mu-Opioid and delta-opioid receptors are expressed in brainstem antinociceptive circuits: studies using immunocytochemistry and retrograde tract-tracing

Opioid-produced antinociception in mammals seems to be mediated in part by pathways originating in the periaqueductal gray (PAG) and the rostroventral medulla (RVM), and these pathways may include serotonergic neurons. In the present study, we examined the relationship of the cloned mu- and delta-receptors (MOR1 and DOR1, respectively) to PAG neurons projecting to the RVM, and RVM neurons projecting to the dorsal spinal cord. This was carried out by combining immunocytochemical staining for MOR1, DOR1, and serotonin with fluorescent retrograde tract-tracing. Of 133 retrogradely labeled cells in the RVM, 31% were immunoreactive for MOR1. Of the double-labeled cells, 41% also were immunoreactive for 5HT. Fifty-three percent of retrogradely labeled cells were apposed by DOR1-ir varicosities; 29% of the apposed cells were immunoreactive for 5HT. In the mesencephalon, cells retrogradely labeled from the RVM were usually surrounded by MOR1-ir structures; however, retrogradely labeled cells were never observed to be immunoreactive for MOR1. Similarly, retrogradely labeled cells in the caudal midbrain were seldom, if ever, labeled for DOR1; however, they frequently were apposed by DOR1-ir varicosities. Of 156 retrogradely labeled profiles from three rats, 52 (33%) were apposed by DOR1-ir varicosities. We conclude that both mu- and delta-opioid receptors could be involved in the antinociception mediated by the PAG-RVM-spinal cord circuit. In addition, opioids seem likely to have both direct and indirect effects on spinally projecting RVM cells in general, and on serotonergic RVM cells in particular.     

Activation of serotonergic neurons in the raphe magnus is not necessary for morphine analgesia

A wealth of pharmacological and behavioral data suggests that spinally projecting serotonergic cells mediate opioid analgesia. A population of medullary neurons, located within raphe magnus (RM) and the neighboring reticular nuclei, contains serotonin and is the source of serotonin in the spinal dorsal horn. To test whether serotonergic neurons mediate opioid analgesia, morphine was administered during recordings from medullary cells that were physiologically characterized as serotonergic (5HTp) by their slow and steady discharge pattern in the lightly anesthetized rat. Selected 5HTp cells (n = 14) were intracellularly labeled, and all contained serotonin immunoreactivity. The discharge of most 5HTp cells was not affected by an analgesic dose of systemic morphine. In a minority of cases, 5HTp cells either increased or decreased their discharge after morphine administration. However, morphine altered the discharge of some 5HTp cells in the absence of producing analgesia and conversely did not alter the discharge of most 5HTp cells in cases in which analgesia occurred. RM cells with irregular discharge patterns and excitatory or inhibitory responses to noxious tail heat were classified as ON and OFF cells, respectively. All ON and OFF cells that were intracellularly labeled (n = 9) lacked serotonin immunoreactivity. All ON cells were inhibited, and most OFF cells were excited by systemic morphine. Because 5HTp cells do not consistently change their discharge during morphine analgesia, they are unlikely to mediate the analgesic effects of morphine. Instead, nonserotonergic cells are likely to mediate morphine analgesia in the anesthetized rat. In light of the sensitivity of morphine analgesia to manipulations of serotonin, serotonin release, although neither necessary nor sufficient for opioid analgesia, is proposed to facilitate the analgesic effects of nonserotonergic RM terminals in the spinal cord.     

GABA(A) receptors in the primate basal ganglia: an autoradiographic and a light and electron microscopic immunohistochemical study of the alpha1 and beta2,3 subunits in the baboon brain

The distribution of gamma-aminobutyric acid(A) (GABA(A)) receptors was investigated in the basal ganglia in the baboon brain by using receptor autoradiography and the immunohistochemical localisation of the alpha1 and beta2,3 subunits of the GABA(A) receptor by light and electron microscopy. In the caudate-putamen, the alpha1 subunit was distributed in high densities in the matrix compartment, and the beta2,3 subunits were more homogeneously distributed; the globus pallidus showed lower levels of the alpha1 and beta2,3 subunits. Four types of alpha1 subunit immunoreactive neurons were identified in the baboon striatum: the most numerous (75%) were type 1 medium-sized aspiny neurons; type 2 (2%) were large aspiny neurons with an indented nuclear membrane located in the ventral striatum; type 3 neurons were the least numerous (1%) and were comprised of large neurons in the ventromedial regions of the striatum; and type 4 (22%) neurons were medium to large aspiny neurons located in striosomes. At the ultrastructural level, alpha1 and beta2,3 subunit immunoreactivity was localised in the neuropil of the striatum in both symmetrical and asymmetrical synaptic contacts. In the globus pallidus, alpha1 and beta2,3 subunits were localised on large neurons and were found in three types of synaptic terminals: type 1 terminals were small and established symmetrical synapses; type 2 terminals were large; and type 3 terminals formed small synaptic terminals with subjunctional dense bodies. These results show that the subunit composition of GABA(A) receptors varies between the striosome and the matrix compartments in the striatum and that there is receptor subunit homogeneity in the globus pallidus.     

GABA- and glutamate-immunoreactive synapses on sympathetic preganglionic neurons projecting to the superior cervical ganglion

Our previous work suggests that virtually all of the synapses on sympathetic preganglionic neurons projecting to the rat adrenal medulla are immunoreactive for either the inhibitory amino acid, gamma-aminobutyric acid (GABA) or the excitatory amino acid, L-glutamate. To investigate whether or not this is true for other groups of sympathetic preganglionic neurons, and to determine whether or not the proportion of inputs containing each type of amino acid neurotransmitter is the same for different groups of sympathetic preganglionic neurons, we retrogradely labelled rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion and used post-embedding immunogold on ultrathin sections to localise GABA- and glutamate-immunoreactivity. The cell bodies and dendrites of both rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion received synapses and direct contacts from nerve fibres immunoreactive for GABA and from nerve fibres immunoreactive for glutamate. In the rat, GABA was present in 48.9% of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion, and glutamate was present in 51.7% of inputs. Double immunogold labelling for glutamate and GABA on the same section, as well as labelling of consecutive serial sections for the two antigens, indicated that GABA and glutamate occur in separate populations of nerve fibres that provide input to rat sympathetic preganglionic neurons projecting to the superior cervical ganglion. We now have shown that GABA or glutamate is present in virtually all of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion and in essentially all of the inputs to sympathetic preganglionic neurons supplying the adrenal medulla. These findings are consistent with the hypothesis that all fast synaptic transmission in central autonomic pathways may be mediated by either excitatory or inhibitory amino acids. Furthermore, we showed a statistically significant difference in the proportion of glutamate-immunoreactive inputs between sympathetic preganglionic neurons projecting to the superior cervical ganglion and sympathoadrenal neurons (data from Llewellyn-Smith et al. [Llewellyn-Smith, I.J., Phend, K.D., Minson, J.B., Pilowsky, P.M., Chalmers, J.P., 1992. Glutamate immunoreactive synapses on retrogradely labelled sympathetic neurons in rat thoracic spinal cord. Brain Res. 581, 67-80]), with preganglionics supplying the adrenal medulla receiving more excitatory inputs than those supplying the superior cervical ganglion. This increased excitatory input to sympathoadrenal neurons may explain the predominant activation of these neurons following baroreceptor unloading.     

Phosphorescent platinum/palladium coproporphyrins for time-resolved luminescence microscopy

The rostral ventromedial medulla (RVM) is an important mediator of the supraspinal component of opioid antinociception. Previous studies have suggested that activation of the cloned mu- and delta-opioid receptors (MOR1 and DOR1 respectively) in the RVM produces the antinociception mediated by spinally projecting neurons. In the present study, we investigated the expression of mRNA encoding either MOR1 or DOR1 in the RVM of rats. In addition, we examined quantitatively the expression of MOR1 and DOR1 mRNAs in spinally projecting RVM neurons including serotonergic (5HT) cells by using in situ hybridization, immunocytochemistry, retrograde tract-tracing, and the physical disector. Brainstem neurons were labeled in 14 male Sprague-Dawley rats by applying Fluoro-Gold (FG) topically to the dorsal surface of the lumbosacral spinal cord. Five-micrometer-thick cryostat sections were cut and in situ hybridization was performed by using full-length cRNA probes labeled with 35S-UTP. We found that 43% of RVM projection neurons expressed MOR1 mRNA and 83% of RVM projection neurons expressed DOR1 mRNA. Of 192 retrogradely labeled cells in the RVM, 51 cells (27%) were immunoreactive for 5HT. Of this population, half appeared to be labeled for the mRNA encoding MOR1 and over three-fourths appeared to be labeled for the mRNA encoding DOR1. Thus, we conclude that bulbospinal neurons express MOR1 and DOR1; moreover, MOR1 and DOR1 are expressed by significant proportions of 5HT neurons projecting to or through the dorsal spinal cord.     

Action of zolpidem on responses to GABA in relation to mRNAs for GABA(A) receptor alpha subunits within single cells: evidence for multiple functional GABA(A) isoreceptors on individual neurons

The relationship between zolpidem sensitivity and GABA(A) receptor alpha subunits was studied in individual dissociated neurons from rat brain. Using whole-cell recording, similar EC50 values were demonstrated for the effect of gamma-aminobutyric acid (GABA) on gated-chloride currents from substantia nigra reticulata (SNR) and lateral septal neurons. Subsequently, many neurons from both the SNR or lateral septum were found to exhibit enhanced GABA-gated chloride currents across concentrations of zolpidem ranging from 10 to 300 nM. Some neurons exhibited a greater than 20% increase in responsiveness to GABA at 30 nM of zolpidem without further increase at higher concentrations of zolpidem. Conversely, zolpidem enhancement of GABA from another group of neurons was not observed at 30 nM zolpidem, but between 100 and 300 nM the response to GABA increased greater than 20%. Finally, a third group of neurons reached both of these criteria for zolpidem enhancement of GABA. This latter spectrum of responses to GABA after varying concentrations of zolpidem was consistent with the presence of either two GABA(A) receptors or a single receptor with differing affinities for zolpidem on an individual neuron. Following determination of the sensitivity of neurons from SNR or lateral septum to zolpidem, cytoplasm was extracted from some individual cells to allow identification of cellular mRNAs for the alpha1, alpha2 and alpha3 GABA(A) receptor subunits with RT-PCR. Those neurons that responded to the 30 nM zolpidem concentration invariably expressed the alpha1-GABA(A) receptor subunit. This result is consistent with the GABA(A) alpha1-receptor subunit being an integral part of a functional high-affinity zolpidem type 1-BZD receptor complex on neurons in brain. Those neurons which showed enhancement of GABA from 100 to 300 nM zolpidem contained mRNAs for the alpha2 and/or the alpha3 receptor subunits, a finding consistent with these alpha subunits forming type 2-BZD receptors. Some individual dissociated SNR neurons were sensitive to both low and high concentrations of zolpidem and contained mRNAs for all three alpha-receptor subunits. These latter individual neurons are proposed to have at least two functional GABA(A) receptor subtypes. Thus, the present investigation emphasizes the importance of characterizing the relationship between endogenous GABA(A) receptor function and the presence of specific structural components forming GABA(A) receptor subtypes on neurons.     

Chronic GABA treatment downregulates the GABAA receptor alpha 2 and alpha 3 subunit mRNAS as well as polypeptide expression in primary cultured cerebral cortical neurons

Chronic GABA exposure of mammalian primary cultured cortical neurons results in a downregulation of the GABA-benzodiazepine receptor complex. In the present study, the mRNA levels, as well as polypeptide expression, for the GABAA receptor alpha 2 and alpha 3 subunits in cultured embryonic mouse cerebral cortical neurons (7 day old) were examined using northern analysis and immunoblotting techniques following chronic GABA treatment. The alpha 1 subunit mRNA or polypeptide could not be detected in these neurons. The steady state levels of mRNA for the GABAA receptor alpha 2 and alpha 3 subunits showed a decrease in comparison with untreated neurons. There was no change in the level of the beta actin or poly(A)+ RNA under the same experimental conditions. This agonist-induced reduction in the GABAA receptor alpha 2 and alpha 3 subunit mRNA was blocked by the concomitant exposure of neurons to R 5135, an antagonist of GABAA receptor. The polypeptide expression for the GABAA receptor alpha 2 and alpha 3 subunits in chronically GABA-treated neurons also showed a decline and this change was also blocked by the concomitant exposure of cells to GABA and R 5135. These results indicate that the chronic exposure of the GABAA receptor complex to agonist downregulates the expression of the alpha subunits of the receptor complex, which may be related to an observed decreases in the number of binding sites and GABA-induced 36Cl-influx in the cortical neurons.     

Base and fog densities of fresh Ektaspeed Plus dental X-ray films

Binding of [3H]ethynylbicycloorthobenzoate ([3H]EBOB) to the gamma-aminobutyric acid type A (GABAA) receptor of cultured cerebellar granule neurons is inhibited by GABA, muscimol and 3-aminopropanesulfonic acid with IC50 values of 69-250 nM. Sensitivity to these GABA mimetics is lower by 3-4-fold for cerebellum and 10-20-fold for cerebral cortex, midbrain, and pons and medulla, a differential sensitivity by brain region and cell type consistent with earlier findings using tert-[35S]butylbicyclophosphorothionate and GABA. In contrast, the inhibitory potencies of two chloride channel blockers, alpha-endosulfan and picrotoxinin, do not differ in these assays. The hypothesis that this pharmacological profile is conferred by the alpha 6 subunit specific to cerebellar granule cells is supported by the finding that forskolin (which downregulates the alpha 6 subunit) but not the inactive dideoxyforskolin markedly decreases the sensitivity of [3H]EBOB binding to GABA without affecting inhibition by alpha-endosulfan.     

Does licensing of drugs in industrialized countries guarantee drug quality and safety for Third World countries? The case of norplant licensing in Finland

A knowledge of the anatomy of medullary serotonergic cells is critical to understanding local and brainstem circuits in which these cells participate. Serotonergic neurons (n = 16) were identified, as previously described (Mason [1997] J. Neurophysiol. 77:1087-1098) by their slow and steady background discharge in halothane anesthetized rats. Neurons were then intracellularly labeled with Neurobiotin and visualized with 3,3'diaminobenzidine. The validity of the physiological identification of serotonergic cells was confirmed by processing two neurons that were physiologically characterized as serotonergic for serotonin immunoreactivity; both tested cells contained immunoreactive serotonin. The dendrites and axon of each labeled cell were reconstructed by using a three-dimensional computerized system. Somata were small or medium in size and had fusiform, triangular, or multipolar shapes. The dendritic arbor was constricted with most dendrites extending for less than 500 microm from the soma. All labeled axons projected caudally and travelled in the ventrolateral medulla, either dorsal or ventral to the lateral reticular nucleus. Most cells had collaterals and/or dense axonal swellings in the nucleus reticularis gigantocellularis, nucleus reticularis magnocellularis, raphe magnus, and the ventrolateral medulla. Non-local collaterals and swellings were also observed in the nucleus reticularis gigantocellularis and in the ventrolateral medulla at all medullary levels. The results demonstrate that 1) the dendrites of serotonergic cells are restricted to raphe magnus and the ventral part of nucleus reticularis magnocellularis; and 2) serotonergic cells project to medullary nuclei that contain bulbospinal cells which project to dorsal, intermediate, and ventral horns. Serotonergic cell projections to brainstem sites may mediate the integration of sensory, autonomic, and motor modulation at the brainstem level.     

Differential regulation of GABA(A) receptor gene expression by ethanol in the rat hippocampus versus cerebral cortex

Previous research has shown that chronic ethanol consumption dramatically alters GABA(A) receptor alpha1 and alpha4 subunit gene expression in the cerebral cortex and GABA(A) receptor alpha1 and alpha6 subunit gene expression in the cerebellum. However, it is not yet known if chronic ethanol consumption produces similar alterations in GABA(A) receptor gene expression in other brain regions. One brain region of interest is the hippocampus because it has recently been shown that a subset of GABA(A) receptors in the hippocampus is responsive to pharmacologically relevant concentrations of ethanol. Therefore, we directly compared the effects of chronic ethanol consumption on GABA(A) receptor subunit gene expression in the hippocampus and cerebral cortex. Furthermore, we investigated whether the duration of ethanol consumption (14 or 40 days) would influence regulation of GABA(A) receptor gene expression in these two brain regions. Chronic ethanol consumption produced a significant increase in the level of GABA(A) receptor alpha4 subunit peptide in the hippocampus following 40 days but not 14 days. The relative expression of hippocampal GABA(A) receptor alpha1, alpha2, alpha3, beta(2/3), or gamma2 was not altered by either period of chronic ethanol exposure. In marked contrast, chronic ethanol consumption for 40 days significantly increased the relative expression of cerebral cortical GABA(A) receptor alpha4 subunits and significantly decreased the relative expression of cerebral cortical GABA(A) receptor alpha1 subunits. This finding is consistent with previous results following 14 days of chronic ethanol consumption. Hence, chronic ethanol consumption alters GABA(A) receptor gene expression in the hippocampus but in a different manner from that in either the cerebral cortex or the cerebellum. Furthermore, these alterations are dependent on the duration of ethanol exposure.     

Expression of GABA(A) receptor subunits in the hippocampus of the rat after kainic acid-induced seizures

The GABA(A) receptor is a ligand gated chloride channel consisting of five membrane spanning proteins for which 13 different genes have been identified in the mammalian brain. The present review summarizes recent work from our laboratory on the characterization of the immunocytochemical distribution of these GABA(A) receptor subunits in the rat brain and changes in immunoreactivity and mRNA expression after kainic acid-induced status epilepticus. A heterogeneous distribution of immunoreactive GABA(A) receptor subunits was observed. The most abundant ones were: alpha1, alpha2, alpha4, alpha5, beta2, beta3, gamma2, and delta. Alpha1, beta2, and gamma2 were about equally distributed in all subfields of the hippocampus; alpha4- and delta-subunits were preferentially found in the dentate molecular layer and in CA1; alpha2 was localized to the dentate molecular layer and CA3; alpha5 was found in the dendritic areas of CA1 to CA3; and beta1 was preferentially seen in CA2. Alpha1, beta2, gamma2 and delta were highly concentrated in interneurons. Kainic acid-induced seizures caused acute and chronic changes in the expression of mRNAs and immunoreactive proteins. Acute changes included decreases in alpha2, alpha5, beta1, beta3, gamma2 and delta mRNA levels (by about 25-50%), accompanied by increases (by about 50%) in alpha1, alpha4, and beta2 messages in granule cells (after 6-12 h). Chronic changes, characterized by losses in mRNA and immunoreactive proteins in CA1 and CA3, are undoubtedly due to seizure-related cell damage. However, compensatory expression of alpha2 and beta3 subunits, especially in CA3b/c, was observed. Furthermore, increases in mRNAs and immunoreactive proteins were seen for alpha1, alpha2 alpha4, beta1, beta2, beta3 and gamma2 in granule cells and in the molecular layer of the dentate gyrus at 7-30 days after kainic acid injection. The changes in the expression of GABA(A) receptor subunits, observed in practically all hippocampal subfields, may reflect altered GABA-ergic transmission during development of the epileptic syndrome. Increased expression of GABA(A) receptor subunits in the dendritic field of granule cells and CA3 suggest that GABA-ergic inhibition may be augmented at these levels. However, the lasting preservation of alpha1-, beta2-, and gamma2-subunits in interneurons could provide a basis for augmented inhibition of GABA-ergic interneurons, leading to net disinhibition.     

The role of the GABA(A) receptor alpha1 subunit N-terminal extracellular domain in propofol potentiation of chloride current

Propofol (2,6-diisopropylphenol), an intravenous general anesthetic in active clinical use today, potentiates the action of gamma-aminobutyric acid (GABA) at the type-A receptor and also directly induces current in the absence of GABA. We expressed different combinations of murine GABA(A) receptor alpha1, beta3 and gamma2 subunits in Xenopus oocytes to investigate the subunit dependence of propofol potentiation of pentobarbital-induced current. Pentobarbital induces current in all beta3-subunit-containing receptors, whereas current gating by GABA requires the presence of both alpha1 and beta3 subunits. Therefore, pentobarbital rather than GABA was used to induce current in order to separate the subunit dependence of current gating from the subunit dependence of potentiating action of propofol. alpha1beta3gamma2, alpha1beta3, beta3gamma2, or beta3 subunit combinations all responded to pentobarbital in a dose-dependent manner. True potentiation was defined as the current magnitude to simultaneous application of pentobarbital and propofol exceeding the additive responses to individual drug applications. A dose-dependent propofol potentiation of pentobarbital-induced current was observed in oocytes injected with alpha1beta3 or alpha1beta3gamma2 but not in beta3gamma2 or beta3 subunits, suggesting that the alpha1 subunit was necessary for this modulatory action of propofol. Further examination of the propofol potentiation in chimeras between the alpha1 and beta3 subunits showed that the extracellular amino-terminal half of the alpha1 subunit was sufficient to support propofol potentiation. The different requirements of the receptor structure for the agonistic (gating) and the potentiating actions suggest that these two actions of propofol are distinct processes mediated through its action at distinct sites.     

Interactions of histaminergic and serotonergic neurons in the hypothalamic regulation of prolactin and ACTH secretion

Serotonergic and histaminergic neuronal systems are both involved in mediation of the stress-induced release of the pituitary hormones prolactin (PRL) and ACTH. We investigated the possibility of an interaction between serotonin (5-HT) and histamine (HA) in regulation of PRL and ACTH secretion in conscious male rats. Animals were pretreated systemically with antagonists to 5-HT1, 5-HT2 or 5-HT3 receptors prior to intracerebroventricular (icv) administration of HA. The 5-HT1 + 2 receptor antagonist methysergide prevented and the 5-HT2 receptor antagonist LY 53857 attenuated the HA-induced PRL release while the 5-HT3 receptor antagonist ondansetron had no effect on this response. None of the three 5-HT receptor antagonists affected the ACTH response to HA. Specific blockade of HA synthesis by alpha-fluoromethylhistidine or blockade of postsynaptic HA receptors by icv infusion of the H1 receptor antagonist mepyramine or the H2 receptor antagonist cimetidine inhibited the PRL response to 5-HT or to the 5-HT precursor 5-hydroxytryptophan (5- HTP) given in combination with the 5-HT reuptake inhibitor fluoxetine (Flx). Blockade of the histaminergic system had no effect on the ACTH response to serotonergic stimulation. The H3 receptors are inhibitory HA receptors. Systemic pretreatment with the H3 receptor agonist R(alpha)methylhistamine, or the H3 receptor antagonist thioperamide had no effect on the hormone response to activation of the serotonergic system by 5-HTP plus Flx. We conclude that the serotonergic and histaminergic neuronal systems interact in their stimulation of PRL secretion, but not in their stimulation of ACTH secretion. This interaction involves serotonergic 5-HT1 and 5-HT2 receptors and histaminergic H1 and H2 receptors. Furthermore, the previously observed inhibitory effect of the H3 receptor agonist R(alpha)methylhistamine on stress-induced PRL and ACTH release seems not to be exerted by activation of presynaptic H3 receptors located on serotonergic neurons but rather on histaminergic neurons.     

Creating an education model for cardiac patients

gamma-Aminobutyric acidA (GABA(A)) gated chloride ion channels were expressed from human recombinant cDNA using the baculovirus/Sf-9 insect cell expression system. The electrophysiological effects in whole-cell currents of 5-(4-piperidyl) isoxazol-3-ol (4-PIOL), a GABA(A) receptor partial agonist, were investigated on GABA(A) receptor complexes of alpha1beta2gamma2S subunits as well as a slightly modified construct of alpha1(valine 121)beta2gamma2S subunits. Here we report that (1)4-PIOL induces an inward whole-cell current in a concentration-dependent manner in both alpha1(val 121)beta2gamma2S and alpha1(ile 121)beta2gamma2S receptor subunit combinations. (2) The 4-PIOL induced whole-cell currents were more pronounced in alpha1(val 121)beta2gamma2S than in alpha1(ile 121)beta2gamma2S receptor subunit combinations. (3) 4-PIOL inhibited GABA-induced responses on alpha1(ile 121)beta2gamma2S and alpha1(val 121)beta2gamma2S receptor combinations with similar potency.     

Properties and sex-specific differences of GABAA receptors in neurons expressing gamma1 subunit mRNA in the preoptic area of the rat

Gamma-aminobutyric acid type A (GABAA) receptors expressed within the medial preoptic area (mPOA) are known to play a critical role in regulating sexual and neuroendocrine functions. In the rat brain, high levels of expression of the gamma1 subunit mRNA of the GABAA receptor are restricted to a limited number of regions that mediate sexual behaviors, including the mPOA. The biophysical and pharmacological profiles of native gamma1-containing receptors in neurons are unknown. Here, we have characterized the properties of GABAA receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) and currents elicited by fast perfusion of GABA to isolated mPOA neurons of juvenile male and female rats. No significant sex-specific differences were evident in the mean peak amplitude, distribution of event amplitudes, kinetics of current decay, or the frequency of sIPSCs. The profile of modulation of sIPSCs by diazepam, beta-CCM and zolpidem, allosteric modulators that act at the benzodiazepine (BZ) site of the GABAA receptor, support the assertion that mPOA neurons of both sexes express functional gamma1-containing receptors. The ability of zolpidem to modulate both sIPSC amplitude and currents elicited by rapid perfusion of GABA to mPOA neurons differed significantly between the sexes. Zolpidem reversibly induced negative modulation of currents in mPOA neurons isolated from male rats, but had no effect in mPOA neurons from female rats. Concentration-response analysis of responses in neurons acutely isolated from male rats indicated an IC50 of 58 nM with maximal decreases of approximately 50% of control peak current amplitude. In situ hybridization analysis demonstrated that levels of the gamma1 subunit mRNA are significantly higher in mPOA neurons from male than female rats. No significant sex-specific differences were detected in the levels of alpha1, alpha2, or alpha5 mRNAs. These results suggest that native gamma1-containing receptors are expressed in primary neurons of the mPOA and that sex-specific differences in the expression of this subunit may contribute to sexual dimorphism in GABAA receptor modulation by compounds acting at the BZ site.     

Functional properties of recombinant GABA(A) receptors composed of single or multiple beta subunit subtypes

GABA(A) receptor (GABAR) isoforms in the central nervous system are composed of combinations of alpha(1-6), beta(1-4), gamma(1-4), delta(1) and epsilon(1) subunit subtypes arranged in a pentamer. Many regions of the brain express high levels of mRNA encoding several different subunits and even multiple subunit subtypes. The stoichiometry of GABAR isoforms is unclear, and the number and identity of individual subunit subtypes that are coassembled remain uncertain. To examine the role of beta subunit subtypes in the functional properties of GABARS and to determine whether multiple beta subtypes can be coassembled in functional GABARs, plasmids containing cDNAs encoding rat beta1 and/or beta3, alpha5 and gamma2L subtypes were cotransfected into L929 fibroblasts. The properties of the expressed receptor populations were determined using whole-cell and single-channel recording techniques. The alpha5beta1gamma2L isoform was less sensitive to GABA than the alpha5beta3gamma2L isoform. alpha5beta1gamma2L isoform currents were also insensitive to the allosteric modulator loreclezole, while alpha5beta3gamma2L isoform currents were strongly potentiated by loreclezole. Fibroblasts transfected with plasmids containing cDNAs for both beta1 and beta3 subtypes along with alpha5 and gamma2L subtypes produced a receptor population with an intermediate sensitivity to GABA which was insensitive to loreclezole. These results suggest that functional GABARs can be formed that contain two different beta1 subunit subtypes with properties different from receptors that contain only a single beta1 subtype and that the beta1 subunit subtypes influence the response of GABARs to GABA and to the allosteric modulator loreclezole.     

SB-205384: a GABA(A) receptor modulator with novel mechanism of action that shows subunit selectivity

1. SB-205384, and its (+) enantiomer (+)-SB-205384 were tested for their modulatory effects on human GABA(A) receptor subunit combinations expressed in Xenopus oocytes by electrophysiological methods. 2. The slowing of the decay rate induced by SB-205384 on native GABA-activated currents in rat neurones was also seen on GABA(A) currents in oocytes expressing human GABA(A) subunits. This temporal effect was observed for the alpha3beta2gamma2 subunit combination with little effect in subunit combinations containing either alpha1 or alpha2. 3. Potentiation of the peak amplitude of the GABA-activated currents by SB-205384 or (+)-SB-205384 was less specific for a particular subunit combination, although the greatest effect at 10 microM drug was seen on the alpha3beta2gamma2 subunit combination. 4. In contrast, zolpidem, a benzodiazepine site modulator, did not significantly slow decay rates of GABA(A) currents in oocytes expressing the alpha3beta2gamma2 subunit combination. Zolpidem, as expected, did selectively potentiate GABA-activated currents on oocytes expressing the gamma2 subunit compared to those containing the gamma1. 5. The results show that the novel kinetic modulatory profile of SB-205384 is selective for the alpha3beta2gamma2 subunit combination. This suggests that the compound is binding to a novel regulatory site on the subunit complex.     

Role of the nucleus raphe magnus in antinociception produced by ABT-594: immediate early gene responses possibly linked to neuronal nicotinic acetylcholine receptors on serotonergic neurons

Recently, a novel cholinergic channel modulator, (R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594), was shown to produce potent analgesia in a variety of rodent pain models when administered either systemically or centrally into the nucleus raphe magnus (NRM). The purpose of the present study was to investigate the possible supraspinal contribution of ABT-594 by assessing its ability to induce expression of the immediate early gene c-fos, a biochemical marker of neuronal activation, in the NRM of rats. Putative serotonergic neurons in the NRM, a medullary nucleus proposed to be involved in descending antinociceptive pathways, were identified immunohistochemically using a monoclonal antibody (mAb) against tryptophan hydroxylase. ABT-594 (0.03-0.3 micromol/kg, i.p.) produced a dose-dependent induction of Fos protein that was blocked by the central nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine (5 micromol/kg, i.p.) but not by the peripheral nAChR antagonist hexamethonium (15 micromol/kg, i.p.). Immunohistological studies using mAb 299 revealed the expression of alpha4-containing nAChRs in the NRM. The alpha4 immunostaining was dramatically reduced by pretreating (30 d) animals with the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), which was previously shown to substantially attenuate the antinociceptive actions of ABT-594. In a double immunohistochemical labeling experiment, coexpression of the serotonin marker tryptophan hxdroxylase and the alpha4 nAChR subunit in NRM neurons was observed. These results suggest that the analgesic mechanism of ABT-594 may in part involve the activation of the NRM, a site where alpha4-containing nAChRs are expressed by serotonergic neurons.