GABA<Subscript>B</Subscript> Receptors in Neuroendocrine Regulation

Gamma-amino butyric acid (GABA), in addition to being a metabolic intermediate and the main inhibitory neurotransmitter in the synaptic cleft, is postulated as a neurohormone, a paracrine signaling molecule, and a trophic factor. It acts through pre- and post-synaptic receptors, named GABA_A and GABA_C (ionotropic receptors) and GABA_B (metabotropic receptor). Here we reviewed the participation of GABA_B receptors in the regulation of the hypothalamic-pituitary-gonadal axis, using physiological, biochemical, and pharmacological approaches in rats, as well as in GABA_B1 knock-out mice, that lack functional GABA_B receptors. Our general conclusion indicates that GABA_B receptors participate in the regulation of pituitary hormone secretion acting both in the central nervous system and directly on the gland. PRL and gonadotropin axes are affected by GABA_B receptor activation, as demonstrated in the rat and also in the GABA_B1 knock-out mouse. In addition, hypothalamic and pituitary GABA_B receptor expression is modulated by steroid hormones. GABA participation in the brain control of pituitary secretion through GABA_B receptors depends on physiological conditions, being age and sex critical factors. These results indicate that patients receiving GABA_B agonists/antagonists should be monitored for possible endocrine side effects.     

Neurochemicals for the Investigation of GABA<Subscript>C</Subscript> Receptors

GABA_C receptors are being investigated for their role in many aspects of nervous system function including memory, myopia, pain and sleep. There is evidence for functional GABA_C receptors in many tissues such as retina, hippocampus, spinal cord, superior colliculus, pituitary and the gut. This review describes a variety of neurochemicals that have been shown to be useful in distinguishing GABA_C receptors from other receptors for the major inhibitory neurotransmitter GABA. Some selective agonists (including (+)-CAMP and 5-methyl-IAA), competitive antagonists (such as TPMPA, (±)-cis-3-ACPBPA and aza-THIP), positive (allopregnanolone) and negative modulators (epipregnanolone, loreclezole) are described. Neurochemicals that may assist in distinguishing between homomeric ρ1 and ρ2 GABA_C receptors (2-methyl-TACA and cyclothiazide) are also covered. Given their less widespread distribution, lower abundance and relative structural simplicity compared to GABA_A and GABA_B receptors, GABA_C receptors are attractive drug targets.     

Pharmacological characterization of GABA<Subscript>A</Subscript> receptors in taurine-fed mice

Background

Taurine is one of the most abundant free amino acids especially in excitable tissues, with wide physiological actions. Chronic supplementation of taurine in drinking water to mice increases brain excitability mainly through alterations in the inhibitory GABAergic system. These changes include elevated expression level of glutamic acid decarboxylase (GAD) and increased levels of GABA. Additionally we reported that GABA_A receptors were down regulated with chronic administration of taurine. Here, we investigated pharmacologically the functional significance of decreased / or change in subunit composition of the GABA_A receptors by determining the threshold for picrotoxin-induced seizures. Picrotoxin, an antagonist of GABA_A receptors that blocks the channels while in the open state, binds within the pore of the channel between the β2 and β3 subunits. These are the same subunits to which GABA and presumably taurine binds.

Methods

Two-month-old male FVB/NJ mice were subcutaneously injected with picrotoxin (5 mg kg^-1) and observed for a) latency until seizures began, b) duration of seizures, and c) frequency of seizures. For taurine treatment, mice were either fed taurine in drinking water (0.05%) or injected (43 mg/kg) 15 min prior to picrotoxin injection.

Results

We found that taurine-fed mice are resistant to picrotoxin-induced seizures when compared to age-matched controls, as measured by increased latency to seizure, decreased occurrence of seizures and reduced mortality rate. In the picrotoxin-treated animals, latency and duration were significantly shorter than in taurine-treated animas. Injection of taurine 15 min before picrotoxin significantly delayed seizure onset, as did chronic administration of taurine in the diet. Further, taurine treatment significantly increased survival rates compared to the picrotoxin-treated mice.

Conclusions

We suggest that the elevated threshold for picrotoxin-induced seizures in taurine-fed mice is due to the reduced binding sites available for picrotoxin binding due to the reduced expression of the beta subunits of the GABA_A receptor. The delayed effects of picrotoxin after acute taurine injection may indicate that the two molecules are competing for the same binding site on the GABA_A receptor. Thus, taurine-fed mice have a functional alteration in the GABAergic system. These include: increased GAD expression, increased GABA levels, and changes in subunit composition of the GABA_A receptors. Such a finding is relevant in conditions where agonists of GABA_A receptors, such as anesthetics, are administered.

     

Homology modeling of the transmembrane domain of the GABA<Subscript>A</Subscript> receptor

The permeability of ion channels for ions and substances that bind inside the pore depends on the cross-sectional area of the pore. We have constructed models of the closed, open, and desensitized α₁β₂γ₂ GABA_A receptor on the basis of known structures of both prokaryotic and eukaryotic ligand-gated channels. We employed Monte Carlo energy minimization to optimize the model structures. We have found significant pore constrictions, whose diameter depends on the functional state of the receptor in the cytoplasmic, middle, and extracellular parts of the pore-forming M2 segments. It is known that the constrictions in the middle (the 9' ring of residues) and cytoplasmic (the 2' ring of residues) parts of the M2 helices form the activation and desensitization gates of the GABA_A receptor. Our computations predict that the constriction in the extracellular part of the M2 helices (the 20' ring of residues) may also serve as a gate in the GABA_A receptor, whose physiological role is still unclear. Our results imply that the structures of a number of prokaryotic and eukaryotic ligand-gated channels that have been found in bacteria and lower organisms can be used for homology modeling of the pore region of the human GABA_A receptor.     

Computational modeling of GABA<Subscript>A</Subscript> receptor-mediated paired-pulse inhibition in the dentate gyrus

Paired-pulse inhibition (PPI) of the population spike observed in extracellular field recordings is widely used as a read-out of hippocampal network inhibition. PPI reflects GABA_A receptor-mediated inhibition of principal neurons through local interneurons. However, because of its polysynaptic nature, it is difficult to assign PPI changes to precise synaptic mechanisms. Here we used a detailed network model of the dentate gyrus to simulate PPI of granule cell action potentials and analyze its network properties. Our computational analysis indicates that PPI results mainly from a combination of perisomatic feed-forward and feedback inhibition of granule cells by basket cells. Feed-forward inhibition mediated by basket cells appeared to be the most significant source of PPI. Our simulations suggest that PPI depends more on somatic than on dendritic inhibition of granule cells. Furthermore, PPI was modulated by changes in GABA_A reversal potential (E_GABA) and by alterations in intrinsic excitability of granule cells. In summary, computer modeling provides a useful tool for determining the role of synaptic and intrinsic cellular mechanisms in paired-pulse field potential responses.     

Developmental expression of amphioxus GABA<Subscript>A</Subscript> receptor-associated protein-like 2 gene

A full-length amphioxus -aminobutyric acid type-A receptor-associated protein-like 2 (GABARAPL2) cDNA was isolated. Its sequence and developmental expression are first described in this paper. The phylogenetic analysis shows that the amphioxus GABARAPL2 and GABARAPL2 in vertebrates are highly homologous. The results of in situ hybridization show that the amphioxus GABARAPL2 gene is expressed in the neural tube, neurenteric canal, notochord, muscle and developing alimentary canal.Edited by P. SimpsonKailong Liang and Yushuang Lin contributed equally to the study     

Hyperthermia-Induced Seizures Modify the GABA<Subscript>A</Subscript> and Benzodiazepine Receptor Binding in Immature Rat Brain

Effects of hyperthermia-induced seizures (HS) on GABA_A and benzodiazepine (BDZ) receptor binding in immature rat brain were evaluated using in vitro autoradiography. HS were induced in 10-days-old rats by a regulated stream of moderately heated air directed 50 cm above the animals. Rats were killed 30 min, 24 h or 20 days after HS and their brains were used for in vitro autoradiography experiments to determine GABA_A and BDZ receptor binding. GABA_A binding was significantly enhanced in all brain areas evaluated 30 min after HS, an effect that endures 24 h and 20 days after seizures. Concerning BDZ receptor binding, a significant increase was detected in entorhinal and perirhinal cortices and decreased in basolateral amygdala 30 min following HS. One day after HS, animals demonstrated enhanced BDZ binding in the cingulate, frontal, posterior parietal, entorhinal, temporal and perirhinal cortices; striatum, accumbens, substantia nigra pars compacta and amygdala nuclei. Twenty days after HS enhanced BDZ binding was restricted in the cingulated, frontal, anterior and posterior parietal cortices, as well as in substantia nigra pars reticulata, whereas decreased values were found in accumbens nucleus and substantia nigra pars compacta. Our data indicate differential effects of HS in GABA_A and BDZ binding in immature brain. HS-induced GABA_A and BDZ changes are different from those previously described in experimental models of temporal lobe epilepsy in adult animals.     

Investigating the Putative Binding-mode of GABA and Diazepam within GABA<Subscript>A</Subscript> Receptor Using Molecular Modeling

The three-dimensional structure of the GABA A receptor that included the ligand/agonist binding site was constructed and validated by using molecular modeling technology. Moreover, the putative binding-mode of GABA and diazepam with GABAA receptor were investigated by means of docking studies. Based on an rmsd-tolerance of 1.0 angstroms, the docking of GABA to alpha1/beta2 interface resulted in three multi-member conformational clusters and model 2 was supported by homologous sequence alignment data and experimental evidence. On the other hand, the docking of diazepam to alpha1/gamma2 interface revealed five multi-member conformational clusters in the binding site and model 1 seemed to represent the correct orientation of diazepam in the binding site.     

Cross-talk between NMDA and GABA<Subscript>A</Subscript> receptors in cultured neurons of the rat inferior colliculus

Neuronal ion channels of different types often do not function independently but will inhibit or potentiate the activity of other types of channels, a process called cross-talk. The N-methyl-D-aspartate receptor (NMDA receptor) and the γ-aminobutyric acid type A receptor (GABA_A receptor) are important excitatory and inhibitory receptors in the central nervous system, respectively. Currently, cross-talk between the NMDA receptor and the GABA_A receptor, particularly in the central auditory system, is not well understood. In the present study, we investigated functional interactions between the NMDA receptor and the GABA_A receptor using whole-cell patch-clamp techniques in cultured neurons from the inferior colliculus, which is an important nucleus in the central auditory system. We found that the currents induced by aspartate at 100 μmol L⁻¹ were suppressed by the pre-perfusion of GABA at 100 μmol L⁻¹, indicating cross-inhibition of NMDA receptors by activation of GABA_A receptors. Moreover, we found that the currents induced by GABA at 100 μmol L⁻¹ (I _GABA) were not suppressed by the pre-perfusion of 100 μmol L⁻¹ aspartate, but those induced by GABA at 3 μmol L⁻¹ were suppressed, indicating concentration-dependent cross-inhibition of GABA_A receptors by activation of NMDA receptors. In addition, inhibition of IGABA by aspartate was not affected by blockade of voltage-dependent Ca²⁺ channels with CdCl₂ in a solution that contained Ca²⁺, however, CdCl₂ effectively attenuated the inhibition of I _GABA by aspartate when it was perfused in a solution that contained Ba²⁺ instead of Ca²⁺ or a solution that contained Ca²⁺ and 10 mmol L⁻¹ BAPTA, a membrane-permeable Ca²⁺ chelator, suggesting that this inhibition is mediated by Ca²⁺ influx through NMDA receptors, rather than voltage-dependent Ca²⁺ channels. Finally, KN-62, a potent inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), reduced the inhibition of I _GABA by aspartate, indicating the involvement of CaMKII in this cross-inhibition. Our study demonstrates a functional interaction between NMDA and GABA_A receptors in the inferior colliculus of rats. The presence of cross-talk between these receptors suggests that the mechanisms underlying information processing in the central auditory system may be more complex than previously believed.     

Decreased GABA<Subscript>A</Subscript> Receptor Function in the Brain Stem during Pancreatic Regeneration in Rats

Gamma amino butyric acid is a major inhibitory neurotransmitter in the central nervous system. In the present study we have investigated the alteration of GABA receptors in the brain stem of rats during pancreatic regeneration. Three groups of rats were used for the study: sham operated, 72 h and 7 days partially pancreatectomised. GABA was quantified by [³H]GABA receptor displacement method. GABA receptor kinetic parameters were studied by using the binding of [³H]GABA as ligand to the Triton X-100 treated membranes and displacement with unlabelled GABA. GABA_A receptor activity was studied by using the [³H]bicuculline and displacement with unlabelled bicuculline. GABA content significantly decreased (P < 0.001) in the brain stem during the regeneration of pancreas. The high affinity GABA receptor binding showed a significant decrease in B _max (P < 0.01) and K _d (P < 0.05) in 72 h and 7 days after partial pancreatectomy. [³H]bicuculline binding showed a significant decrease in B _max and K _d (P < 0.001) in 72 h pancreatectomised rats when compared with sham where as B _max and K _d reversed to near sham after 7 days of pancreatectomy. The results suggest that GABA through GABA receptors in brain stem has a regulatory role during active regeneration of pancreas which will have immense clinical significance in the treatment of diabetes.     

Role of GABA<Subscript>B</Subscript> receptor in the regulation of excitatory synaptic transmission in trigeminal motoneurons

The aim of the present study was to determine if excitatory synaptic transmission onto trigeminal motoneurons is subject to a presynaptic modulation by gamma-aminobutyric acid (GABA) via GABA(B) receptor in this system. Whole cell recordings were made from trigeminal motoneurons in longitudinal brain stem slices taken from 8-day-old rats. Monosynaptic excitatory postsynaptic potential (EPSP) activity was evoked by placing bipolar stainless steel electrodes dorsal-caudal to the trigeminal motor nucleus. Bath application of the GABA(B) receptor agonist, baclofen, produced a marked reduction in the mean amplitude and variance of evoked EPSPs and also increased the portion of transmission failures. It also produced a decrease in the frequency, but not in the mean amplitude, of spontaneous miniature EPSPs. Bath application of GABA(B) receptor antagonists 6-hydroxy-saclofen and CGP35348 increased both the amplitude and frequency of miniature EPSP activity. Taken together the above results suggest that the excitatory synaptic inputs onto trigeminal motoneurons are controlled by tonic presynaptic modulation by GABA(B) receptor.     

Plasticity of GABA<Subscript>A</Subscript> Receptors in Brains of Rats Treated with Chronic Intermittent Ethanol

The study of alcohol dependence mechanisms has been aided by work in rodents, where regimens of intermittent chronic administration with repeated episodes of intoxication and withdrawal can be coupled with controlled timing of in vitro studies and the possibility of relating them to behavior. The chronic intermittent ethanol (CIE) model in the rat has been found to be a good model of human alcohol dependence, showing persistent signs of withdrawal and self-administration. Studies in CIE rats suggest that plastic changes in GABA-mediated inhibition involving the GABA_A receptor system may be responsible for the behavioral alterations. Here we summarize a combination of evidence that the alcoholic rat CIE model demonstrates changes in GABA_A receptor subunit levels, in receptor localization, and in physiology and pharmacology, leading to alterations in behavior that contribute to the hyperexcitable alcohol withdrawal state (anxiety, insomnia, seizure susceptibility) and alcohol dependence. Special Issue dedicated to Dr. Simo S. Oja