Localization of NADPH diaphorase in the thoracolumbar and sacrococcygeal spinal cord of the dog

The distribution of NADPH-d activity and NOS-immunoreactivity in the spinal cord of the dog was studied to evaluate the role of nitric oxide in lumbosacral afferent and spinal autonomic pathways. At all levels of the spinal cord examined, NADPH-d staining and NOS-immunoreactivity were present in neurons and fibers in the superficial dorsal horn, dorsal commissure and in neurons around the central canal. Sympathetic preganglionic neurons in the rostral lumbar segments identified by choline acetyl transferase (ChAT) immunoreactivity exhibited prominent NADPH-d and and NOS-immunoreactive staining; whereas the ChAT-immunoreactive parasympathetic preganglionic neurons in the sacral segments were not stained. The most prominent NADPH-d activity in the sacral segments occurred in fibers extending form Lissauer's tract through lamina I along the lateral edge of the dorsal horn to the region of the sacral parasympathetic nucleus. These fibers were prominent in the S1-S3 segments but not in adjacent segments (L5-L7 and Cx1 or in thoracolumbar segments. The NADPH-d fibers were not NOS-immunoreactive, but did overlap with a prominent fiber bundle containing vasoactive intestinal polypeptide immunoreactivity in the sacral spinal cord. These results indicate that nitric oxide may function as a transmitter in thoracolumbar sympathetic preganglionic neurons, but not in sacral parasympathetic preganglionic neurons. The functional significance of the NADPH-d positive, NOS-negative fiber bundle on the lateral edge of the sacral dorsal horn remains to be determined. However, based on anatomical studies in other species it seems reasonable to speculate that the fiber tract represents, in part, visceral afferent projections to the sacral parasympathetic nucleus.     

Sprouting of primary afferent fibers after spinal cord transection in the rat

After spinal cord injury, hyper-reflexia can lead to episodic hypertension, muscle spasticity and urinary bladder dyssynergia. This condition may be caused by primary afferent fiber sprouting providing new input to partially denervated spinal interneurons, autonomic neurons and motor neurons. However, conflicting reports concerning afferent neurite sprouting after cord injury do not provide adequate information to associate sprouting with hyper-reflexia. Therefore, we studied the effect of mid-thoracic spinal cord transection on central projections of sensory neurons, quantified by area measurements. The area of myelinated afferent arbors, immunolabeled by cholera toxin B, was greater in laminae I-V in lumbar, but not thoracic cord, by one week after cord transection. Changes in small sensory neurons and their unmyelinated fibers, immunolabeled for calcitonin gene-related peptide, were assessed in the cord and in dorsal root ganglia. The area of calcitonin gene-related peptide-immunoreactive fibers in laminae III-V increased in all cord segments at two weeks after cord transection, but not at one week. Numbers of sensory neurons immunoreactive for calcitonin gene-related peptide were unchanged, suggesting that the increased area of immunoreactivity reflected sprouting rather than peptide up-regulation. Immunoreactive fibers in the lateral horn increased only above the lesion and in lumbar segments at two weeks after cord transection. They were not continuous with dorsal horn fibers, suggesting that they were not primary afferent fibers. Using the fluorescent tracer DiI to label afferent fibers, an increase in area could be seen in Clarke's nucleus caudal to the injury two weeks after transection. In conclusion, site- and time-dependent sprouting of myelinated and unmyelinated primary afferent fibers, and possibly interneurons, occurred after spinal cord transection. Afferent fiber sprouting did not reach autonomic or motor neurons directly, but may cause hyper-reflexia by increasing inputs to interneurons.     

Oxytocinergic and serotonergic innervation of identified lumbosacral nuclei controlling penile erection in the male rat

Penile erection is due to activation of proerectile neurons located in the sacral parasympathetic nucleus of the L6-S1 spinal cord in the rat. Contraction of the ischiocavernosus and bulbospongiosus striated muscles, controlled by motoneurons located in the ventral horn of the L5-L6 spinal cord, reinforces penile erection. Physiological and pharmacological arguments have been provided for a role of oxytocin and serotonin in the spinal regulation of penile erection. Immunohistochemistry of oxytocinergic and serotonergic fibres was performed at the lumbosacral level of the male rat spinal cord, and combined with retrograde tracing from the pelvic nerve or from the ischiocavernosus and bulbospongiosus muscles using wheat germ agglutinin-horseradish peroxidase. Sacral preganglionic neurons retrogradely labelled from the pelvic nerve formed a homogeneous population, predominant at the L6 level. Motoneurons retrogradely labelled from the ischiocavernosus and bulbospongiosus muscles were observed in the medial part of the dorsolateral and in the dorsomedial nuclei. Fibres immunoreactive for oxytocin were mainly distributed in the superficial layers of the dorsal horn, the dorsal gray commissure and the sacral parasympathetic nucleus. Some of these fibres were apposed to retrogradely-labelled sacral preganglionic neurons and at the ultrastructural level, some synapses were evidenced. Fibres immunoreactive for serotonin were largely and densely distributed in the dorsal horn, the dorsal gray commissure, the sacral parasympathetic nucleus and the ventral horn. Some serotonergic fibres occurred in close apposition with retrogradely-labelled sacral preganglionic neurons and motoneurons, and synapses were demonstrated at the ultrastructural level. This study provides morphological support for a role of oxytocin and serotonin on sacral preganglionic neurons innervating pelvic organs and motoneurons innervating the ischiocavernosus and bulbospongiosus muscles.     

Endomorphin-2 is an endogenous opioid in primary sensory afferent fibers

Evidence is presented that the recently discovered endogenous mu-selective agonist, endomorphin-2, is localized in primary sensory afferents. Endomorphin-2-like immunoreactivity was found to be colocalized in a subset of substance P- and mu opiate receptor-containing fibers in the superficial laminae of the spinal cord and spinal trigeminal nucleus. Disruption of primary sensory afferents by mechanical (deafferentation by dorsal rhizotomy) or chemical (exposure to the primary afferent neurotoxin, capsaicin) methods virtually abolished endomorphin-2-like immunoreactivity in the dorsal horn. These results indicate that endomorphin-2 is present in primary afferent fibers where it can serve as the endogenous ligand for pre- and postsynaptic mu receptors and as a major modulator of pain perception.     

Nitric oxide in the stress axis

Calcitonin gene-related peptide in sensory primary afferent neurons has an excitatory effect on postsynaptic neurons and potentiates the effect of substance P in the rat spinal dorsal horn. It has been established that calcitonin gene-related peptide expression in dorsal root ganglion neurons is depressed, and the effect of calcitonin gene-related peptide on dorsal horn neurons is attenuated, following peripheral nerve injury. We report here that a subpopulation of injured dorsal root ganglion neurons show increased expression of calcitonin gene-related peptide. Using in situ hybridization and the retrograde tracer, FluoroGold, we detected an increased number of medium- to large-sized rat dorsal root ganglion neurons projecting to the gracile nucleus that expressed alpha-calcitonin gene-related peptide messenger RNA following spinal nerve transection. Immunohistochemistry revealed a significant increase in calcitonin gene-related peptide immunoreactivity in the gracile nucleus and in laminae III-IV of the spinal dorsal horn. These results indicate that a subpopulation of dorsal root ganglion neurons express alpha-calcitonin gene-related peptide messenger RNA in response to peripheral nerve injury, and transport this peptide to the gracile nucleus and to laminae III-IV of the spinal dorsal horn. The increase of the excitatory neuropeptide, calcitonin gene-related peptide, in sites of primary afferent termination may affect the excitability of postsynaptic neurons, and have a role in neuronal plasticity following peripheral nerve injury.     

Localization of dopamine D2 receptor in rat spinal cord identified with immunocytochemistry and in situ hybridization

In the present study the distribution of dopamine D2 receptors in rat spinal cord was determined by means of immunocytochemistry using an anti-peptide antibody, directed against the putative third intracellular loop of the D2 receptor and in situ hybridization (ISH) using a [35S]UTP labelled anti-sense riboprobe. With the immunocytochemical technique, labelling was confined to neuronal cell bodies and their proximal dendrites. Strongest labelling was present in the parasympathetic area of the sacral cord and in two sexually dimorphic motor nuclei of the lumbosacral cord, the spinal nucleus of the bulbocavernosus and the dorsolateral nucleus. Moderately labelled cells were present in the intermediolateral cell column, the area around the central canal and lamina I of the dorsal horn. Weak labelling was present in the lateral spinal nucleus and laminae VII and VIII of the ventral horn. Except for the two sexually dimorphic motornuclei of the lumbosacral cord labelled motoneurons were not encountered. With the ISH technique radioactive labelling was present in many neurons, indicating that they contained D2 receptor mRNA. The distribution of these neurons was very similar to the distribution obtained with immunocytochemistry, but with ISH additional labelled cells were detected in laminae III and IV of the dorsal horn, which were never labelled with immunocytochemistry. The present study shows that the D2 receptor is expressed in specific areas of the rat spinal cord. This distribution provides anatomical support for the involvement of D2 receptors in modulating nociceptive transmission and autonomic control. Our data further indicate that D2 receptors are not directly involved in modulating motor functions with the exception, possibly, of some sexual motor functions.     

Activation of coeruleospinal noradrenergic inhibitory controls during withdrawal from morphine in the rat

We previously reported that withdrawal from morphine induces the expression of Fos, a marker of neuronal activity, in spinal cord neurons, particularly in laminae I and II of the superficial dorsal horn, and that the magnitude of Fos expression is increased in rats with a midthoracic spinal transection. We suggested that loss of withdrawal-associated increases in descending inhibitory controls that arise in the brainstem underlie the increased Fos expression after spinal transection. Here, we addressed the origin of the supraspinal inhibition. We injected rats intracerebroventricularly with saline or anti-dopamine-beta-hydroxylase-saporin, a toxin that destroys noradrenergic neurons of the locus coeruleus. Eleven days later, we implanted rats with morphine or placebo pellets, and after 4 d, we precipitated withdrawal with naltrexone. One hour later, the rats were killed, their brains and spinal cords were removed, and transverse sections of the brains and spinal cords were immunoreacted with an antibody to Fos. In placebo-pelleted rats, the toxin injection did not alter behavior and did not induce expression of the Fos protein. However, compared with saline-injected withdrawing rats, the toxin-treated rats that underwent withdrawal demonstrated an intense withdrawal behavior rarely seen in the absence of toxin, namely forepaw fluttering. The rats also had significantly increased Fos-like immunoreactivity in all laminae of the cervical cord and in laminae I and II and the ventral horn of the lumbar cord. No differences were recorded in the sacral cord. We conclude that the effects of spinal transection in rats that withdraw from morphine in part reflect a loss of coeruleospinal noradrenergic inhibitory controls.     

Reduced nicotinamide adenine dinucleotide phosphate diaphorase in the spinal cord of dogs

The distribution of somatic, fibre-like and punctate, non-somatic reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity was examined in dog spinal cord using horizontal, sagittal and transverse sections. The morphological features of NADPH diaphorase exhibiting neurons divided into six different neuronal types (N1-N6) were described and their laminar distribution specified. Major cell groups were identified in the superficial dorsal horn and around the central canal at all spinal levels, and in the intermediolateral cell column at thoracic level. NADPH diaphorase exhibiting neurons of the pericentral region were distributed in a thin subependymal cell column containing longitudinally-arranged small bipolar neurons with processes penetrating deeply into the intermediolateral cell column and/or running rostrocaudally in the subependymal layer. The second pericentral cell column located more laterally in lamina X contains large, intensely-stained NADPH diaphorase exhibiting neurons with long dendrites radiating in the transverse plane. Neurons of the sacral parasympathetic nucleus seen in segments S1-S3 exhibited prominent NADPH diaphorase activity accompanied by heavily-stained fibres extending from Lissauer's tract through lamina I along the lateral edge of the dorsal horn to lamina V. A massive dorsal gray commissure, with high NADPH diaphorase activity, was found in segments S1-S3. At the same segmental level a prominent group of moderately-stained motoneurons was detected in the dorsolateral portion of the anterior horn. Fibre-like NADPH diaphorase activity was found in the superficial dorsal horn and pericentral region in all segments studied. Punctate, non-somatic NADPH diaphorase activity was detected in the superficial dorsal horn, in the pericentral region all along the rostrocaudal axis and in the nucleus phrenicus (segments C4-C5), nucleus dorsalis (segments Th2-L2), nucleus Y (segments S1-S3), and the dorsal part of the dorsal gray commissure (S1-S3). A schematic diagram documenting the segmental and laminar distribution of NADPH diaphorase activity is given.     

Sensory axons are guided by local cues in the developing dorsal spinal cord

During development, different classes of sensory neurons establish distinctive central projections within the spinal cord. Muscle spindle afferents (Ia fibers) grow ventrally through the dorsal horn to the ventral cord, whereas cutaneous sensory collaterals remain confined to the dorsal horn. We have studied the nature of the cues used by Ia fibers in establishing their characteristic projections within the dorsal horn. An organotypic culture preparation of embryonic chicken spinal cord and sensory ganglia was used to test the influence of ventral spinal cord and local cues within the dorsal spinal cord on the growing Ia afferents. When the ventral half of the spinal cord was replaced with an inverted duplicate dorsal half, Ia fibers entering through the dorsal columns still grew ventrally within the host dorsal horn. After the fibers entered the duplicate dorsal half, they continued growing in the same direction. With respect to the duplicate dorsal tissue, this was in an opposite, ventral-to-dorsal, direction. In both cases, however, Ia collaterals remained confined to the medial dorsal laminae. Restriction to these laminae was maintained even when the fibers had to change their direction of growth to stay within them. These results show that cues from the ventral cord are not required for the development of correct Ia projections within the dorsal horn. Local, rather than long-range directional, cues appear to determine the pattern of these projections. When the ventral half of the spinal cord was left intact but sensory axons were forced to enter the dorsal gray matter growing rostrally or caudally, their collateral axons grew in random directions, further showing the absence of directional cues even when the ventral cord was present. Taken together, these observations suggest that Ia fibers are guided by local positional cues that keep them confined to the medial gray matter within the dorsal horn, but their direction of growth is determined primarily by their orientation and position as they enter the dorsal gray matter.     

Localization of bradykinin-like immunoreactivity in the rat spinal cord: effects of capsaicin, melittin, dorsal rhizotomy and peripheral axotomy

A putative role for bradykinin has been proposed in the processing of sensory information at the level of the spinal cord. Autoradiographic studies have demonstrated the presence of B2 kinin receptor binding sites in superficial laminae of the dorsal horn and a down-regulation of those receptors in rat models of pain injury. In this study, classical immunocytochemistry and confocal microscopy immunofluorescence were used first to localize bradykinin-like immunoreactivity in all major spinal cord segments of naive rats; second, to assess bradykinin-like immunoreactivity changes that occur in animals subjected to various chemical treatments and surgical lesions. High densities of bradykinin-like immunoreactivity were observed in motoneuron of the ventral horn, deeper laminae and nucleus dorsalis of the dorsal horn. Higher magnification of ventral horn showed strong immunostaining of motoneuron perikaryas and their proximal processes. Two types of bradykinin-like immunoreactivity immunostained cellular bodies were observed in deeper laminae of the dorsal horn. These interneurons, morphologically corresponding to islets and antenna-type cells project dendrites to adjacent laminae. Furthermore, numerous strongly marked dendrites, transversally cut, suggest the presence of projection neurons to higher cervical centres. Following unilateral lumbar dorsal rhizotomy (L1-L6) or peripheral lesion of the sciatic nerve, important increases of bradykinin-like immunoreactivity were found in laminae III and IV of the ipsilateral dorsal horn. In contrast, significant decreases of immunodeposits were observed in both cell bodies and numerous dendrites of motoneuron surrounding neuropil. Specific destructions of sensory afferent fibres with capsaicin or selective activation of kallikreins with melittin caused increases of bradykinin-like immunoreactivity in both the dorsal and ventral horns of the spinal cord. These results which demonstrate the cellular localization of bradykinin-like immunoreactivity in both dorsal and ventral horns of the rat spinal cord, further reveal the plasticity of this non-sensory peptidergic system following various chemical and surgical treatments. Hence, these anatomical findings along with earlier functional and receptor autoradiographic studies reinforce the putative role of bradykinin in sensory function.     

Reconstruction of trajectory of primary afferent collaterals in the dorsal horn of the cat spinal cord, using Golgi-stained serial sections

Using Golgi-stained serial sections obtained at the sacro-caudal levels of the cat spinal cord, it was possible to reconstruct the trajectory of primary afferents. They were classified into two groups: reliable primary afferents directly traced from the dorsal root and probable primary afferents traced from the dorsal funiculus or Lissauer's tract. The diameters of the reliable primary afferents vary from 0.88-1.88 mum. According to their courses, reliable primary afferents as well as probable primary afferents were classified into three groups: the first is distributed to both medial and lateral halves of the dorsal horn, the second to the medial half, and the third to the lateral half. Commissural fibers were also observed among the probable primary afferents. The rostro-caudal and medio-lateral extents of reliable primary afferents are found to be between 250 and 950 mum and 270 and 700 mum respectively, while those of the probable primary afferents were between 125 and 670 mum and 270 and 1,640 mum respectively. These primary afferent fibers are connected with at least two or more laminae of the dorsal horn gray matter.     

Free alpha-subunit and intact TSH secretion in vitro are closely associated in human somatotroph adenomas

B-50(GAP-43) is a phosphoprotein mainly found in the nervous system which plays a major role in neurite growth during development and regeneration as well as in synaptic remodelling. In the mature intact central nervous system, intense B-50 immunoreactivity (B-50-IR) can still be detected in regions which maintain residual capacity for structural re-organization. B-50 expression has been studied extensively in laboratory animals; however, its distribution and regulation in the human spinal cord is largely unknown. As a first step to analyze lesion-induced structural alterations, we investigated the distribution of B-50 protein and mRNA in the normal adult human spinal cord and dorsal root ganglia. Intense B-50-IR was localized to the superficial laminae of the dorsal horn at all segmental levels, the intermediolateral nucleus at thoracic levels and Onuf's nucleus at sacral levels. Scattered neurons, particularly in the ventral horn of lumbar and sacral segmental levels (and occasionally also in Clarke's nucleus) displayed intense B-50-IR in close apposition to the perikaryal and proximal dendritic surfaces. Nonradioactive in situ hybridization indicated that B-50 mRNA could also be detected in neurons of the ventral horn and also in the intermediolateral nucleus. The distribution of B-50 mRNA and protein in the normal human spinal cord shows a marked similarity to that reported in experimental animals, including the selective labelling of Onuf's nucleus. However, the strong B-50-IR on the surface of some large anterior horn motor neurons has not been observed in other mammals. This finding might reflect a particular state of readiness for synaptic plasticity.     

Increased uptake and transport of cholera toxin B-subunit in dorsal root ganglion neurons after peripheral axotomy: possible implications for sensory sprouting

In the present study we show that, in contrast to the rat, injection of cholera toxin B-subunit (CTB) into the intact sciatic nerve of Macaca mulatta monkey gives rise to labelling of a sparse network of fibers in laminae I-II of spinal cord and of some mainly small dorsal root ganglion (DRG) neurons. Twenty days after sciatic nerve cut, the percentage of CTB-positive lumbar 5 (L5) DRG neuron profiles increased from 11% to 73% of all profiles. In the spinal cord, a marked increase in CTB labelling was seen in laminae I, II, and the dorsal part of lamina III. In the rat L5 DRGs, 18 days after sciatic nerve cut, the percentage of CTB- and CTB conjugated to horseradish peroxidase (HRP)-labelled neuron profiles increased from 45% to 81%, and from 54% to 87% of all neuron profiles, respectively. Cell size measurements in the rat showed that most of the CTB-positive neuron profiles were small in size after axotomy, whereas most were large in intact DRGs. In the rat spinal dorsal horn, a dense network of CTB-positive fibers covered the whole dorsal horn on the axotomized side, whereas CTB-labelled fibers were mainly seen in laminae III and deeper laminae on the contralateral side. A marked increase in CTB-positive fibers was also seen in the gracile nucleus. The present study shows that in both monkey and rat DRGs, a subpopulation of mainly small neurons acquires the capacity to take up CTB/CTB-HRP after axotomy, a capacity normally not associated with these DRG neurons. These neurons may transganglionically transport CTB and CTB-HRP. Thus, after peripheral axotomy, CTB and CTB-HRP are markers not only for large but also for small DRG neurons and, thus, possibly also for both myelinated and unmyelinated primary afferents in the spinal dorsal horn. These findings may lead to a reevaluation of the concept of sprouting, considered to take place in the dorsal horn after peripheral nerve injury.     

Distribution of trigeminohypothalamic and spinohypothalamic tract neurons displaying substance P receptor-like immunoreactivity in the rat

Primary afferent neurons containing substance P (SP) are apparently implicated in the transmission of noxious information from the periphery to the central nervous system, and SP released from primary afferent neurons acts on second-order neurons with the SP receptor (SPR). In the rat, nociceptive information reached the hypothalamus not only through indirect pathways but also directly through trigeminohypothalamic and spinohypothalamic pathways. Thus, in the present study, the distribution pattern of trigeminohypothalamic and spinohypothalamic tract neurons showing SPR-like immunoreactivity (SPR-LI) was examined in the rat by a retrograde tract-tracing method combined with immunofluorescence histochemistry for SPR. A substantial number of trigeminal and spinal neurons with SPR-LI were retrogradely labeled with Fluoro-Gold (FG) injected into the hypothalamic regions. These neurons were distributed mainly in lamina I of the medullary and spinal dorsal horns, lateral spinal nucleus, regions around the central canal of the spinal cord, and the lateral aspect of the deep part of the spinal dorsal horn. A number of SPR-LI neurons in the spinal parasympathetic nucleus were labeled with FG injected into the area around the paraventricular hypothalamic nucleus. Some SPR-LI neurons in the lateral spinal nucleus and the lateral aspect of the deep part of the spinal dorsal horn were also labeled with FG injected into the septal region. On the basis of the distribution areas of SPR-LI trigeminal and spinal neurons projecting to the hypothalamic and septal regions, it is likely that these neurons are involved in the transmission of somatic and/or visceral noxious information.     

Inflammation-induced Fos protein expression in the rat spinal cord is enhanced following dorsolateral or ventrolateral funiculus lesions

Previous studies have shown an enhanced expression of Fos protein-like immunoreactivity in the lumbar spinal cord of rats with complete spinal transection following persistent hindpaw inflammation. To further locate the spinal pathways responsible for these effects, we compared the inflammation-evoked Fos expression in rats with bilateral lesions of the dorsolateral (DLFX) or ventrolateral (VLFX) funiculus, and with rats with a sham operation. The results indicate that the number of Fos-labeled neurons was significantly increased in all laminae of the dorsal horn ipsilateral to the inflamed hindpaw and in contralateral deep dorsal horn in both DLFX and VLFX rats compared to sham-operated rats. Moreover, when comparing DLFX and VLFX rats, in the ipsilateral spinal cord, DLFX resulted in more Fos expression in the deep dorsal horn; in contrast, a larger number of Fos-labeled cells in superficial laminae was observed in VLFX rats. These results suggest that modulatory systems, which descend in both DLF and VLF pathways, mediate the enhanced net descending nociceptive inhibition after persistent inflammation, although the supraspinal sites of origin of each pathway are likely functionally diverse.     

How to manage chest pain in patients with normal coronary angiograms

Sympathetic nerve activity is maintained after high spinal injury through circuits that remain in question. We evaluated patterns of c-fos gene induction as a monitor of spinal neurons responding to high spinal cord transection in the rat. Rats were anesthetized with isofluorane. Lower cervical or upper thoracic spinal segments were exposed, immersed in warm mineral oil and transected. Spinal cords were exposed but not transected in anesthetized controls. After 2.5 h, spinalized and control rats were perfused for immunocytochemistry. Cervical and thoracolumbar spinal segments and dorsal root ganglia were sectioned coronally. Tissues were incubated in primary, polyclonal antisera raised in rabbit or sheep against a peptide sequence unique to the N-terminal domain of Fos, and processed immunocytochemically. Neurons were induced to express Fos-like immunoreactivity (FLI), bilaterally, in the spinal gray, but not in primary sensory ganglia. Spinal cord transection induced neurons to express FLI in thoracic laminae I, IIo (outer substantia gelatinosa), Vre (lateral reticulated division), VII (lamina intermedia) and X, and the intermediolateral cell column. Lamina VIII was also labeled in spinal-injured but not in control animals. Immunolabeled nuclei were prominent in lumbar segments and were concentrated in the medial third of laminae I and IIo, and in laminae VII and X. Few cells were labeled in upper cervical or sacral segments. FLI was sparse in the spinal gray of controls and expressed mainly within the dorsal root entry zone of upper thoracic segments. Patterns of c-fos gene expression were site-specific and correlated with laminae that respond predominantly to noxious stimulation and that contain sympathetic interneurons. Laminae that are responsive to non-noxious stimuli and activated by walking, IIi, nucleus proprius, medial V and layer VI were not induced to express FLI. We conclude that neurons in specific spinal laminae that process high threshold afferents and that harbor neurons with sympathetic nerve-related activity are activated selectively by spinal cord transections. We hypothesize that peripheral afferents processed by spinal-sympathetic circuit neurons may regulate sympathetic discharge in the absence of supraspinal drive.     

Partial and complete sciatic nerve injuries induce similar increases of neuropeptide Y and vasoactive intestinal peptide immunoreactivities in primary sensory neurons and their central projections

Partial nerve injury is more likely to cause neuropathic pain than complete nerve injury. We have compared the changes in neuropeptide expression in primary sensory neurons which follow complete and partial injuries to determine if these might be involved. Since more neurons are damaged by complete injury, we expected that complete sciatic nerve injury would simply cause greater increases in neuropeptide Y and vasoactive intestinal peptide than partial injury. We examined neuropeptide Y and vasoactive intestinal peptide immunoreactivities in L4 and L5 dorsal root ganglia, the dorsal horn of L4-L5 spinal cord, and the gracile nuclei of rats killed 14 days after unilateral complete sciatic nerve transection, partial sciatic nerve transection and chronic constriction injury of the sciatic nerves. In all three groups of rats, neuropeptide Y- and vasoactive intestinal peptide-immunoreactive neurons were increased in the ipsilateral L4 and L5 dorsal root ganglion when compared with the contralateral side. Most neuropeptide Y-immunoreactive neurons were of medium and large size, but a few were small. Neuropeptide Y-immunoreactive axonal fibers were increased from laminae I to IV, and vasoactive intestinal peptide-immunoreactive axonal fibers were increased in laminae I and II, of the ipsilateral dorsal horn of L4-L5 spinal cord. The increases of neuropeptide Y and vasoactive intestinal peptide immunoreactivities in the dorsal horn were similar among the three groups. However, only after constriction injury were some vasoactive intestinal peptide-immunoreactive neurons seen in the deeper laminae of the ipsilateral dorsal horn. Robust neuropeptide Y-immunoreactive axonal fibers and some neuropeptide Y-immunoreactive cells were seen in the ipsilateral gracile nuclei of all three groups of animals, but neuropeptide Y-immunoreactive cells were more prominent after constriction injury. Contrary to our expectations, partial and complete sciatic nerve injuries induced similar increases in neuropeptide Y and vasoactive intestinal peptide in lumbar dorsal root ganglion neurons and their central projections in the dorsal horn and the gracile nuclei two weeks after injury. Some neurons whose axons were spared by partial injury may also increase neuropeptide Y or vasoactive intestinal peptide expression. Altered neuropeptide release from these functional sensory neurons may play a role in neuropathic pain.     

A longitudinal study of mothers'' and preschool children''s aversive behaviors during dyadic interactions

The neuronal organization of the spinal cord in red stingray was studied using the rapid Golgi method. The gray matter of the spinal cord was divided into seven laminae: RS-I, RS-II, RS-III, RS-IV, RS-V, RS-VI and RS-VII. RS-I is cell dense lamina which occupies the major part of the dorsal horn and corresponds to laminae I and II of the spinal cord of mammals, birds and reptiles. The neurons of the lamina I are interspersed with those of lamina II, without forming a discrete lamina. RS-II is located at the base of the dorsal horn and is considered to correspond to the nucleus proprius. RS-III and IV form the intermediate zone and are highly reticulated. A few neurons of various shapes and sizes are distributed among the numerous fibers. The nuclei such as the intermediolateral, intermediomedial or Clarke's nucleus cannot be identified in the intermediate zone. RS-V and VI constitute the ventral horn. RS-V occupies the major part of the ventral horn and contains motoneurons which are distributed diffusely, without forming any distinct cell groups. RS-VI is located in the ventromedial part of the ventral horn, contains commissural neurons and correspond to lamina VIII. RS-VII is a small area surrounding the central canal and corresponds to lamina X. Thus, while the major features of the spinal cord of the red stingray can be correlated with those of the spinal cord of mammals, birds and reptiles, the neuronal organization of the spinal cord of the red stingray remains in an undifferentiated state.     

Segmental and propriospinal projection systems of frog lumbar interneurons

Spinal interneuronal networks have been implicated in the coordination of reflex behaviors and limb postures in the spinal frog. As a first step in defining these networks, retrograde transport of horseradish peroxidase (HRP) was used to examine the anatomical organization of interneuronal circuitry in the lumbar spinal cord of the frog. Following neuronal degeneration induced by spinal transection and section of the dorsal and ventral roots, HRP was placed at different locations in the spinal cord and the positions of labeled neuronal cell bodies plotted using a Eutectics Neuron Tracing System. We describe four spinal interneuronal systems, three with cell bodies located in the lumbar cord and one with descending projections to the lumbar cord. Interneurons with cell bodies located in the lumbar cord include: (1) Lumbar neurons projecting rostrally. Those projecting to thoracic segments tended to be located in the lateral and ventrolateral gray and in the lower two-thirds of the dorsal horn, with projections that were predominantly uncrossed. Those projecting to the brachial plexus and beyond were located in the dorsal part of the dorsal horn (uncrossed) and in the lateral, ventrolateral, and ventromedial gray (crossed). (2) Lumbar neurons with segmental projections within the lumbar cord. These neurons, which were by far the most numerous, had both uncrossed and crossed projections and were distributed throughout the dorsal, lateral, ventrolateral, and ventromedial gray matter. (3) Lumbar neurons projecting to the sacral cord. This population, which arose mainly from the dorsal horn and lateral or ventrolateral gray, was much smaller than in the other systems. Neuronal density of some of these populations of lumbar interneurons appeared to vary with rostrocaudal level. Finally, a population of neurons with cell bodies in the brachial and thoracic segments that projects to the lumbar cord is described. The most rostral of these neurons were multipolar cells with uncrossed projections, while those with crossed projections were confined almost exclusively to the ventral half of the cord. The distribution of spinal interneurons reported here will provide guidance for future studies of the role of interneuronal networks in the control of movements using the spinal frog as a model system.     

ATP P2x receptors and sensory synaptic transmission between primary afferent fibers and spinal dorsal horn neurons in rats

ATP P2x receptors and sensory synaptic transmission between primary afferent fibers and spinal dorsal horn neurons in rats. J. Neurophysiol. 80: 3356-3360, 1998. Glutamate is a major fast transmitter between primary afferent fibers and dorsal horn neurons in the spinal cord. Recent evidence indicates that ATP acts as another fast transmitter at the rat cervical spinal cord and is proposed to serve as a transmitter for nociception and pain. Sensory synaptic transmission between dorsal root afferent fibers and neurons in the superficial dorsal horn of the lumbar spinal cord were examined by whole cell patch-clamp recording techniques. Experiments were designed to test if ATP could serve as a transmitter at the lumbar spinal cord. Monosynaptic excitatory postsynaptic currents (EPSCs) were completely abolished after the blockade of both glutamatergic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate and N-methyl--aspartate receptors. No residual current was detected, indicating that glutamate but not ATP is a fast transmitter at the dorsal horn of the lumbar spinal cord. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a selective P2x receptor antagonist, produced an inhibitory modulatory effect on fast EPSCs and altered responses to paired-pulse stimulation, suggesting the involvement of a presynaptic mechanism. Intrathecal administration of PPADS did not produce any antinociceptive effect in two different types of behavioral nociceptive tests. The present results suggest that ATP P2x2 receptors modulate excitatory synaptic transmission in the superficial dorsal horn of the lumbar spinal cord by a presynaptic mechanism, and such a mechanism does not play an important role in behavioral responses to noxious heating. The involvement of other P2x subtype receptors, which is are less sensitive to PPADS, in acute nociceptive modulation and persistent pain remains to be investigated.