Does ethanol activate G-protein coupled inwardly rectifying K+ channels?

G-protein coupled inwardly rectifying K+ (GIRK) channels have been reported to be targets of ethanol actions. We investigated if ethanol affects native GIRK channels in rat brain tissues at clinically relevant concentrations using brain slices containing the ventrolateral periaqueductal gray (PAG), an area related to pain regulation. Ethanol did not affect the membrane current elicited by hyperpolarization ramps at concentrations up to 150 mM. However, at 200-300 mM, which is above the lethal level, it activated a barium-sensitive GIRK current in 30-57% of neurons. In neurons unresponsive to ethanol, baclofen, the mu-opioid or nociceptin successfully activated GIRK channels. It is suggested that GIRK channels of the ventrolateral PAG are unlikely to be targets of the analgesic action of ethanol.     

Opiate withdrawal increases ProTRH gene expression in the ventrolateral column of the midbrain periaqueductal gray

The midbrain periaqueductal gray matter (PAG) has a critical role in the modulation of behavioral and autonomic manifestations of the opiate withdrawal syndrome. We report a nearly 5-fold increase in proTRH gene expression in neurons of the ventrolateral column of the PAG following naltrexone precipitated morphine withdrawal. The accumulation of immunoreactive proTRH-derived peptides, but not the mature TRH tripeptide was concomitantly observed in these cells. These findings indicate that proTRH-derived peptides synthesized in neurons of the ventrolateral PAG may function as modifiers of opiate withdrawal responses.     

Efferent projections of ProTRH neurons in the ventrolateral periaqueductal gray.

Our previous study has shown that prothyrotropin-releasing hormone (proTRH) gene expression is increased in the ventrolateral periaqueductal gray (PAG) neurons following precipitated morphine withdrawal and continues to be activated even 24 h after withdrawal. We have hypothesized that peptide products of proTRH may participate in the recovery from morphine withdrawal. To identify neuroanatomical substrates of the proposed action of proTRH-derived peptides originating from the ventrolateral PAG proTRH neurons, projections of these neurons were investigated by a series of anterograde and retrograde tract-tracing experiments. First, Phaseolus vulgaris-leucoagglutinin (PHA-L) was injected in the ventrolateral PAG in Sprague-Dawley rats. Following transport of the tracer, simultaneous immunolabeling for PHA-L and proTRH peptides was performed and mapped in discrete brain regions. PHA-L-immunoreactive (IR) fibers showing preterminal and terminal-like arborization that contained proTRH were identified in the dorsolateral and lateral PAG, deep layer of superior colliculus (CS), parafascicular nucleus (PF), ventromedial zona incerta (ZI) and at the border of the locus coeruleus (LC) and Barrington's nucleus. Scattered double-labeled fibers were present in the lateral septal nucleus, ventromedial preoptic nucleus, lateral hypothalamus, perifornical area and in the periventricular region at the diencephalon/midbrain junction. The retrogradely transported marker, cholera toxin beta-subunit (CTb) was then injected in the dorsolateral PAG, CS, PF, ZI and medial to the LC. Double-labeled perikarya for both CTb and proTRH in the ventrolateral PAG were found for each region injected with CTb, corroborating the findings by the anterograde tracing experiment. These studies demonstrate that proTRH neurons in the ventrolateral PAG project to several regions of the brain that are involved in autonomic and behavioral regulation and thereby, may function as an integrating center to coordinate responses to opiate withdrawal.     

Tolerance to morphine microinjections in the periaqueductal gray (PAG) induces tolerance to systemic, but not intrathecal morphine

The acquisition and retention of tolerance to the antinociceptive effect of supraspinal morphine on the tail withdrawal reflex was assessed in rats implanted with unilateral cannulae in the periaqueductal gray (PAG). Development of tolerance to daily microinjections of morphine was indicated by the return of the tail flick response within 4 days, followed by the recovery of analgesic sensitivity one week later. After tolerance had developed, the effect of an acute systemic (1.5-4.5 mg/kg) or intrathecal (5-15 micrograms) morphine injection was determined. 'Cross-tolerance' was observed between systemic and supraspinal morphine but not between intrathecal and supraspinal morphine. The data indicate that tolerance to chronic intracerebral morphine produces the same behavioral consequences as tolerance to systemic morphine.     

Potassium channels underlie postsynaptic but not presynaptic GABA(B) receptor-mediated inhibition on ventrolateral periaqueductal gray neurons

γ-Aminobutyric acid (GABA) is the principle inhibitory neurotransmitter in adult mammalian brain. GABA receptors B subtype (GABA(B)Rs) are abundantly expressed at presynaptic and postsynaptic neuronal structures in the rat ventrolateral periaqueductal gray (PAG), an area related to pain regulation. Activation of GABA(B)Rs by baclofen, a selective agonist, induces presynaptic inhibition by decreasing presynaptic glutamate release. At the same time, baclofen induces a postsynaptic inhibitory membrane current or potential. We here report that in the ventrolateral PAG, the postsynaptic inhibition is mediated by activation of G protein-coupled inwardly rectifying K(+) (GIRK) channels. Blockade of K(+) channels largely prevents postsynaptic action of baclofen. In contrast, presynaptic inhibition of baclofen is insensitive to K(+) channel blockade. The data indicate that potassium channels play different roles in GABA(B)R-mediated presynaptic and postsynaptic inhibition on PAG neurons.     

Direct evidence for the activation of phospholipase C gamma 1 by in vivo treatment with morphine in the mouse periaqueductal gray matter

The aim of the present study was to investigate whether in vivo morphine treatment could participate in the activation of phospholipase Cgamma1 (PLCgamma1) isoform in the mouse periaqueductal gray matter (PAG) which can be accompanied by antinociceptive responses induced by morphine. As well as mu-opioid receptor-like immunoreactivity (MOR-IR), moderate PLCgamma1-like immunoreactivity (PLCgamma1-IR) was noted in the mouse PAG section. After s.c. treatment with morphine, the intensive PLCgamma1-IR was detected in the cell surface of the positive cells. Treatment s.c. with morphine produced a robust increase in the number of phosphorylated-PLCgamma1 (p-PLCgamma1) expressing cells in the PAG. Deletion of PLCgamma1 gene by i.c.v. pretreatment with antisense oligodeoxynucleotide against PLCgamma1 revealed a significant inhibition of supraspinal antinociception induced by a selective mu-opioid receptor agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO). Furthermore, i.c.v. pretreatment with a specific antibody to PLCgamma1 caused a concentration-dependent attenuation of antinociception produced by i.c.v. treatment with either morphine or DAMGO. These findings suggest that in vivo morphine treatment can activate PLCgamma1 isoform in the mouse PAG which can be, at least in part, associated with the expression of supraspinal antinociception induced by mu-opioid receptor agonists in the mouse.     

Defensive behaviors evoked from the ventrolateral periaqueductal gray of the rat: comparison of opioid and GABA disinhibition

Microinjection of morphine into the ventrolateral periaqueductal gray (PAG) disinhibits output neurons resulting in immobility and antinociception. Disinhibition also can be produced by microinjection of the GABA antagonist bicuculline. If morphine and bicuculline disinhibit the same class of neurons, then the behavioral effects evoked should be the same. Microinjection of morphine (5 microg/0.4 microl) into the ventrolateral PAG produced antinociception in 46 of 85 rats (54%). Subsets of rats with and without morphine antinociception were subsequently injected with bicuculline (2.5, 5, 10, and 25 ng/0.4 microl) into the same PAG site. Microinjection of bicuculline produced an increase in hot plate latency that was independent of the effect of the prior morphine microinjection. Bicuculline administration also produced an increase in locomotor activity in most rats, not immobility as with morphine microinjections. These differences between morphine and bicuculline microinjections indicate three things: (a) disinhibition of PAG neurons whether by morphine or bicuculline is an effective means of producing antinociception; (b) the circuitry underlying the behavioral effects of morphine and bicuculline differ; (c) the ventrolateral PAG appears capable of supporting a range of defensive behaviors from immobility to flight.     

Role of the dorsal raphe nucleus in morphine-induced immediate early gene expression in the rat striatum

Serotonin (5-HT) is thought to be involved in morphine action in the brain. To determine if the periaqueductal gray (PAG) and the dorsal raphe nucleus (DRN) are involved in morphine-induced c-Fos and JunB expression in the caudate-putamen (CPu), the mu receptor antagonist, beta-funaltrexamine (beta-FNA), was unilaterally infused into the PAG adjacent to DRN prior to morphine. Behaviorally, beta-FNA prevented morphine-induced loss of righting and Straub tail. In the CPu of beta-FNA treated rats, morphine-induced c-Fos and JunB were attenuated compared to vehicle-infused rats. These results suggest that morphine acts within the PAG-DRN to exert rapid behavioral effects and to induce c-Fos and JunB in the striatum.     

Serotonergic projections from the midbrain periaqueductal gray and nucleus raphe dorsalis to the nucleus parafascicularis of the thalamus.

By a double-labeling method combining the retrograde tracing of horseradish peroxidase and the immunocytochemical technique, serotonin-like immunoreactive neurons in the midbrain periaqueductal gray (PAG) and nucleus raphe dorsalis (DR) of the rat were observed to send projection fibers to the nucleus parafascicularis of the thalamus bilaterally with an ipsilateral dominance. These serotonin-containing projecting neurons were observed mainly at the middle-caudal levels of the ventrolateral subdivision of the PAG and less at the middle-rostral levels of the DR.     

Serotonergic projections from the midbrain periaqueductal gray and nucleus raphe dorsalis to the nucleus parafasccicularis of the thalamus

By a double-labeling method combining the retrograde tracing of horseradish peroxidase and the immunocytochemical technique, serotonin-like immunoreactive neurons in the midbrain periaqueductal gray (PAG) and nucleus raphe dorsalis (DR) of the rat were observed to send projection fibers to the nucleus parafascicularis of the thalamus bilaterally with an ipsilateral dominance. These serotonin-containing projecting neurons were observed mainly at the middle-caudal levels of the ventrolateral subdivision of the PAG and less at the middle-rostral levels of the DR.     

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. Bicuculline 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 PAG which modulate nociceptive transmission at the spinal level through actions on neurons in the RVM.     

Lumbar intrathecal morphine alters activity of putative nociceptive modulatory neurons in rostral ventromedial medulla

Two physiologically and pharmacologically distinct classes of putative nociceptive modulatory neurons have been identified in the rostral ventral medulla (RVM) of the lightly anesthetized rat: on-cells and off-cells. We have previously shown that administration of morphine either systemically or by microinjection into the periaqueductal gray (PAG) produces an increase in the activity of all off-cells and a depression of the activity of all on-cells concomitant with inhibition of the tail flick reflex. We now demonstrate that morphine applied intrathecally has effects on RVM neurons that are indistinguishable from those of systemic or PAG administration. This may contribute to the known multiplicative effects of concurrent administration of opioids at spinal and supraspinal sites.     

Neurons containing a N-terminal sequence of the TRH-prohormone (preproTRH53–74) are present in a unique location of the midbrain periaqueductal gray of the rat

Using an antiserum (no. 373) raised against a tyrosinated analog of preproTRH_53–74([Tyr¹]preproTRH_53–74 or pYT 22), we have demonstrated the presence of a discrete population of immunoreactive neurons in the midbrain periaqueductal gray (PAG). Relative to the distribution of serotonin, somatostain, peptide histidine isoleucine (PHI), methionine enkephalin, substance P and neurotensin-containing neuronal perikarya in the PAG, neurons containing immunoreactive pYT 22 occupied a unique location in the ventrolateral PAG. In contrast, terminal fields containing these neuroactive substances with the exception of PHI, were seen in abundance in the region of the ventrolateral PAG neurons. These studies indicate that a non-TRH sequence contained within the N-terminal portion of the TRH prohormone are expressed in a distinct group of neurons in the ventrolateral PAG. The location of these neurons in the PAG in a region richly innervated by nerve terminals containing analgesia-mediating substances, suggests a possible role for proTRH-derived peptides in the modulation of nociception.     

Neurons containing a N-terminal sequence of the TRH-prohormone (preproTRH53-74) are present in a unique location of the midbrain periaqueductal gray of the rat

Using an antiserum (no. 373) raised against a tyrosinated analog of preproTRH53-74 [( Tyr1]preproTRH53-74 or pYT 22), we have demonstrated the presence of a discrete population of immunoreactive neurons in the midbrain periaqueductal gray (PAG). Relative to the distribution of serotonin, somatostatin, peptide histidine isoleucine (PHI), methionine enkephalin, substance P and neurotensin-containing neuronal perikarya in the PAG, neurons containing immunoreactive pYT 22 occupied a unique location in the ventrolateral PAG. In contrast, terminal fields containing these neuroactive substances with the exception of PHI, were seen in abundance in the region of the ventrolateral PAG neurons. These studies indicate that a non-TRH sequence contained within the N-terminal portion of the TRH prohormone are expressed in a distinct group of neurons in the ventrolateral PAG. The location of these neurons in the PAG in a region richly innervated by nerve terminals containing analgesia-mediating substances, suggests a possible role for proTRH-derived peptides in the modulation of nociception.     

Features of the cytoarchitectonics of the periaqueductal gray of the cat midbrain

When studying cytoarchitectonics of the midbrain periaqueductal gray (PAG) in cat four areas were distinguished: medial, dorsal, lateral rostral and lateral caudal. They differ in size and distribution density of neurons.     

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.     

An evaluation of stimulation-produced analgesia in the cat

The analgesia produced by focal electrical brain stimulation of the periaqueductal central gray matter (PAG) was examined in 24 cats. The cats were chronically implanted with PAG and radial nerve bipolar stimulating electrodes. After the cats were trained to escape noxious radial nerve stimulation by crossing a low barrier in a shuttle box, the effects of focal electrical stimulation of the PAG, morphine, and chlorpromazine were evaluated on the escape threshold and latency. It was found that analgesic brain stimulation loci in the cat PAG were localized in the caudal ventrolateral PAG. The most efficacious analgesia-producing loci were observed to be at the ventrolateral edge of the PAG, suggesting a catecholaminergic involvement. Tolerance to repeated analgesia-producing electrical brain stimulation was apparent. Naloxone was not found to attenuate or antagonize the analgesia produced by focal electrical stimulation of the cat PAG.     

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 revealed 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.     

Sex differences in the anatomical and functional organization of the periaqueductal gray-rostral ventromedial medullary pathway in the rat: a potential circuit mediating the sexually dimorphic actions of morphine

Previous studies have demonstrated that morphine, administered systemically or directly into the periaqueductal gray (PAG), produces a significantly greater degree of antinociception in males in comparison with females. Because the midbrain PAG and its descending projections to the rostral ventromedial medulla (RVM) constitute an essential neural circuit for opioid-based analgesia, the present studies were conducted to determine whether sex differences in the anatomical organization of the PAG-RVM pathway, and its activation during persistent inflammatory pain, could account for sex-based differences in opioid analgesia. In the rat, retrograde tracing was combined with Fos immunocytochemistry to investigate sexual dimorphism in the organization of the PAG-RVM circuit and its activation by persistent inflammatory pain induced by intraplantar injection of complete Freund's adjuvant (CFA). The ability of morphine to suppress the activation of the PAG-RVM circuit was also examined. Sexually dimorphic retrograde labeling was observed within the dorsomedial and lateral/ventrolateral PAG at all rostrocaudal levels, with females having significantly more PAG-RVM output neurons in comparison with males. While no sex differences were noted in the activation of the PAG by persistent inflammatory pain, significantly more PAG-RVM cells were activated in males in comparison with females. Systemic administration of morphine significantly suppressed CFA-induced Fos in the PAG in males only. The results of these studies demonstrate that both the anatomical organization and the functional activation of the PAG-RVM circuit are sexually dimorphic and may provide the anatomical substrate for sex-based differences in morphine analgesia.