In Vivo Calcium Imaging Visualizes Incision-Induced Primary Afferent Sensitization and Its Amelioration by Capsaicin Pretreatment.

Previous studies have shown that infiltration of capsaicin into the surgical site can prevent incision-induced spontaneous pain like behaviors and heat hyperalgesia. In the present study, we aimed to monitor primary sensory neuron Ca2+ activity in the intact dorsal root ganglia (DRG) using Pirt-GCaMP3 male and female mice pretreated with capsaicin or vehicle before the plantar incision. Intraplantar injection of capsaicin (0.05%) significantly attenuated spontaneous pain, mechanical, and heat hypersensitivity after plantar incision. The Ca2+ response in in vivo DRG and in in situ spinal cord was significantly enhanced in the ipsilateral side compared with contralateral side or naive control. Primary sensory nerve fiber length was significantly decreased in the incision skin area in capsaicin-pretreated animals detected by immunohistochemistry and placental alkaline phosphatase (PLAP) staining. Thus, capsaicin pretreatment attenuates incisional pain by suppressing Ca2+ response because of degeneration of primary sensory nerve fibers in the skin.SIGNIFICANCE STATEMENT Postoperative surgery pain is a major health and economic problem worldwide with ∼235 million major surgical procedures annually. Approximately 50% of these patients report uncontrolled or poorly controlled postoperative pain. However, mechanistic studies of postoperative surgery pain in primary sensory neurons have been limited to in vitro models or small numbers of neurons. Using an innovative, distinctive, and interdisciplinary in vivo populational dorsal root ganglia (DRG) imaging (>1800 neurons/DRG) approach, we revealed increased DRG neuronal Ca2+ activity from postoperative pain mouse model. This indicates widespread DRG primary sensory neuron plasticity. Increased neuronal Ca2+ activity occurs among various sizes of neurons but mostly in small-diameter and medium-diameter nociceptors. Capsaicin pretreatment as a therapeutic option significantly attenuates Ca2+ activity and postoperative pain.

Nucleus paragigantocellularis afferents in male and female rats: organization, gonadal steroid receptor expression, and activation during sexual behavior.

The supraspinal regulation of genital reflexes is poorly understood. The brainstem nucleus paragigantocellularis (nPGi) of rats is a well-established source of tonic inhibition of genital reflexes. However, the organization, gonadal steroid receptor expression, and activity of nPGi afferents during sex have not been fully characterized in male and female rats. To delineate the anatomical and physiological organization of nPGi afferents, the retrograde tracer Fluoro-Gold (FG) was injected into the nPGi of sexually experienced male and female rats. Animals engaged in sexual behavior 1 hour before sacrifice. Cells containing FG, estrogen receptor-alpha (ER(alpha)), androgen receptor (AR), and the immediate-early gene product Fos were identified immunocytochemically. Retrograde labeling from the nPGi was prominent in the bed nucleus of the stria terminalis, paraventricular nucleus (PVN), posterior hypothalamus, precommissural nucleus, deep mesencephalic nucleus, and periaqueductal gray (PAG) of both sexes. Sex differences were observed in the caudal medial preoptic area (MPO), with significantly more FG+ cells observed in males, and in the PAG and inferior colliculus, where significantly more FG+ cells were observed in females. The majority of regions that contained FG+ cells also contained ER(alpha) or AR, indicating sensitivity to gonadal steroids. The proportions of FG+ cells that co-localized with sex-induced Fos was high in the PVN of both sexes and high in the MPO of males but low in the PAG of both sexes despite the large number of PAG-nPGi output neurons and Fos+ cells in both sexes. The characterization of these afferents will lead to a further understanding of the neural regulation of genital reflexes.

Genetic manipulation of intraspinal plasticity after spinal cord injury alters the severity of autonomic dysreflexia.

Severe spinal cord injuries above mid-thoracic levels can lead to a potentially life-threatening hypertensive condition termed autonomic dysreflexia, which is often triggered by painful distension of pelvic viscera (bladder or bowel) and consequent sensory fiber activation, including nociceptive C-fibers. Interruption of tonically active medullo-spinal pathways after injury causes disinhibition of thoracolumbar sympathetic preganglionic neurons, and intraspinal sprouting of nerve growth factor (NGF)-responsive primary afferent fibers is thought to contribute to their hyperactivity. We investigated spinal levels that are critical for eliciting autonomic dysreflexia using a model of noxious colorectal distension (CRD) after complete spinal transection at the fourth thoracic segment in rats. Post-traumatic sprouting of calcitonin gene-related peptide (CGRP)-immunoreactive primary afferent fibers was selectively altered at specific spinal levels caudal to the injury with bilateral microinjections of adenovirus encoding the growth-promoting NGF or growth-inhibitory semaphorin 3A (Sema3a) compared with control green fluorescent protein (GFP). Two weeks later, cardio-physiological responses to CRD were assessed among treatment groups before histological analysis of afferent fiber density at the injection sites. Dysreflexic hypertension was significantly higher with NGF overexpression in lumbosacral segments compared with GFP, whereas similar overexpression of Sema3a significantly reduced noxious CRD-evoked hypertension. Quantitative analysis of CGRP immunostaining in the spinal dorsal horns showed a significant correlation between the extent of fiber sprouting into the spinal segments injected and the severity of autonomic dysreflexia. These results demonstrate that site-directed genetic manipulation of axon guidance molecules after complete spinal cord injury can alter endogenous circuitry to modulate plasticity-induced autonomic pathophysiology.

Tumor-induced injury of primary afferent sensory nerve fibers in bone cancer pain.

Bone is the most common site of chronic pain in patients with metastatic cancer. What remains unclear are the mechanisms that generate this pain and why bone cancer pain can be so severe and refractory to treatment with opioids. Here we show that following injection and confinement of NCTC 2472 osteolytic tumor cells within the mouse femur, tumor cells sensitize and injure the unmyelinated and myelinated sensory fibers that innervate the marrow and mineralized bone. This tumor-induced injury of sensory nerve fibers is accompanied by an increase in ongoing and movement-evoked pain behaviors, an upregulation of activating transcription factor 3 (ATF3) and galanin by sensory neurons that innervate the tumor-bearing femur, upregulation of glial fibrillary acidic protein (GFAP) and hypertrophy of satellite cells surrounding sensory neuron cell bodies within the ipsilateral dorsal root ganglia (DRG), and macrophage infiltration of the DRG ipsilateral to the tumor-bearing femur. Similar neurochemical changes have been described following peripheral nerve injury and in other non-cancerous neuropathic pain states. Chronic treatment with gabapentin did not influence tumor growth, tumor-induced bone destruction or the tumor-induced neurochemical reorganization that occurs in sensory neurons or the spinal cord, but it did attenuate both ongoing and movement-evoked bone cancer-related pain behaviors. These results suggest that even when the tumor is confined within the bone, a component of bone cancer pain is due to tumor-induced injury to primary afferent nerve fibers that innervate the tumor-bearing bone. Tumor-derived, inflammatory, and neuropathic mechanisms may therefore be simultaneously driving this chronic pain state.

The role of the vagal nerve in peripheral PYY3-36-induced feeding reduction in rats.

Peptide YY (PYY), an anorectic peptide, is secreted postprandially from the distal gastrointestinal tract. PYY(3-36), the major form of circulating PYY, binds to the hypothalamic neuropeptide Y Y2 receptor (Y2-R) with a high-affinity, reducing food intake in rodents and humans. Additional gastrointestinal hormones involved in feeding, including cholecystokinin, glucagon-like peptide 1, and ghrelin, transmit satiety or hunger signals to the brain via the vagal afferent nerve and/or the blood stream. Here we determined the role of the afferent vagus nerve in PYY function. Abdominal vagotomy abolished the anorectic effect of PYY(3-36) in rats. Peripheral administration of PYY(3-36) induced Fos expression in the arcuate nucleus of sham-operated rats but not vagotomized rats. We showed that Y2-R is synthesized in the rat nodose ganglion and transported to the vagal afferent terminals. PYY(3-36) stimulated firing of the gastric vagal afferent nerve when administered iv. Considering that Y2-R is present in the vagal afferent fibers, PYY(3-36) could directly alter the firing rate of the vagal afferent nerve via Y2-R. We also investigated the effect of ascending fibers from the nucleus of the solitary tract on the transmission of PYY(3-36)-mediated satiety signals. In rats, bilateral midbrain transections rostral to the nucleus of the solitary tract also abolished PYY(3-36)-induced reductions in feeding. This study indicates that peripheral PYY(3-36) may transmit satiety signals to the brain in part via the vagal afferent pathway.

Bladder and cutaneous sensory neurons of the rat express different functional P2X receptors.

The expression and functional responses of P2X receptors in bladder and cutaneous sensory neurons of adult rats and mice have been studied using immunohistochemistry and patch clamp techniques. Cell bodies of bladder pelvic afferents were identified in L6 and S1 dorsal root ganglia (DRG), following Fast Blue injection into the muscle wall of the urinary bladder. Similarly, cutaneous sensory neurons were identified in L3 and L4 DRG, following Fast Blue injection into the saphenous nerve innervating the skin. Bladder sensory neurons contained only weak to moderate P2X(3)-immunoreactivity (IR), in contrast to strong P2X(3)-IR observed in a sub-population of cutaneous afferents. Whole-cell patch-clamp recordings revealed that approximately 90% of bladder afferent neurons responded to alpha beta-methylene ATP (alpha beta meATP) and ATP (30 microM) with persistent currents, which were inhibited by 2',3'-O-trinitrophenyl-ATP (TNP-ATP) (0.3 microM) to 6.4+/-1.9% and 8.0+/-2.6% of control, respectively (n=8). The remaining bladder sensory neurons demonstrated biphasic, transient or no response to P2X agonists. In contrast, only 24% of cutaneous afferent neurons gave persistent currents to alpha beta meATP (30 microM), with 66% of cells giving transient or biphasic currents and the remaining 10% being non-responsive. Our results suggest that, in contrast to DRG neurons in general, bladder sensory neurons projecting via pelvic nerves express predominantly P2X(2/3) heteromeric receptors, which are likely to mediate the important roles of ATP as a signaling molecule of urinary bladder filling and nociception.

Sensory fibres expressing capsaicin receptor TRPV1 in patients with rectal hypersensitivity and faecal urgency.

BACKGROUND: Faecal urgency and incontinence with rectal hypersensitivity is a distressing, unexplained disorder that is inadequately treated. We aimed to determine whether expression of the heat and capsaicin receptor vanilloid receptor 1 (TRPV1 or VR1) was changed in rectal sensory fibres, and to correlate nerve fibre density with sensory abnormalities. METHODS: We compared full-thickness rectal biopsy samples from nine patients with physiologically characterised rectal hypersensitivity with tissue samples from 12 controls. Sensory thresholds to rectal balloon distension and heating the rectal mucosa were measured before biopsy. We assessed specimens with immunohistochemistry and image analysis using specific antibodies to TRPV1; nerve growth factor (NGF) receptor tyrosine kinase A; glial cell line-derived neurotrophic factor (GDNF); neuropeptides calcitonin gene-related peptide (CGRP) and substance P; the related vanilloid receptor-like protein (VRL) 2; glial markers S-100 and glial fibrillary acid protein (GFAP); and the nerve structural marker peripherin. FINDINGS: In rectal hypersensitivity, nerve fibres immunoreactive to TRPV1 were increased in muscle, submucosal, and mucosal layers: in the mucosal layer, the median% area positive was 0.44 (range 0.30-0.59) in patients who were hypersensitive and 0.11 (0.00-0.21) in controls (p=0.0005). The numbers of peripherin-positive fibres also increased in the mucosal layer (hypersensitive 3.00 [1.80-6.50], controls 1.20 [0.39-2.10]: (p=0.0002). The increase in TRVP1 correlated significantly with the decrease in rectal heat (p=0.03) and the distension (p=0.02) sensory thresholds. The thresholds for heat and distension were also significantly correlated (p=0.0028). Expression of nerve fibres positive for GDNF (p=0.001) and tyrosine kinase A (p=0.002) was also increased, as were cell bodies of the submucosal ganglia immunoreactive to CGRP (p=0.0009). INTERPRETATION: Faecal urgency and rectal hypersensitivity could result from increased numbers of polymodal sensory nerve fibres expressing TRPV1. The triggering factor or factors remain uncertain, but drugs that target nerve terminals that express this receptor, such as topical resiniferatoxin, deserve consideration.

Fos expression in afferents to the rat midline thalamus following immobilization stress.

The paraventricular thalamic nucleus (PVT), the most dorsal component of the thalamic midline, is known to be strongly activated following a variety of stressors and thus might be suggested to play a role as a relay for stress-related information targeted for viscerolimbic areas in the brain. This thalamic midline nucleus, however, lacks significant direct connections with the paraventricular hypothalamic nucleus (PVH), which is a key player in the hypothalamic-pituitary-adrenal (HPA) axis whose activation and subsequent glucocorticoid secretion are clearly crucial for homeostasis under 'stressful' conditions. The present study was designed to identify afferents of the PVT, which are activated by an immobilization stress, one type of the 'neurogenic' stress paradigms, using combined Fos immunohistochemistry and retrograde tracing experiments with cholera toxin B subunit. Dual immunohistochemistry revealed that immobilization stress induced expression of Fos immunoreactive nuclei was constantly observed in many regions of the neuraxis. Dually-labeled neurons in the cerebral cortex were mainly observed in the hippocampus, exclusively in the pyramidal layer of the caudal part of the ventral subiculum. In diencephalons a small number of dually labeled neurons was observed in the rostromedial zona incerta. In the midbrain, many of the retrogradely labeled neurons in the dorsal raphe nucleus were also immunoreactive for Fos protein. Mesencephalic periaqueductal gray contained a substantial number of dually labeled neurons. In the pons, the parabrachial nuclei, locus ceruleus, Barrington's nucleus and raphe nucleus contained only small numbers of dually labeled neurons. Within the medulla, nearly all of the retrogradely labeled neurons in the caudal part of the ventrolateral medulla were also immunoreactive for Fos antigen. Dually labeled neurons in the medulla were also observed in the nucleus of the solitary tract, exclusively in its commissural part. Given the known fact that most of the regions mentioned above provide important inputs to the HPA axis, our results suggest that a diencephalic network, presumably implicated in behavioral responses to given stress, might be activated by the parallel projection system that activate the HPA axis and might add some important insights to the understanding of animal and human stress-related HPA pathology.

Evidence for a localization of [(3)H]nociceptin binding sites on medullar primary afferent fibers.

The ORL1 receptor (opioid receptor-like 1) and its endogenous ligand, nociceptin, are involved in nociperception. We have studied, in a deafferented animal model, the modification of medullar [(3)H]nociceptin binding site density. A rhizotomy was carried out in rats at the cervicothoracic level, and the dorsal afferent fibers from C5 to T1 were lesioned. Seven days after surgery, animals were sacrificed, and the binding of [(3)H]nociceptin (2 nM) was then performed on spinal cord sections. An autoradiographic analysis revealed a significant reduction (-18%) of [(3)H]nociceptin binding site density in the dorsal horn ipsilateral to the deafferentation compared with the contralateral side of the lesion. In the ventral horn, no significant difference (-5%) of binding was observed in the ipsilateral side of the deafferentation compared with the contralateral side. Thus, [(3)H]nociceptin binding sites appear to be located mainly on either interneurons or deutoneurons of the spinal cord, because the bulk of the labeling is spared by the lesion. However, the significant reduction of labeling that occurs on the dorsal part of the ipsilateral side to the lesion indicates that [(3)H]nociceptin binding sites are also present on these dorsal afferent fibers.

Nerve growth factor induces P2X(3) expression in sensory neurons.

Glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) are neuroprotective for subpopulations of sensory neurons and thus are candidates for pain treatment. However, delivering these factors to damaged neurons will invariably result in undamaged systems also being treated, with possible consequences for sensory processing. In sensory neurons the purinergic receptor P2X(3) is found predominantly in GDNF-sensitive nociceptors. ATP signalling via the P2X(3) receptor may contribute to pathological pain, suggesting an important role for this receptor in regulating nociceptive function. We therefore investigated the effects of intrathecal GDNF or NGF on P2X(3) expression in adult rat spinal cord and dorsal root ganglia (DRG). In control spinal cords, P2X(3) expression was restricted to a narrow band of primary afferent terminals within inner lamina II (II(i)). Glial cell line-derived neurotrophic factor treatment increased P2X(3) immunoreactivity within lamina II(i) but not elsewhere in the cord. Nerve growth factor treatment, however, induced novel P2X(3) expression, with intense immunoreactivity in axons projecting to lamina I and outer lamina II and to the ventro-medial afferent bundle beneath the central canal. In the normal DRG, we found a greater proportion of P2X(3)-positive neurons at cervical levels, many of which were large-diameter and calcitonin gene-related peptide-positive. In both cervical and lumbar DRG, the number of P2X(3)-positive cells increased following GDNF or NGF treatment. De novo expression of P2X(3) in NGF-sensitive nociceptors may contribute to chronic inflammatory pain.

Neurochemistry of superficial spinal neurones projecting to nucleus of the solitary tract that express c-fos on chemical somatic and visceral nociceptive input in the rat.

We have investigated the presence of three neurochemical markers, glutamate, calbindin-D28k, and nitric oxide synthase, in spinal neurones that transmit chemical noxious inputs from both the skin and the viscera, by combining retrograde labelling with the fluorescent tracer Fluorogold with dual labelling immunohistochemistry. Neurones projecting to the nucleus of the solitary tract (NTS) that expressed Fos protein in response to cutaneous or visceral noxious stimulation were concentrated in lamina I of the cervical and lumbosacral segments, respectively. Although both labelled neuronal populations were numerous, the spino-solitary cells that transmit visceral nociceptive input predominated over those transmitting cutaneous nociceptive input. Calbindin-D28k-immunoreactivity was observed in neurones of three morphological types (fusiform, flattened, and pyramidal) projecting to the NTS that were activated by somatic or visceral nociceptive neurones. Nitric oxide synthase and glutamate immunoreactivities were present only in viscerally activated nociceptive neurones projecting to the NTS. Glutamate-immunopositive NTS-projecting cells were exclusively of the flattened type, and the nitric oxide synthase-immunolabelled NTS-projecting cells comprised 75%/fusiform cells and 25% flattened cells. These data suggest that the involvement of excitatory spinal lamina I projection neurones in the transmission of peripheral chemical nociceptive inputs to the NTS may be restricted to information of visceral origin.

Pituitary adenylate cyclase-activating polypeptide innervation of the mudpuppy cardiac ganglion.

The presence and potential origin of the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) was determined in cardiac ganglia of the mudpuppy, Necturus maculosus. Although PACAP has been implicated in the regulation of cardiac function in several mammalian species, the presence of this peptide in the autonomic nervous system (ANS) of other species is unclear. Thus, this study is the first to characterize this highly conserved peptide in the ANS of a non-mammalian species. PACAP-immunoreactivity was observed in nerve fibers throughout the mudpuppy cardiac ganglia and often was co-localized with the sensory neuropeptides substance P and calcitonin gene-related peptide. Removal of all extrinsic inputs to the ganglia by organ culture eliminated PACAP-immunoreactivity in the cardiac ganglia, whereas bilateral vagotomies only partially reduced PACAP-labeling. PACAP-immunoreactive neurons were observed in both high thoracic dorsal root ganglia and in vagal sensory ganglia. While no PACAP-positive neurons were observed in caudal medulla brainstem regions, PACAP-containing nerve fibers were found in the region of the nucleus solitarius. These results suggest that, in the mudpuppy, PACAP is found primarily in visceral afferent fibers, originating from cells in either the dorsal root ganglia or vagal sensory ganglia. Based on their anatomic localization, these afferent fibers may function to transmit important sensory information to cardiovascular centers in the brain as well as serving as local reflex inputs to modulate postganglionic parasympathetic output within the cardiac ganglion itself.

Effects of acute and chronic gonadectomy on the catecholamine innervation of the cerebral cortex in adult male rats: insensitivity of axons immunoreactive for dopamine-beta-hydroxylase to gonadal steroids, and differential sensitivity of axons immunoreactive for tyrosine hydroxylase to ovarian and testicular hormones.

Previous studies have shown that gonadectomy in adult male rats induces a complex series of region- and time-specific changes in the density of presumed cerebral cortical dopamine axons that are immunoreactive for tyrosine hydroxylase. The present study asked whether noradrenergic cortical afferents also show hormone sensitivity by assaying axons immunoreactive for the enzyme dopamine-beta-hydroxylase in representative areas of acutely and chronically gonadectomized and sham-operated adult male rats. Catecholamine afferents (both tyrosine hydroxylase-immunoreactive and dopamine-beta-hydroxylase-immunoreactive) were also quantified in gonadectomized rats supplemented with testosterone propionate, with 17-beta-estradiol, or with 5-alpha-dihydrotestosterone. Analyses of noradrenergic (dopamine-beta-hydroxylase) afferents revealed no differences in axon appearance or density among the hormonally intact and hormonally manipulated groups. However, analyses of tyrosine hydroxylase immunoreactivity revealed an unexpected division of labor among ovarian and testicular hormones in ameliorating the effects of acute verses chronic hormone deprivation on these afferents. Estradiol replacement attenuated the decreases in immunoreactivity induced by acute gonadectomy, but was ineffective in suppressing changes in immunoreactivity stimulated by chronic gonadectomy. In contrast, supplementing gonadectomized animals with dihydrotestosterone provided no protection from acute decreases in innervation, but fully attenuated both the supragranular decreases and infragranular increases in tyrosine hydroxylase-immunoreactive axon density that mark the association cortices of chronically gonadectomized rats. Together these findings indicate both long- and short-term effects of gonadectomy on cortical catecholamines, principally target dopamine afferents, and that chronic gonadectomy, which selectively disturbs dopamine innervation in the prefrontal cortices, involves a compromise in androgen signaling pathways.

Calcitonin gene-related peptide and spinal afferents partly mediate postoperative colonic ileus in the rat.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a widely distributed neuropeptide contained in intrinsic and extrinsic neurons of the gastrointestinal wall that has been shown to be released by noxious stimulation, to be involved in nociception, to inhibit gastrointestinal motility, and to partly mediate postoperative gastric ileus. We hypothesized that abdominal surgery-induced release of CGRP might inhibit postoperative colonic motility and food intake. METHODS: Colonic transit, stool pellet number, stool pellet weight, and food intake were measured for 48 hours after induction of postoperative ileus in rats. CGRP was immunoneutralized by preoperative injection of CGRP monoclonal antibody, or visceral afferent nerve fibers containing CGRP were functionally ablated by topical capsaicin treatment of the vagus nerves or of the celiac/superior mesenteric ganglia before abdominal surgery. RESULTS: Abdominal surgery increased colonic transit time and decreased 24-hour cumulative stool pellet number, stool pellet weight, and food intake. CGRP immunoneutralization reversed postoperative inhibition of colonic transit, 24-hour cumulative stool pellet number, stool pellet weight, and food intake by 77%, 82%, 80%, and 52%, respectively. Whereas ablation of vagal afferent nerve fibers had no effect, spinal afferent nerve fiber ablation reversed postoperative inhibition of 24-hour cumulative stool pellet number, stool pellet weight, and food intake by 41%, 38%, and 19%, respectively. CONCLUSIONS: CGRP and spinal afferent nerve fibers partly mediate postoperative colonic ileus and inhibition of food intake in the rat. By the magnitude of reversal of postoperative ileus, CGRP seems to be an important mediator of postoperative colonic ileus. Our results for the first time show involvement of a neuropeptide and spinal afferents in the mediation of postoperative colonic ileus and postoperative inhibition of food intake in rats.

Innervation of VIP-immunoreactive neurons by the ventroposteromedial thalamic nucleus in the barrel cortex of the rat.

We investigated the synaptic terminals of fibers originating in the ventroposteromedial thalamic nucleus (VPM) and projecting to the main input layers (IV/III) of the rat posteromedial barrel subfield. It was our aim to determine whether or not the subpopulation of vasoactive intestinal polypeptide (VIP)-immunoreactive neurons in these layers are directly innervated by the sensory thalamus. Anterograde tracing with Phaseolus vulgaris leucoagglutinin (PHA-L) and immunohistochemistry for VIP were combined for correlated light and electron microscopic examination. Columns of cortical tissue were well defined by barrel-like patches of PHA-L-labeled fibers and boutons in layers IV and III. Within these columns VIP-immunoreactive perikarya were located mainly in supragranular layers. Marked perikarya were also seen in infragranular layers, but their immunoreactivity was often weaker. Granular layer IV, which is the main terminal field for thalamic fibers, contained fewer VIP neurons than supragranular layers. In the light microscope, however, PHA-L-labeled fibers appeared to contact the somata or proximal dendrites of 60-86% of the layer IV VIP neurons . By contrast, only 18-35% of the VIP neurons in the supragranular layers, which receive a moderately dense projection from the VPM, appeared to be contacted. PHA-L-labeled boutons were seen close to 13-25% of infragranular VIP-positive cells. Electron microscopy showed that thalamic fibers formed at most four asymmetric synapses on a single layer IV, VIP-positive neuron. Although the proportion of VIP-positive neurons with labeled synapses was lower in supragranular layers, most of them shared multiple asymmetric synapses with labeled thalamic fibers. Up to six labeled synapses were seen on individual VIP neurons in layer III. We conclude that subpopulations of VIP-immunoreactive neurons, located in layers IV, III, and II are directly innervated by the VPM. These neurons may be involved in the initial stages of cortical processing of sensory information from the large, mystacial vibrissae. Since VIP is known to be colocalized with the inhibitory transmitter GABA, it is likely that VIP neurons participate in the shaping of the receptive fields in the barrel cortex.

Noxious heat-evoked Fos-like immunoreactivity in the rat medulla, with emphasis on the catecholamine cell groups.

The objectives of the present study were 1) to utilize Fos immunohistochemistry as a marker for neuronal activity in order to examine the population of neurons in the medulla that is engaged by activation of nociceptive peripheral afferents and 2) to determine whether catecholamine-containing neurons in the medulla also express noxious heat-evoked Fos-like immunoreactivity. Noxious heating of the hindpaw evoked specific patterns of Fos-like immunoreactivity in the medulla in regions known to be involved in both nociceptive processing and cardiovascular regulation. Noxious heating of the hindpaw significantly increased the mean number of neurons expressing Fos-like immunoreactivity in the contralateral ventrolateral medulla. Increased numbers of Fos-positive neurons also were observed in both the ipsilateral and the contralateral A1 catecholamine cell groups. Similarly, in the contralateral medullary dorsal reticular fields, noxious heating of the hindpaw significantly increased the mean number of neurons expressing Fos-like immunoreactivity. In contrast, in the paramedian reticular nucleus, noxious heating of the hindpaw resulted in a significant decrease in the mean number of neurons expressing Fos-like immunoreactivity. No significant differences in the mean numbers of neurons expressing Fos-like immunoreactivity were noted in the A2, C1, or C2/C3 medullary catecholamine cell groups. These results suggest that noxious stimuli affect pools of neurons in the medulla with multiple physiological functions.

Somatostatin-like immunoreactivity in primary afferents of the medial articular nerve and colocalization with substance P in the cat.

The proportion of somatostatin-containing dorsal root ganglion cells innervating the knee joint of the cat via the medial articular nerve was determined by using retrograde labeling with fast blue and immunohistochemistry. Immunoreactivity was found in 8.6% of labeled cell bodies. In colchicine-treated ganglia, the proportion increased to 16.8%. Only small and intermediate-sized perikarya showed somatostatin-like immunoreactivity, indicating that this neuropeptide is synthesized predominantly in primary afferent units with unmyelinated sensory axons but may also be present in primary afferents with thinly myelinated sensory fibers. Colchicine treatment had no influence on the cell size distribution. Colocalization of somatostatin with substance P was determined by comparing the proportions of immunopositive dorsal root ganglion cells after incubation with antibodies against substance P or somatostatin or with a mixture of both. Substance P-like immunoreactivity was found in 18.1% (untreated ganglia) and 19.6% (colchicine treated ganglia) of the labeled neurons. After incubation with a mixed antibody solution, 18.2% of joint afferents in untreated and 19.9% of the cells in colchicine-treated ganglia were immunopositive. Comparing this result with the results obtained using somatostatin and substance P antibodies alone, one can calculate that both neuropeptides are colocalized in about 17% of the cat's knee joint afferents. About 3% of the neurons contain only substance P, whereas almost none of the neurons contain only somatostatin. Based on this fact, one can assume that both neuropeptides are coreleased in peripheral tissue as well as in the central nervous system.

The distribution of GAP-43 in normal rat spinal cord.

We have studied the distribution of the growth-associated protein GAP-43 in the spinal cord of adult rats by light and electron microscopy, using a new antiserum raised against GAP-43/beta-galactosidase fusion protein. We show that GAP-43 is present at all vertebral levels but is more concentrated in cervical and thoracic regions. In addition to heavy staining in the corticospinal tracts of the white matter, staining can be seen at the light microscopic level throughout the grey matter and is particularly heavy around the central canal and in the superficial dorsal horn. Electron microscopic examination revealed that GAP-43 immunostaining is confined to a subpopulation of axons and axon terminals. Staining occurs in small myelinated and unmyelinated fibres and in terminals which are mainly small and make single axodendritic or axosomatic synapses. Staining in such terminals occurs in the axoplasm but is heaviest immediately adjoining the axolema. Staining was not observed in dendrites, nor in large myelinated axons or large axon terminals. Our results indicate that GAP-43 is expressed in adult rat spinal cord in a subpopulation of small diameter fibres and axon terminals. The distribution and morphology of these terminals is consistent with several different possible origins including corticospinal projection neurons, small diameter primary afferent neurons, and descending raphe-spinal serotonin containing neurons.

Organization of medullary adrenergic and noradrenergic projections to the periaqueductal gray matter in the rat.

The periaqueductal or midbrain central gray matter (CG) in the rat contains a dense network of adrenergic and noradrenergic fibers. We examined the origin of this innervation by using retrograde and anterograde axonal tracers combined with immunohistochemistry for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), and phenylethanolamine N-methyltransferase (PNMT). Following injections of the fluorescent tracers Fast Blue or Fluorogold into the CG, double-labeled neurons in the medulla were identified mainly in the noradrenergic A1 group in the caudal ventrolateral medulla (VLM) and A2 group in the medial part of the nucleus of the solitary tract (NTS); and in the adrenergic C1 group in the rostral ventrolateral medulla and C3 group in the rostral dorsomedial medulla. Injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) into these cell groups resulted in a distinct pattern of axonal labeling in various subdivisions of the CG. Anterogradely labeled fibers originating in the medial NTS were predominantly found in the lateral portion of the dorsal raphe nucleus and in the adjacent part of the lateroventral CG (CGlv). Following PHA-L injections into the C3 region the anterogradely labeled fibers were diffusely distributed in the CGlv and the dorsal raphe nucleus at caudal levels, but rostrally tended to be located laterally in the CGlv. In contrast, ascending fibers from the caudal and rostral VLM terminated in the rostral dorsal part of the CGlv and in the dorsal nucleus of the CG, whereas ventral parts of the CG, including the dorsal raphe nucleus, contained few afferent fibers. Double-label studies with antisera against DBH and PNMT confirmed that noradrenergic neurons in the A1 and A2 groups and adrenergic neurons in the C1 and C3 groups contributed to these innervation patterns in the CGlv. Noradrenergic and adrenergic projections from the medulla to the CG may play an important role in a variety of autonomic, sensory and behavioral processes.