific autonomic responses to physiological and psychological stimuli.     

Differential projections of B and C sympathetic axons in peripheral nerves of the bullfrog

Accumulating evidence indicates that electrophysiologically distinct subsets of sympathetic neurons selectively innervate different classes of targets. The organization of this system may therefore be reflected in the sympathetic fiber contents of peripheral nerves. To test this possibility, we have mapped the pathways followed by three groups of postganglionic sympathetic axons in the bullfrog by recording compound action potentials and by retrograde tracing with horseradish peroxidase (HRP). The axons that were studied arise from fast B, slow B, and C-type neurons in ganglia 9 and 10 at the lumbar end of the paravertebral sympathetic chain. They project to peripheral targets primarily by way of the sciatic nerve and can be distinguished by the velocities with which they conduct action potentials. Action potentials were recorded with suction electrodes from isolated preparations composed of paravertebral chain ganglia 7-10, the sciatic nerve, and branches of the sciatic nerve that supply striated muscles, skin, and the bladder. Preganglionic B fibers were selectively activated by stimulating the paravertebral chain rostral to ganglion 7, and preganglionic C fibers were selectively activated by stimulating spinal nerves 7 and 8 at points central to their rami communicantes. Compound action potentials recorded from the sciatic, peroneal, tibial, and sural nerves and from the primary trunk of the pelvic nerve were each found to contain three components produced, respectively, by fast B, slow B, and C-type sympathetic axons. Similarly, action potentials recorded from cutaneous branches of the sciatic tree were found to contain three sympathetic components. By contrast, when compound action potentials were recorded from branches of the sciatic tree that directly enter and innervate striated muscles and also the bladder, the sympathetic responses were found to arise solely from C-type axons. HRP was used to label the sympathetic neurons that project to the sartorius muscle and into the cutaneous lateral crural nerve. Retrograde transport of HRP from the sartorius muscle labeled 17 +/- 4 (mean +/- s.d.) sympathetic neurons and 27 +/- 3 spinal motoneurons while transport from the lateral crural nerve labeled 68 +/- 47 sympathetic neurons but no spinal neurons. The average somatic diameter of ganglion cells projecting to the sartorius muscle was significantly smaller than that of cells projecting to the lateral crural nerve. The electrophysiological results indicate that fast B and slow B sympathetic axons in the sciatic trunk and its primary branches project selectively into cutaneous nerves while sympathetic C axons project into all peripheral nerves.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantitative analysis of bulbospinal projections from the rostral ventrolateral medulla: contribution of C1-adrenergic and nonadrenergic neurons.

The contribution of C1-adrenergic and nonadrenergic neurons to the spinal projection from the rostral ventrolateral medulla (RVLM) and their relative innervation density throughout thoracic spinal segments were examined by combining the Fluorogold (FG) retrograde tracing technique with immunofluorescent labeling for the epinephrine-synthesis enzyme phenylethanolamine N-methyltransferase (PNMT). The results indicate that the RVLM-spinal projection is comprised of both PNMT-positive and PNMT-negative neurons located in the subretrofacial area of the RVLM, approximately 1 to 1.7 mm rostral to obex. The bulbospinal projection from the RVLM is predominantly ipsilateral, and bulbospinal neurons do not appear to be organized within the RVLM in a manner indicating their segmental termination site. Eighty-one percent (+/- 4%, n = 2) of the PNMT-positive cells in the ipsilateral subretrofacial RVLM were retrogradely labeled after unilateral FG injections into multiple thoracic levels of the intermediolateral cell column (IML). Following single level FG injections, the number of retrogradely labeled PNMT-positive neurons in the subretrofacial RVLM decreased with injections in more caudal thoracic segments, indicating a heavier innervation of the upper thoracic IML by C1 neurons. PNMT-negative neurons were the main component of the RVLM-spinal population with 63 +/- 8% (n = 7) of the non-PNMT-containing neurons within the ipsilateral subretrofacial RVLM innervating all thoracic levels of the IML. The results indicate that both C1-adrenergic and nonadrenergic neurons in the RVLM make a substantial contribution to the innervation of the IML.     

Morphology of sympathetic preganglionic neurons in the thoracic spinal cord of the cat: an intracellular horseradish peroxidase study

Horseradish peroxidase was intracellularly injected into sympathetic preganglionic neurons (SPN) of the third thoracic segment in cats. Seven neurons were reconstructed from serial horizontal or parasagittal sections of the spinal cord. The cell bodies of all neurons were located in the n. intermediolateralis pars principalis (ILp). They were spindle-shaped with the long axis in craniocaudal direction or large and multipolar or small and oval in shape. Preferentially on the cranial and caudal pole of the cell body, five to eight primary dendrites arose from the cell body. Dendritic branches were traced to their terminations at distances up to 1,330 microns from the cell body. The dendritic fields of all SPNs were strictly oriented in the longitudinal direction with a total length of 1,500-2,540 microns. The cranial and caudal dendritic fields were about equal in length but, with one exception, the degree of branching was always greater in the cranial than in the caudal dendritic field. The dendritic fields of all SPNs were primarily restricted to the ILp. In the mediolateral direction it extended from 130 to 360 microns and in the dorsoventral direction from 50 to 180 microns. Only rarely, a higher-order dendrite left the boundaries of the ILp and projected dorsolaterally or laterally into the white matter or ventromedially or medially into the adjacent n. intercalatus. All dendrites showed various forms of spines. At a distance of 132-437 microns from the cell body the axon arose as a direct extension of a process which closely resembled a primary or second-order dendrite. The axons projected ventrally and mostly caudally along the lateral border of the gray matter until they turned laterally at the end of the ventral horn. No axon collaterals were observed.     

Variability in the occurrence of nitric oxide synthase immunoreactivity in different populations of rat sympathetic preganglionic neurons

The proportion of sympathetic preganglionic neurons (SPN) showing nitric oxide synthase (NOS) immunoreactivity appears to vary with innervation target and blood pressure level. For normotensive Sprague‐Dawley rats (SD), we evaluated peroxidase immunolabelling for choline acetyltransferase (ChAT) plus NOS in spinal cord segments T1–L2 and assessed NOS immunofluorescence in SPN retrogradely labelled with cholera toxin B subunit from the adrenal medulla (AM) or superior cervical (SCG), coeliac (CG), or major pelvic (MPG) ganglia. We also compared the distributions and numbers of NOS‐positive and NOS‐negative/ChAT‐positive lateral horn neurons in SD with those in normotensive Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHR). In SD, WKY, and SHR, rostrocaudal, dorsoventral, and mediolateral differences occurred in the distributions of NOS‐positive and NOS‐negative/ChAT‐positive neurons in the intermediolateral cell column (IML), whereas the two groups were similarly distributed throughout the central autonomic area (CAA). Among the four retrogradely labelled populations of SPN, the percentages showing NOS immunoreactivity differed (CG‐projecting, 54.8% ± 0.7%; SCG‐projecting, 75.3% ± 1.2%; MPG‐projecting, 89% ± 1.1% and AM‐projecting, 98.6% ± 0.2%). Within each retrogradely labelled group of SPN, the NOS‐positive proportion also varied with subnuclear location (e.g., 25.5% ± 4.0% of CG‐projecting SPN in the CAA vs. 82.7% ± 7.6% of CG‐projecting SPN in the dorsolateral funiculus). The numbers of NOS‐positive and NOS‐negative/ChAT‐positive neurons in T9–T11 were the same in SD and SHR but differed in WKY. Our results show that the expression of NOS within SPN varies depending on the target that they innervate and also on their subnuclear location. Our data indicate that there are no anatomical differences between nitric oxide‐synthesizing SPN in normotensive SD and hypertensive SHR. J. Comp. Neurol. 514:492–506, 2009. © 2009 Wiley‐Liss, Inc.     

Development of differential preganglionic projections to pre- and paravertebral sympathetic ganglia

Sympathetic preganglionic axons project to spatially distinct targets in the periphery. A precise topographic pattern exists within the thoracic preganglionic cell column relative to the direction of axonal projections within the sympathetic chain. In this study, the time course and pattern of axonal outgrowth from different populations of preganglionic neurons in the chicken embryo is examined in detail to clarify the origin of the topography in this system. Projections to prevertebral targets are established by development of the splanchnic nerves by stage 25, well after the earliest somatic motor projections at stage 19 but at least two stages before the reported onset of paravertebral projections. Further, preganglionic axons that project rostrally into the sympathetic chain may do so earlier than those that project caudally in the chain. The separation of preganglionic axons into prevertebral, rostral paravertebral or caudal paravertebral directions occurs at a common site in the ventral mesenchyme, established by the initial ventromedial projection of the splanchnic nerves. Analysis of the axonal trajectories of rostrally and caudally projecting cells reveals that preganglionic axons are not selectively fasciculated before their point of separation at the sympathetic chain. The patterning of the preganglionic cell column is specified before the establishment of functional connections within the chain, indicating that target contact is not a determinant of the segmental pattern. We suggest that the differential outgrowth of preganglionic axons to peripheral targets is determined by the unique identities of underlying subpopulations of preganglionic axons. J. Comp. Neurol. 382:1-18, 1997. © 1997 Wiley-Liss, Inc.     

Gamma-aminobutyric acid-containing sympathetic preganglionic neurons in rat thoracic spinal cord send their axons to the superior cervical ganglion

Gamma-aminobutyric acid (GABA)-containing fibers have been observed in the rat superior cervical ganglion (SCG) and, to a lesser extent, in the stellate ganglion (STG). The aim of present study is to clarify the source of these fibers. No cell body showed mRNAs for glutamic acid decarboxylases (GADs) or immunoreactivity for GAD of 67 kDa (GAD67) in the cervical sympathetic chain. Thus, GABA-containing fibers in the ganglia are suggested to be of extraganglionic origin. GAD67-immunoreactive fibers were found not in the dorsal roots or ganglia, but in the ventral roots, so GABA-containing fibers in the sympathetic ganglia were considered to originate from the spinal cord. Furthermore, almost all GAD67-immunoreactive fibers in the sympathetic ganglia showed immunoreactivity for vesicular acetylcholine transporter, suggesting that GABA was utilized by some cholinergic preganglionic neurons. This was confirmed by the following results. 1) After injection of Sindbis/palGFP virus into the intermediolateral nucleus, some anterogradely labeled fibers in the SCG were immunopositive for GAD67. 2) After injection of fluorogold into the SCG, some retrogradely labeled neurons in the thoracic spinal cord were positive for GAD67 mRNA. 3) When the ventral roots of the eighth cervical to the fourth thoracic segments were cut, almost all GAD67- and GABA-immunoreactive fibers disappeared from the ipsilateral SCG and STG, suggesting that the vast majority of GABA-containing fibers in those ganglia were of spinal origin. Thus, the present findings strongly indicate that some sympathetic preganglionic neurons are not only cholinergic but also GABAegic.     

Spinal origin of sympathetic preganglionic neurons in the rat

The segmental distribution of sympathetic preganglionic neurons (SPNs) and dorsal root ganglion cells (DRGs) was studied after Fluoro-gold injections into the major sympathetic ganglia and adrenal gland in rats. A quantitative assessment of the segmental and nuclear locations was made. Four general patterns of innervation were apparent: (1) a large number of SPNs (1000–2000/ganglion) innervate the sympathetic ganglia which control head or thoracic organs and a relatively small number of SPNs (100–400/ganglion) innervate the sympathetic ganglia controlling the gut, kidney, and pelvic organs; this difference in density of innervation probably relates to the level of fine control that can occur in these end organs by the SPNs; (2) the reverse pattern is seen in the DRG labeling where a large number of DRGs were labeled after Fluoro-gold injections into the preaortic ganglia (celiac, superior, and inferior mesenteric) and a small number were labeled after injections into the cervical sympathetic ganglia; (3) the intermediolateral cell column is the main source of SPNs except for the inferior mesenteric ganglion which is innervated predominantly by SPNs originating in the central autonomic nucleus (75%); the lateral funiculus is a source of SPNs mainly for the cervical sympathetic ganglia; and (4) each sympathetic ganglion and the adrenal gland receives a multisegmental SPN and DRG input with one segment being the predominant source of the innervation. The adrenal gland shows an intermediate position in terms of the density of SPN input (800 cells) and dorsal root input (300 cells); it has a widespread segmental input (T₄-T₁₂) with the T₈ segment being the major source.     

Responses of adrenal sympathetic preganglionic neurons to stimulation of cardiopulmonary receptors

The current study examined whether or not the activation of Bezold-Jarisch reflex with administration of phenylbiguanide (PBG, 100 microg/kg) into right atrium elicits differential responses in the two populations of adrenal sympathetic preganglionic neurons (SPNs) regulating the release of epinephrine (EPI ADR SPNs) and norepinephrine (NE ADR SPNs), respectively, from adrenal medullary chromaffin cells. Extracellular activity of 48 adrenal SPNs in the intermediolateral cell column (IML) were recorded in urethane/chloralose-anesthetized rats. Twenty-three EPI ADR SPNs and 25 NE ADR SPNs were antidromically activated by stimulation of left adrenal nerve and orthodromically activated by rostral ventrolateral medulla (RVLM) stimulation. At a mean arterial pressure (MAP) of 99. 6+/-2.8 mmHg, the mean spontaneous discharge rates of EPI ADR SPNs and NE ADR SPNs were 6.2+/-0.5 and 4.3+/-0.5 spikes/s, respectively. Intra-atrial PBG markedly inhibited 96% of EPI ADR SPNs (by 3.8+/-0. 4 spikes/s; n=22) and 76% of NE ADR SPNs (by 2.9+/-0.5 spikes/s; n=19) with hypotensive responses (DeltaMAP=33.2+/-5.3 and 26.4+/-5.0 mmHg, respectively). The remaining SPNs were weakly excited or unaffected. We conclude that both groups of SPNs regulating catecholamine release are primarily inhibited by stimulation of cardiopulmonary receptors and that these responses parallel the sympathoinhibitory and hypotensive components of the Bezold-Jarisch reflex.     

Propriospinal input to thoracolumbar sympathetic nuclei from cervical and lumbar lamina I neurons in the cat and the monkey

The possibility that specific thermoreceptive and nociceptive influences on sympathetic outflow are conveyed directly to spinal sympathetic regions by lamina I neurons was investigated anatomically with the immunofluorescent PHA-L technique in the cat and the cynomolgus monkey. Iontophoretic injections made with physiological guidance were restricted to lamina I or to laminae I–II in the cervical (C6–8) or lumbar (L6–7) enlargement. Bilateral (symmetric) terminal arborizations were observed (with an ipsilateral predominance) in the intermdiolateral, intermediomedial, and intervening regions of the thoracolumbar intermediate zone. In serial horizontal sections, patches of terminal labeling appeared at regular longitudinal intervals in the intermediolateral region. Longitudinally coursing fibers that had multiple varicosities and gave off small terminal branches were observed in the intermediolateral and the intermediomedial regions. Mediolateral strips of labeling that extended from labeling in the intermediolateral region to labeling in the intermediomedial region occurred at fairly regular longitudinal intervas. Because the longitudinal distribution of these terminations corresponds very well with the characteristic (ladder-like) longitudinal pattern of organization of the neuropil of the thoracolumbar sympathetic nuclei, i.e., the principal part of the intermediolateral cell column, the central autonomic n., and the intervening n. intercalatus, it is inferred that these lamina I terminations occur within these nuclei. After cervical injections, the labeling was most dense in the upper thoracic T2–4 spinal cord segments in both the cat and the monkey; labeling was also present in the T10–12 segments. After lumbar injections, labeling in the cat was located in the L4 segment; labeling in the monkey was present in the T4–6 and T10–12 segments. The labeling obtained was much more dense in the monkey than in the cat. These observations reveal a spinal lamina I projection that could provide a direct pathway for the somatosympathetic reflex effects of thermal and noxious stimuli. Considered together with reports that lamina I and the sympathetic nuclei both receive descending input from certain key autonomic regions, this result emphasizes the importance of lamina I for homeostasis, in addition to its probable roles in behavioral arousal, affect, and sensation. These observations thus support the proposed concept that lamina I processes and distributes in a functionally specific manner the sensory input relevant to the physiological status of the tissues and organs of the entire organism. © 1993 Wiley-Liss, Inc.     

Different synaptic channel kinetics in sympathetic B and C neurons of the bullfrog

Sympathetic ganglia of the frog contain two types of principal neurons, B and C cells, which are innervated by two distinct classes of cholinergic preganglionic axons, B and C fibers. This study examined and compared the kinetics of nicotinic ACh-gated channels that produce the fast excitatory postsynaptic currents (EPSCs) in B and C cells. Neurons were identified and voltage-clamped at -50 mV, 22 degrees C. The EPSC decayed as a single exponential for both cell types, but the decay time constants differed considerably; 10.2 +/- 2.3 msec (mean +/- SD) for C cells and 5.5 +/- 1.1 msec for B cells. Analysis of ACh-induced membrane current fluctuations revealed spectra that fit single-Lorentzian functions and gave estimates of the mean channel open time of 9.8 +/- 2.0 msec and 5.2 +/- 0.9 msec for C and B cells, respectively. Thus, the clear difference in EPSC decay rate for these two types of ganglion cells is most likely due to the differences in the mean lifetimes of their synaptic channels.     

Afferent and sympathetic neurons projecting into lumbar visceral nerves of the male rat.

The cell bodies of thoracolumbar sensory and sympathetic pre- and postganglionic neurons that project to the colon and pelvic organs of the male rat were labeled retrogradely with horseradish peroxidase (HRP) in order to study numbers, segmental distribution, and location of the somata of these neurons quantitatively. HRP was applied to one hypogastric nerve (HGN), to the lumbar colonic nerves (LCN) and to the intermesenteric nerve (IMN). In order to estimate the significance of the branching of one axon into both hypogastric nerves a double-labeling technique with fluorogold and HRP was used. About 2640 neurons project into the two HGN added together (800 afferent, 1320 pre-, and 520 postganglionic), 4650 neurons into the LCN (360 afferent, 0 pre- and 4290 postganglionic), and 5990 into the IMN (1500 afferent, 1250 pre-, and 3240 postganglionic). About 4190 sympathetic postganglionic prevertebral neurons innervate the colon and pelvic organs, 1900 are located in the inferior mesenteric ganglion and 2290 in ganglia of the IMN. Considering the efferent component, the HGN mainly are preganglionic and the LCN exclusively postganglionic nerves. Branching of one axon into both HGN is a rare event and quantitatively negligible (less than 3%). Afferent neurons of all three nerves were found in the dorsal root ganglia (DRG) T12-L2 with the maximum in L1 and L2. The distribution of afferent neurons projecting into the LCN is shifted slightly more rostrally compared to neurons projecting into the HGN. The IMN distribution is located in a position in between. Preganglionic neurons projecting into the IMN are located in the spinal cord segments T12-L3 with the maximum in L1 and L2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development and migration of avian sympathetic preganglionic neurons.

Modern neuronanatomical techniques were used to investigate the development of the avian sympathetic preganglionic cell column in the spinal cord of the chick embryo. [3H]thymidine autoradiography indicated that the majority of these preganglionic, or "Terni column" neurons are generated between stages 18 and 24 (days 2-4). This coincides with the genesis of the somatic motoneurons in the thoracic levels of the cord, and therefore differences in the time of origin cannot explain the divergent fates of these two neuronal populations. Data obtained from short-survival autoradiographic experiments indicated that many early born cells remain close to the ventral region of the ventricular epithelium until day 5 of incubation. Ventral root injections used to label retrogradely neurons projecting an axon into the ventral root (Terni cells and somatic motoneurons) have labeled neurons next to the ventricular epithelium at the same early stages. Thus, it seems likely that some Terni cells, if not all, maintain medial positions and do not migrate laterally to join a common motor column before initiating a dorsal migration. Analysis of a closely staged series of embryos, whose Terni column neurons were retrogradely labeled with wheat germ agglutinin-horseradish peroxidase (WGA-HRP), revealed that between days 5 and 8 of incubation, Terni column neurons migrated dorsally to attain their adult position adjacent to the central canal. These changes in position were reflected in the changing morphology of the Terni column neurons, visualized by the Golgi-like HRP labeling. The positions of the migrating Terni cells differed from those of commissural cells, indicating that these fibers are not the substrate for the dorsal migration. The dorsal migration of Terni column cells was not disrupted by the surgical removal of the sympathetic ganglia, the synaptic targets of these neurons, nor by disruption of spinal afferents. Taken together, these results suggest that the migratory behavior of Terni cells in distinctive when compared to that of somatic motoneurons, and that local and/or intrinsic cues within the spinal cord guide the dorsal migration of Terni column cells.     

Basal ganglia organization in amphibians: Catecholaminergic innervation of the striatum and the nucleus accumbens

The aim of the present study was to determine the origin of the catecholaminergic inputs to the telencephalic basal ganglia of amphibians. For that purpose, retrograde tracing techniques were combined with tyrosine hydroxylase immunohistochemistry in the anurans Xenopus laevis and Rana perezi and the urodele Pleurodeles waltl. In all three species studied, a topographically organized dopaminergic projection was identified arising from the posterior tubercle/mesencephalic tegmentum and terminating in the striatum and the nucleus accumbens. Although essentially similar, the organization of the mesolimbic and mesostriatal connections in anurans seems to be more elaborate than in urodeles. The present study has also revealed the existence of a noradrenergic projection to the basal forebrain, which has its origin in the locus coeruleus. Additional catecholaminergic afferents to the striatum and the nucleus accumbens arise from the nucleus of the solitary tract, where catecholaminergic neurons appear to give rise to the bulk of the projections to the basal forebrain. In other regions, such as the olfactory bulb, the anterior preoptic area, the suprachiasmatic nucleus, and the thalamus, retrogradely labeled neurons (after basal forebrain tracer-applications) and catecholaminergic cells were intermingled, but none of these centers contained double-labeled cell bodies. It is concluded that the origin of the catecholaminergic innervation of the striatum and the nucleus accumbens in amphibians is largely comparable to that in amniotes. The present study, therefore, strongly supports the existence of a common pattern in the organization of the catecholaminergic inputs to the basal forebrain among tetrapod vertebrates. J. Comp. Neurol. 378:50–69, 1997. © 1997 Wiley-Liss, Inc.     

Identification of spinal neurons involved in the urethrogenital reflex in the female rat

The urethrogenital (UG) reflex is a spinal sexual reflex that consists of autonomic and somatic nerve activity and vaginal, uterine, and anal sphincter contractions. The UG reflex is under tonic descending inhibition by neurons in the region of the nucleus paragigantocellularis (nPGi). The location of spinal neurons activated by the UG reflex was examined in the female rat using the immediate early gene, c-fos. In addition, the descending inputs from the nPGi onto fos-activated neurons was examined using the anterograde tracer biotin dextran amine injected into the nPGi. The UG reflex resulted in a significant increase in fos-positive nuclei in segments T12-S1, compared with experimental 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. Efferent output includes preganglionic neurons located in the lateral gray of L5-S1 and lateral and medial gray of T13-L2. Spinal interneurons involved in the UG reflex were found close to the preganglionic neurons and in the dorsal horn and intermediate and medial gray of T12-S1. NPGi inputs were found primarily on the autonomic efferents and interneurons in the medial and intermediate gray. These studies demonstrate multisegmental spinal circuits activated with the UG reflex and demonstrate that the descending inhibition from the nPGi is by means of preganglionic and somatic efferents and spinal interneurons closely associated with the efferent output.     

Localization of NADPH-diaphorase-containing neurons in sensory ganglia of the rat

The presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity was studied histochemically in the sensory ganglia of the rat. Supraspinally, the trigeminal ganglion possessed only a few cells positively stained for NADPH-diaphorase, while a large number of positive neurons was found in the nodose ganglion. In the dorsal root ganglia, the distribution of positive cells showed a peculiar pattern in relation to spinal levels. Very minor populations (less than 2% of the total ganglionic cells) exhibited positive reaction in ganglia at levels ranging from the first cervical (C1) to fourth thoracic (T4) and from the second lumber (L2) through the entire sacral levels. In the middle to lower thoracic levels (from T5 to L1), however, abundant diaphorase-positive cells were observed. From these positive neurons it was possible to trace intensely stained nerve fibers. In the lower thoracic level, for example, dense positive fibers were seen in the ramus communicans. Retrograde tracing studies revealed that diaphorase-containing neurons in the lower thoracic level project at least partly to the gastric wall and the celiac ganglion. These results indicate that the diaphorase-positive ganglionic neurons in the thoracicolumbar levels may carry autonomic visceral afferent information. Double staining with NADPH-diaphorase histochemistry and peptide immunohistochemistry revealed that NADPH-diaphorase colocalizes with calcitonin gene-related peptide and substance P in many of these visceral afferent neurons.     

Insulin-like growth factor-I counteracts bFGF-induced survival of nitric oxide synthase (NOS)-positive spinal cord neurons after target-lesion in vivo.

We have used nitric oxide synthase (NOS) histochemistry as a perikaryal viability marker to trace the retrograde reaction of spinal cord intermediolateral (IML) sympathoadrenal projection (SAP)-neurons to target-removal, i.e., selective adrenomedullectomy and local administration of either insulin-like growth factor-I (IGF-I), basic fibroblast growth factor (bFGF) or a combination of both. Counting of NOS-positive preganglionic spinal cord neurons 4 weeks post surgery indicated that more than 80% of stained neurons were lost from the IML-cell column. This percentage loss corresponds to the numerical loss of NOS-stained SAP-neurons labeled retrogradely with Fast-blue prior to adrenomedullectomy. Basic FGF-supplementation at the site of lesion resulted in maintenance of the majority of NOS-positive IML-neurons, a finding confirmed by the survival rate of Fast-blue prelabeled SAP-neurons. Thus, besides maintenance of the structural integrity of SAP-neurons, bFGF prevents loss of intracellular NOS-activity which may reflect unaltered cell metabolism (and function) of these neurons following target-removal in vivo. By contrast, IGF-I failed to alter the rate of disappearance of NOS-staining and labeling index of neurons within the IML-cell column postlesion, suggesting that IGF-I is not neurotrophic for SAP-neurons by itself. Combined treatment with both factors resulted in a more widespread loss of NOS-stained and Fast-blue-prelabeled SAP-neurons than registered after bFGF-only treatment. No co-trophic effect of bFGF and IGF-I was evident; rather, the pronounced bFGF-induced rescuing effect was significantly suppressed by exogenous IGF-I in vivo, supporting the idea that this or another molecule induced by the treatment enhances rather than prevents retrograde degeneration and neuronal death within the adult lesioned IML-adrenal pathway.     

Double labeling of the paravertebral sympathetic C system in the bullfrog with antisera to LHRH and NPY

The specificity of synaptic contacts between pre- and postganglionic cells in the sympathetic C system has been examined by immunocytochemical localization of two neuropeptides. Sections of bullfrog paravertebral sympathetic ganglia were stained with antibodies to luteinizing hormone releasing hormone (LHRH) and neuropeptide Y (NPY). Preganglionic synaptic boutons containing LHRH immunoreactivity were found to make contact with a subpopulation of postganglionic cell bodies and with some clusters of small intensely fluorescent (SIF) cells. In ganglia 9 and 10, 95.8% of the neurons contacted by LHRH-containing boutons were also positive for NPY-like immunoreactivity and conversely, 99.3% of the neurons that contained NPY-like immunoreactivity were contacted by LHRH-containing boutons. Qualitatively similar results were found in most other paravertebral ganglia. These observations support the conclusions that preganglionic C axons selectively innervate C-type ganglion cells and that virtually all C-type ganglion cells and some SIF cells receive a direct LHRH input. Moreover, they suggest that a pattern of specific connections between two sets of peptidergic neurons is expressed throughout most of the paravertebral sympathetic chain of the bullfrog.