GABAB Receptor Expression Research Articles

GABAergic Afferents Activate Both GABAAand GABABReceptors in Mouse Substantia Nigra Dopaminergic NeuronsIn Vivo

Most in vivo electrophysiological studies of substantia nigra have used rats. With the recent proliferation of the use of mice for in vitro neurophysiological studies because of the availability of various genetically modified strains to identify the roles of various channels and proteins in neuronal function, it is crucial to obtain data on in vivo responses in mice to verify that the in vitro results reflect functioning of systems comparable with those that have been well studied in rat. Inhibitory responses of rat nigral dopaminergic neurons by stimulation of afferents from striatum, globus pallidus, or pars reticulata have been shown to be mediated predominantly or exclusively by GABA(A) receptors. This is puzzling given the substantial expression of GABA(B) receptors and the ubiquitous appearance of GABA(B) synaptic responses in rat dopaminergic neurons in vitro. In the present study, we studied electrically evoked GABAergic inhibition in nigral dopaminergic neurons in C57BL/6J mice. Stimulation of the three major GABAergic inputs elicited stronger and longer-lasting inhibitory responses than those seen in rats. The early inhibition was GABA(A) mediated, whereas the later component, absent in rats, was GABA(B) mediated and selectively enhanced by GABA uptake inhibition. Striatal-evoked inhibition exhibited a slower onset and a weaker initial component compared with inhibition from globus pallidus or substantia nigra pars reticulata. These results are discussed with respect to differences in the size and neuronal density of the rat and mouse brain and the different sites of synaptic contact of the synapses from the three GABAergic afferents.

Angiotensin II increases GABAB receptor expression in nucleus tractus solitarii of rats

Increasing evidence indicates that both the angiotensin II (ANG II) and gamma-aminobutyric acid (GABA) systems play a very important role in the regulation of blood pressure (BP). However, there is little information concerning the interactions between these two systems in the nucleus tractus solitarii (NTS). In the present study, we examined the effects of ANG II on GABAA and GABAB receptor (GAR and GBR) expression in the NTS of Sprague-Dawley rats. The direct effect of ANG II on GBR expression was determined in neurons cultured from NTS. Treatment of neuronal cultures with ANG II (100 nM, 5 h) induced a twofold increase in GBR1 expression, as detected with real-time RT-PCR and Western blots, but had no effect on GBR2 or GAR expression. In electrophysiological experiments, perfusion of neuronal cultures with the GBR agonist baclofen decreased neuronal firing rate by 39% and 63% in neurons treated with either PBS (control) or ANG II, respectively, indicating that chronic ANG II treatment significantly enhanced the neuronal response to GBR activation. In contrast, ANG II had no significant effect on the inhibitory action of the GAR agonist muscimol. In whole animal studies, intracerebroventricular infusion of ANG II induced a sustained increase in mean BP and an elevation of GBR1 mRNA and protein levels in the NTS. These results indicate that ANG II stimulates GBR expression in NTS neurons, and this could contribute to the central nervous system actions of ANG II that result in dampening of baroreflexes and elevated BP in the central actions of ANG II.

Symposium: ‘A celebration of neuroanatomy in the centennial of Cajal's Nobel Prize’

By 1889, at the age of 37 years, Cajal had published two papers on neuroanatomy in the Revista trimestral de Histologia normal y patologica, which he had founded (and subsidised) the previous year. However, his ideas received hardly any attention from his colleagues and he became a ‘little alarmed by their silence’ (Ramon y Cajal, 1981). He admitted that most histologists had not read his papers since ‘it is true that Spanish is an unknown language to the educated’ and he thought that the ‘direct objective demonstration’ of facts would be more convincing. Therefore, he first translated his principal papers into French and then decided to show his histological preparations to scientific colleagues, in order to demonstrate his findings and ideas. To this end, Cajal applied for membership of the Anatomische Gesellschaft, asked his Rector for permission to travel, collected all of his savings and set out for Berlin, where the Anatomische Gesellschaft met in October 1889.

From neuroanatomy to gene therapy: searching for new ways to manipulate the supraspinal endogenous pain modulatory system.

The endogenous pain modulatory system is a complex network of brain areas that control nociceptive transmission at the spinal cord by inhibitory and facilitatory actions. The balance between these actions ensures effective modulation of acute pain, while during chronic pain the pronociceptive effects appear to prevail. The mechanisms underlying this imbalance were studied as to the role of two medullary components of the pain modulatory system: the dorsal reticular nucleus and the caudal ventrolateral medulla, which function primarily as pronociceptive and antinociceptive centres, respectively. Both areas are connected with the spinal dorsal horn by closed reciprocal loops. In the spino-dorsal reticular nucleus loop, the ascending branch is strongly inhibited by spinal GABAergic neurons, which may act as a buffering system of the dorsal reticular nucleus-centred amplifying effect. In the spino-caudal ventrolateral medulla loop, the ascending branch is under potent excitation of substance P (SP) released from primary afferents, which is likely to trigger the intense descending inhibition detected in acute pain. During chronic pain, the activity in the lateral reticular formation of the caudal ventrolateral medulla changes, so that the action of the caudal ventrolateral medulla upon SP-responsive spinal neurons shifts from inhibitory to excitatory. The mechanisms of this modulatory shift are unknown but probably relate to the decreased expression of micro-opioid, delta-opioid and GABAB receptors. Normalizing receptor expression in the caudal ventrolateral medulla or controlling noci-evoked activity at the dorsal reticular nucleus or caudal ventrolateral medulla by interfering with neurotransmitter release is now possible by the use of gene therapy, an approach that stands out as a unique tool to manipulate the supraspinal endogenous pain control system.

Characterization of neuronal subsets surrounded by perineuronal nets in the rhesus auditory brainstem

The distribution of perineuronal nets and the potassium channel subunit Kv3.1b was studied in the subdivisions of the cochlear nucleus, the medial nucleus of the trapezoid body, the medial and lateral superior olivary nuclei, the lateral lemniscal nucleus and the inferior colliculus of the rhesus monkey. Additional sections were used for receptor autoradiography to visualize the patterns of GABAA and GABAB receptor distribution. The Kv3.1b protein and perineuronal nets [visualized as Wisteria floribunda agglutinin (WFA) binding] were revealed, showing corresponding region-specific patterns of distribution. There was a gradient of labelled perineuronal nets which corresponded to that seen for the intensity of Kv3.1b expression. In the cochlear nucleus intensely and faintly stained perineuronal nets were intermingled, whereas in the medial nucleus of the trapezoid body the pattern changed to intensely stained perineuronal nets in the medial part and weakly labelled nets in its lateral part. In the inferior colliculus, intensely labelled perineuronal nets were arranged in clusters and faintly labelled nets were arranged in sheets. Using receptor autoradiography, GABAB receptor expression in the anterior ventral cochlear nucleus was revealed. The medial part of the medial nucleus of the trapezoid body showed a high number of GABAA binding sites whereas the lateral part exhibited more binding sites for GABAB. In the inferior colliculus, we found moderate GABAB receptor expression. In conclusion, intensely WFA-labelled structures are those known to be functionally involved in high-frequency processing.

GABAB Receptor Association with the PDZ Scaffold Mupp1 Alters Receptor Stability and Function

gamma-Aminobutyric acid, type B (GABA(B)) receptors are heterodimeric G protein-coupled receptors that mediate slow inhibitory synaptic transmission in the central nervous system. To identify novel interacting partners that might regulate GABA(B) receptor (GABA(B)R) functionality, we screened the GABA(B)R2 carboxyl terminus against a recently created proteomic array of 96 distinct PDZ (PSD-95/Dlg/ZO-1 homology) domains. The screen identified three specific PDZ domains that exhibit interactions with GABA(B)R2: Mupp1 PDZ13, PAPIN PDZ1, and Erbin PDZ. Biochemical analysis confirmed that full-length Mupp1 and PAPIN interact with GABA(B)R2 in cells. Disruption of the GABA(B)R2 interaction with PDZ scaffolds by a point mutation to the carboxyl terminus of the receptor dramatically decreased receptor stability and attenuated the duration of GABA(B) receptor signaling. The effects of mutating the GABA(B)R2 carboxyl terminus on receptor stability and signaling were mimicked by small interference RNA knockdown of endogenous Mupp1. These findings reveal that GABA(B) receptor stability and signaling can be modulated via GABA(B)R2 interactions with the PDZ scaffold protein Mupp1, which may contribute to cell-specific regulation of GABA(B) receptors in the central nervous system.

Interaction between Ang II and GABA systems in NTS: Central resetting of blood pressure regulation

There is little information concerning the interactions between Ang II and GABA systems in the NTS. Our previous studies, using gene microarrays, demonstrated that the GABA-B receptor (GBR) expression was increased following treatment of cultured NTS neurons with Ang II. In the present study, we examined the effects of Ang II on GBR expression in the NTS of SD rats. Ang II or saline control was infused subcutaneously for 2 weeks with osmotic minipumps. Ang II infusion induced a sustained increase in mean BP. GBR mRNA levels in NTS brain tissue were detected with real-time PCR showing GBR1 mRNA levels in the NTS were significantly increased in Ang II-infused rats. To rule out the possibility that the alterations of GBR expression observed in the above experiment is a result of the elevated BP, we examined the effect of Ang II on GBR expression in neurons cultured from NTS. Ang II treatment (100 nM, 5 hrs) induced a 2-fold increase in GBR mRNA levels. In addition, Superfusion of baclofen decreased the neuronal firing rate by 39% and 63% respectively in control and Ang II-treated neurons. In summary, these experiments indicate that Ang II stimulates GBR expression, consequently enhances GABAergic neurotransmission in the NTS. This enhanced GABAergic neurotransmission may lead to dampening of baroreflexes and consequently central resetting of long-term blood pressure regulation to a higher level in Ang II-related hypertension. Supported by AHA grant 0635050N

Coordinated action of NSF and PKC regulates GABAB receptor signaling efficacy

The obligatory heterodimerization of the GABAB receptor (GBR) raises fundamental questions about molecular mechanisms controlling its signaling efficacy. Here, we show that NEM sensitive fusion (NSF) protein interacts directly with the GBR heterodimer both in rat brain synaptosomes and in CHO cells, forming a ternary complex that can be regulated by agonist stimulation. Inhibition of NSF binding with a peptide derived from GBR2 (TAT-Pep-27) did not affect basal signaling activity but almost completely abolished agonist-promoted GBR desensitization in both CHO cells and hippocampal slices. Taken with the role of PKC in the desensitization process, our observation that TAT-Pep-27 prevented both agonist-promoted recruitment of PKC and receptor phosphorylation suggests that NSF is a priming factor required for GBR desensitization. Given that GBR desensitization does not involve receptor internalization, the NSF/PKC coordinated action revealed herein suggests that NSF can regulate GPCR signalling efficacy independently of its role in membrane trafficking. The functional interaction between three bona fide regulators of neurotransmitter release, such as GBR, NSF and PKC, could shed new light on the modulation of presynaptic GBR action.

Endocytosis of GABAB receptors modulates membrane excitability in the single-celled organism Paramecium

GABAB receptors modulate swimming behavior in Paramecium by inhibiting dihydropyridine-sensitive Ca2+ channels via G-proteins. Prolonged occupancy of GABAB receptors by baclofen results in a decrease in GABAB receptor functions. Since changes in the number of cell-surface GABAA receptors have been postulated to be of importance in modulating inhibitory synaptic transmission in neurons, we have studied the cell-surface expression and maintenance of GABAB receptors in P. primaurelia. In this study, we use immunostaining in electron and confocal microscopy to demonstrate that constitutive internalization of GABAB receptors in P. primaurelia is mediated by clathrin-dependent and -independent endocytosis. Indeed, GABAB receptors colocalize with the adaptin complex AP2, which is implicated in the selective recruitment of integral membrane proteins to clathrin-coated vesicles, and with caveolin 1, which is associated with uncoated membrane invaginations. Furthermore, when endocytosis is blocked with hypertonic medium, cytosol acidification, filipin or with a peptide that disrupts the association between amphiphysin and dynamin, the effect of baclofen on swimming is increased. These results suggest that GABAB receptor endocytosis into clathrin-coated and -uncoated vesicles represents an important mechanism in the modulation of swimming behavior in Paramecium.

Inhibition of nociceptive responses of spinal cord neurones during hypertension involves the spinal GABAergic system and a pain modulatory center located at the caudal ventrolateral medulla

The mechanisms of hypertension-induced hypoalgesia were studied in a model of hypertension induced by adenosine receptors blockade with the non-selective antagonist 1,3-dipropyl-8-sulfophenylxanthine (DPSPX) during 7 days. Based on the positive correlation between pain thresholds and noxious-evoked expression of the c-fos protooncogene in spinal cord neurones, we used this marker of nociceptive activation of spinal neurones to evaluate the involvement of the spinal GABAergic system and the caudal ventrolateral medulla (VLM), an important inhibitory component of the supraspinal endogenous pain modulatory system. In DPSPX-treated animals, a 20% increase in blood pressure was achieved along with a decrease in Fos expression in the superficial (laminae I-II) and deep (laminae III-VII) dorsal horn. In these animals, lower percentages of neurones labeled for GABAB receptors that expressed Fos were obtained in the superficial dorsal horn. Lesioning the VLMlat with quinolinic acid prevented the decrease in Fos expression at the spinal cord of DPSPX-hypertensive rats whereas in normotensive animals, no changes in Fos expression were detected. The present results support previous findings that hypertension is associated with a decrease of nociceptive activation of spinal cord neurones, through descending inhibition exerted by the VLMlat. This study further shows that during hypertension a decrease in the expression of GABAB receptors in nociceptive spinal neurones occurs, probably due to changes in the local GABAergic inhibitory system.

Spinal nerve ligation does not alter the expression or function of GABAB receptors in spinal cord and dorsal root ganglia of the rat

Loss of GABA-mediated inhibition in the spinal cord is thought to mediate allodynia and spontaneous pain after nerve injury. Despite extensive investigation of GABA itself, relatively little is known about how nerve injury alters the receptors at which GABA acts. This study examined levels of GABAB receptor protein in the spinal cord dorsal horn, and in the L4 and L5 (lumbar designations) dorsal root ganglia one to 18 weeks after L5 spinal nerve ligation. Mechanical allodynia was maximal by 1 week and persisted at blunted levels for at least 18 weeks after injury. Spontaneous pain behaviors were evident for 6 weeks. Western blotting of dorsal horn detected two isoforms of the GABAB(1) subunit and a single GABAB(2) subunit. High levels of GABAB(1a) and low levels of GABAB(1b) protein were present in the dorsal root ganglia. However, GABAB(2) protein was not detected in the dorsal root ganglia, consistent with the proposed existence of an atypical receptor composed of GABAB(1) homodimers. The levels of GABAB(1a), GABAB(1b), and GABAB(2) protein in the ipsilateral dorsal horn were unchanged at any time after injury. Immunohistochemical staining also did not detect a change in GABAB(1) or GABAB(2) subunits in dorsal horn segments having a robust loss of isolectin B4 staining. The levels of GABAB(1a) protein were also unchanged in the L4 or L5 dorsal root ganglia at any time after spinal nerve ligation. Levels of GABAB(2) remained undetectable. Finally, baclofen-stimulated binding of guanosine-5′-(γ-O-thio)triphosphate in dorsal horn did not differ between sham and ligated rats. Collectively, these results argue that a loss of GABAB receptor-mediated inhibition, particularly of central terminals of primary afferents, is unlikely to mediate the development or maintenance of allodynia or spontaneous pain behaviors after spinal nerve injury.

Subtype-selective Interaction with the Transcription Factor CCAAT/Enhancer-binding Protein (C/EBP) Homologous Protein (CHOP) Regulates Cell Surface Expression of GABAB Receptors

The metabotropic gamma-aminobutyric acid, type B (GABA(B)) receptors mediate the slow component of GABAergic transmission in the brain. Functional GABA(B) receptors are heterodimers of the two subunits GABA(B1) and GABA(B2), of which GABA(B1) exists in two main isoforms, GABA(B1a) and GABA(B1b). The significance of the structural heterogeneity of GABA(B) receptors, the mechanism leading to their differential targeting in neurons as well as the regulation of cell surface numbers of GABA(B) receptors, is poorly understood. To gain insights into these processes, we searched for proteins interacting with the C-terminal domain of GABA(B2). Here, we showed that the transcription factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) directly interacts with GABA(B) receptors in a subtype-selective manner to regulate cell surface expression of GABA(B1a)/GABA(B2) receptors upon co-expression in HEK 293 cells. The interaction of CHOP with GABA(B1a)/GABA(B2) receptors resulted in their intracellular accumulation and in a reduced number of cell surface receptors. This regulation required the interaction of CHOP via two distinct domains with the heterodimeric receptor; its C-terminal leucine zipper associates with the leucine zipper present in the C-terminal domain of GABA(B2), and its N-terminal domain associates with an as yet unidentified site on GABA(B1a). In conclusion, the data indicated a subtype-selective regulation of cell surface receptors by interaction with the transcription factor CHOP.

Expression of GABAA and GABAB receptors in rat growth plate chondrocytes: Activation of the GABA receptors promotes proliferation of mouse chondrogenic ATDC5 cells

Our previous study showed the local production of gamma-aminobutyrate (GABA) in hypertrophic-zone chondrocytes of the rat tibial growth plate, an important long bone growth site. The aim of this study was to identify the presence of GABA receptors in growth plate chondrocytes by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Chondrocytes expressed both GABA(A) and GABA(B) receptor subunit mRNAs as well as the corresponding proteins necessary for the assembly of functional receptors. The GABA(A) receptor subunits detected included alpha1-alpha4, alpha6, beta1-beta3, and delta, and both R1 and R2 subunits of GABA(B) receptors were detected. All receptor subunits were expressed in chondrocytes of the proliferative and hypertrophic zones. These results suggest that GABA is an autocrine/paracrine factor that regulates the physiological state of the growth plate. Subsequent studies with the mouse chondrogenic cell line ATDC5 showed the presence of mRNAs and the corresponding proteins for GABA(A) receptor alpha1, beta2, and beta3 subunits and GABA(B) receptor R1 and R2 subunits. GABA, muscimol (a GABA(A) receptor agonist), and baclofen (a GABA(B) receptor agonist) increased 5-bromodeoxyuridine (BrdU) incorporation into ATDC5 cells. The effect of muscimol was blocked by bicuculline (a GABA(A) receptor antagonist), and the effect of baclofen was blocked by CGP 35348 (a GABA(B) receptor antagonist). These results suggest that GABA contributes to the ATDC5 cell proliferation via GABA(A) and GABA(B) receptors and these mechanisms may be involved in cartilaginous cell growth.

Expression of GABAB receptor in the avian auditory brainstem: Ontogeny, afferent deprivation, and ultrastructure

Nucleus magnocellularis (NM), nucleus angularis (NA), and nucleus laminaris (NL), second- and third-order auditory neurons in the avian brainstem, receive GABAergic input primarily from the superior olivary nucleus (SON). Previous studies have demonstrated that both GABA(A) and GABA(B) receptors (GABA(B)Rs) influence physiological properties of NM neurons. We characterized the distribution of GABA(B)R expression in these nuclei during development and after deafferentation of the excitatory auditory nerve (nVIII) inputs. We used a polyclonal antibody raised against rat GABA(B)Rs in the auditory brainstem during developmental periods that are thought to precede and include synaptogenesis of GABAergic inputs. As early as embryonic day (E)14, dense labeling is observed in NA, NM, NL, and SON. At earlier ages immunoreactivity is present in somas as diffuse staining with few puncta. By E21, when the structure and function of the auditory nuclei are known to be mature, GABA(B) immunoreactivity is characterized by dense punctate labeling in NM, NL, and a subset of NA neurons, but label is sparse in the SON. Removal of the cochlea and nVIII neurons in posthatch chicks resulted in only a small decrease in immunoreactivity after survival times of 14 or 28 days, suggesting that a major proportion of GABA(B)Rs may be expressed postsynaptically or on GABAergic terminals. We confirmed this interpretation with immunogold TEM, where expression at postsynaptic membrane sites is clearly observed. The characterization of GABA(B)R distribution enriches our understanding of the full complement of inhibitory influences on central auditory processing in this well-studied neuronal circuit.

CAMP response element-binding protein, activating transcription factor-4, and upstream stimulatory factor differentially control hippocampal GABABR1a and GABABR1b subunit gene expression through alternative promoters.

Expression of metabotropic GABA(B) receptors is essential for slow inhibitory synaptic transmission in the CNS, and disruption of GABA(B) receptor-mediated responses has been associated with several disorders, including neuropathic pain and epilepsy. The location of GABA(B) receptors in neurons determines their specific role in synaptic transmission, and it is believed that sorting of subunit isoforms, GABA(B)R1a and GABA(B)R1b, to presynaptic or postsynaptic membranes helps to determine this role. GABA(B)R1a and GABA(B)R1b are thought to arise by alternative splicing of heteronuclear RNA. We now demonstrate that alternative promoters, rather than alternative splicing, produce GABA(B)R1a and GABA(B)R1b isoforms. Our data further show that subunit gene expression in hippocampal neurons is mediated by the cAMP response element-binding protein (CREB) by binding to unique cAMP response elements in the alternative promoter regions. Double-stranded oligonucleotide decoys selectively alter levels of endogenous GABA(B)R1a and GABA(B)R1b in primary hippocampal neurons, and CREB knock-out mice show changes in levels of GABA(B)R1a and GABA(B)R1b transcripts, consistent with decoy competition experiments. These results demonstrate a critical role of CREB in transcriptional mechanisms that control GABA(B)R1 subunit levels in vivo. In addition, the CREB-related factor activating transcription factor-4 (ATF4) has been shown to interact directly with GABA(B)R1 in neurons, and we show that ATF4 differentially regulates GABA(B)R1a and GABA(B)R1b promoter activity. These results, together with our finding that the depolarization-sensitive upstream stimulatory factor (USF) binds to a composite CREB/ATF4/USF regulatory element only in the absence of CREB binding, indicate that selective control of alternative GABA(B)R1 promoters by CREB, ATF4, and USF may dynamically regulate expression of their gene products in the nervous system.

Activation of postsynaptic GABAB receptors modulate the firing activity of supraoptic oxytocin and vasopressin neurones: role of calcium channels.

Oxytocin and vasopressin release from neurohypophysial terminals is closely related to the firing activity of magnocellular neurones in the supraoptic (SON) and paraventricular nuclei. It is well established that activation of GABAA receptors potently inhibits the activity of SON neurones and, thus, hormone release. However, whether postsynaptic GABAB receptors are expressed in magnocellular neurones, and the role they play in controlling their firing activity, is still controversial. In the present work, we combined immunohistochemical and electrophysiological techniques to determine whether activation of GABAB receptors in identified oxytocin and vasopressin neurones modulates their firing activity. Patch-clamp recordings from SON neurones were obtained either in the slice preparation or from acutely dissociated neurones. Activation of GABAB receptors with the selective agonist baclofen (10 micro m) inhibited voltage-gated Ca2+ currents, reduced the duration of individual action potentials, as well as the magnitude of the hyperpolarizing after-potential. SON firing activity was reduced by baclofen, and effect that was accompanied by a small membrane hyperpolarization. The inhibition of firing discharge persisted in the presence of synaptic blockade media, and was also observed in acutely dissociated SON neurones. Finally, GABAB-mediated modulation of firing activity was largely blocked by the Ca2+ channel blocker Co2+ (2 mm). In general, baclofen modulatory actions were significantly larger, or observed more predominantly, in vasopressin neurones. In summary, these results support the expression of functional postsynaptic GABAB receptors in SON neurones, activation of which efficiently modulates neuronal excitability, in a Ca2+- and cell-type dependent manner.

GABAB(1) mRNA expression in hippocampal sclerosis associated with human temporal lobe epilepsy

GABAB receptors act to inhibit neurotransmitter release from presynaptic terminals, and mediate the late inhibitory postsynaptic potential. Studies of GABAB receptor function in rodent models of temporal lobe epilepsy (TLE) suggest that GABAB receptor expression and/or function may be perturbed. GABAB(1) mRNA levels were investigated in 10 hippocampal resection samples obtained at surgery from intractable hippocampal sclerosis (HS) associated TLE patients and five neurologically normal post-mortem (PM) control samples. In situ hybridisation with a 35S-dATP-labelled oligonucleotide was carried out to measure mRNA levels, along with three-dimensional cell counting, for assessment of neuronal density in hippocampal subregions. GABAB(1) mRNA was significantly up-regulated in the subiculum of HS samples as compared with PM controls. When adjusted for the characteristic neuronal density changes observed in HS, GABAB(1) mRNA was significantly up-regulated in CA1, hilus and dentate gyrus granule cell layer of HS samples as compared with PM controls. The possibility of increased GABAB(1) expression suggests that changes in GABAB receptor mechanisms may be involved in the pathogenesis of human HS-associated TLE.

Role of GABAB receptors in intracellular Ca2+ homeostasis and possible interaction between GABAA and GABAB receptors in regulation of transmitter release in cerebellar granule neurons

The expression of GABAB receptors in cultured mouse cerebellar granule cells was investigated in binding experiments using [3H](S,R)-baclofen as well as in functional assessment of the ability of (R)-baclofen to interact with depolarization (15-40 mM KCl) coupled changes in intracellular Ca2+ homeostasis and neurotransmitter release. In the latter case a possible functional coupling between GABAA and GABAB receptors was investigated. The binding studies showed that the granule cells express specific binding sites for (R)-baclofen. The number of binding sites could be increased by exposure of the cells to the GABAA receptor agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol) during the culture period. Pretreatment of the neurons with pertussis toxin showed that the GABAB receptors are coupled to G-proteins. This coupling was, however, less pronounced when the cells had been cultured in the presence of THIP. When 45Ca2+ uptake was measured or the intracellular Ca2+ concentration ([Ca2+]i) determined using the fluorescent Ca2+ chelator Fluo-3 it could be demonstrated that culturing the neurons in THIP influences intracellular Ca2+ homeostasis. Moreover, this homeostasis was found to be functionally coupled to the GABAB receptors as (R)-baclofen inhibited depolarization-induced increases in 45Ca2+ uptake and [Ca2+]i. (R)-Baclofen also inhibited K(+)-induced transmitter release from the neurons as monitored by the use of [3H]D-aspartate which labels the neurotransmitter pool of glutamate. Using the selective GABAA receptor agonist isoguvacine it could be demonstrated that the GABAB receptors are functionally coupled to GABAA receptors in the neurons leading to a disinhibitory action of GABAB receptor agonists.

GABA uptake into isolated retinal Müller glial cells of the guinea-pig detected electrophysiologically.

In order to investigate GABA uptake into Müller glial cells electrophysiologically, we used the whole-cell voltage-clamp technique. Since Müller cells were insensitive to application of muscimol and baclofen, the expression of GABAA and GABAB receptors can be excluded. Therefore, the observed GABA current must be due to an electrogenic GABA transporter. This transporter is driven by the transmembrane Na+ gradient, as replacement of the extracellular Na+ by choline inhibits the GABA current. Currents evoked by cis-4-aminocrotonic acid, a GABAC specific agonist, are also inhibited by replacement of extracellular Na+, indicating that this compound is a substrate for the uptake. The competitive GABA uptake blockers beta-alanine and nipecotic acid are substrates of the transporter as well, and produce 42% and 65% of the currents elicited by the same GABA concentration, respectively. Affinity of the transporter for GABA is high, the Km value being 5 microM at -80 mV.

Expression of the GABAB receptor in Xenopus oocytes and desensitization by activation of protein kinase C.

The γ-aminobutyric acid (GABA) receptors have been classified into two subtypes, termed GABAA and GABAB receptors, in the basis of their pharmacological properties (Bowery et al., 1984; 1989; Bormann, 1988). The GABAA receptor with its integrated Cl-channel is now well characterized. Studies revealed the amino acid sequence of the GABAA receptor (Schofield et al., 1987; Levitan et al., 1988). Stimulation of the GABAB receptor has been shown to induce two membrane effects, namely reduction in Ca2+ conductance and increase in K+ conductance (Bormann, 1988; Bowery, 1989), however, the GABAB receptor has not been purified. The expression of GABAB receptor in the Xenopus oocytes may provide information on molecular mechanisms of GABAB receptor-mediated responses.KeywordsGABAA ReceptorXenopus OocytePhorbol EsterPertussis ToxinGABAB ReceptorThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.