Maureen Riedl

Immunohistochemical study of the P2X2 and P2X3 receptor subunits in rat and monkey sensory neurons and their central terminals

Of the cloned P2X receptor subunits, six are expressed in sensory neurons, suggesting that the native channels may be heteromultimers with diverse composition. It has been proposed that P2X2 and P2X3 form heteromultimers in sensory neurons. We further tested this hypothesis by examining the relationship of P2X2 and P2X3 immunocytochemically. In rat dorsal root and nodose ganglia, P2X2- and P2X3-immunoreactivity (-ir) were highly colocalized, although single-labeled cells were also present. In dorsal root ganglia (DRG), in some cases P2X2-ir appeared to be present in satellite cells. In dorsal horn of spinal cord, at low magnification the laminar localization of P2X2- and P2X3-ir overlapped, but at high magnification colocalization was rarely observed. In contrast, in the solitary tract and its nucleus (NTS), colocalization of P2X2- and P2X3-ir was seen at low and high magnification. These results suggest that the relationship of P2X2- and P2X3-ir is different in nodose and dorsal root ganglia and might reflect differences in the targeting of P2X receptors in different sensory neurons. In monkey, P2X2-ir was observed in DRG neurons and satellite cells and in dorsal horn of spinal cord. P2X3-ir was also seen in DRG neurons. However, the presence of P2X2-ir in NTS as well as the presence of P2X3-ir in spinal cord and NTS could not be established definitively. These results suggest species differences, although a more extensive study of primate sensory systems is necessary.

Vesicular acetylcholine transporter (VAChT) protein: A novel and unique marker for cholinergic neurons in the central and peripheral nervous systems

Acetylcholine (ACh) is synthesized in nerve terminals from choline and acetyl coenzyme A by the cytoplasmic enzyme choline acetyltransferase (ChAT). The neurotransmitter is thereafter transported into synaptic vesicles, where it is stored until release. cDNA clones encoding a vesicular ACh transporter (VAChT) were recently isolated. In this paper, we report on the generation of highly specific goat polyclonal antisera to the rat VAChT protein by using a synthetic carboxy‐terminal 20‐amino‐acid peptide sequence as an immunogen. Characterization of the antisera revealed recognition of VAChT, but not vesicular monoamine transporter (VMAT) protein, in transfected CV‐1 cells. VAChT immunoreactivity was also detected in cells that endogenously express the protein, such as in PC12 cells and in primary cultures of spinal motoneurons. Absorption controls showed that the VAChT antisera could be completely blocked at the 10 5 M concentration by cognate peptide used for immunization. The antisera cross‐reacted with the VAChT protein in rat and mouse but not in guinea pig, rabbit, or cat. Immunohistochemistry and confocal laser microscopy, using the goat VAChT antisera, showed strong immunoreactivity in discrete fibers and neuronal cell bodies of the central and peripheral nervous systems. Within cell bodies and axonal nerve terminals, as well as in dendrites, the staining appeared granular, presumably representing labeling of synaptic vesicles containing ACh. In the rat central nervous system, VAChT‐positive cell bodies were demonstrated in the cerebral cortex, striatum, septum, nucleus basalis, medial habenula, mesopontine complex, cranial, and autonomic and spinal motor nuclei and in the intermediomedial region near the central canal. High densities of VAChT‐immunoreactive axonal fibers were encountered in areas such as the olfactory bulb, cerebral cortex, striatum, basal forebrain, amygdala, thalamus, hypothalamus including median eminence, hippocampal formation, superior colliculus, interpeduncular nucleus, and pedunculopontine and laterodorsal tegmental nuclei. In cranial and spinal motor nuclei, particularly large varicosities were seen in close proximity to the motoneuron cell somata and their proximal dendrites. In the peripheral nervous system, VAChT immunoreactivity was also detected in motor endplates of skeletal muscle as well as in fibers of sympathetic and parasympathetic abdominal ganglia, heart atrium, respiratory tract, gastrointestinal tract, pancreas, adrenal medulla, male genitourinary tract, and salivary and lacrimal glands. Direct double labeling revealed colocalization of VAChT and ChAT immunoreactivity in neurons. The results show that VAChT antisera represent novel and unique tools for the study of cholinergic neurons in the central and peripheral nervous systems. J. Comp. Neurol. 378:454–467, 1997.

P2X3 is expressed by DRG neurons that terminate in inner lamina II

The P2X3 receptor subunit, a member of the P2X family of ATP‐gated ion channels, is almost exclusively localized in sensory neurons. In the present study, we sought to gain insight into the role of P2X3 and P2X3‐containing neurons in sensory transmission, using immunohistochemical approaches. In rat dorsal root ganglia (DRG), P2X3‐immunoreactivity (‐ir) was observed in small‐ and medium‐sized neurons. Approximately 40% of DRG neuronal profiles in normal rats contained P2X3‐ir. In rats that had received neonatal capsaicin treatment, the number of P2X3‐positive neurons was decreased by approximately 70%. Analysis of the colocalization of P2X3‐ir with cytochemical markers of DRG neurons indicated that approximately 94% of the P2X3‐positive neuronal profiles were labelled by isolectin B4 from Bandeiraea simplicifolia, while only 3% contained substance P‐ir, and 7% contained somatostatin‐ir. In dorsal horn of rat spinal cord, P2X3‐ir was observed in the inner portion of lamina II and was reduced subsequent to dorsal rhizotomy, as well as subsequent to neonatal capsaicin treatment. Finally, P2X3‐ir accumulated proximal to the site of sciatic nerve ligation, and was seen in nerve fibres in skin and corneal epithelium. In summary, our results suggest that P2X3 is expressed by a functionally heterogeneous population of BSI‐B4‐binding sensory neurons, and is transported into both central and peripheral processes of these neurons.

Differential Distribution of α2A and α2C Adrenergic Receptor Immunoreactivity in the Rat Spinal Cord

α2-Adrenergic receptors (α2-ARs) mediate a number of physiological phenomena, including spinal analgesia. We have developed subtype-selective antisera against the C termini of the α2A-AR and α2C-AR to investigate the relative distribution and cellular source or sources of these receptor subtypes in the rat spinal cord. Immunoreactivity (IR) for both receptor subtypes was observed in the superficial layers of the dorsal horn of the spinal cord. Our results suggest that the primary localization of the α2A-AR in the rat spinal cord is on the terminals of capsaicin-sensitive, substance P (SP)-containing primary afferent fibers. In contrast, the majority of α2C-AR-IR was not of primary afferent origin, not strongly colocalized with SP-IR, and not sensitive to neonatal capsaicin treatment. Spinal α2C-AR-IR does not appear to colocalize with the neurokinin-1 receptor, nor is it localized on astrocytes, as evidenced by a lack of costaining with the glial marker GFAP. However, some colocalization was observed between α2C-AR-IR and enkephalin-IR, suggesting that the α2C-AR may be expressed by a subset of spinal interneurons. Interestingly, neither subtype was detected on descending noradrenergic terminals. These results indicate that the α2-AR subtypes investigated are likely expressed by different subpopulations of neurons and may therefore subserve different physiological functions in the spinal cord, with the α2A-AR being more likely to play a role in the modulation of nociceptive information.

An acid sensing ion channel (ASIC) localizes to small primary afferent neurons in rats

THE acid sensing ion channel (ASIC) identified in rat brain and spinal cord is potentially involved in the transmission of acid-induced nociception. We have developed polyclonal antisera against ASIC, and used them to screen rat brain and spinal cord using immunocyto-chemistry. ASIC-immunoreactivity (-ir) is present in but not limited to the superficial dorsal horn, the dorsal root ganglia (DRG) and the spinal trigeminal nucleus, as well as peripheral nerve fibers. These observations, combined with the disappearance of ASIC-ir following dorsal rhizotomy, suggest localization of ASIC to primary afferents. DRG ASIC-ir co-localizes with substance P (SP) and calcitonin gene-related peptide (CGRP)-ir in small capsaicin-sensitive cell bodies, suggesting that ASIC is poised to play a role in the transduction of noxious stimuli.

Cytotoxic targeting of isolectin IB4-binding sensory neurons

The isolectin I-B4 (IB4) binds specifically to a subset of small sensory neurons. We used a conjugate of IB4 and the toxin saporin to examine in vivo the contribution of IB4-binding sensory neurons to nociception. A single dose of the conjugate was injected unilaterally into the sciatic nerve of rats. The treatment resulted in a permanent selective loss of IB4-binding neurons as indicated by histological analysis of dorsal root ganglia, spinal cord, and skin from treated animals. Behavioral measurements showed that 7-10 days after the injection, conjugate-treated rats had elevated thermal and mechanical nociceptive thresholds. However, 21 days post-treatment the nociceptive thresholds returned to baseline levels. These results demonstrate the utility of the IB4-saporin conjugate as a tool for selective cytotoxic targeting and provide behavioral evidence for the role of IB4-binding neurons in nociception. The decreased sensitivity to noxious stimuli associated with the loss of IB4-binding neurons indicates that these sensory neurons are essential for the signaling of acute pain. Furthermore, the unexpected recovery of nociceptive thresholds suggests that the loss of IB4-binding neurons triggers changes in the processing of nociceptive information, which may represent a compensatory mechanism for the decreased sensitivity to acute pain.

Spinal opioid mu receptor expression in lumbar spinal cord of rats following nerve injury

Previous studies in rats have shown that spinal morphine loses potency and efficacy to suppress an acute nociceptive stimulus applied to the tail or the paw following injury to peripheral nerves by tight ligation of the L5/L6 spinal nerves. Additionally, intrathecal (i.th.) morphine is ineffective in suppressing tactile allodynia at fully antinociceptive doses in these animals. The molecular basis for this loss of morphine potency and efficacy in nerve injury states is not known. One possible explanation for this phenomenon is a generalized, multi-segmental loss of opioid mu (mu) receptors in the dorsal horn of the spinal cord after nerve injury. This hypothesis was tested here by determining whether nerve injury produces (a) a decrease in mu receptors in the lumbar spinal cord; (b) a decrease in the affinity of ligand-receptor interaction, (c) a decrease in the fraction of high-affinity state of the mu receptors and (d) a reduced ability of morphine to activate G-proteins via mu receptors. Lumbar spinal cord tissues were examined 7 days after the nerve injury, a time when stable allodynia was observed. At this point, no differences were observed in the receptor density or affinity of [3H]DAMGO (mu selective agonist) or [3H]CTAP (mu selective antagonist) in the dorsal quadrant of lumbar spinal cord ipsilateral to nerve injury. Additionally, no change in morphines potency and efficacy in activating G-proteins was observed. In contrast, staining for mu opioid receptors using mu-selective antibodies revealed a discrete loss of mu opioid receptors localized ipsilateral to the nerve injury and specific for sections taken at the L6 level. At these spinal segments, mu opioid receptors were decreased in laminae I and II. The data indicate that the loss of mu opioid receptors are highly localized and may contribute to the loss of morphine activity involving input at these spinal segments (e.g., foot-flick response). On the other hand, the lack of a generalized loss of opioid mu receptors across spinal segments makes it unlikely that this is the primary cause for the loss of potency and efficacy of mu opioids to suppress multi-segmental reflexes, such as the tail-flick response.

Orphanin FQ/nociceptin-immunoreactive nerve fibers parallel those containing endogenous opioids in rat spinal cord.

Antisera were developed that specifically recognize orphanin FQ/nociceptin, the 17 amino acid peptide reported to be the endogenous ligand for the orphan opioid receptor. Immunocytochemical localizations in rat spinal cord demonstrated that orphanin FQ /nociceptin-immunoreactivity (-ir) was abundant in superficial dorsal horn, lateral spinal nucleus and the region dorsal to the central canal, areas that also exhibit prominent enkephalin-and dynorphin-ir. Orphanin FQ/nociceptin-ir was not affected by dorsal rhizotomy, indicating that in spinal cord the peptide is produced by central rather than primary afferent neurons. thus, the distribution of orphanin FQ/nociceptin-ir appeared in neuronal circuits that parallel those containing enkephalin- and dynorphin-ir, with only modest co-existence of these peptides.

Effects of peripheral nerve injury on alpha-2A and alpha-2C adrenergic receptor immunoreactivity in the rat spinal cord

Neuropathic pain resulting from peripheral nerve injury can often be relieved by administration of alpha-adrenergic receptor antagonists. Tonic activation of alpha-adrenergic receptors may therefore facilitate the hyperalgesia and allodynia associated with neuropathic pain. It is currently unclear whether alpha2A- or alpha2c-adrenergic receptor subtypes are involved in the pro-nociceptive actions of alpha-adrenergic receptors under neuropathic conditions. We therefore investigated the effects of peripheral nerve injury on the expression of these subtypes in rat spinal cord using immunohistochemical techniques. In addition, neuropeptide Y immunoreactivity was examined as an internal control because it has previously been shown to be up-regulated following nerve injury. We observed a decrease in alpha2A-adrenergic receptor immunoreactivity in the spinal cord ipsilateral to three models of neuropathic pain: complete sciatic nerve transection, chronic constriction injury of the sciatic nerve and L5/L6 spinal nerve ligation. The extent of this down-regulation was significantly correlated with the magnitude of injury-induced changes in mechanical sensitivity. In contrast, alpha2c-adrenergic receptor immunoreactivity was only increased in the spinal nerve ligation model; these increases did not correlate with changes in mechanical sensitivity. Neuropeptide Y immunoreactivity was up-regulated in all models examined. Increased expression of neuropeptide Y correlated with changes in mechanical sensitivity. The decrease in alpha2A-adrenergic receptor immunoreactivity and the lack of consistent changes in alpha2C-adrenergic receptor immunoreactivity suggest that neither of these receptor subtypes is likely to be responsible for the abnormal adrenergic sensitivity observed following nerve injury. On the contrary, the decrease in alpha2A-adrenergic receptor immunoreactivity following nerve injury may result in an attenuation of the influence of descending inhibitory noradrenergic input into the spinal cord resulting in increased excitatory transmitter release following peripheral stimuli.

Distribution of neuropeptide receptors: New views of peptidergic neurotransmission made possible by antibodies to opioid receptors

The cloning of receptors for neuropeptides made possible studies that identified the neurons that utilize these receptors. In situ hybridization can detect transcripts that encode receptors and thereby identify the cells responsible for their expression, whereas immunocytochemistry enables one to determine the region of the plasma membrane where the receptor is located. We produced antibodies to portions of the predicted amino acid sequences of delta, mu, and kappa opioid receptors and used them in combination with antibodies to a variety of neurotransmitters in multicolor immunofluorescence studies visualized by confocal microscopy. Several findings are notable: First, the cloned delta opioid receptor appears to be distributed primarily in axons, and therefore most likely functions in a presynaptic manner. Second, the cloned mu and kappa opioid receptors are found associated with neuronal plasma membranes of dendrites and cell bodies and therefore most likely function in a postsynaptic manner. However, in certain, discrete populations of neurons, mu and kappa opioid receptors appear to be distributed in axons. Third, enkephalin-containing terminals are often found in close proximity (although not necessarily synaptically linked) to membranes containing either the delta or mu opioid receptors, whereas dynorphin-containing terminals are often found in proximity to kappa opioid receptors. Finally, a substantial mismatch between opioid receptors and their endogenous ligands was observed in some brain regions. However, this mismatch was characterized by complementary zones of receptor and ligand, suggesting underlying principles of organization that underlie long-distance, nonsynaptic neurotransmission.