Activation of the spinal sigma-1 receptor enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NR1 subunit in mice.

BACKGROUND AND PURPOSE: Previously we demonstrated that the spinal sigma-1 receptor (Sig-1 R) plays an important role in pain transmission, although the exact mechanism is still unclear. It has been suggested that Sig-1 R agonists increase glutamate-induced calcium influx through N-methyl-D-aspartate (NMDA) receptors. Despite data suggesting a link between Sig-1 Rs and NMDA receptors, there are no studies addressing whether Sig-1 R activation directly affects NMDA receptor sensitivity. EXPERIMENTAL APPROACH: We studied the effect of intrathecal (i.t.) administration of Sig-1 R agonists on protein kinase C (PKC) and protein kinase A (PKA) dependent phosphorylation of the NMDA receptor subunit NR1 (pNR1) as a marker of NMDA receptor sensitization. In addition, we examined whether this Sig-1 R mediated phosphorylation of NR1 plays an important role in sensory function using a model of NMDA-induced pain. KEY RESULTS: Both Western blot assays and image analysis of pNR1 immunohistochemical staining in the spinal cord indicated that i.t. injection of the Sig-1 R agonists, PRE-084 or carbetapentane dose dependently enhanced pNR1 expression in the murine dorsal horn. This increased pNR1 expression was significantly reduced by pretreatment with the specific Sig-1 R antagonist, BD-1047. In another set of experiments Sig-1 R agonists further potentiated NMDA-induced pain behaviour and pNR1 immunoreactivity and this was also reversed with BD-1047. CONCLUSIONS AND IMPLICATIONS: The results of this study suggest that the activation of spinal Sig-1 R enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NMDA receptor NR 1 subunit.

The relationship of bone-tumor-induced spinal cord astrocyte activation and aromatase expression to mechanical hyperalgesia and cold hypersensitivity in intact female and ovariectomized mice.

Recently, our group established a relationship between tumor-induced spinal cord astrocyte activation and aromatase expression and the development of bone tumor nociception in male mice. As an extension of this work, we now report on the association of tumor-induced mechanical hyperalgesia and cold hypersensitivity to changes in spinal cord dorsal horn GFAP and aromatase expression in intact (INT) female mice and the effect of ovariectomy on these parameters. Implantation of fibrosarcoma cells produced robust mechanical hyperalgesia in INT animals, while ovariectomized (OVX) females had significantly less mechanical hyperalgesia. Cold hypersensitivity was apparent by post-implantation day 7 in INT and OVX females compared to their saline-injected controls and increased throughout the experiment. The decrease in mechanical hyperalgesia in OVX females was mirrored by significant decreases in spinal astrocyte activity in laminae I-II, III-IV, V-VI and X and aromatase expression in laminae V-VI and X in the dorsal horn of tumor-bearing animals. Administration of the aromatase inhibitor letrozole reduced tumor-induced hyperalgesia in INT females only suggesting that the tumor-induced increase in aromatase expression and its associated increase in spinal estrogen play a role in the development of bone tumor-induced hyperalgesia. Finally, intrathecal (i.t.) administration of 17ß-estradiol caused a significant increase in tumor-induced hyperalgesia in INT tumor-bearing females. Since i.t. 17ß-estradiol increases tumor pain and ovariectomy significantly decreases tumor pain, as well as spinal aromatase, estrogen may play a critical role in the spinal cord response to the changing tumor environment and the development of tumor-induced nociception.

Role of peripheral sigma-1 receptors in ischaemic pain: Potential interactions with ASIC and P2X receptors.

BACKGROUND: The role of peripheral sigma-1 receptors (Sig-1Rs) in normal nociception and in pathologically induced pain conditions has not been thoroughly investigated. Since there is mounting evidence that Sig-1Rs modulate ischaemia-induced pathological conditions, we investigated the role of Sig-1Rs in ischaemia-induced mechanical allodynia (MA) and addressed their possible interaction with acid-sensing ion channels (ASICs) and P2X receptors at the ischaemic site. METHODS: We used a rodent model of hindlimb thrombus-induced ischaemic pain (TIIP) to investigate their role. Western blot was performed to observe changes in Sig-1R expression in peripheral nervous tissues. MA was measured after intraplantar (i.pl.) injections of antagonists for the Sig-1, ASIC and P2X receptors in TIIP rats or agonists of each receptor in naïve rats. RESULTS: Sig-1R expression significantly increased in skin, sciatic nerve and dorsal root ganglia at 3 days post-TIIP surgery. I.pl. injections of the Sig-1R antagonist, BD-1047 on post-operative days 0-3 significantly attenuated the development of MA during the induction phase, but had no effect on MA when given during the maintenance phase (days 3-6 post-surgery). BD-1047 synergistically increased amiloride (an ASICs blocker)- and TNP-ATP (a P2X antagonist)-induced analgesic effects in TIIP rats. In naïve rats, i.pl. injection of Sig-1R agonist PRE-084 alone did not produce MA; but it did induce MA when co-administered with either an acidic pH solution or a sub-effective dose of αßmeATP. CONCLUSION: Peripheral Sig-1Rs contribute to the induction of ischaemia-induced MA via facilitation of ASICs and P2X receptors. Thus, peripheral Sig-1Rs represent a novel therapeutic target for the treatment of ischaemic pain.

Functional interactions between tumor and peripheral nerve: morphology, algogen identification, and behavioral characterization of a new murine model of cancer pain.

This paper describes a model of tumor-induced bone destruction and hyperalgesia produced by implantation of fibrosarcoma cells into the mouse calcaneus bone. Histological examination indicates that tumor cells adhere to the bone edge as early as post-implantation day (PID) 3, but osteolysis does not begin until PID 6, correlating with the development of hyperalgesia. C3H/He mice exhibit a reproducible hyperalgesia to mechanical and cold stimuli between PID 6 and 16. These behaviors are present but significantly reduced with subcutaneous implantation that does not involve bone. Systemic administration of morphine (ED(50) 9.0 mg/kg) dose-dependently attenuated the mechanical hyperalgesia. In contrast, bone destruction and hypersensitivity were not evident in mice implanted with melanoma tumors or a paraffin mass of similar size. A novel microperfusion technique was used to identify elevated levels of the putative algogen endothelin (ET) in perfusates collected from the tumor sites of hyperalgesic mice between PID 7 and 12. Increased ET was evident in microperfusates from fibrosarcoma tumor-implanted mice but not from melanoma tumor-implanted mice, which are not hyperalgesic. Intraplantar injection of ET-1 in naive and, to a greater extent, fibrosarcoma tumor-bearing mice produced spontaneous pain behaviors, suggesting that ET-1 activates primary afferent fibers. Intraplantar but not systemic injection of the ET-A receptor antagonist BQ-123 partially blocked tumor-associated mechanical hyperalgesia, indicating that ET-1 contributes to tumor-induced nociception. This model provides a unique approach for quantifying the behavioral, biochemical, and electrophysiological consequences of tumor-nerve interactions.

Nociceptive characteristics of tumor necrosis factor-alpha in naive and tumor-bearing mice.

A nociceptive role for tumor necrosis factor-alpha (TNF-alpha) in naive mice and in mice with fibrosarcoma tumor-induced primary hyperalgesia was investigated. The presence of TNF-alpha mRNA was confirmed in tumor site homogenates by reverse transcription-polymerase chain reaction (RT-PCR), and examination of TNF-alpha protein levels in tumor-bearing mice indicated a significantly higher concentration of this cytokine in tumor microperfusates and tumor site homogenates compared with that obtained from a similar site on the contralateral limb or in naive mice. Intraplantar injection of TNF-alpha into naive or fibrosarcoma tumor-bearing mice induced mechanical hypersensitivity, as measured by withdrawal responses evoked by von Frey monofilaments. This hypersensitivity suggests that TNF-alpha can excite or sensitize primary afferent fibers to mechanical stimulation in both naive and tumor-bearing mice. In addition, the hyperalgesia produced by TNF-alpha was completely eliminated when the injected TNF-alpha was pre-incubated with the soluble receptor antagonist TNFR:Fc. Importantly, pre-implantation systemic as well as post-implantation intra-tumor injection of TNFR:Fc partially blocked the mechanical hyperalgesia, indicating that local production of TNF-alpha may contribute to tumor-induced nociception.

Colocalization of aromatase in spinal cord astrocytes: differences in expression and relationship to mechanical and thermal hyperalgesia in murine models of a painful and a non-painful bone tumor.

While spinal cord astrocytes play a key role in the generation of cancer pain, there have been no studies that have examined the relationship of tumor-induced astrocyte activation and aromatase expression during the development of cancer pain. Here, we examined tumor-induced mechanical hyperalgesia and cold allodynia, and changes in Glial fibrillary acid protein (GFAP) and aromatase expression in murine models of painful and non-painful bone cancer. We demonstrate that implantation of fibrosarcoma cells, but not melanoma cells, produces robust mechanical hyperalgesia and cold allodynia in tumor-bearing mice compared to saline-injected controls. Secondly, this increase in mechanical hyperalgesia and cold allodynia is mirrored by significant increases in both spinal astrocyte activity and aromatase expression in the dorsal horn of fibrosarcoma-bearing mice. Importantly, we show that aromatase is only found within a subset of astrocytes and not in neurons in the lumbar spinal cord. Finally, administration of an aromatase inhibitor reduced tumor-induced hyperalgesia in fibrosarcoma-bearing animals. We conclude that a painful fibrosarcoma tumor induces a significant increase in spinal astrocyte activation and aromatase expression and that the up-regulation of aromatase plays a role in the development of bone tumor-induced hyperalgesia. Since spinal aromatase is also upregulated, but to a lesser extent, in non-painful melanoma bone tumors, it may also be neuroprotective and responsive to the changing tumor environment.

The midbrain periaqueductal gray in the rat. I. Nuclear volume, cell number, density, orientation, and regional subdivisions.

The midbrain periaqueductal gray is a functionally heterogeneous region which plays an important role in pain modulation. Despite the heterogeneity considerable controversy exists regarding the presence or absence of morphological subdivisions within the region. The present study was designed to evaluate the possibility of morphological subdivisions within the rat periaqueductal gray by using a statistical cluster analysis system. In addition both qualitative and quantitative data concerning neuronal size, shape, and density were obtained. On the basis of measurements of over 12,000 neurons in two planes of section, the mean neuronal length of cell bodies in this region was 14.82 microns and the mean neuronal area was 95.59 microns squared . The mean neuronal density was found to be 16,284 cells per mm3. Neuronal density decreased from rostral to caudal in the periaqueductal gray. The data obtained from cluster maps suggest the presence of four subdivisions within this midbrain region. The medial subdivision contains the smallest neurons and exhibits the lowest cell density. The dorsolateral and ventrolateral divisions contain the largest neurons while the dorsal division displays the highest packing density. These results are discussed in light of recent receptor binding and immunohistochemical studies of this region.

Colocalization of fixative-modified glutamate and glutaminase but not GAD in rubrospinal neurons.

In an attempt to identify putative neurotransmitters of rubrospinal neurons, immunocytochemical procedures were utilized in combination with retrograde tracing techniques in 15 adult male rats. Following injections of horseradish peroxidase (HRP) or wheat germ agglutinin conjugated to HRP (WGA-HRP) into the spinal cord, midbrain sections were processed with a combined procedure that allowed visualization of both the retrograde tracer and one or more antigens including glutamate, glutaminase, and glutamatic acid decarboxylase (GAD). Initial colocalization studies demonstrated that glutamatelike and glutaminaselike immunoreactivities were cocontained within the same neurons. Following injections of HRP or WGA-HRP into the spinal cord approximately 53% of retrogradely labeled neurons contained glutamate immunoreactivity. Triple-labeling experiments indicated that glutamatelike immunoreactivity was colocalized with glutaminase immunoreactivity in retrogradely labeled rubrospinal neurons. Retrogradely labeled neurons did not contain GAD immunoreactivity. Moreover, triple labeling experiments verified that glutamatelike immunoreactive retrogradely labeled cells did not cocontain GAD immunoreactivity. These studies demonstrate that glutamate and its synthesizing enzyme, glutaminase, are present in some rubrospinal neurons and raise the possibility that a component of the rubrospinal projection may be glutamatergic. GAD, on the other hand, is not present in rubrospinal neurons. This finding supports the hypothesis that GABAergic neurons play a role as interneurons in the red nucleus.

The midbrain periaqueductal gray in the rat. II. A Golgi analysis.

This study consists of a detailed analysis of neurons in the midbrain periaqueductal gray of the rat utilizing four variants of the Golgi technique. Neurons were classified into three major categories based on soma shape, number of primary dendrites, number of dendritic bifurcations, interspinous distance, axonal origin, and axon trajectory. Neurons in each category were further subdivided into large and small varieties based predominantly on soma size and dendritic patterns. Both quantitative and qualitative data concerning each neuronal type is provided as well as data relating to its relative distribution among the four periaqueductal gray subdivisions. The small bipolar neuron, characterized by its small size and spindle-shaped soma, was the most prominent cell type observed, composing 37% of the impregnated neurons in our material. This cell type was most numerous in the medial subdivision and least prominent in the dorsolateral subdivision. The small triangular neuron composed 23% of the neuronal population and was relatively evenly distributed through the periaqueductal gray. The remaining four cell types include the large and small multipolar neurons, the large fusiform neurons, and the large triangular neurons. Axons originated from either the perikaryon or a proximal dendrite, with a dendritic origin being most common for large and small triangular neurons and large fusiform neurons. The trajectory of axons in single thick coronal sections originating from periaqueductal gray neurons is typically away from the mesencephalic aqueduct. The exact trajectory is dependent on the location of the neuron. Axons arising from cells in the dorsal subdivision usually project in a dorsal or dorsolateral direction while axons of ventrolateral neurons may project dorsally, laterally, or ventrally. In sum, these data indicate a complex level of internal organization of the periaqueductal gray. The results are discussed in terms of previous immunohistochemical studies of neurons in this region.

Altered c-fos expression in the parabrachial nucleus in a rodent model of CFA-induced peripheral inflammation.

Increases in the expression of immediate early genes have been shown to occur in the lumbar spinal cord dorsal horn after peripheral inflammation. Given that the pontine parabrachial nucleus has been implicated in nociceptive as well as antinociceptive processes and is reciprocally connected with the spinal cord dorsal horn, it seems likely that peripheral inflammation will cause alterations in immediate early gene expression in this nucleus. To test this hypothesis we examined cFos-like immunoreactivity in a rodent complete Freund's adjuvant-induced peripheral inflammatory model of persistent nociception. Unilateral hind paw injections of complete Freund's adjuvant produced inflammation, hyperalgesia of the affected limb, and alterations in open field behaviors. Immunocytochemical analysis demonstrated a bilateral increase in cFos-like immunoreactivity in the lateral and Kolliker-Fuse subdivisions of the parabrachial nucleus at 6 and 24 hours postinjection and an ipsilateral decrease below basal levels in the Kolliker-Fuse subdivision at 96 hours postinjection when compared to saline controls. Taken together, these results suggest that select parabrachial neurons are activated by noxious somatic inflammation. These active parabrachial neurons are likely to participate in ascending nociceptive and/or descending antinociceptive pathways.

An electron microscopic description of glutamate-like immunoreactive axon terminals in the rat principal sensory and spinal trigeminal nuclei.

The spinal and principal sensory trigeminal nuclei relay noxious and nonnoxious stimuli from the orofacial region to the thalamus. Physiological studies have implicated glutamate as an important neurotransmitter in this region. Despite its importance as a potential transmitter, few studies have examined the anatomical distribution of glutamate within these nuclei. We therefore chose to use a monoclonal antibody raised against glutamate conjugated to a carrier protein to identify and describe glutamate-like immunoreactive processes at the electron microscopic level. Glutamate-like immunoreactive axon terminals were identified throughout the spinal trigeminal and principal sensory trigeminal nucleus. In subnucleus caudalis glutamate-like immunoreactive terminals occurred frequently in all laminae and were morphologically heterogeneous. In lamina I, glutamate-like immunoreactive terminals were primarily ovoid, contained spherical synaptic vesicles, and participated in synaptic complexes with both dendritic and axonal profiles. In laminae II and III many glutamate-like immunoreactive axon terminals were identified as the central element in synaptic glomeruli. Within discrete patches of lamina II, large numbers of glutamate-like immunoreactive terminals contained dense core vesicles. The majority of glutamate-like immunoreactive terminals in subnucleus interpolaris, subnucleus oralis, and principal sensory trigeminal nucleus were similar in morphology and synaptic interaction to the glutamate-like immunoreactive terminals found in subnucleus caudalis. Glutamate-like immunoreactive terminals that were the central presynaptic element in glomerular complexes were seen in all subnuclei. In sections from subnucleus interpolaris and subnucleus oralis central glutamate-like immunoreactive terminations within glomerular complexes had much smoother profiles, and in subnucleus interpolaris participated primarily in axodendritic synaptic junctions. In the principal sensory trigeminal nucleus central glutamate-like immunoreactive terminations were highly scalloped and participated in numerous axoaxonic synaptic junctions. The above observations are consistent with the hypothesis that glutamate-like immunoreactivity is present in some primary afferent terminations and functions as an important excitatory transmitter involved in the relay of sensory information to the spinal trigeminal and principal sensory trigeminal nucleus.

Induction of NADPH-diaphorase/nitric oxide synthase in the brainstem trigeminal system resulting from cerebellar lesions.

Recent evidence indicates that NADPH-diaphorase (NADPH-d) and nitric oxide synthase (NOS) can be induced in cerebellar afferent neurons following mechanical, thermal, or chemical damage to the cerebellar cortex (Saxon and Beitz [1994] Neuroreport 5:809-812). The present study reports on the induction of NADPH-d/NOS in neurons of the brainstem trigeminal complex (BVC). Three groups of rats were used: Group I received a unilateral glass micropipette lesion into the vermal/paravermal region of the cerebellar cortex, group II received a concurrent injection of fluoro-gold along with the pipette lesion, and in group III the cerebellar cortex on one side was aspirated. Following survival times of 7-120 days, animals were processed for NADPH-d histochemistry. All three groups showed projection-specific induction of NADPH-d in different regions of the brainstem trigeminal complex. Induced neurons were distributed throughout the ipsilateral subnucleus interpolaris, principal trigeminal nucleus, and intertrigeminal nucleus. Subnucleus oralis contained a small number of induced neurons localized to the ipsilateral dorsomedial portion of the subnucleus. Projection-specific induction was confirmed by the presence of neurons double-labeled for NADPH-d and Fluoro-Gold. Although the functional consequences of NADPH-d/NOS induction remain to be elucidated, the induction of these enzymes in precerebellar neurons suggests that nitric oxide may play a role in the neuronal response to target specific lesions.

The normal distribution and projections of constitutive NADPH-d/NOS neurons in the brainstem vestibular complex of the rat.

The vestibular system is a highly conserved sensory system in vertebrates that is largely responsible for maintenance of one's orientation in space, posture, and balance and for visual fixation of objects during motion. In light of the considerable literature indicating an involvement of nitric oxide (NO) in sensory systems, it is important to determine whether NO is associated with vestibular pathways. To study the relationship of NO to vestibular pathways, we first examined the normal distribution of constitutive NADPH-diaphorase (NADPH-d), a marker for nitric oxide synthase (NOS), in the vestibular complex (VC) and then examined its association with selected vestibular projection neurons. Survey of the four major vestibular nuclei revealed that only the medial vestibular nucleus contained significant numbers of perikarya stained for NADPH-d/NOS. By contrast, all the vestibular nuclei contained a network of fine processes that stained positive for NADPH-d, although the density of this network varied among the individual nuclei. To determine whether NADPH-d/NOS neurons project to vestibular efferent targets, injections of the retrograde tracer Fluoro-Gold were made into known targets of second-order vestibular neurons. Vestibular neurons containing constitutive NADPH-d/NOS were found to project predominantly to the oculomotor nucleus. A small number of neurons also participate in vestibulothalamic and intrinsic vestibular connections. These results indicate that NADPH-d/NOS neurons are prevalent in the MVN and that a subpopulation of these neurons project to the oculomotor complex. Nitric oxide is probably released locally from axons located throughout the vestibular complex but may play a particularly important role in vestibulo-ocular pathways.

Rat tooth pulp projections to spinal trigeminal subnucleus caudalis are glutamate-like immunoreactive.

It has been shown that glutamate-like immunoreactive axon terminals are present within the spinal trigeminal nucleus, including subnucleus caudalis. The morphology of many of these terminations is consistent with their identification as primary afferents. To establish whether primary afferent projections to subnucleus caudalis are glutamate-like immunoreactive, we injected an anterograde tract tracer into rat incisor tooth pulp, histochemically visualized this tracer within subnucleus caudalis, and then used an immunocytochemical technique to label glutamate-like immunoreactive profiles within these same sections. The anterograde tract tracer used, the B subunit of cholera toxin conjugated to horseradish peroxidase (B-HRP), is transported transganglionically and can be used to localize tooth pulp projection fibers in the spinal trigeminal nucleus. A majority of B-HRP projection fibers from rat lower incisors terminated ipsilaterally in axon terminals in the dorsal region of subnucleus caudalis. Labeled axon terminals were both scallop-shaped and smooth in profile. Small numbers of fibers containing B-HRP extended into laminae I-III caudally and were present in both the border zone between laminae IV and V and the most lateral region of lamina V rostrally. Approximately 75% of the B-HRP-labeled projection fibers were glutamate-like immunoreactive, providing evidence that the excitatory amino acid glutamate functions as a neurotransmitter in a subpopulation of these fibers. Terminals reactive for both B-HRP and glutamate-like immunoreactivity contained small, spherical round vesicles, formed asymmetric synapses, and participated in axoaxonic and axodendritic synaptic junctions. These results support the hypothesis that glutamate may be a transmitter of A delta and C fibers involved in relaying nociceptive information from the tooth pulp.

A quantitative light and electron microscopic analysis of taurine-like immunoreactivity in the dorsal horn of the rat spinal cord.

Taurine has been proposed as an inhibitory neurotransmitter or neuromodulator in the vertebrate central nervous system. Within the spinal cord, taurine has been shown to have a direct inhibitory effect on spinal neurons and to have a selective antinociceptive effect on chemically induced nociception. Although sufficient data exists to suggest that taurine plays a neurotransmitter or neuromodulatory role in the spinal cord, it is not known whether this amino acid is present in axon terminals nor if this amino acid has a unique pattern of distribution within spinal tissue. To address these questions a monoclonal antibody against taurine was employed to localize taurine-like immunoreactivity in the dorsal horn of the rat spinal cord by using both light and electron microscopic techniques. Taurine-like immunoreactivity was most dense and most prominent in laminae I and II of the dorsal horn. A moderate amount of immunoreactivity was also present in laminae VIII and IX and X while the remaining laminae were only lightly stained. In laminae I and II taurine-like immunostaining was evident within neuronal cell bodies, dendrites, myelinated and unmyelinated axons, axon terminals, and astrocytes and their processes. Cell counts of these two laminae indicated that approximately 30% of neuronal perikarya at the C2 level, 52% of neuronal perikarya at the T6 level, and 18% of neuronal perikarya at the L2 level of the cord exhibited taurine-like immunoreactivity. With preembedding diaminobenzidine staining, approximately 20% of the axons examined in laminae I and II were found to be immunoreactive for taurine. Using postembedding immunogold staining in combination with quantitative procedures, the highest densities of gold particles were found in axon terminals containing pleomorphic vesicles and forming symmetrical synapses (36.8 particles/micron2), in a subpopulation of myelinated axons (34.2 particles/micron2), in a subpopulation of neuronal dendrites (32.6 particles/micron2), and in capillary endothelial cells (39.8 particles/micron2). Moderate labeling occurred in astrocytes (20.9 particles/micron2) and neuronal perikarya (18.7 particles/micron2). The localization of taurine to presumptive inhibitory axon terminals provides anatomical support for the hypothesis that taurine may serve an inhibitory neurotransmitter role in the superficial dorsal horn of the spinal cord. On the other hand, its localization to astrocytes and endothelial cells within both the dorsal ventral horns implies that it serves other nonneuronal functions as well.

Co-localization of fixative-modified glutamate and glutaminase in neurons of the spinal trigeminal nucleus of the rat: an immunohistochemical and immunoradiochemical analysis.

The spinal trigeminal nucleus (STN) is involved in processing orofacial sensory information, including tactile, thermal and nociceptive input, and relaying this information to higher brain centers, such as the thalamus. Very little information is available regarding the major excitatory neurotransmitters of this nucleus. The amino acid glutamate has been proposed as a major excitatory neurotransmitter in the central nervous system. In the present study, a novel monoclonal antibody, specific for fixative-modified glutamate, was utilized in conjunction with polyclonal antisera against glutaminase and aspartate aminotransferase (AATase) in an attempt to identify and map the locations of possible glutamatergic neurons in the STN. Co-localization experiments were performed by radiolabeling our monoclonal antibody and using this antibody in conjunction with the polyclonal antisera against glutaminase and AATase to evaluate the possible coexistence of glutamate with glutaminase or AATase in STN neurons. In all three subnuclei of the STN, immunohistochemically labeled neuronal profiles were observed with both of the polyclonal antisera and with the monoclonal antibody. Subnucleus caudalis contained the greatest number of labeled profiles per coronal section followed by subnucleus interpolaris and subnucleus oralis. The number and the distribution of immunoreactive profiles observed after the use of the glutaminase antiserum was comparable to that obtained with the monoclonal antibody. Co-localization experiments demonstrated that all glutaminase-like immunoreactive neurons also contained fixative-modified glutamate-like immunoradioactivity. These results suggest that glutamatergic neurons are present in the spinal trigeminal nucleus. The AATase antiserum labeled more neuronal profiles in each of the three subnuclei than did the glutaminase antiserum or the monoclonal antibody. In addition, co-localization experiments indicated that glutamate-like immunoreactivity was present in only two-thirds of AATase-like immunoreactive neuronal profiles. These findings suggest that glutaminase may be a more reliable marker of glutamatergic function than AATase.

Immunocytochemical localization of serotonin in the rat periaqueductal gray: a quantitative light and electron microscopic study.

The distribution of serotonin-like immunoreactivity in five regions of the rodent midbrain periaqueductal gray (PAG) was studied by using light and electron microscopic immunohistochemistry in combination with quantitative analysis. Light microscopic analysis revealed the presence of serotonin-like immunoreactive cell bodies located in the ventrolateral and ventromedial regions of the caudal PAG and serotonin-like immunoreactive processes throughout the PAG. Ultrastructural analysis showed dendritic profiles that stained positively for serotonin primarily in ventral regions, although an occasional profile was seen dorsally. Numerous synaptic contacts between unstained axon terminals and ventral dendritic profiles were seen. Axonal profiles that contained reaction product were identified throughout the PAG, but were rarely observed to make any type of specialized contact. Ultrastructural quantification of serotonin-like immunoreactive processes indicated that the highest volume fraction of serotonin immunoreactivity occurred caudoventrally where stained processes constituted 2.6% of the neuropil volume. Rostroventrally stained processes constituted only 0.14% of the neuropil volume at the level of the posterior commissure. By contrast the amount of serotonin-like immunoreactivity found dorsally remained relatively constant at all rostrocaudal levels. Analysis of serotonin staining among PAG regions demonstrated the lowest overall volume fraction in the dorsal region and the highest overall volume fraction in the ventromedial region. No significant differences were observed between medial and lateral regions. A comparison of the results of light microscopic quantitative analysis of serotoninergic processes with electron microscopic quantitative analysis indicated that both techniques produce comparable results.

Differential origin of brainstem serotoninergic projections to the midbrain periaqueductal gray and superior colliculus of the rat.

Previous studies have shown that both the midbrain periaqueductal gray (PAG) and the superior colliculus receive a significant serotoninergic (5-HT) innervation. In the present study the origins of these 5-HT projections to the rodent PAG and superior colliculus were analyzed by using a combined immunohistochemical-retrograde transport technique. Thirteen brainstem regions were found to contain double-labelled 5-HT-like immunoreactive neurons following HRP injections into the PAG while only four brainstem nuclei contained double-labelled neurons following superior collicular injections. After HRP deposits into the ventral PAG, the largest percentage of double-labelled neurons was identified in nucleus raphe magnus, pars alpha of the nucleus gigantocellularis, and the paragigantocellular nucleus. The dorsal PAG, on the other hand, received the largest percentage of its 5-HT projections from nuclei raphe dorsalis, raphe obscurus, raphe pontis, and raphe medianis. The 5-HT input to the superior colliculus was found to arise exclusively from nuclei raphe dorsalis, raphe medianis, and raphe pontis and from the contralateral periaqueductal gray. Raphe nuclei were found to contribute serotoninergic projections to both the PAG and the superior colliculus while reticular nuclei contributed 5-HT projections only to the PAG. Injections of the fluorescent retrograde tracers true blue and nuclear yellow were then made into the PAG and superior colliculus to ascertain if neurons located in raphe nuclei that projected to both structures provided axon collaterals to both areas. Generally, less than 10% of raphe neurons projecting to the superior colliculus were identified as providing axon collaterals to the PAG. The present results demonstrate major quantitative and qualitative differences in the origin of 5-HT projections to the ventral PAG and superior colliculus. The origin of 5-HT input to the dorsal PAG, on the other hand, showed many similarities to the origin of 5-HT innervation of the superior colliculus. These data also indicate that approximately 35% of raphe neurons provide nonserotoninergic projections to the PAG and superior colliculus.

Distribution of nitric oxide synthase-immunoreactive interneurons in the spinal trigeminal nucleus.

The spinal trigeminal nucleus is involved in the transmission of orofacial sensory information. Neither the distribution of the neuromessenger, nitric oxide, within the trigeminal system nor the possible relationship of this simple gas with trigeminothalamic neurons has been carefully studied. Using immunocytochemical (against nitric oxide synthase) and histochemical (NADPH-diaphorase staining) techniques, we have found that nitric oxide neurons and processes are more prominent in the nucleus caudalis and the dorsomedial aspect of the nucleus oralis than in other spinal trigeminal regions. To study the relationship of nitric oxide to trigeminothalamic neurons and intertrigeminal interneurons of the spinal trigeminal nucleus, spinal trigeminal neurons were retrogradely labeled with fluorogold by thalamic injections or by injections into the junction of the nucleus interpolaris and nucleus caudalis. Medullary sections were subsequently processed with NADPH-diaphorase histochemistry. None of the diaphorase-stained neurons in the spinal trigeminal nucleus was found to contain fluorogold; however, some diaphorase-stained processes were found in close proximity to trigeminothalamic neurons. Following spinal trigeminal nucleus injections, many diaphorase-stained neurons were found to contain fluorogold, especially in the nucleus caudalis, suggesting that nitric oxide-containing neurons in the spinal trigeminal nucleus are intertrigeminal interneurons. Collectively, these data indicate that nitric oxide is most prominent in interneurons located in nucleus caudalis and that these interneurons give rise to processes that appose trigeminothalamic neurons, raising the possibility that they may indirectly influence orofacial nociceptive processing at the level of the spinal trigeminal nucleus via nitric oxide production.

Chronic pain and immunity: mononeuropathy alters immune responses in rats.

In order to investigate the possible relationship between chronic pain and the immune system, delayed-type hypersensitivity (DTH) and humoral immunity were assessed in Sprague-Dawley rats subjected to unilateral peripheral mononeuropathy induced by sciatic ligation. Paw withdrawal latency (PWL) time was measured twice during the experiment in animals subjected to sciatic nerve ligation or sham surgery. Sciatic nerve-ligated animals showed hyperalgesia in the leg subjected to neural ligation when compared to the contralateral leg. No differences in PWL times existed in sham-operated animals. In order to exclude possible alterations in immune response due to the surgical procedure or to the hyperalgesia testing, a group of control animals, not subjected to surgical procedures or hyperalgesia testing, was also included in the experiment. Three days post-sciatic ligation or sham surgery, both experimental and control animals were sensitized to keyhole limpet hemocyanin (KLH). A secondary sensitization followed 1 week after the initial immunization. Fourteen days after the initial sensitization, KLH was injected into the hind foot pad and vehicle into the contralateral foot pad in order to assess DTH. One group of rats subjected to sciatic nerve ligation was tested for DTH in the hind foot pad ipsilateral to the ligated nerve, while another group was tested in the contralateral foot pad. Twenty-four hours following foot pad injections, the thickness of both paws was measured and animals were bled to test for anti-KLH immunoglobulins. Animals in which mononeuropathy was induced, but not sham-operated or control animals, exhibited an enhanced DTH response to KLH.(ABSTRACT TRUNCATED AT 250 WORDS)