Differential localization of GABAA receptor subunits in relation to rat striatopallidal and pallidopallidal synapses

As a central integrator of basal ganglia function, the external segment of the globus pallidus (GP) plays a critical role in the control of voluntary movement. The GP is composed of a network of inhibitory GABA-containing projection neurons which receive GABAergic input from axons of the striatum (Str) and local collaterals of GP neurons. Here, using electrophysiological techniques and immunofluorescent labeling we have investigated the differential cellular distribution of a1, a2 and a3 GABAA receptor subunits in relation to striatopallidal (Str-GP) and pallidopallidal (GP-GP) synapses. Electrophysiological investigations showed that zolpidem (100 nm; selective for the a1 subunit) increased the amplitude and the decay time of both Str-GP and GP-GP IPSCs, indicating the presence of the a1 subunits at both synapses. However, the application of drugs selective for the a2, a3 and a5 subunits (zolpidem at 400 nm, L-838,417 and TP003) revealed differential effects on amplitude and decay time of IPSCs, suggesting the nonuniform distribution of non-a1 subunits. Immunofluorescence revealed widespread distribution of the a1 subunit at both soma and dendrites, while double- and triple-immunofluorescent labeling for parvalbumin, enkephalin, gephyrin and the ?2 subunit indicated strong immunoreactivity for GABAAa3 subunits in perisomatic synapses, a region mainly targeted by local axon collaterals. In contrast, immunoreactivity for synaptic GABAAa2 subunits was observed in dendritic compartments where striatal synapses are preferentially located. Due to the kinetic properties which each GABAAa subunit confers, this distribution is likely to contribute differentially to both physiological and pathological patterns of activity.

Identification of GABA receptors in chick cornea

Purpose: The cornea has an important role in vision, is highly innervated and many neurotransmitter receptors are present, e.g., muscarine, melatonin, and dopamine receptors. γ-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the retina and central nervous system, but it is unknown whether GABA receptors are present in cornea. The aim of this study was to determine if GABA receptors are located in chick cornea. Methods: Corneal tissues were collected from 25, 12-day-old chicks. Real time PCR, western blot, and immunohistochemistry were used to determine whether alpha1 GABAA, GABAB, and rho1 GABAC receptors were expressed and located in chick cornea. Results: Corneal tissue was positive for alpha1 GABAA and rho1 GABAC receptor mRNA (PCR) and protein (western blot) expression but was negative for GABAB receptor mRNA and protein. Alpha1 GABAA and rho1 GABAC receptor protein labeling was observed in the corneal epithelium using immunohistochemistry. Conclusions: These investigations clearly show that chick cornea possesses alpha1 GABAA, and rho1 GABAC receptors, but not GABAB receptors. The purpose of the alpha1 GABAA and rho1 GABAC receptors in cornea is a fascinating unexplored question.

Investigation of the role of the GABRG2 gene variant in migraine

Migraine is the most common neurological disorder worldwide affecting about 12% of the worldwide population. This disorder has been classed into two main types of migraine—with and without aura. While a number of factors can influence the onset of migraine, a major factor is that of genetics. The GABAA gene encodes for the GABAA receptor. Along with other receptors, the GABAA receptor is involved in the mediation of neuronal activities. In this study, a GABRG2 gene (GABAA receptor gamma-2-subunit) SNP (rs211037) was genotyped on a migraine case–control population of 546 (273 affected and an equal number of healthy) individuals. Using specifically designed primers, a high resolution melt (HRM) assay was carried out in the genotyping process. After genotyping, results were compared in the case and control populations. Analysis of results showed no significant differences in the allele frequencies between case and control populations. Similarly no differences were detected for subtypes or for a specific gender of migraine (p > 0.05). Although this gene has been previously found to be involved in febrile seizures and there is some co-morbidity between epilepsy and migraine, we decided to investigate this marker for involvement in migraine. The results did not support a role for the tested GABRG2 variant in migraine.

The effects and interactions of GABAergic and dopaminergic agents in the prevention of form deprivation myopia by brief periods of normal vision

Intravitreal injections of GABA antagonists, dopamine agonists and brief periods of normal vision have been shown separately to inhibit form-deprivation myopia (FDM). Our study had three aims: (i) establish whether GABAergic agents modify the myopia protective effect of normal vision, (ii) investigate the receptor sub-type specificity of any observed effect, and (iii) consider an interaction with the dopamine (DA) system. Prior to the period of normal vision GABAergic agents were applied either (i) individually, (ii) in combination with other GABAergic agents (an agonist with an antagonist), or (iii) in combination with DA agonists and antagonists. Water injections were given to groups not receiving drug treatments so that all experimental eyes received intravitreal injections. As shown previously, constant form-deprivation resulted in high myopia and when diffusers were removed for 2 h per day the period of normal vision greatly reduced the FDM that developed. GABA agonists inhibited the protective effect of normal vision whereas antagonists had the opposite effect. GABAA/C agonists and D2 DA antagonists when used in combination were additive in suppressing the protective effect of normal vision. A D2 DA agonist restored some of the protective effect of normal vision that was inhibited by a GABA agonist (muscimol). The protective effect of normal vision against form-deprivation is modifiable by both the GABAergic and DAergic pathways.

Identification of GABA receptors in chick retinal pigment epithelium

Purpose: The retinal pigment epithelium (RPE) is a multifunctional, monolayer of cells located between the neural retina and the choroicapillaris. γ-Aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the retina and GABA receptors are known to be present in chick retina, sclera and cornea. There is a report of genes involved in GABA receptor signaling being expressed in human RPE, however, whether GABA receptors are present in chick RPE is unknown. Methods: Real time PCR and western blot were used to determine the expression of GABA receptors (alpha1 GABAA, GABABR2, and rho1 GABAC receptors) in isolated chicken RPE. Immunofluorescence using antibodies against one of the GABA receptor sub-types was used to determine receptor localization. Results: Both real-time PCR and western blot demonstrated that alpha1 GABAA, GABABR2 and rho1 GABAC receptors were expressed in isolated chick RPE. Immunofluorescence further demonstrated that GABA receptors were localized to the cell membrane and plasma of RPE cells. Conclusions: Alpha1 GABAA, GABABR2 and rho1 GABAC receptors were expressed in chick RPE. The purpose of the GABA receptors within the RPE remains to be explored.

Expression of functional GABAc receptors in the brain

γ-aminobutyric acid (GABA) is the main inhibitory transmitter in the nervous system and acts via three distinct receptor classes: A, B, and C. GABAC receptors are ionotropic receptors comprising ρ subunits. In this work, we aimed to elucidate the expression of ρ subunits in the postnatal brain, the characteristics of ρ2 homo-oligomeric receptors, and the function of GABAC receptors in the hippocampus. In situ hybridization on rat brain slices showed ρ2 mRNA expression from the newborn in the superficial grey layer of the superior colliculus, from the first postnatal week in the hippocampal CA1 region and the pretectal nucleus of the optic tract, and in the adult dorsal lateral geniculate nucleus. Quantitative RT-PCR revealed expression of all three ρ subunits in the hippocampus and superior colliculus from the first postnatal day. In the hippocampus, ρ2 mRNA expression clearly dominated over ρ1 and ρ3. GABAC receptor protein expression was confirmed in the adult hippocampus, superior colliculus, and dorsal lateral geniculate nucleus by immunohistochemistry. From the selective distribution of ρ subunits, GABAC receptors may be hypothesized to be specifically involved in aspects of visual image motion processing in the rat brain. Although previous data had indicated a much higher expression level for ρ2 subunit transcripts than for ρ1 or ρ3 in the brain, previous work done on Xenopus oocytes had suggested that rat ρ2 subunits do not form functional homo-oligomeric GABAC receptors but need ρ1 or ρ3 subunits to form hetero-oligomers. Our results demonstrated, for the first time, that HEK 293 cells transfected with ρ2 cDNA displayed currents in whole-cell patch-clamp recordings. Homomeric rat ρ2 receptors had a decreased sensitivity to, but a high affinity for picrotoxin and a marked sensitivity to the GABAC receptor agonist CACA. Our results suggest that ρ2 subunits may contribute to brain function, also in areas not expressing other ρ subunits. Using extracellular electrophysiological recordings, we aimed to study the effects of the GABAC receptor agonists and antagonists on responses of the hippocampal neurons to electrical stimulation. Activation of GABAC receptors with CACA suppressed postsynaptic excitability and the GABAC receptor antagonist TPMPA inhibited the effects of CACA. Next, we aimed to display the activation of the GABAC receptors by synaptically released GABA using intracellular recordings. GABA-mediated long-lasting depolarizing responses evoked by high-frequency stimulation were prolonged by TPMPA. For weaker stimulation, the effect of TPMPA was enhanced after GABA uptake was inhibited. Our data demonstrate that GABAC receptors can be activated by endogenous synaptic transmitter release following strong stimulation or under conditions of reduced GABA uptake. The lack of GABAC receptor activation by less intensive stimulation under control conditions suggests that these receptors are extrasynaptic and activated via spillover of synaptically released GABA. Taken together with the restricted expression pattern of GABAC receptors in the brain and their distinctive pharmacological and biophysical properties, our findings supporting extrasynaptic localization of these receptors raise interesting possibilities for novel pharmacological therapies in the treatment of, for example, epilepsy and sleep disorders.

Cellular and network mechanisms generating spontaneous population events in the immature rat hippocampus

Distinct endogenous network events, generated independently of sensory input, are a general feature of various structures of the immature central nervous system. In the immature hippocampus, these type of events are seen as "giant depolarizing potentials" (GDPs) in intracellular recordings in vitro. GABA, the major inhibitory neurotransmitter of the adult brain, has a depolarizing action in immature neurons, and GDPs have been proposed to be driven by GABAergic transmission. Moreover, GDPs have been thought to reflect an early pattern that disappears during development in parallel with the maturation of hyperpolarizing GABAergic inhibition. However, the adult hippocampus in vivo also generates endogenous network events known as sharp (positive) waves (SPWs), which reflect synchronous discharges of CA3 pyramidal neurons and are thought to be involved in cognitive functions. In this thesis, mechanisms of GDP generation were studied with intra- and extracellular recordings in the neonatal rat hippocampus in vitro and in vivo. Immature CA3 pyramidal neurons were found to generate intrinsic bursts of spikes and to act as cellular pacemakers for GDP activity whereas depolarizing GABAergic signalling was found to have a temporally non-patterned facilitatory role in the generation of the network events. Furthermore, the data indicate that the intrinsic bursts of neonatal CA3 pyramidal neurons and, consequently, GDPs are driven by a persistent Na+ current and terminated by a slow Ca2+-dependent K+ current. Gramicidin-perforated patch recordings showed that the depolarizing driving force for GABAA receptor-mediated actions is provided by Cl- uptake via the Na-K-C1 cotransporter, NKCC1, in the immature CA3 pyramids. A specific blocker of NKCC1, bumetanide, inhibited SPWs and GDPs in the neonatal rat hippocampus in vivo and in vitro, respectively. Finally, pharmacological blockade of the GABA transporter-1 prolonged the decay of the large GDP-associated GABA transients but not of single postsynaptic GABAA receptor-mediated currents. As a whole the data in this thesis indicate that the mechanism of GDP generation, based on the interconnected network of bursting CA3 pyramidal neurons, is similar to that involved in adult SPW activity. Hence, GDPs do not reflect a network pattern that disappears during development but they are the in vitro counterpart of neonatal SPWs.