5-Hydroxytryptamine, substance P and thyrotropin-releasing hormone synapses in the intermediolateral cell column of the rat thoracic spinal cord.

Serotonin-, substance P-, and thyrotropin-releasing hormone-immunoreactive profiles were studied in the intermediolateral cell column at the thoracic level of the rat spinal cord with light- and electron-microscopic immunocytochemistry. For each transmitter, a dense immunoreactive deposit was observed with the light microscope. At ultrastructural level, morphologically identified synapses amounted to 47% of all serotonergic varicosities, to 49% for substance P and 50% for thyrotropin-releasing hormone. Synapses appeared both symmetrical and asymmetrical. In each case, these synapses were mainly axodendritic (98%). These synaptic connections could mediate the physiological influence of these 3 substances in the spinal cord on the cardiovascular system.     

Neuropeptide Y-immunoreactive synapses in the intermediolateral cell column of rat and rabbit thoracic spinal cord

Neuropeptide Y (NPY)-immunoreactive nerve fibers in the intermediolateral cell column of rat spinal cord segments T2-T3 and T8-T10 and rabbit segments T3-T6 were studied with light and electron microscopic immunocytochemistry. Plexuses of NPY-immunoreactive nerve fibers were found by light microscopy. NPY-positive synapses were present electron microscopically but non-immunoreactive synapses greatly outnumbered NPY-immunoreactive ones. In the lateral horn of rat T9, 30% of the vesicle-containing NPY-positive axon profiles formed synapses, 95% of which were axodendritic. These synaptic connections may mediate the effects of brainstem NPY neurons on the activity of sympathetic preganglionic neurons.     

Characterization of trkB immunoreactive cells in the intermediolateral cell column of the rat spinal cord

The objective of the present study was to characterize the trkB receptor immunoreactive (-ir) cells in the intermediolateral cell column (IML) of the upper thoracic spinal cord. Small trkB-ir cells (area = 56.1 ± 4.4 μm²) observed in the IML showed characteristics of oligodendrocytes and were frequently observed in close apposition to choline acetyltransferase (ChAT)-ir cell bodies. Large trkB-ir cells (area = 209.3 ± 25.2 μm²) showed immunoreactivity for the neuronal marker NeuN, indicating their neuronal phenotype, as well as for ChAT, a marker for preganglionic neurons. TrkB and ChAT were co-localized in IML neurons primarily in cases that had received in vivo administration of nerve growth factor (NGF). These findings reveal two different cell types, oligodendrocytes and neurons, in the IML of the spinal cord that show trkB immunoreactivity, suggesting their regulation by brain derived neurotrophic factor (BDNF) and/or neurotrophin-4 (NT-4). In addition, there is evidence that NGF may play a role in the regulation of trkB-ir preganglionic neurons in the IML.     

Evidence for co-existence of thyrotropin-releasing hormone, substance P and serotonin in ventral medullary neurons that project to the intermediolateral cell column in the rat.

The present study was conducted to determine if substance P-, thyrotropin-releasing hormone- and/or serotonin-immunoreactivities coexist in ventral medullary neurons that project to the intermediolateral cell column in the rat. Neurons that projected to the intermediolateral cell column were identified by the presence of retrogradely transported rhodamine bead-labeled microspheres in the cell body after an injection of the microspheres into the intermediolateral cell column of the third thoracic spinal cord segment. Co-existence was determined by using a combination of dual color immunohistochemistry and serial 4-microns sections that were immunostained with different antibodies. Antibodies to substance P, serotonin, and pre-pro-thyrotropin releasing hormone160-169 were used to identify substance P, serotonin and thyrotropin-releasing hormone, respectively. Neurons that contained substance P-, thyrotropin-releasing hormone- and/or serotonin-immunoreactivities and that projected to the intermediolateral cell column were present in the nucleus raphe magnus, the nucleus raphe pallidus, the nucleus reticularis magnocellularis pars alpha, the paragigantocellular reticular nucleus and the parapyramidal region. Neurons that projected to the intermediolateral cell column, in each of these regions, were found to contain each of the following combinations of immunoreactive neurochemicals: substance P and thyrotropin-releasing hormone: substance P and serotonin; thyrotropin-releasing hormone and serotonin; or substance P, thyrotropin-releasing hormone and serotonin. In addition, most of the regions also contained neurons that appeared to contain only one of the neurochemicals and that also projected to the intermediolateral cell column. The greatest number of neurons that projected to the intermediolateral cell column and that also contained two or more co-existing neurochemicals was present in the midline regions. This study demonstrates the presence of neurons in the ventral medulla that project to the intermediolateral cell column and contain three co-existing neurochemicals. This study also demonstrates the use of a new method for the localization of three neurochemicals in single projection-specific neurons.     

Bulbospinal thyrotropin-releasing hormone projections to the intermediolateral cell column: a double fluorescence immunohistochemical-retrograde tracing study in the rat

Whereas the neurochemistry of the peripheral autonomic nervous system has been well characterized, less is known concerning the neurotransmitters utilized by medullary projections onto sympathetic preganglionic neurons residing in the thoracolumbar spinal intermediolateral cell column. Retrograde transport of rhodamine-labeled fluorescent microspheres following discrete microinjection into the thoracic intermediolateral cell column was combined with immunohistochemistry to determine neuroanatomic location of thyrotropin-releasing hormone-immunoreactive neurons which project to the intermediolateral cell column in the rat. The ventromedial group of raphe nuclei including the nucleus raphe pallidus, obscurus, and magnus possessed the greatest number of medullary thyrotropin-releasing hormone-immunoreactive neurons which also contained rhodamine-labeled microspheres. High numbers of intermediolateral cell column-projecting thyrotropin-releasing hormone-immunoreactive neurons were also observed in nucleus reticularis paragigantocellularis lateralis and magnocellularis, the lateral reticular nucleus, and the superficial ventral (arcuate) medullary surface. Despite the observations that nucleus reticularis gigantocellularis, paramedianus, and ventralis pars beta project to the intermediolateral cell column, double-labeled cells were not observed in these nuclei. Furthermore, whereas the nucleus reticularis magnocellularis and gigantocellularis, and the lateral reticular nucleus displayed strong ipsilateral predominance in projecting to the intermediolateral cell column, other medullary reticular and raphe nuclei displayed bilateral projections. The present findings support the hypothesis that thyrotropin-releasing hormone-containing neurons in the ventral medulla project to the intermediolateral cell column, and may influence sympathetic preganglionic neurons.     

Evidence for entorhinal and parietal cortices involvement in path integration in the rat

Rats with lesions of the entorhinal or parietal cortex were tested in a homing task on a circular platform containing food cups and surrounded by curtains. The animals had to leave a refuge, explore the platform to find a hidden piece of food and carry it back to the refuge. Once the rats were proficient at performing the procedural aspects of the task, they were tested in two successive types of trials in which the food pellet was either always located in the central cup (food at center, FAC trials) or placed in a randomly chosen cup (food at random, FAR trials). Except in the first FAC trials, all groups displayed similar outward paths in FAC and FAR trials, showing that both types of trials involved equivalent path integration demand. Analysis of the homing accuracy showed that rats with entorhinal cortex or parietal cortex lesions exhibited inaccurate returns to the starting hole, suggesting that these two cortical areas are part of a neural network mediating path integration.     

Calcitonin gene-related peptide-like immunoreactivity in spinal cord and superior cervical ganglion of the djungarian hamster (Phodopus sungorus)

The indirect immunofluorescent method was employed to investigate the distribution of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in the spinal cord and superior cervical ganglion of the Djungarian hamster Phodopus sungorus. In cross-sections of the spinal cord, immunoreactive fibres and terminals were found in laminae 1 and 2 in high density, in the dorsolateral (Lissauer's) tract, in ventral and lateral horns, and in the area surrounding the central canal. A few CGRP-LI perikarya were seen in the ventral but not the dorsal horn. CGRP-LI was further observed in preganglionic sympathetic neurons which were labelled by retrograde axonal transport of fluoro-gold (FG) following injection of the substance unilaterally into the superior cervical ganglion. Preganglionic sympathetic neurons (PSN) were localized ipsilateral to the injection site mainly in the intermediolateral nucleus and the lateral funiculus of the upper thoracic segments. Most PSN exhibited CGRP-LI. Immunoreactive PSN were not seen contralaterally to the site of FG application nor in animals that did not receive injections. When the preganglionic fibres were ligated 4 days before perfusion. CGRP-LI cell bodies were found in preganglionic sympathetic neurons similar to the situation seen upon FG treatment.

In the superior cervical ganglia of untreated hamsters, immunoreactive fibres were seen to enter the ganglion in which they terminated at non-immunoreactive principal ganglion cells.

The present study, the first in a hamster species, describes the widespread distribution of CGRP in the spinal cord of P. sungorus and supports the view that considerable interspecies differences exist in occurrence and location of this neuropeptide.     

Presence of neurons with GABA-like immunoreactivity in the superior cervical ganglion of the rat

Using antibodies raised against gamma-aminobutyric acid (GABA)-glutaraldehyde complexes, we have found neurons with GABA-like immunoreactivity in the superior cervical ganglion of adult rats. The processes of these neurons formed pericellular networks around the principal ganglion cells. Electron microscopy revealed that the immunoreactive dendrites were innervated by non-reactive axon terminals which formed asymmetrical synapses and probably originated from the preganglionic nerve. Axons with GABA-like immunoreactivity, especially axonal varicosities filled with synaptic vesicles, were found in direct apposition to principal ganglion cells. The GABA-positive axons and axon varicosities persisted in experimentally decentralized (deafferented) ganglia, suggesting that the perikarya of the immunoreactive neurons were intrinsic to the superior cervical ganglion. Taken together with data on inhibitory effects of GABA in sympathetic ganglia, these findings suggest that the superior cervical ganglion of rats contains a subpopulation of inhibitory interneurons which is GABAergic. This would indicate that GABAergic neurons do not only occur in the central but also in the peripheral nervous system.     

Properties of interneurones in the intermediolateral cell column of the rat spinal cord: role of the potassium channel subunit Kv3.1

Sympathetic preganglionic neurones located in the intermediolateral cell column (IML) are subject to inputs descending from higher brain regions, as well as strong influences from local interneurones. Since interneurones in the IML have been rarely studied directly we examined their electrophysiological and anatomical properties. Whole cell patch clamp recordings were made from neurones in the IML of 250 microM slices of the thoracic spinal cord of the rat at room temperature. Action potential durations of interneurones (4.2+/-0.1 ms) were strikingly shorter than those of sympathetic preganglionic neurones (9.4+/-0.2 ms) due to a more rapid repolarisation phase. Low concentrations of tetraethylammonium chloride (TEA) (0.5 mM) or 4-aminopyridine (4-AP) (30 microM) affected interneurones but not sympathetic preganglionic neurones by prolonging the action potential repolarisation as well as decreasing both the afterhypolarisation amplitude and firing frequency. Following recordings, neurones sensitive to TEA and 4-AP were confirmed histologically as interneurones with axons that ramified extensively in the spinal cord, including the IML and other autonomic regions. In contrast, all cells that were insensitive to TEA and 4-AP were confirmed as sympathetic preganglionic neurones. Both electrophysiological and morphological data are therefore consistent with the presence of the voltage-gated potassium channel subunit Kv3.1 in interneurones, but not sympathetic preganglionic neurones. Testing this proposal immunohistochemically revealed that Kv3.1b was localised in low numbers of neurones within the IML but in higher numbers of neurones on the periphery of the IML. Kv3.1b-expressing neurones were not sympathetic preganglionic neurones since they were not retrogradely labelled following intraperitoneal injections of Fluorogold. Since Kv3.2 plays a similar role to Kv3.1 we also tested for the presence of Kv3.2 using immunohistochemistry, but failed to detect it in neuronal somata in the spinal cord. These studies provide electrophysiological and morphological data on interneurones in the IML and indicate that the channels containing the Kv3.1 subunit are important in setting the firing pattern of these neurones.     

Effects of combined entorhinal cortex-hippocampal lesions on locomotor behavior, spontaneous alternation and spatial maze learning in the rat

Male Sprague-Dawley rats with combined lesions to the dorsal and lateral aspects of the entorhinal cortex in one hemisphere and of the contralateral dorsolateral hippocampal formation were compared with both operated and unoperated controls on three different behaviors, monitored across a 53 day postoperative period. The rats with the combined entorhinal cortex-hippocampal lesions (EH) showed transient hyperactivity in the open field, transient reduction in spontaneous alternation levels in an unbaited T-maze and persistent deficits in learning spatial maze problems. The results of the present experiment are discussed in comparison with those from experiments on rats with bilateral hippocampal lesions and those from experiments on rats with bilateral entorhinal cortex lesions. Although some similarities among these findings suggest that these two brain regions probably function in a coordinated fashion with respect to these behaviors, differences in the various syndromes are also discussed.     

Somatostatin immunoreactive cell bodies in the dorsal horn and the parasympathetic intermediolateral nucleus of the rat spinal cord

Relative changes in the amount of galactocerebroside (GC) during development were measured in mouse brain cell cultures at different stages of development. For this purpose we used an ¹²⁵I-labelled protein A indirect assay modified in the respect that the total amount of cellular proteins was evaluated before counting the radioactivity. The amount of GC greatly increased between the 10th and the 14th day of culture, then a steady state was reached between the 14th and the 20th day of culture. This change correlates well with the dynamics of the number of oligodendrocytes we observed earlier. These data suggests that the increase of the GC amount in culture during development corresponds to the increase in the number of GC-positive oligodendrocytes rather than to the increase in the number of GC molecules per cell.     

Immunohistochemical localization of serotonin nerve terminals in the lateral entorhinal cortex of the rat: Demonstration of two separate patterns of innervation from the midbrain raphe

Summary The distribution of serotonin (5-hydroxytryptamine, 5HT) containing nerve terminals in the lateral entorhinal cortex (LEC) has been studied using antibodies against 5-HT in combination with fluorescence histochemistry. Thin, varicose, branching fibers were found to be distributed in a relatively even, diffuse pattern throughout all layers of the LEC. The largest amount of this type of 5HT innervation was in Layer I. This diffuse pattern of 5-HT terminals was supplemented by a dense network of 5HT terminals restricted to Layer III of a small (1 mm) strip of the LEC. The fibers in this layer were thicker and more convoluted and contained larger varicosities than fibers in any other layer. The existence of a distinct innervation by 5-HT terminals of only a small portion of the LEC demonstrates a hitherto unrecognized and important principle of heterogeneity in 5-HT innervation of cortex. It suggests that 5-5HT neurons in the raphe can selectively influence specific, narrow regions of the lateral perforant path system, which, in turn, affect the intrinsic hippocampal circuits.This work was supported in part by U.S. Public Health Service Grants NS 10536, NS 14740, a Louise Harkness Ingalls Fellowship in Research in Parkinson's Disease and an Alfred P. Sloan Foundation Fellowship in Neuroscience, and a White House Fellowship at the Department of Defense of the U.S. Government (VC-P)     

Effects of guanethidine on paraganglionic cells in the superior cervical ganglion of the rat.

Paraganglionic cells in the rat superior cervical ganglion were investigated by fluorescence and electron microscopy following treatment with guanethidine for 5-30 days. Control animals received saline and guanidine. Fluorescence cytophotometric measurements revealed a general decrease in the catecholamine content of paraganglionic cells in guanethidine-treated animals. However a few cell clusters showed focal increases. Similarly by electron microscopy there was a general decrease of cell clusters showing increases. -- guanethidine -- as well as guanidine--treated animals showed non-specific cytological alterations such as mitochondrial swelling and increase of cytoplasmic glycogen. However no changes of catecholamine contents and of dense core vesicles were noted in control animals. These results confirm the conception that in rat paraganglionic cells the dense core vesicles are the main storage site of catecholamines. The marked difference in the response of some cell culsters to the experimental treatment can be considered as evidence of functional heterogeneity of this cell population in the rat superior cervical ganglion.     

Coexistence of serotonin- and substance P-like immunoreactivity in nerve fibers apposing identified sympathoadrenal preganglionic neurons in rat intermediolateral cell column

In this study we examined the possibility that serotonin (5-HT) and substance P (SP) coexist in fibers and terminals afferent to sympathoadrenal preganglionic (SAP) neurons in the intermediolateral cell column (IML) of the spinal cord. SAP neurons in the IML were identified by retrograde labeling with either Fast Blue or True Blue injected into the adrenal medulla of rats. A simultaneous immunofluorescent double labeling technique was used to identify both 5-HT- and SP-like immunoreactivity in single tissue sections. Labeled SAP neurons were observed which were apposed by fibers immunoreactive for either neurotransmitter, as well as SAP neurons apposed by neither 5-HT- nor SP-like immunoreactive structures. In addition, 5-HT- and SP-like immunoreactivity were observed in separate fibers apposing the same labeled neuron and coexisting in fibers and terminal appearing in apposition to labeled SAP neurons. These data suggest a complex interaction by these neurotransmitters in regulating sympathetic outflow and may provide a model for interpreting conflicting observations concerning the effects of local 5-HT administration on sympathetic nerve activity.     

Effects of ryanodine on the spike after-hyperpolarization in sympathetic neurones of the rat superior cervical ganglion

Effects of ryanodine on sympathetic neurones of the rat superior cervical ganglion were investigated by means of intracellular recording. Ryanodine (1 M) significantly shortened the after-hyperpolarization (AH) following the spike evoked by current injection or pre-ganglionic stimulation without affecting the configuration of the spikes. The shortening of AH caused by ryanodine was dose-dependent at concentrations between 0.1 and 1 M and was slowly recovered by washing the tissue over 1 h. A partial inhibition of the apamin-sensitive slow component of AH was the maximal effect obtained at 1 M. Although the input membrane resistance was not changed, ryanodine evoked repetitive discharges at long intervals in response to long depolarizing current pulses applied across the cell membrane. Ryanodine (5 M) did not depress the Ca-spike but shortened the following AH in a lesser degree than that following the normal spike. Spontaneous small fluctuations of the resting membrane potential were occasionally observed under normal conditions. They were facilitated by caffeine and abolished by ryanodine. Caffeine also enhanced the slow component of the AH but did not affect it in the presence of ryanodine. These results suggest that ryanodine inhibits Ca release from intracellular store sites. The released Ca may contribute to generating the long-lasting AH and to regulating the excitability of rat sympathetic neurones.     

Zinc modulates primary afferent fiber-evoked responses of ventral roots in neonatal rat spinal cord in vitro

Zinc ions (Zn(2+)) are known to modulate the functions of a variety of channels, receptors and transporters. We examined the effects of Zn(2+) on the reflex potentials evoked by electrical stimulation and responses to depolarizing agents in the isolated spinal cord of the neonatal rat in vitro. Zn(2+) at low concentrations (0.5-2 microM) inhibited, but at high concentrations (5 and 10 microM) augmented, a slow depolarizing component (slow ventral root potential). Zn(2+) had no effect on fast components (monosynaptic reflex potential; fast polysynaptic reflex potential). Unlike Zn(2+), strychnine (5 microM), a glycine receptor antagonist, and (S),9(R)-(-)-bicuculline methobromide (10 microM), a GABA(A) receptor antagonist, potentiated both fast polysynaptic reflex potential and slow ventral root potential. Zn(2+) (5 microM) did not affect depolarizing responses to glutamate and N-methyl-D-aspartate. Zn(2+) enhanced the substance P-evoked depolarization in the absence of tetrodotoxin (0.3 microM) but not in its presence. The dorsal root potential was inhibited by (S),9(R)-(-)-bicuculline methobromide (10 microM) but not by Zn(2+) (5 microM). The Zn(2+)-potentiated slow ventral root potential was inhibited by the N-methyl-D-aspartate receptor antagonists, ketamine (10 microM) and DL-2-amino-5-phosphaonovaleric acid (50 microM) but not by P2X receptor antagonists, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (30 microM) and 2',3'-O-(2,4,6-trinitrophenyl)ATP (10 microM). Ketamine (10 microM) and DL-2-amino-5-phosphaonovaleric acid (50 microM) almost abolished spontaneous activities increased by Zn(2+). It is concluded that Zn(2+) potentiated slow ventral root potential induced by primary afferent stimulation, which was mediated by the activation of N-methyl-D-aspartate receptors but not by activation of P2X receptors or blockade of glycinergic and GABAergic inhibition. Zn(2+) does not seem to directly affect N-methyl-D-aspartate receptors. The release of glutamate from interneurons may play an important role in Zn(2+)-induced potentiation of slow ventral root potential in the spinal cord of the neonatal rat.     

Interaction of thyrotropin-releasing hormone and methysergide on the monosynaptic reflex in isolated mammalian spinal cord

The interaction between thyrotropin-releasing hormone (TRH) and methysergide (MeSG) on reflex activity was examined in spinal cords from neonatal rats. MeSG depressed the monosynaptic reflex (MSR) by nearly 90% at 0.03 microM but had no effect on the dorsal root reflex at 0.003-3.0 microM. Neither spiperone, ketanserin, cyproheptadine nor ICS 205-930 (3-tropanyl-indole-3-carboxylate) depressed the MSR nor did they affect the potentiation elicited by TRH. TRH reversed the depression of the MSR by MeSG in a concentration-dependent manner without affecting the dorsal root reflex. MeSG-induced depression of the MSR was also reversed by 3,4-diaminopyridine which simultaneously increased the magnitude and duration of both reflexes. It appears that neither MeSG-induced depression nor TRH-induced potentiation of the MSR involves the spinal serotonergic system or blockade of K+ channels.     

Voltage-sensitivity of motoneuron NMDA receptor channels is modulated by serotonin in the neonatal rat spinal cord

Both N-methyl-D-aspartate (NMDA) and serotonin (5-HT) receptors contribute to the generation of rhythmic motor patterns in the rat spinal cord. Co-application of these chemicals is more effective at producing locomotor-like activity than either neurochemical alone. In addition, NMDA application to rat spinal motoneurons, synaptically isolated in tetrodotoxin, induces nonlinear membrane behavior that results in voltage oscillations which can be blocked by 5-HT antagonists. However, the mechanisms underlying NMDA and 5-HT receptor interactions pertinent to motor rhythm production remain to be determined. In the present study, an in vitro neonatal rat spinal cord preparation was used to examine whether NMDA receptor-mediated nonlinear membrane voltage is modulated by 5-HT. Whole-cell recordings of spinal motoneurons demonstrated that 5-HT shifts the region of NMDA receptor-dependent negative slope conductance (RNSC) of the current-voltage relationship to more hyperpolarized potentials and enhances whole-cell inward current. The influence of 5-HT on the RNSC was similar to the effect on the RNSC of decreasing the extracellular Mg(2+)concentration. The results suggest that 5-HT may modulate this form of membrane voltage nonlinearity by regulating Mg(2+) blockade of the NMDA ionophore.     

Direct and indirect actions of thyrotropin-releasing hormone on neonatal rat motoneurons in vitro

In addition to causing a slow depolarization, thyrotropin-releasing hormone (TRH, 5-10 microM) evoked synaptic activity in antidromically identified motoneurons in thin transverse neonatal rat spinal cord slices. The synaptic activity but not the slow depolarization was reversibly abolished by TTX or low Ca2+/high Mg2+ solution. Furthermore, the TRH-induced synaptic activity was eliminated by the glutamate receptor antagonist kynurenic acid (0.5 or 1 mM) and/or the glycine receptor antagonist strychnine (1 microM). Our results indicate that in addition to depolarizing motoneurons, TRH modifies the activity of spinal interneurons which may secondarily alter the activity of motoneurons.