Effects of spinal lesions on substance P levels in the rat sympathetic preganglionic cell column: evidence for local spinal regulation

Substance P has been localized to the neuropil of sympathetic preganglionic neurons in light and electron microscopic studies. Two recent reports have suggested that the majority of substance P in the rat intermediolateral cell column was contained in synaptic terminals of bulbospinal axons. However, previous investigations in our laboratory indicated the presence of major substance P spinal-sympathetic preganglionic neuron circuitry in pigeon. The present study used radioimmunoassay and immunohistochemistry to examine substance P levels in rat intermediolateral cell column following various spinal lesions in order to assess the relative contributions of bulbospinal and intraspinal substance P neurons to the substance P content of the intermediolateral cell column. The results from these experiments support the existence of both bulbospinal and intraspinal substance P-containing projections to the rat intermediolateral cell column. In addition, characterization of spinal cord substance P-like immunoreactivity by combined high performance liquid chromatography and radioimmunoassay, revealed that substance P in rat intermediolateral cell column was indistinguishable from synthetic substance P. Following transection of thoracic spinal cord, substance P-immunoreactive staining was still evident in the intermediolateral cell column caudal to the lesion. These substance P-positive fibers were studded with bouton-like swellings and appeared normal. Following high cervical hemisection, depletion of substance P (radioimmunoassay measurements) was bilateral and equal in the intermediolateral cell column: 25% depletion was observed after 7 days and 35% depletion after 14 days. However, rats which were hemisected at low cervical and/or mid-thoracic levels contained normal or elevated amounts of substance P in the intermediolateral cell column. Since substance P remains in the intermediolateral cell column following total transection, substance P spinal-sympathetic preganglionic neuron circuitry must exist. Additionally, depletion of substance P following high cervical hemisection suggests the existence of a substance P-containing, bilateral bulbospinal pathway to the intermediolateral cell column. The observation that substance P levels were normal or elevated following low cervical lesions raises the possibility that intraspinal substance P neurons can compensate for loss of substance P in the spinal cord. Sprouting or altered substance P metabolism and/or release by intraspinal substance P neurons could be responsible, suggesting an important homeostatic mechanism for maintaining substance P content within the intermediolateral cell column.     

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.     

Enkephalin-containing sympathetic preganglionic neurons projecting to the inferior mesenteric ganglion: Evidence from combined retrograde tracing and immunohistochemistry

The origin of fibers containing enkephalin immunoreactivity in the inferior mesenteric ganglion of the guinea-pig was studied by combining retrograde axonal tracing and indirect immunofluorescence techniques. Fast Blue was applied into the inferior mesenteric ganglion. Three days later colchicine was administered into the subarachnoid space in order to increase the peptide content of the spinal cord cell bodies. The drug was injected through a catheter which was inserted into the cisterna magna and moved to the appropriate spinal cord levels. After the colchicine injection the animals were perfused with formalin and the L₂-L₃ spinal cord segments were dissected. Cryostat sections of the spinal cord were analyzed in a fluorescence microscope and subsequently processed for indirect immunohistochemistry using antiserum against enkephalin. Several sympathetic pregnanglionic neurons containing both Fast Blue and enkephalin-like immunoreactivity were seen mainly in the intermediolateral cell column of the cord.The observations strongly support the view that at least some of the enkephalin-containing fibers in the inferior mesenteric ganglion originate in the sympathetic preganglionic nuclei of the spinal cord. These findings are discussed in view of recent physiological studies which have shown that enkephalin may have a presynaptic inhibitory action on preganglionic neurons as well as on substance P containing primary afferent neurons in the inferior mesenteric ganglion.     

Oxytocinergic innervation of autonomic nuclei controlling penile erection in the rat.

In the rat, spinal autonomic neurons controlling penile erection receive descending pathways that modulate their activity. The paraventricular nucleus of the hypothalamus contributes oxytocinergic fibers to the dorsal horn and preganglionic sympathetic and parasympathetic cell columns. We used retrograde tracing techniques with pseudorabies virus combined with immunohistochemistry against oxytocin and radioligand binding detection of oxytocinergic receptors to evidence the oxytocinergic innervation of thoracolumbar and lumbosacral spinal neurons controlling penile erection. Spinal neurons labelled with pseudo-rabies virus transsynaptically transported from the corpus cavernosum were present in the intermediolateral cell column and the dorsal gray commissure of the thoracolumbar and lumbosacral spinal cord. Confocal laser scanning microscopic observation of the same preparations revealed close appositions between oxytocinergic varicosities and pseudorabies virus-infected neurons, suggesting strongly the presence of synaptic contacts. Electron microscopy confirmed this hypothesis. Oxytocin binding sites were present in the superficial layers of the dorsal horn, the dorsal gray commissure and the intermediolateral cell column in both the thoracolumbar and lumbosacral segments. In rats, stimulation of the paraventricular nucleus induces penile erection, but the link between the nucleus and penile innervation remains unknown. Our findings support the hypothesis that oxytocin, released by descending paraventriculo-spinal pathways, activates proerectile spinal neurons.     

Studies on the cellular localization of spinal cord substance P receptors

Substance P-immunoreactivity and specific substance P binding sites are present in the spinal cord. Receptor autoradiography showed the discrete localization of substance P binding sites in both sensory and motor regions of the spinal cord and functional studies suggested an important role for substance P receptor activation in autonomic outflow, nociception, respiration and somatic motor function. In the current studies, we investigated the cellular localization of substance P binding sites in rat spinal cord using light microscopic autoradiography combined with several lesioning techniques. Unilateral injections of the suicide transport agent, ricin, into the superior cervical ganglion reduced substance P binding and cholinesterase-stained preganglionic sympathetic neurons in the intermediolateral cell column. However, unilateral electrolytic lesions of ventral medullary substance P neurons which project to the intermediolateral cell column did not alter the density of substance P binding in the intermediolateral cell column. Likewise, 6-hydroxydopamine and 5,7-dihydroxytryptamine, which destroy noradrenergic and serotonergic nerve terminals, did not reduce the substance P binding in the intermediolateral cell column. It appears, therefore, that the substance P binding sites are located postsynaptically on preganglionic sympathetic neurons rather than presynaptically on substance P-immunoreactive processes (i.e. as autoreceptors) or on monoamine nerve terminals. Unilateral injections of ricin into the phrenic nerve resulted in the unilateral destruction of phrenic motor neurons in the cervical spinal cord and caused a marked reduction in the substance P binding in the nucleus. Likewise, sciatic nerve injections of ricin caused a loss of associated motor neurons in the lateral portion of the ventral horn of the lumbar spinal cord and a reduction in the substance P binding. Sciatic nerve injections of ricin also destroyed afferent nerves of the associated dorsal root ganglia and increased the density of substance P binding in the dorsal horn. Capsaicin, which destroys small diameter primary sensory neurons, similarly increased the substance P binding in the dorsal horn. These studies show that the cellular localization of substance P binding sites can be determined by analysis of changes in substance P binding to discrete regions of spinal cord after selective lesions of specific groups of neurons. The data show the presence of substance P binding sites on preganglionic sympathetic neurons in the intermediolateral cell column and on somatic motor neurons in the ventral horn, including the phrenic motor nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of substance P on sympathetic preganglionic neurones

Antidromically identified sympathetic preganglionic neurones, located in the second thoracic segment of the rat spinal cord, were tested for their response to iontophoretically applied substance P. Substance P increased the firing rate of both 'spontaneously' active and glutamate-activated neurones. As substance P-like immunoreactivity is present in the intermediolateral cell column of the thoracic spinal cord, it is concluded that substance P may be an excitatory transmitter or modulator involved in mediating excitatory drive to sympathetic preganglionic neurones.     

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.     

大白鼠脊髓中间带内5-羟色胺免疫反应纤维的分布

本文用免疫细胞化学卵白素-生物素-过氧化物酶复合体(Avidin-Biotin PeroxidaseComplex, ABC)法显示大白鼠脊髓中间带内5-羟色胺(5-HT)免疫反应纤维的分布。5-HT纤维见于交感节前神经元所在的各亚核区。在含中间外侧核的侧角内,5-HT纤维呈簇状分布,各簇之间以纵行纤维联系。纤维簇向外连于外侧索内的5-HT纤维,向内成横束经中间灰质至中央管周围。5-HT纤维在中央管背方较多,靠中线者趋于纵行。中央管室管膜下也有5-HT纤维,紧贴中央管两旁则形成两条纵向的5-HT纤维束。在含交感节前神经元的各节段内,5-HT纤维在中间带的分布形式虽然基本相似,但在各节段侧角内5-HT纤维簇的大小和疏密,以及中间带内横行纤维束的粗细和间隔大小等又有各自的特点。这些特点与各节段中交感节前神经元的分布形式和多寡是相互一致的。     

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.     

Intraspinal substance P-containing projections to the sympathetic preganglionic neuropil in pigeon, Columba livia: high-performance liquid chromatography, radioimmunoassay and electron microscopic evidence

The present study uses quantitative and electron microscopic methods to investigate the hypothesis that intraspinal substance P-sympathetic preganglionic neuron circuitry exists in vertebrates. Radioimmunoassay and high-performance liquid chromatography were used to: (1) characterize the chemical nature of the substance P-like immunoreactivity in the sympathetic preganglionic neuropil; and (2) quantify the relative contributions of brain stem, primary sensory and intraspinal neurons to the substance P content within the sympathetic preganglionic neuropil. Electron microscopic observations on the localization of substance P-like immunoreactivity within the preganglionic neuropil caudal to complete thoracic spinal cord transections are also reported. High-performance liquid chromatographic analyses demonstrate that pigeon substance P-like immunoreactivity co-migrates with synthetic substance P, suggesting that the substance P-like material is authentic substance P content within the sympathetic preganglionic neuropil. Electron microscopic observations on the localization of substance P-like immunoreactivity within the preganglionic neuropil caudal to complete preganglionic cell column (inclusive of intermediate spinal laminae V and VII as well as preganglionic neurons located within nucleus intercalatus spinalis); (2) cutting the dorsal rootlets entering the last cervical (C14) and first two thoracic (T1, T2) spinal segments resulted in massive depletion of substance P content in dorsal horn of T1, but no detectable losses within the preganglionic cell column or ventral horn of T1; and (3) total mid-thoracic (T3-4) spinal cord transection significantly depleted the substance P content in the preganglionic cell column (T3-4) as well as in the dorsal (T1-4) and ventral horns (T2-4). Ultrastructural examination of the sympathetic preganglionic neuropil caudal to spinal transections (survival times of 3-14 days) revealed the presence of numerous, intact, normal appearing substance P-like immunoreactive terminals. Immunolabeled terminals formed asymmetric contacts on medium-sized and small caliber dendrites. Extensive degeneration was evident in this material as well. The ultrastructural features of degenerating processes were distinctive and quite dissimilar in appearance from those exhibiting substance P-like immunoreactive staining. No evidence for damage-induced sequestration of substance P-like material into glial elements was found. The above observations are consistent with earlier findings in rat and pigeon, and provide new quantitative and qualitative evidence to support the hypothesis that intraspinal substance P-containing interneurons contribute t     

Secretoneurin-immunoreactivity in nerve terminals apposing identified preganglionic sympathetic neurons in the rat: colocalization with substance P and enkephalin

Preganglionic sympathetic neurons projecting to the superior cervical ganglion are innervated by nerve fibers containing classical neurotransmitters as well as neuropeptides. In this study we examined the possible participation of a novel peptide, secretoneurin (a cleavage product of secretogranin II), in regulation of sympathetic outflow to head and neck by using a retrograde labelling-technique combined with immunohistochemistry. In addition, the coexistence of secretoneurin with substance P and leu-enkephalin, peptides known to innervate preganglionic neurons, was investigated. The majority of retrogradely labeled neurons were localized in the nucleus intermediolateralis of spinal cord segments T1–T3 (maximum at T2). Nearly all of Fast Blue positive neuronal perikarya were apposed by nerve fibers and terminals exhibiting immunoreactivity to secretoneurin. The main secretoneurin-immunoreactive form found in the upper thoracic segments corresponded to the free peptide secretoneurin as revealed by chromatography and radioimmunoassay. More than half of labeled neurons were surrounded by nerve endings containing in addition substance P or leu-enkephalin which were also, however, less frequently colocalized. Our results suggest that secretoneurin influences the activity of preganglionic sympathetic neurons projecting to the superior cervical ganglion. Regarding their frequent colocalization with substance P and leu-enkephalin, functional interactions of these peptides on preganglionic sympathetic nerve activity have to be considered.     

alpha(2a) and alpha(2c) adrenoceptors on spinal neurons controlling penile erection

The thoracolumbar and lumbosacral spinal cord contain respectively sympathetic and parasympathetic preganglionic neurons that supply the organs of the pelvis including the penis. These neurons are influenced by supraspinal information and receive aminergic projections from the brainstem. The presence of the alpha(1)- and alpha(2)-adrenoceptor subtypes has been demonstrated in the rat spinal cord. In this species, we looked for the presence of alpha(2a)- and alpha(2c)-adrenoceptor subtypes in the sympathetic and parasympathetic preganglionic neurons controlling erection. In adult male rats, transsynaptic axonal transport of pseudorabies virus injected into the penis was combined with immunohistochemistry against alpha(2a)- and alpha(2c)-adrenoceptor subtypes. At 4 days survival time, neurons infected with the pseudorabies virus were solely found in the intermediolateral cell column and dorsal gray commissure of segment T12-L2 and in the intermediolateral cell column of segment L6-S1. Neurons and fibers immunoreactive for alpha(2a)- and alpha(2c)-adrenoceptor subtypes were mainly present in the intermediolateral cell column, the dorsal gray commissure and the ventral horn of the T12-L2 and L5-S1 spinal cord, the dorsal horn displayed only immunoreactive fibers. Pseudorabies virus-infected neurons in the autonomic nuclei were both immunoreactive for alpha(2a)- and alpha(2c)-adrenoceptor subtypes and closely apposed by alpha(2a)- and alpha(2c)-immunoreactive fibers.The results suggest an intraspinal modulation of the noradrenergic and adrenergic control of the autonomic outflow to the penis by pre- and postsynaptic alpha(2) adrenoceptors.     

Distribution of five peptides,three general neuroendocrine markers,and two synaptic-vesicle-associated proteins in the spinal cord and dorsal root ganglia of the adult and newborn dog: An immunocytochemical study

This study describes the immunocytochemical distribution of five neuropeptides (calcitonin gene-related peptide [CGRP], enkephalin, galanin, somatostatin, and substance P), three neuronal markers (neurofilament triplet proteins, neuron-specific enolase [NSE], and protein gene product 9.5), and two synaptic-vesicle-associated proteins (synapsin I and synaptophysin) in the spinal cord and dorsal root ganglia of adult and newborn dogs. CGRP and substance P were the only peptides detectable at birth in the spinal cord; they were present within a small number of immunoreactive fibers concentrated in laminae I–II. CGRP immunoreactivity was also observed in motoneurons and in dorsal root ganglion cells. In adult animals, all peptides under study were localized to varicose fibers forming rich plexuses within laminae I–III and, to a lesser extent, lamina X and the intermediolateral cell columns. Some dorsal root ganglion neurons were CGRP- and/or substance P-immunoreactive. The other antigens were present in the spinal cord and dorsal root ganglia of both adult and newborn animals, with the exception of NSE, which, at birth, was not detectable in spinal cord neurons. Moreover, synapsin I/synaptophysin immunoreactivity, at birth, was restricted to laminae I–II, while in adult dogs, immunostaining was observed in terminal-like elements throughout the spinal neuropil. These results suggest that in the dog spinal cord and dorsal root ganglia, peptide-containing pathways complete their development during postnatal life, together with the full expression of NSE and synapsin I/synaptophysin immunoreactivities. In adulthood, peptide distribution is similar to that described in other mammals, although a relative absence of immunoreactive cell bodies was observed in the spinal cord.     

Prenatal development of transient receptor potential vanilloid 1-expressing primary sensory projections to sacral autonomic preganglionic neurons

The visceral reflexes of the pelvic organs are mediated by connections between primary afferents innervating the pelvic organs and parasympathetic preganglionic neurons in the intermediolateral column of the sacral spinal cord. The present immunohistochemical study revealed many varicosities expressing transient receptor potential vanilloid 1 (TRPV1) that were closely apposed to the preganglionic neuronal perikarya at embryonic day 16 in mice. Many, but not all, varicosities expressing TRPV1 in the intermediolateral column were also immunopositive for calcitonin gene-related peptide. In contrast, no nerve fibers expressing TRPV1 projected to the sympathetic preganglionic cell column in the lumbar spinal cord in prenatal stages. The results of the present study raised the possibility that the primary afferents transmit signals elicited by the activation of TRPV1 receptors to the sacral parasympathetic preganglionic neurons. Thus, the functional circuit for pelvic spinal reflexes, such as micturition induced by urine influx, might develop in the prenatal stages in mice.     

A subpopulation of preganglionic parasympathetic neurons in the rat contain adenosine deaminase

Immunohistochemical staining and retrograde fluorescent tracing techniques were used to demonstrate the presence of adenosine deaminase in preganglionic parasympathetic neurons. Both brainstem and sacral spinal cord parasympathetic nuclei were found to contain a subpopulation of neurons immunoreactive for adenosine deaminase. Immunostaining of preganglionic neurons in brainstem was restricted to a group of cells which were shown by retrograde tracing with Fast Blue to project exclusively to the sphenopalatine ganglion. This group was defined as the lacrimo-nasopalatine parasympathetic nucleus. Neurons in all other cranial preganglionic centers were devoid of adenosine deaminase immunoreactivity. In spinal cord adenosine deaminase-immunoreactive neurons were found in the intermediolateral gray matter in the region of the sacral parasympathetic nucleus. Injections of Fast Blue into the pelvic ganglion labeled large numbers of neurons in this nucleus, only some of which contained adenosine deaminase. The majority of neurons immunoreactive for adenosine deaminase were also shown to be immunoreactive for choline acetyltransferase in both brainstem and sacral parasympathetic nuclei. The present results show that a subclass of preganglionic parasympathetic neurons are among the few structures in the central nervous system that express what appear to be high levels of adenosine deaminase. This observation together with evidence suggesting that purines serve as neurotransmitters in some sacral parasympathetic neurons supports the notion that adenosine deaminase may constitute a marker for adenine nucleoside and/or nucleotide neurotransmission.     

The pattern of distribution of serotoninergic fibers in the anterior horn of the chick spinal cord

Summary The pattern of distribution of serotonin positive fibers in the motor nuclei of the chick spinal cord was examined immunohistochemically by using an antiserum against serotonin. A dense aggregation of serotoninergic fibers was located around anterior horn cells in the cervical spinal cord. In the brachial spinal cord, serotoninergic fibers were densely aggregated in the medial motor column and in the parts of the lateral motor column. There were two regions of serotonin immunoreactivity in the lateral motor column of the brachial spinal cord; one located in the ventromedial regions where a dense aggregation of serotoninergic fibers was found, and the reminder of the lateral motor column where only a few serotoninergic fibers were observed. The region containing a dense cluster of serotoninergic fibres around profiles of motoneuron somata and proximal dendrites appears to correspond to motor neuron pools of flexor muscles. In the thoracic spinal cord a high density of serotoninergic fibers was found in the motor nucleus. In the lumbosacral spinal cord (segments LS1–LS8) serotoninergic fibers were not observed in the medial motor column. However, there were five regions in the lateral motor column, where a high density of serotoninergic fibers was found. These very likely correspond to motor neuron pools of muscles which extend the hip joint.     

Immunohistochemical evidence for separate populations of somatostatin-containing and substance P-containing primary afferent neurons in the rat

The localization of two small peptides, somatostatin and substance P, has been studied with the indirect immunofluorescence technique. Both peptides were present in small neuronal cell bodies in spinal ganglia, in fibers in the dorsal horn of the spinal cord and in fibers in the intestinal wall. By comparing consecutive sections incubated with antisera to somastostatin and to substance P respectively, it was established that somatostatin, or somatostatin-like immunoreactivity and substance P, or substance P-like immunoreactivity are present in different cells. This is possibly indicated also by a somewhat differential distribution of the immunoreactive fibers in the dorsal horn: the highest concentration of somatostatin-positive fibers was observed in lamina II, whereas abundant substance P-positive fibers were present also in lamina I. Furthermore, numerous substance P-, but no somatostatin-positive fibers, were found around the central canal and in the ventral horns. In the intestinal wall more substance P-positive than somatostatin-positive fibers were seen.The present results indicate that two subpopulations of primary sensory neurons exist, one containing somatostatin, or somatostatin-like immunoreactivity, and the other containing substance P, or substance P-like immunoreactivity.     

Nitric oxide synthase immunoreactivity in rat spinal cord.

Immunoreactivity to nitric oxide synthase (NOS-IR) and choline acetyltransferase (ChAT-IR) was detected in the adult rat spinal cord using the avidin-biotin-peroxidase technique. Intensely stained NOS-positive neurons with cell processes were observed in the intermediolateral cell column of the thoracic and sacral segments and around the central canal of all segments. These areas also contained ChAT-IR neurons. A number of small- to medium-sized NOS-IR cells were noted in the superficial and deeper laminae throughout the entire cord. NOS-IR was not detected in the ventral horn motoneurons, which were, however, ChAT-IR. The results indicate that NOS-IR is present in autonomic preganglionic neurons and in selected neurons in the dorsal horn and lamina X, but appears to be absent in motoneurons.     

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.     

Distribution and identity of neurons expressing the oxytocin receptor in the mouse spinal cord

Oxytocin can influence various spinal functions. However, little is known about the spinal neuronal networks responsible for oxytocin effects. The aim of this study was to localize and characterize spinal neurons expressing oxytocin receptors. We used an oxytocin receptor-reporter mouse in which the fluorescent protein Venus is expressed under the control of the oxytocin receptor gene promoter. At all segmental levels, Venus-expressing neurons were most numerous in the substantia gelatinosa, mingled with protein kinase Cγ interneurons in the innermost layer of the inner lamina II, which, in contrast to the outer two thirds of this layer, does not receive nociceptive input. Venus-expressing neurons were also observed in the intermediolateral and sacral parasympathetic nuclei, where they represented about 5% of presumed preganglionic neurons identified by choline acetyltransferase immunoreactivity. Finally, Venus immunoreactivity was detected in lumbar and sacral dorsal gray commissures as well as in isolated neurons scattered in different regions of the dorsal horn. Altogether, our results establish the location of neurons putatively involved in oxytocin modulation of spinal functions, in particular of sexual functioning and nociception.