A Central Amygdala-Ventrolateral Periaqueductal Gray Matter Pathway for Pain in a Mouse Model of Depression-like Behavior.

BACKGROUND: The mechanisms underlying depression-associated pain remain poorly understood. Using a mouse model of depression, the authors hypothesized that the central amygdala-periaqueductal gray circuitry is involved in pathologic nociception associated with depressive states. METHODS: The authors used chronic restraint stress to create a mouse model of nociception with depressive-like behaviors. They then used retrograde tracing strategies to dissect the pathway from the central nucleus of the amygdala to the ventrolateral periaqueductal gray. The authors performed optogenetic and chemogenetic experiments to manipulate the activity of this pathway to explore its roles for nociception. RESULTS: The authors found that γ-aminobutyric acid-mediated (GABAergic) neurons from the central amygdala project onto GABAergic neurons of the ventrolateral periaqueductal gray, which, in turn, locally innervate their adjacent glutamatergic neurons. After chronic restraint stress, male mice displayed reliable nociception (control, mean ± SD: 0.34 ± 0.11 g, n = 7 mice; chronic restraint stress, 0.18 ± 0.11 g, n = 9 mice, P = 0.011). Comparable nociception phenotypes were observed in female mice. After chronic restraint stress, increased circuit activity was generated by disinhibition of glutamatergic neurons of the ventrolateral periaqueductal gray by local GABAergic interneurons via receiving enhanced central amygdala GABAergic inputs. Inhibition of this circuit increased nociception in chronic restraint stress mice (median [25th, 75th percentiles]: 0.16 [0.16, 0.16] g to 0.07 [0.04, 0.16] g, n = 7 mice per group, P < 0.001). In contrast, activation of this pathway reduced nociception (mean ± SD: 0.16 ± 0.08 g to 0.34 ± 0.13 g, n = 7 mice per group, P < 0.001). CONCLUSIONS: These findings indicate that the central amygdala-ventrolateral periaqueductal gray pathway may mediate some aspects of pain symptoms under depression conditions.

A determination of submicromolar concentrations of glycine in periaqueductal gray matter microdialyzates using capillary zone electrophoresis with contactless conductivity detection.

CE with contactless conductivity detection has been used to determine the glycine neurotransmitter in periaqueductal gray matter (PAG) of rats. The LOD for glycine has been decreased to a value of 0.2 microM by adding 75% v/v of ACN to the samples and increasing the sample zone introduced to a value of 20% of the overall capillary length. The repeatabilities of the analyte migration times and the zone areas amount to 2.1 and 2.7%, respectively. The optimized CE/contactless conductivity detection method makes it possible to determine the micromolar concentrations of glycine in PAG microdialyzates without the necessity of sample derivatization. It follows from a pharmacological study that a local inflammation initiated by an application of carrageenan increased the glycine concentration in the rat PAG seven times, compared with a control. The glycine level in PAG can be decreased and the pain suppressed by administering paracetamol.

Effects of Salmon Calcitonin on the Concentrations of Monoamines in Periaqueductal Gray in Formalin Test

Background: The receptors of salmon calcitonin, located on certain areas of the brain such as the periaqueductal gray matter, are responsible for pain modulation. Aims: The effects of intracerebroventricular injection of salmon calcitonin on the behavioral response to pain and on the levels of monoamines in the periaqueductal gray were explored using a biphasic animal model of pain. Study Design: Animal experiment. Methods: A total of 45 male rats were divided into four groups (n=6). Salmon calcitonin was injected into the lateral ventricle of the brain (1.5 nmol, with a volume of 5 µL). After 20 min, 2.5% formalin was subcutaneously injected into the right leg claw, and pain behavior was recorded on a numerical basis. At the time of the formalin test, the periaqueductal gray area was microdialized. High-performance liquid chromatography method was used to gauge the levels of monoamines and their metabolites. Results: Intracerebroventricular injections of salmon calcitonin resulted in pain reduction in the formalin test (p<0.05). The dialysate concentrations of serotonin, dopamine, norepinephrine, 5-hydroxyindoleacetic acid, 3,4-dihydroxyphenylacetic, and 4-hydroxy-3-methoxyphenylglycol increased in the periaqueductal gray area in different phases of the formalin pain test (p<0.05). Conclusion: Salmon calcitonin reduced pain by increasing the concentrations of monoamines and the metabolites derived from them in the periaqueductal gray area.

Periaqueductal Gray Sheds Light on Dark Areas of Psychopathology.

Neurons in the periaqueductal gray (PAG) integrate negative emotions with the autonomic, neuroendocrine, and immune systems to facilitate responses to threat. Modern functional track tracing in animals and optogenetic and chemogenetic techniques show that the PAG is a rich substrate for the integration of active and passive responses to threat. In humans, the same regions of the PAG that give rise to adaptive anger/fight, fear/panic, depression/shutdown, pain, and predatory behaviors in response to challenging situations or overwhelming threats can become activated pathologically, resulting in symptoms that resemble those of psychiatric disorders. This review coalesces human and animal studies to link PAG neuropathways to specific elements of psychiatric diagnoses. The insights gained from this overview may eventually lead to new therapeutic interventions.

Tac1-Expressing Neurons in the Periaqueductal Gray Facilitate the Itch-Scratching Cycle via Descending Regulation.

Uncontrollable itch-scratching cycles lead to serious skin damage in patients with chronic itch. However, the neural mechanism promoting the itch-scratching cycle remains elusive. Here, we report that tachykinin 1 (Tac1)-expressing glutamatergic neurons in the lateral and ventrolateral periaqueductal gray (l/vlPAG) facilitate the itch-scratching cycle. We found that l/vlPAG neurons exhibited scratching-behavior-related neural activity and that itch-evoked scratching behavior was impaired after suppressing the activity of l/vlPAG neurons. Furthermore, we showed that the activity of Tac1-expressing glutamatergic neurons in the l/vlPAG was elevated during itch-induced scratching behavior and that ablating or suppressing the activity of these neurons decreased itch-induced scratching behavior. Importantly, activation of Tac1-expressing neurons induced robust spontaneous scratching and grooming behaviors. The scratching behavior evoked by Tac1-expressing neuron activation was suppressed by ablation of spinal neurons expressing gastrin-releasing peptide receptor (GRPR), the key relay neurons for itch. These results suggest that Tac1-expressing neurons in the l/vlPAG promote itch-scratching cycles.

Sexually dimorphic activation of the periaqueductal gray-rostral ventromedial medullary circuit during the development of tolerance to morphine in the rat.

The midbrain periaqueductal gray (PAG) and its descending projections to the rostral ventromedial medulla (RVM) provides an essential neural circuit for the antinociceptive effects of opiates, and has been implicated in the development of tolerance to morphine. Systemic morphine activates a greater proportion of PAG-RVM neurons in male vs female rats, and induces tolerance to a greater degree in males. The present studies tested the hypothesis that if the PAG-RVM pathway is essential for the development of tolerance, then: (i) morphine activation of the PAG-RVM pathway should decline as tolerance develops; and (ii) sex differences in the development of tolerance to morphine should be reflected as a greater decline in the activation of this pathway in males. These hypotheses were tested in male and female rats using behavioral testing (hot-plate) and immunohistochemistry to map the activation of the PAG-RVM pathway following repeated morphine administration (5 mg/kg; s.c.). In males, morphine potency decreased from 3.0 to 6.3 mg/kg, indicating tolerance, and this was paralleled by a steady decline in the percentage of PAG-RVM output neurons activated by morphine. In contrast, in females the shift in morphine potency was significantly attenuated (D(50) 6-8.3 mg/kg), and no significant difference in the activity of PAG-RVM output neurons was noted. These results demonstrate that the greater development of tolerance to morphine administration in male rats corresponds with a significant reduction in the activation of the PAG-RVM circuit and suggest a central role for the PAG in the development of tolerance to morphine.

Concentrations of oxidized linoleic acid derived lipid mediators in the amygdala and periaqueductal grey are reduced in a mouse model of chronic inflammatory pain.

Chronic pain is both a global public health concern and a serious source of personal suffering for which current treatments have limited efficacy. Recently, oxylipins derived from linoleic acid (LA), the most abundantly consumed polyunsaturated fatty acid in the modern diet, have been implicated as mediators of pain in the periphery and spinal cord. However, oxidized linoleic acid derived mediators (OXLAMs) remain understudied in the brain, particularly during pain states. In this study, we employed a mouse model of chronic inflammatory pain followed by a targeted lipidomic analysis of the animals' amygdala and periaqueductal grey (PAG) using LC-MS/MS to investigate the effect of chronic inflammatory pain on oxylipin concentrations in these two brain nuclei known to participate in pain sensation and perception. From punch biopsies of these brain nuclei, we detected twelve OXLAMs in both the PAG and amygdala and one arachidonic acid derived mediator, 15-HETE, in the amygdala only. In the amygdala, we observed an overall decrease in the concentration of the majority of OXLAMs detected, while in the PAG the concentrations of only the epoxide LA derived mediators, 9,10-EpOME and 12,13-EpOME, and one trihydroxy LA derived mediator, 9,10,11-TriHOME, were reduced. This data provides the first evidence that OXLAM concentrations in the brain are affected by chronic pain, suggesting that OXLAMs may be relevant to pain signaling and adaptation to chronic pain in pain circuits in the brain and that the current view of OXLAMs in nociception derived from studies in the periphery is incomplete.

Identification of wake-active dopaminergic neurons in the ventral periaqueductal gray matter.

Recent evidence suggests that dopamine plays an important role in arousal, but the location of the dopaminergic neurons that may regulate arousal remains unclear. It is sometimes assumed that the dopaminergic neurons in the ventral tegmental area that project to the prefrontal cortex and striatum may regulate the state of arousal; however, the firing of these dopaminergic neurons does not correlate with overall levels of behavioral wakefulness. We identified wake-active dopaminergic neurons by combining immunohistochemical staining for Fos and tyrosine hydroxylase (TH) in awake and sleeping rats. Approximately 50% of the TH-immunoreactive (TH-ir) cells in the ventral periaqueductal gray matter (vPAG) expressed Fos protein during natural wakefulness or wakefulness induced by environmental stimulation, but none expressed Fos during sleep. Fos immunoreactivity was not seen in the substantia nigra TH-immunoreactive cells in either condition. Injections of 6-hydroxydopamine into the vPAG, which killed 55-65% of wake-active TH-ir cells but did not injure nearby serotoninergic cells, increased total daily sleep by approximately 20%. By combining retrograde and anterograde tracing, we showed that these wake-active dopaminergic cells have extensive reciprocal connections with the sleep-wake regulatory system. The vPAG dopaminergic cells may provide the long-sought ascending dopaminergic waking influence. In addition, their close relationship with the dorsal raphe nucleus will require reassessment of previous studies of the role of the dorsal raphe nucleus in sleep, because many of those experiments may have been confounded by the then-unrecognized presence of intermingled wake-active dopaminergic neurons.

Anxiogenic effects of activation of NK-1 receptors of the dorsal periaqueductal gray as assessed by the elevated plus-maze, ultrasound vocalizations and tail-flick tests.

Ultrasound vocalizations (USVs) known as 22kHz are usual components of the defensive responses of rats exposed to threatening conditions. The amount of emission of 22kHz USVs depends on the intensity of the aversive stimuli. While moderate fear causes an anxiolytic-sensitive enhancement of the defensive responses, high fear tended to reduce the defensive performance of the animals to aversive stimuli. The dorsal periaqueductal gray (dPAG) is an important vocal center and a crucial structure for the expression of defensive responses. Substance P (SP) is involved in the modulation of the defensive response at this midbrain level, but the type of neurokinin receptors involved in this action is not completely understood. In this study we examined whether local injections of the selective NK-1 agonist SAR-MET-SP (10-100 pmol/0.2microL) into the dPAG (i) cause anxiogenic effects in the elevated plus-maze (EPM) (Exp. I), (ii) influence the novelty-induced 22kHz USVs recorded within the frequency range of 20-26kHz (Exp. II) and (iii) change the nociceptive reactivity to heat applied to the rat's tail (Exp III). The data obtained showed that SAR-MET-SP elicited significant "anxiety-like" behaviors, as revealed by the decrease in the number of entries into and time spent onto the open arms of the EPM. These anxiogenic effects were accompanied with antinociception and disruption of the novelty-induced increase in the number and duration of 22kHz USVs. These findings are in agreement with the notion that NK-1 receptors of the dPAG may be an important neurochemical target for new selective drugs aimed at the control of pathological anxiety states.

Mu Opioid Receptor Modulation of Dopamine Neurons in the Periaqueductal Gray/Dorsal Raphe: A Role in Regulation of Pain.

The periaqueductal gray (PAG) is a brain region involved in nociception modulation, and an important relay center for the descending nociceptive pathway through the rostral ventral lateral medulla. Given the dense expression of mu opioid receptors and the role of dopamine in pain, the recently characterized dopamine neurons in the ventral PAG (vPAG)/dorsal raphe (DR) region are a potentially critical site for the antinociceptive actions of opioids. The objectives of this study were to (1) evaluate synaptic modulation of the vPAG/DR dopamine neurons by mu opioid receptors and to (2) dissect the anatomy and neurochemistry of these neurons, in order to assess the downstream loci and functions of their activation. Using a mouse line that expresses eGFP under control of the tyrosine hydroxylase (TH) promoter, we found that mu opioid receptor activation led to a decrease in inhibitory inputs onto the vPAG/DR dopamine neurons. Furthermore, combining immunohistochemistry, optogenetics, electrophysiology, and fast-scan cyclic voltammetry in a TH-cre mouse line, we demonstrated that these neurons also express the vesicular glutamate type 2 transporter and co-release dopamine and glutamate in a major downstream projection structure-the bed nucleus of the stria terminalis. Finally, activation of TH-positive neurons in the vPAG/DR using Gq designer receptors exclusively activated by designer drugs displayed a supraspinal, but not spinal, antinociceptive effect. These results indicate that vPAG/DR dopamine neurons likely play a key role in opiate antinociception, potentially via the activation of downstream structures through dopamine and glutamate release.

Contribution of brainstem GABAergic circuitry to descending antinociceptive controls: II. Electron microscopic immunocytochemical evidence of GABAergic control over the projection from the periaqueductal gray to the nucleus raphe magnus in the rat.

Pharmacological, physiological, and behavioral studies suggest that inhibitory GABAergic neurons influence the projection from the midbrain periaqueductal gray matter to the medullary nucleus raphe magnus. The present study used electron microscopic immunocytochemical techniques to examine the morphology and synaptic relationships of GABA-immunoreactive terminals in the ventrolateral periaqueductal gray. These putative GABAergic terminals comprise almost 40% of all axon terminals in the periaqueductal gray. GABA-immunoreactive terminals contain small, clear, pleomorphic or round, vesicles, and 46% also contain some dense-cored vesicles. In some experiments we also used a colloidal gold-conjugated retrograde tracer to label periaqueductal gray neurons that project to the nucleus raphe magnus. About half of the synaptic inputs onto the cell bodies and proximal dendrites of retrogradely labeled neurons are GABA-immunoreactive; these putative GABAergic synapses, which directly control activity in neurons projecting from the periaqueductal gray to the nucleus raphe magnus, might mediate the antinociception-related effects of exogenous GABAA receptor ligands.

Lateral cervical nucleus projections to periaqueductal gray matter in cat.

The midbrain periaqueductal gray matter (PAG) integrates the basic responses necessary for survival of individuals and species. Examples are defense behaviors such as fight, flight, and freezing, but also sexual behavior, vocalization, and micturition. To control these behaviors the PAG depends on strong input from more rostrally located limbic structures, as well as from afferent input from the lower brainstem and spinal cord. Mouton and Holstege (2000, J Comp Neurol 428:389-410) showed that there exist at least five different groups of spino-PAG neurons, each of which is thought to subserve a specific function. The lateral cervical nucleus (LCN) in the upper cervical cord is not among these five groups. The LCN relays information from hair receptors and noxious information and projects strongly to the contralateral ventroposterior and posterior regions of thalamus and to intermediate and deep tectal layers. The question is whether the LCN also projects to the PAG. The present study in cat, using retrograde and anterograde tracing techniques, showed that neurons located in the lateral two-thirds of the LCN send fibers to the lateral part of the PAG, predominantly at rostrocaudal levels A0.6-P0.2. This part of the PAG is known to be involved in flight behavior. A concept is put forward according to which the LCN-PAG pathway alerts the animal about the presence of cutaneous stimuli that might represent danger, necessitating flight. J. Comp. Neurol. 471:434-445, 2004.

Immunocytochemical localization of substance P receptor in rat periaqueductal gray matter: a light and electron microscopic study.

The distribution of substance P receptor (SPR) protein in the rat periaqueductal gray matter (PAG) was investigated with a polyclonal antibody in the four subdivisions obtained by cytochrome-oxidase histochemistry (Co-hi). At light microscopic analysis, immunoreactivity appeared particularly dense in the dorsal subdivision of the PAG, was less intense in the other subdivisions, and formed several longitudinally organized columns. SPR-like immunoreactivity (SP(R-i)) was localized mostly to cell bodies and dendrites of small and medium-sized neurons, which constituted about 6% of the total neuronal population of the PAG. At the electron microscopic level, SP(R-i) could be observed on postsynaptic as well as on nonsynaptic regions of both cell bodies and dendrites. A small proportion of axons (4.2%) and axon terminals (5.3%) showed SP(R-i), the majority of labeled axon terminals, amounting to about 70% of synapsing elements, formed asymmetric synapses with dendrites. Rare astroglial processes displaying SP(R-i) were also observed scattered throughout the neuropil of all PAG subdivisions. Our observations suggest that 1) also in the PAG, SP may act in a diffuse, nonsynaptic manner, probably on targets that are distant from its sites of release; and 2) SP may modulate excitatory neurotransmission acting presynaptically on those labeled axons that form asymmetric synapses.

A multiple Golgi analysis of the periaqueductal gray in the rabbit.

Four variants of the Golgi method have been used, in the rabbit, to reveal the morphological attributes of neurons within the periaqueductal gray. Of these methods, the Golgi-Cox version provided the most satisfactory results in terms of both quality and quantity of cell impregnation. In order to make comparison with other descriptions of Golgi characteristics of the periaqueductal gray, statistical analysis was carried out on the distinguishing features of individual neurons, following two different rationales. One method, primarily based on soma characteristics (shape, area, length, and width) and basic features of the primary dendrites (number, length, and number of end-points) resulted in nine different categories of neurons being recognized: round, ovoid, spindle, pyriform, triangular, pyramidal, rhomboidal, polygonal, and stellate shaped cells. The alternative method principally characterized neurons by an assessment of the degree of dendritic spine development and prominent features of the dendritic tree (number of primary dendrites, length, number of branches, end-points, and degree of spine development). This approach resulted in nine subgroups within three major classes being identified: three spiny, four moderately spiny, and two aspiny classes (subdivision of each of the groups being resultant on neuronal size and/or the degree of dendritic branching). There was no similarity between the nine groups found by the two methods. Some, though little, correlation of neuron type was evidenced with respect to four zonal subdivisions of the periaqueductal gray complex. It remains to be seen how any of these readily recognizable morphological features, or the subgroups (derived on a statistical basis) into which they fall, might be shown to relate to function.

Quantitative Nissl study of the neuronal types, and recognition of cytoarchitectural subdivisions, within the rabbit periaqueductal gray.

A quantitative analysis of 4,621 Nissl-stained neurons within the periaqueductal gray of the rabbit found that there were four main cell types (stellate/round, ovoid, spindle, and triangular) distributed throughout this complex. Further statistical analysis on these neurons confirmed that there were morphological grounds to subdivide the periaqueductal gray into four cytologically distinct regions: ventral, lateral, dorsal, and medial. Neurons in the narrow medial zone, which completely surrounds the aqueduct, were orientated essentially parallel to the aqueduct. The majority of these neurons were small, ovoid, or spindle in shape, and highly basophilic. The cells in this region had the lowest packing density of those in any periaqueductal gray subdivision. The dorsal subdivision, a small midline region, contains the largest cells of any division and the highest packing density of glial cells. The neurons in this region show no preference for orientation, tend to be round, and are moderately basophilic. Cells in the lateral zone are also quite large and demonstrate a preferred orientation either parallel or perpendicular to the aqueduct. The average cell density within lateral PAG is considerably higher than in other regions. Most of these neurons are round or ovoid, and moderately basophilic. Neurons in the ventral zone are mainly ovoid, of medium size, highly basophilic, and lie fairly sparsely arrayed and are orientated essentially parallel to the aqueductal surface.

Retrograde tracing of projections between the nucleus submedius, the ventrolateral orbital cortex, and the midbrain in the rat.

The fluorescent tracers fluoro-gold and 1,1'-dioctadecyl-3,3,3,3-tetramethyl indocarbocyanine perchlorate were used as retrograde markers to examine reciprocal connections between the rat nucleus submedius and the ventrolateral orbital cortex. In addition, midbrain projections to each of these regions were examined. In the prefrontal cortex, we found that input from the nucleus submedius terminates rostrally within the lateral and ventral areas of the ventrolateral orbital cortex. Conversely, the cortical input to the nucleus submedius originates from the medial and dorsal parts of the ventrolateral orbital cortex. Our data also demonstrated that neurons from the ventrolateral periaqueductal gray and the raphe nuclei project to the midline nuclei of the thalamus, including a small projection to the nucleus submedius. We further determined that regions within the ventrolateral periaqueductal gray and raphe nuclei project to the ventrolateral orbital cortex, and that these regions overlap with those that project to the nucleus submedius. These findings suggest that the nucleus submedius might be part of a neural circuit involved in the activation of endogenous analgesia.

Contribution of brainstem GABAergic circuitry to descending antinociceptive controls: I. GABA-immunoreactive projection neurons in the periaqueductal gray and nucleus raphe magnus.

The fact that GABA receptor agonists and antagonists influence nociceptive thresholds when microinjected into the rostroventral medulla or in the spinal cord may reflect the involvement of GABAergic neuronal elements in endogenous antinociceptive pathways. In the present study we used immunocytochemistry and retrograde tract tracing to investigate the contribution of GABAergic projection neurons to the antinociceptive network linking the midbrain periaqueductal gray matter (PAG), the nucleus raphe magnus (NRM), and the spinal cord dorsal horn. The tracer, WGAapoHRP-Au was injected into either the NRM or the spinal cord and the distribution of labeled neurons in sections of the PAG and medulla, respectively, was studied. The same sections were immunostained to demonstrate GABA-immunoreactive neurons. Although GABA-immunoreactive neurons were abundant in the PAG, only 1.5% were retrogradely labeled from the NRM. Similarly, very few GABA-immunoreactive neurons within the cytoarchitectural boundaries of the NRM were retrogradely labeled from the spinal cord. A much higher proportion of GABA-immunoreactive neurons in the region lateral to the NRM, however, were retrogradely labeled from the spinal cord. Eighteen percent of GABA-immunoreactive neurons were retrogradely labeled in the nucleus reticularis paragigantocellularis; conversely, 15% of the retrogradely labeled neurons in this region were GABA-immunoreactive. These results indicate that GABAergic projections constitute a very minor component of the PAG-NRM-spinal cord pathway; however, there is a significant contribution of GABAergic neurons to the spinal projections that originate lateral to the NRM. The majority of GABAergic neurons in the PAG and NRM are presumed to be inhibitory interneurons that directly or indirectly regulate activity in efferent pathways from these regions.

Distribution of gonadal steroid receptor-containing neurons in the preoptic-periaqueductal gray-brainstem pathway: a potential circuit for the initiation of male sexual behavior.

The present study used anterograde and retrograde tract tracing techniques to examine the organization of the medial preoptic-periaqueductal gray-nucleus paragigantocellularis pathway in the male rat. The location of neurons containing estrogen (alpha subtype; ER alpha) and androgen receptors (AR) were also examined. We report here that injection of the anterograde tracer biotinylated dextran amine (BDA) into the medial preoptic (MPO) produced dense labeling within the periaqueductal gray (PAG); anterogradely labeled fibers terminated in close juxtaposition to neurons retrogradely labeled from the nucleus paragigantocellularis (nPGi). Dual immunostaining for Fluoro-Gold (FG) and ER alpha or FG and AR showed that over one-third of MPO efferents to the PAG contain receptors for either estrogen or androgen. In addition, approximately 50% of PAG neurons retrogradely labeled from the nPGi were immunoreactive for either ER alpha or AR. These results are the first to establish an MPO-->PAG-->nPGi circuit and further indicate that gonadal steroids can influence neuronal synaptic activity within these sites. We reported previously that nPGi reticulospinal neurons terminate preferentially within the motoneuronal pools of the lumbosacral spinal cord that innervate the pelvic viscera. Together, we propose that the MPO-->PAG-->nPGi circuit forms the final common pathway whereby MPO neural output results in the initiation and maintenance of male copulatory reflexes.

Laryngeal afferent stimulation enhances Fos immunoreactivity in periaqueductal gray in the cat.

The main functions of the larynx are protection of the airways, respiration, and vocalization. Previous studies have suggested a link between the mechanisms controlling vocalization and afferent feedback from the larynx. We inquired whether stimulation of the laryngeal afferents that run in the internal branch of the superior laryngeal nerve (ISLN) activates neurons of the periaqueductal gray (PAG), a midbrain region implicated in vocalization. We counted the number of neurons expressing Fos, the protein product of the immediate early gene c-fos, in the PAG. The counts were done both in experimental cats after electrical stimulation of the ISLN and nonstimulated controls. We also investigated the possible presence of nitric oxide synthase, an enzyme that synthesizes nitric oxide, in PAG neurons that respond to laryngeal afferent stimulation by double labeling for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase and Fos. Fos expression was significantly greater (P < or = 0.00714) in the lateral and dorsolateral regions of the PAG in the experimental group than in the controls. The Fos-immunoreactive neurons did not contain NADPH-diaphorase, a marker for nitric oxide synthase. Our study suggests that laryngeal afferent stimulation activates neurons in discrete longitudinal columns of the PAG including the regions that have previously been shown to be involved in vocalization, and that these neurons do not contain nitric oxide synthase.

ORL-1 and mu opioid receptor antisera label different fibers in areas involved in pain processing.

Mu opioid receptors (MOR) mediate the analgesic effects of opioid drugs such as morphine. The opioid receptor-like (ORL-1) receptor is structurally related to opioid receptors and the ORL-1 receptor agonist, orphanin FQ/nociceptin, induces analgesia at the spinal level, but appears to recruit different circuitry than that used by mu opioids. When administered intracerebroventricularly, orphanin FQ/nociceptin produces hyperalgesia and/or reverses opioid analgesia. The functionally distinct actions elicited by MOR and ORL-1 receptors, which activate similar intracellular signaling systems and show similar regional distributions, could be explained by their differential cellular localization. By using double label immunohistochemistry and confocal microscopy, the present study investigates the distribution of MOR and ORL-1 receptors in regions of the rat nervous system that are involved with nociceptive processing. In general co-localization of MOR and ORL-1 receptor immunoreactivity was not observed in either perikarya or neuropil in the dorsal root ganglia, nor in the Lissauer's tract and superficial laminae of the spinal cord. Likewise, there was no evidence for co-localization of these receptors within the periaqueductal gray, the nucleus raphe magnus, the gigantocellular reticular nucleus, and the nucleus of the solitary tract. These observations indicate that MOR and ORL-1 receptors are expressed predominantly on different fiber systems in these regions. This differential distribution is consistent with the distinct pharmacology of ORL-1 and MOR receptor agonists and suggests that the antisera to MOR and ORL-1 receptors may provide useful markers for further investigations of analgesic and counteranalgesic pathways modulating pain perception.