Caudal Periaqueductal Gray Research Articles

Increased fos-like immunoreactivity in the periaqueductal gray of anaesthetised rats during opiate withdrawal

Staining of c-fos-like-immunoreactivity (CFIR) in neurones was used to study neuronal activation within subdivisions of periaqueductal gray (PAG), and in locus coeruleus and ventral tegmental area during opiate withdrawal in awake and anaesthetised, morphine-dependent rats. The number of CFIR containing neurones was significantly increased during naloxone-precipitated withdrawal in lateral and ventrolateral, particularly the caudal ventrolateral PAG. No changes were observed in dorsal-intermediate or dorsal-caudal PAG. In awake rats, a similar but more generalised increase in CFIR was observed in PAG following naloxone-precipitated withdrawal. Increases in ventral tegmental area and locus coeruleus during naloxone-precipitated withdrawal under anaesthesia varied greatly between animals. Induction of c-fos in lateral and ventrolateral PAG during withdrawal is consistent with known functions of these regions, involving the integration of autonomic and somatic components of defensive and escape behaviours which are characteristic signs of opiate withdrawal.

Identification of periaqueductal gray and dorsal raphe nucleus neurons projecting to both the trigeminal sensory complex and forebrain structures: a fluorescent retrograde double-labeling study in the rat

The midbrain periaqueductal gray (PAG) including the dorsal raphe nucleus (DR) has been known to contain serotoninergic neurons projecting to many brain regions. Employing fluorescent retrograde double labeling combined with immunofluorescence histochemistry for serotonin (5-HT), we examined in the rat whether or not single PAG/DR neurons with 5-HT send their axons to both the trigeminal sensory complex and forebrain regions. Stereotaxic injections of Diamidino Yellow (DY) and Fast Blue (FB) were performed unilaterally; DY was injected into the caudal spinal trigeminal nucleus or principal sensory trigeminal nucleus, and FB into the ventrolateral orbital cortex, nucleus accumbens or amygdala. A small percentage of PAG/DR neurons were doubly labeled with DY and FB, and the majority of them showed 5-HT-like immunoreactivity (5-HT-LI). Most of these 5-HT-LI PAG/DR neurons that were indicated to send their axons simultaneously to both the trigeminal sensory complex and forebrain regions were distributed in the ventrolateral PAG subdivision and ventral aspects of the medial PAG subdivision at the middle and caudal PAG levels, bilaterally with a predominant distribution on the side ipsilateral to the injections. This indicates a possible role of these PAG/DR neurons in the limbic or affective-motivational aspect of the pain-related neural system.

Chronic pain increases brainstem proneurotensin/neuromedin-N mRNA expression: a hybridization-histochemical and immunohistochemical study using three different rat models for chronic nociception

The role of neurotensin in the central nervous system is poorly understood. Exogenous neurotensin has potent antinociceptive effects when injected into the midbrain periaqueductal gray (PAG). Although it is present in terminals, fibers and perikarya within the PAG and other midbrain regions known for their antinociceptive circuits, it is not known whether endogenous neurotensin modulates nociception. We examined the midbrain in three different rat models for nociception to learn whether acute or chronic pain altered neuronal levels of the proneurotensin/neuromedin N mRNA (neurotensin mRNA). The models were: adjuvant-induced polyarthritis, adjuvant-induced unilateral paw inflammation, and unilateral peripheral mononeuropathy caused by ligation of the sciatic nerve. Behavioral observations confirmed that the expected symptoms developed as previously described. Within each of the three experimental models, we performed in situ hybridization histochemistry on coronal sections from three midbrain levels that included the rostral one-third of the PAG, the middle one-third of the PAG, and the caudal one-third of the PAG. At the level of the rostral PAG, we found that neither chronic nor acute nociception altered the frequencies or distributions of neurons containing neurotensin mRNA. In contrast, at the levels of the mid- and caudal one third of the PAG, the early effects of the nociceptive lesions differed from the chronic effects. During the acute phase of each model, increases in either the frequency or field area of neurons that were hybridization-positive for neurotensin mRNA were confined to the ventromedial PAG and the dorsal raphe nucleus. As the nociceptive stimuli became chronic, the early increases in neurotensin mRNA-containing neurons at the level of the middle third of the ventral PAG were diminished but remained above control levels, while increases in neurotensin mRNA began to occur in the midbrain tegmentum lateral to the PAG. The most striking increases in neurotensin mRNA expression were observed 16-17 days after the onset of nociceptive stimuli. At the level of the mid-PAG and caudal PAG, increased hybridization signal intensities and neuron frequencies occurred within the nucleus cuneiformis and the lateral tegmental nuclei, including the pedunculopontine and microcellular tegmental nuclei, as well as the deep mesencephalic nuclei. Hybridization-positive neurons in the tegmental nuclei were not observed at early stages of lesion development, but were a consistent feature of caudal midbrains after nociception became chronic.(ABSTRACT TRUNCATED AT 400 WORDS)

Actions of epinephrine on neurons in the rat midbrain periaqueductal gray maintained in vitro

The effect of epinephrine (EPI) on the activity of 150 periaqueductal gray (PAG) neurons was examined using extracellular recordings in an in vitro slice preparation. Drop application of EPI inhibited 45%, excited 35%, and had no effect on 20% of PAG neurons. Both the excitatory and inhibitory effects of EPI were of long duration; excitatory responses averaged 17 min and inhibitory responses averaged 11 min in duration. EPI responses could be blocked by specific alpha-1 and alpha-2 receptor antagonists. In 35% of the neurons tested, blockade of synaptic transmission by perfusion with low calcium-high magnesium physiological saline blocked responses to EPI. The effects of EPI were site specific: 77% of the cells in the caudal ventrolateral region of the PAG were inhibited by EPI; in all other regions of PAG equal numbers of cells were excited and inhibited by EPI. It is concluded that: 1. (a) EPI has potent effects on a majority (80%) of PAG neurons; 2. (b) EPI responses are mediated by presynaptic as well as postsynaptic mechanisms; 3. (c) EPI preferentially inhibits neurons in the ventrolateral subdivision of caudal PAG. As this part of PAG contains many neurons that project to the ventral medulla, it is possible that EPI modulates the PAG-medullary functions such as analgesia, autonomic regulation, defense reactions, and sexual behaviors.

Ultrastructural morphometric analysis of enkephalin-immunoreactive terminals in the ventrocaudal periaqueductal gray: Analysis of their relationship to periaqueductal gray-raphe magnus projection neurons

The periaqueductal gray plays an important role in the descending modulation of nociception. While the importance of endogenous opioids to periaqueductal gray circuits that modulate nociception is supported by many studies, the ultrastructural relationships between enkephalin-immunoreactive axon terminals and the surrounding periaqueductal gray neuropil have not been quantitated in the rat. Further, the possible interaction between enkephalin-immunoreactive axon terminals and periaqueductal gray neurons that project to the rostroventral medulla has not been described. The present study utilized electron microscopic immunocytochemistry to quantitate the normal neuronal associations of enkephalinimmunoreactive terminals in the caudal periaqueductal gray of the rat. A primary focus of this analysis was to ascertain whether any interaction exists between enkephalin-immunoreactive axon terminals and periaqueductal gray neurons that were retrogradely-labeled from the nucleus raphe magnus and adjacent medullary reticular nuclei. We examined the ventrolateral periaqueductal gray and the ventral periaqueductal gray immediately subjacent to the aqueduct and found that both the average terminal diameters and the volume fractions of enkephalin-immunoreactive terminals were very similar. In these two regions, most terminals were observed to be in close apposition to either two or three dendrites that were neither retrogradely-labeled nor enkephalin-immunoreactive, although axonal and perikaryal associations were also observed. In the ventrolateral periaqueductal gray, 22% of all enkephalin-immunoreactive terminals were adjacent to periaqueductal gray-nucleus raphe magnus and periaqueductal gray-reticular nucleus projection neurons. In the periaqueductal gray subjacent to the aqueduct, 32% of all enkephalin-immunoreactive terminals were adjacent to periaqueductal gray-nucleus raphe magnus and periaqueductal gray-reticular nucleus projection neurons. Symmetrical synapses with these retrogradelylabeled neurons were formed by 5.5% of enkephalin-immunoreactive terminals in the ventrolateral periaqueductal gray, and by 4.3% of enkephalin-immunoreactive terminals located subjacent to the aqueduct. We also noted that enkephalin-immunoreactive terminals formed symmetrical synapses with non-retrogradely-labeled, enkephalin-immunoreactive dendrites in the periaqueductal gray. Direct opioid input onto putative excitatory periaqueductal gray output neurons that are hypothesized to modulate nociception was an unexpected finding. While presynaptic mechanisms for enkephalin action on periaqueductal gray output neurons may potentially exist, the low frequency of terminal-terminal interactions compared with the high frequency of terminal-dendrite interactions suggested a significant role of enkephalins via either disinhibitory or direct inhibitory mechanisms. These results are discussed in light of current theories about nociception-modulating circuits within the periaqueductal gray.

Anatomical study of the final common pathway for vocalization in the cat.

Vocalization, the nonverbal production of sound, can be elicited in many vertebrates by stimulation in several regions of the limbic system but most easily in the caudal periaqueductal gray (PAG). This study shows that a specific cell group in the lateral part of the caudal PAG and in the tegmentum just lateral to it projects bilaterally to the nucleus retroambiguus (NRA) in the caudal medulla oblongata. Similar but much weaker projections are derived from the dorsal PAG. Neurons in the NRA in turn project via a contralateral pathway through the ventral funiculus of the spinal cord to motoneuronal cell groups, innervating intercostal and abdominal muscles. These projections are stronger on the contralateral side, although at lower thoracic and upper lumbar levels, many fibers recross to terminate in the ipsilateral motoneuronal cell groups. In the brainstem NRA neurons project to the motoneuronal cell groups innervating mouth-opening and perioral muscles as well as to motoneurons innervating the pharynx, soft palate, and tongue, and probably to the larynx. All these muscles are active in vocalization. The present anatomical results, combined with the physiological results of others, indicate that the projections from PAG via NRA to vocalization motoneurons form the final common pathway in vocalization. The role of this pathway in the total framework of emotional behavior is discussed.

Opiate and serotonergic mechanisms of stimulation-produced analgesia within the periaqueductal gray

These studies investigated the distribution of analgesia-producing sites within the periaqueductal gray (PAG), and their potential reversal by naloxone and methysergide. The PAG is not differentiable in its ability to elicit stimulation-produced analgesia (SPA) until the point of stimulation is caudal to the dorsal raphe nucleus, where analgesia was not obtained. Naloxone, however, was found to have a differential effect at specific loci, significantly reducing SPA from ventral but not dorsal sites. In contrast, methysergide was effective in reversing analgesia both at ventral and dorsal sites. The site of stimulation was critical to whether motor effects were elicited: Motor effects accompanied by analgesia were most often produced rostrally, while motor effects without analgesia were most frequently produced in the middle PAG. Null effects for both motor activity and analgesia were obtained from caudal PAG sites.

Calcitonin gene-related peptide (CGRP)-positive neurons and fibers in the cat periaqueductal grey matter.

The morphology and topographical distribution of neurons and terminals containing calcitonin gene-related peptide (CGRP) immunoreactivity in the cat periaqueductal grey (PAG) were studied using a rabbit antiserum raised against the C-terminal region of rat alpha-CGRP. In normal cats, numerous fibers, but rarely immunoreactive neurons, were observed in the PAG. CGRP-containing fibers showed bouton-like swellings along their length and expanded in terminal clusters of boutons. In many cases, CGRP-positive fibers were also observed in close association with small blood vessels. Immunoreactive fibers were particularly numerous at caudal PAG levels, mostly in its ventrolateral portion. In colchicine-treated cats, the pattern of CGRP-containing fibers was basically unchanged, despite a reduction of both the number of fibers and the intensity of fiber staining; in addition, numerous CGRP-positive neurons were found, mostly in the ventrolateral portion of the caudal PAG. These neurons were fusiform, spheroidal, and triangular in shape. The selective distribution of CGRP-positive elements in the PAG suggests a functional specialization of these neurons in the activation of pain-modulating mechanisms.

Autoradiographic localization of substance P ligand binding sites and distribution of immunoreactive neurons in the periaqueductal gray of the rat

The autoradiographic localization of substance P (SP) binding sites and the distribution of SP immunoreactive (SP-ir) neurons in the periaqueductal gray (PAG) of the rat were studied. The autoradiograms reveled an uneven distribution of specific SP binding sites in the PAG. Throughout the rostrocaudal extent, the densest ligand binding sites were observed in the medial PAG adjacent to the aqueduct, and extended into the dorsal medullary region and to the dorsal raphe nucleus midline region. The distribution of binding sites were denser in the dorsal PAG than the ventral half. In the cuneiform nucleus, a lesser and a denser binding site were observed in the medial and lateral halves respectively. Optical density readings of autoradiograms also supported the differences between these areas. The distribution of SP-ir neurons was also found uneven. In the rostral PAG, SP-ir neurons were found in the entire dorsoventral region. In the caudal PAG, SP-ir neurons were found as 3 clusters: in the dorsomedial, dorsolateral and ventrolateral regions. The present study revealed more SP-ir neurons in the PAG than previously reported.

A blood pressure lowering effect of lesions of the caudal periaqueductal gray: relationship to basal pressure

Basal mean arterial pressure (MAP) measured one week following placement of pontine lesions was markedly lower (−27.85 mm Hg) in cats with bilateral lesions of the caudal periaqueductal gray than in cats with bilateral lesions of the area anteroventral to the locus coeruleus. Regression models of the relationship between basal arterial pressure (MAP basal) and the change in arterial pressure (MAP change) after the lesions indicate that lesions of the caudal periaqueductal gray led to a marked decrease in MAP in animals with an elevated basal MAP (MAP change = MAP basal× (−1.182) + 139.433; r = −0.902; P < 0.002). In contrast, lesions of the area anteroventral to the locus coeruleus had no such effect (MAP change = MAP basal× (−0.363) + 56.49; r = −0.375; P > 0.1). The region of the caudal periaqueductal gray affecting MAP appears anterior to the locus coeruleus and through intrinsic neurons or fibers of passage may play a critical role in control of arterial pressure.

Evidence for GABA involvement in midbrain control of medullary neurons that modulate nociceptive transmission

The effects of GABA-related compounds microinjected into the midbrain periaqueductal gray (PAG) on the tail-flick reflex (TF) and on the activity of tail-flick related neurons in the rostral ventromedial medulla (RVM), were studied in barbiturate anesthesized rats. Neurons whose activity either decreased (off-cells) or increased (on-cells) immediately prior to TF were examined. Biouculline and picrotoxin microinjected into the ventrolateral aspect of the caudal PAG inhibited the TF, increased the spontaneous activity of the off-cells and decreased that of the on-cells. Concomitant with the increase in TF latency, the TF-related deceleration of the off-cells and acceleration of the on-cells were reduced. These effects were reversed by a microinjection of muscimol (MUS) into the PAG. The analgesic effect of morphine microinjected into the PAG was also reversed by a MUS microinjection at the same site. These results support the hypothesis that a GABAergic synapse inhibits cells in the PGA which modulate nociceptive transmission at the spinal level through actions on neurons in the RVM.

A projection from the periaqueductal grey to the lateral reticular nucleus in the cat.

A projection from the periaqueductal grey (PAG) to the lateral reticular nucleus (NRL) in the cat was demonstrated by means of retrograde transport of the wheat germ agglutinin-horseradish peroxidase complex. The connection has its main origin ipsilaterally in the ventral part of the caudal PAG, but scanty projections from other parts of the PAG were also found. The neurons projecting to the NRL are of varying shapes and sizes, but most cells have a maximum diameter of less than 20 micron. The findings are discussed in relation to the other afferent and efferent connections of the NRL.

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.

Cortical projections to the periaqueductal grey in the cat: A retrograde horseradish peroxidase study

Stereotaxic fluid microinjections of horseradish peroxidase into different parts of the rostral and caudal periaqueductal grey (PAG) in cats have provided substantial retrograde evidence that the somatosensory cortex (I and II), frontal cortex, insular and cingular cortex are the principal sources of cortical-PAG projections. The somatosensory cortex II projects to all the regions of the rostral and caudal PAG. The frontal cortex projects to dorso-lateral quadrant of the PAG. The same projections were determined from insular and cingular cortex to PAG. The findings revealed a morphological substratum of corticofugal effects on PAG.

The fine structure of the caudal periaqueductal gray of the cat: Morphology and synaptic organization of normal and immunoreactive enkephalin-labeled profiles

Although the midbrain periaqueductal gray (PAG) is thought to have a major role in an endorphin-mediated analgesia system, little is known about its neuroanatomical organization. To determine the microcircuitry within the PAG through which exogenous and endogenous opiates may act, we analyzed the synaptic organization of normal and immunoreactive enkephalin (ENK)-labeled profiles in the caudal PAG, a region of particular interest because of its effectiveness in generating analgesia. Examination of the normal fine structure of this region demonstrated that there is no characteristic synaptic morphology that distinguishes individual regions of the caudal PAG (ventromedial, ventrolateral and dorsolateral) from one another. In all 3 regions of the caudal PAG, axodendritic synapses are the predominant form of synaptic interaction making up 93–97% of all synapses counted. Axosomatic synapses are much less common, as are presumed axoaxonic and dendrodendritic synapses. In the caudal ventral PAG, the largest population of ENK-labeled axonal boutons are found presynaptic to unlabeled, centrally placed dendrites. Much less frequently, immunoreactive ENK-containing boutons are found presynaptic to neuronal perikarya or vesicle-containing profiles. Thus, these results suggest that the dendrites of neurons intrinsic to the PAG are the most probable site of opiate action in the caudal ventral PAG.

The neural basis of footshock analgesia: The effect of periaqueductal gray lesions and decerebration

Previous studies have demonstrated that brief front paw shock and brief hind paw shock produce potent opiate and non-opiate analgesia, respectively. Front paw footshock-induced analgesia (FSIA) and hind paw FSIA are similar in that each is mediated by a medullospinal pathway. A question which arises is whether these opiate and non-opiate descending pathways are activated in direct response to afferent information from the spinal cord or whether indirect activation via more rostral centers is required. The first experiment examined the effect of lesions of the rostral periaqueductal gray (PAG) and caudal PAG on front paw (opiate) FSIA and hind paw (non-opiate) FSIA. In no case did PAG lesions markedly reduce the magnitude of these pain inhibitory states. Since this result raised the possibility that rostral centers may not have any major involvement in the production of these phenomena, the second experiment examined the effect of decerebration on front paw FSIA and hind paw FSIA. Decerebration had no effect on hind paw FSIA and, at most, produced only a very modest decrease in front paw FSIA. The fact that potent and prolonged analgesia can still be elicited after decerebration clearly demonstrates that limbic, cortical, thalamic, and rostral midbrain structures are not critical to the production of these pain inhibitory effects. Thus this work provides the first demonstration of opiate and non-opiate analgesia systems within the caudal brainstem and spinal cord which can be activated by environmental stimuli.

Periventricular system lesions and stimulation-produced analgesia

Three areas within the periventricular system were studied: caudal periaqueductal gray (PAG), rostral PAG, and caudal midline thalamus. Rats were chronically prepared with a bipolar stimulating electrode in one of these areas and two lesion electrodes in another. Current thresholds for stimulation-produced analgesia in the tail-flick test were assessed. Then, lesions were made and thresholds for analgesia re-assessed. Destruction of the caudal PAG consistently produced large increases in thresholds for analgesia at rostral stimulation sites; however, destruction of the rostral areas did not affect thresholds at caudal PAG sites. Lesions in all 3 areas yielded significant reductions in baseline (pre-brain stimulation) tail-flick latencies. Both sham lesioned control animals and animals with small lesions maintained stable baseline latencies and analgesia thresholds. The data support the view that all 3 brain areas studied contribute to the same pain-inhibitory system. They further suggest that stimulation at rostral sites activates elements which connect to or pass through the caudal PAG.

Analgesia produced by microinjection of baclofen and morphine at brain stem sites

Microinjection of either baclofen (1.5 μg) or morphine (2.5 μg), in equimolar doses (14 mM), at sites located in the caudal periaqueductal gray (PAG) resulted in a delay in tail flick latency (analgesia). The relative analgesic potency of baclofen among caudal PAG sites, however, did not correlate with that of morphine. Application of either drug into the caudal aspect of the cerebral aqueduct also produced analgesia, but neither agent caused analgesia when applied at PAG sites rostral to the interaural line. Baclofen alos produced analgesia when microinjected in the lower brain stem at sites lateral to the midline in or near the nucleus gigantocellularis, but did not produce analgesia when applied on the midline at sites located within or near the raphe magnus. Conversely, morphine produced analgesia when applied locally at midline sites but not at sites located lateral to the midline. These data suggest that the analgesia produced by systemic administration of baclofen and morphine involves activation of different neuronal substrates.