Biochemical identification of multiple GABAB binding sites: association with noradrenergic terminals in rat forebrain

GABAA (bicuculline-sensitive) and GABAB (bicuculline-insensitive) receptor binding were examined in an attempt to determine whether either site was present on noradrenergic terminals in rat brain cerebral cortex. Equilibrium binding saturation studies revealed the presence of two GABAB binding sites in brain tissue, with affinity constants of 60 and 229 nM. Regional distribution studies in the cow brain revealed that the ratio of the high and low affinity GABAB binding sites differed among the areas examined. Both GABAA and GABAB binding were examined in the rat brain cortex 12 days following a unilateral lesion of the dorsal noradrenergic bundle. While GABAA and high affinity GABAB binding were unaffected by the lesion, the number of low affinity GABAB sites was significantly reduced. These results indicate that high and low affinity GABAB binding sites may be anatomically distinct, with only the latter being associated with cerebral cortical noradrenergic nerve terminals.     

GABAA and GABAB receptors modulating basal and footshock-induced nitric oxide releases in rat prefrontal cortex

Using an in vivo brain microdialysis technique, we measured extracellular levels of nitric oxide (NO) metabolites (NO_x⁻) in the medial prefrontal cortex (mPFC) upon perfusion of γ-aminobutyric acid (GABA) receptor antagonists as well as agonists, and also examined the effects of GABA receptor agonists on mild intermittent footshock-induced NO releases in the mPFC in conscious rats. Perfusion of either bicuculline methiodide, a GABA_A receptor antagonist, or saclofen, a GABA_B receptor antagonist, through a microdialysis probe resulted in dose-dependent increases in NO_x⁻ levels. Higher-dose perfusion of either muscimol (50 μM), a GABA_A receptor agonist, or baclofen (250 μM), a GABA_B receptor agonist resulted in a significant decrease in NO_x⁻ levels. The elevated levels of NO_x⁻ after mild intermittent footshock were attenuated by perfusion of either muscimol (10 μM) or baclofen (50 μM), either of which alone did not affect basal NO_x⁻ levels. These findings are likely to provide helpful clues to our understanding of the inhibitory modulation of basal and footshock-induced NO metabolites releases by GABA_A and GABA_B receptors in the mPFC.     

Zinc modulates GABAB binding in rat brain

The effects of ZnCl₂ on [³H]GABA binding to GABA_A and GABA_B binding sites were investigated using receptor autoradiography. At concentrations exceeding 100 μM, zinc non-competitively inhibited GABA_B binding in a dose dependent fashion. GABA_A binding was not inhibited significantly by zinc eliminating the possibility of a non-specific effect of zinc. Increased calcium concentrations up to 10 MM enhanced total GABA_B binding but did not prevent zinc induced inhibition of GABA_B binding, indicating a separate site of action for these cations at the GABA_B binding site. In some regions, zinc modulates GABA_B binding in a biphasic manner as concentrations of 10–100 μM zinc significantly enhanced GABA_B binding in the hippocampus and the molecular layer of the cerebellum but not in the thalamus. These results provide further evidence for a neuromodulatory role for zinc in the central nervous system.     

Evidence for pharmacologically distinct subsets of GABAB receptors

Activation of GABA_B receptors augments neurotransmitter-stimulated cyclic AMP accumulation while inhibiting forskolin-mediated second messenger production. Previous studies have revealed that GABA_B receptors are associated with a pertussis toxin sensitive G protein, such as G_i. While such a linkage is consistent with the finding that GABA_B receptor activation inhibits forskolin-mediated second messenger accumulation, it fails to explain how GABA_B agonists are capable of augmenting receptor-mediated cyclic AMP production. The present experiments were undertaken to explore the possible existence of pharmacologically distinct GABA_B receptors in an attempt to explain this apparent discrepancy. For the study, a variety of agents were examined for their ability to inhibit GABA_B binding to brain membranes and to modify isoproterenol- or forskolin-stimulated second messenger production in rat brain slices. Of the compounds studied, only 3-aminopropylphosphonic acid and 4-aminobutylphosphonic acid were found to inhibit GABA_B binding. However, 4-aminobutylphosphonic acid failed to influence either isoproterenol- or forskolin-stimulated cyclic AMP production. On the other hand, while 3-aminopropylphosphonic acid also failed to affect isoproterenol-stimulated second messenger accumulation, it inhibited the forskolin-mediated response. Given this finding, and the fact that some of the agents tested are known to influence GABA_B receptor function in other systems, the results indicate a multiplicity of pharmacologically distinct GABA_B receptor recognition sites. This discovery paves the way for the development of more selective GABA_B receptor agonists and antagonists possessing different therapeutic potentials.     

Modulation of acetylcholine release via GABAA and GABAB receptors in rat striatum

In order to investigate whether changes in acetylcholine (ACh) release induced by GABA receptors are due to a direct or indirect effect on cholinergic neurons in the striatum, GABA_A and GABA_B receptor bindings were assayed in the striatum microinjected with ethylcholine mustard aziridinium ion (AF64A), a cholinergic neurotoxin. Intra-striatal injection of a selective concentration of AF64A (10 nmol) reduced GABA_A receptor binding without significantly altering GABA_B receptor binding. Treatment with a higher, less selective concentration of AF64A (20 nmol) reduced all markers examined. These results suggest that GABA_A, but not GABA_B receptors, are located on cholinergic neurons in the striatum, and that GABA can directly modulate ACh release through stimulation of GABA_A receptors. Findings further suggest that GABA can also indirectly modulate ACh release through stimulation of GABA_B receptors located on non-cholinergic neuronal elements in the striatum.     

Localization of GABAA and GABAB receptor subtypes on serotonergic neurons

The effect of selective destruction of serotonin (5-HT)-containing neurons with 5,7-dihydroxytryptamine (5,7-DHT) on [3H] muscimol and (-)-[3H]baclofen binding was investigated in various rat brain regions. Ten days after intracerebroventricular 5,7-DHT, serotonin levels and [3H]imipramine binding were markedly decreased. 5,7-DHT reduced [3H]muscimol binding only in the mesencephalon, and (-)-[3H]baclofen binding was unmodified in all the areas considered. These results suggest that except in the mesencephalon GABA receptors may not be localized on serotonergic nerve terminals.     

Evidence for pharmacologically distinct GABAB receptors associated with cAMP production in rat brain

γ-Aminobutyric acid-B (GABA_B) receptors mediate a variety of cellular functions, suggesting the possibility of pharmacologically and molecularly distinct receptors. To explore this possibility a number of GABA_B receptor agonists and antagonists were examined for their ability to influence cAMP production in rat brain cerebral cortical slices. While the agonists did not differentiate between receptors associated with the augmentation of isoproterenol-induced cAMP production and those mediating inhibition of forskolin-stimulated second messenger accumulation, significant differences were noted between the potencies of some antagonists to inhibit these GABA_B receptor-mediated responses. The results suggest at least two pharmacologically distinct subclasses of GABA_B receptors regulate cAMP production in brain.     

Progesterone regulates gamma-aminobutyric acid B (GABAB) receptors in the neocortex of female rats

After discovering that binding to GABA_B receptors in rat neocortex varied as a function of the estrous cycle of the rat, we asked whether either or both of the major ovarian steroids could affect binding to GABA_B receptors in the same way, namely, by regulating the apparent density (B_max) of GABA_B receptors. We report here that in ovariectomized rats, subcutaneous injection of progesterone alone, without the necessity of estrogen priming, increased the Borax of baclofen binding to GABA_B receptors in the neocortex. Radioimmunoassay of plasma progesterone before and after progesterone injections revealed that plasma progesterone levels similar to those reached during the progesterone surge in proestrus were associated with increased baclofen binding. The effect of progesterone upon baclofen binding was evident 4 h but not 1 h following progesterone treatment. There was some specificity with respect to the cortical receptors affected by progesterone in that under our conditions, progesterone did not increase agonist binding to 5-HT_1A or GABA_A receptors. We interpret our results to indicate that progesterone variation during the estrous cycle could be responsible for a component of the regulation of GABA_B receptors that occurs in neocortex during the estrous cycle of the rat.     

Pharmacology of the GABAB receptor in amphibian retina

Amacrine and ganglion cells in the amphibian retina contain GABA_B, as well as GABA_A, receptors. Baclofen, a GABA_B agonist, hyperpolarizes the dark membrane potential of these third order neurons and makes their light responses more transient. GABA_B receptors in the retina have a similar agonist profile to GABA_B receptors described at other sites in the brain. Namely, preferential activation by the R-enantiomer of baclofen, and agonist sensitivity in the order 3-aminopropylphosphinic acid > baclofen > 3-aminopropylphosphonic acid. The GABA_B receptor was not activated by 4-aminobutylphosphonic acid. Several antagonists, such as phaclofen, saclofen, and 2-hydroxysaclofen, were ineffective in the amphibian retina. However, CGP35348 blocked the action of applied baclofen and produced effects on the light response that were opposite to those of baclofen. Applied agonists and antagonists support the hypothesis that GABA_Breceptors serve to regulate the balance of sustained and transient signals to the inner retina.     

Effects of GABAB receptor antagonism on the development of Pentylenetetrazol-induced kindling in mice

Pentylenetetrazol (PTZ) administered chronically in rodents induces kindling which is considered to be a model of chronic epilepsy mediated through a specific interaction with the GABA-gated chloride ionophore. PTZ kindling also impairs shuttle-box learning indicating a possible modulation of memory storage [A. Becker, G. Grecksch, H. Mathies. The influence of diazepam on learning processes impaired by pentylenetetrazol kindling. Naunyn-Schmiedeberg's Arch. Pharmacol. 349 (1994) 429-496]. Since GABA_B receptor antagonism has been shown to improve cognitive performance in rodents and primates we have examined the effects of 3 antagonists: CGP 36742 (3-amino-propyl-n-butyl-phosphinic acid), CGP 56433 ([3-{1-(S)-[{3-(cyclohexylmethyl) hydroxyphosphinyl]-2-(S)-hydroxypropyl]-amino]ethyl]benzoic acid) and CGP 61334 ([3-{[3[(diethoxymethyl)hydroxyphosphinyl]-propyl]-amino}methyl]-benzoic acid) on the induction of PTZ kindling in mice at 48 h intervals for 8 weeks. Subsequently the mice were tested in an active avoidance paradigm. At the end of the experiment GABA_B receptor autoradiography was performed on brain sections from these animals. Seizure intensity increased progressively in control mice reaching by 8 weeks a mean score which corresponded to clonic seizures. The GABA_B antagonists suppressed kindling during the first 4 weeks and after that restored the seizure intensity to the level of control animals. The level of kindling was proportional to the avoidance score. The density of GABA_B receptor binding in brain sections from PTZ kindled mice was significantly greater than in controls. This was not altered by pretreatment with the GABA_B antagonists except in the cerebellum.     

GABAB receptor-mediated inhibition of spontaneous action potential discharge in rat supraoptic neurons in vitro

To elucidate the role of GABA_B receptors in the regulation of the electrical activity of magnocellular neurons of the supraoptic nucleus (SON), the effects of GABA_B agonist and antagonist on the firing rate of spontaneous action potentials were studied in SON slice preparations of rats by extracellular recordings. In the presence of the γ-amino butyric acid (GABA)-gated chloride channel blocker, picrotoxin, the selective GABA_B agonist, baclofen, reduced the firing rate of action potentials in both phasic and non-phasic neurons in a dose-dependent manner. The reduction in the firing rate induced by baclofen was reversed by the selective GABA_B antagonist, 2-hydroxy saclofen (2OH-saclofen), also in a dose-dependent manner. In non-phasic neurons, 2OH-saclofen significantly increased the firing rate and the effect was additive to the effect of picrotoxin. In phasic neurons, 2OH-saclofen alone did not increase the firing rate, but it reversed suppression of the firing induced by increasing extracellular Ca²⁺ concentration to 2.1 mM. Baclofen also reduced the firing rate of non-phasic neurons of virgin and lactating female rats, indicating that the GABA_B receptor-mediated inhibition is not confined to SON neurons of male rats. The evidence indicates that activation of GABA_B receptors inhibits electrical activity of SON neurons of both male and female rats and that GABA_B receptors may play an important role in the inhibitory regulation of the electrical activity of SON neurons by GABA.     

Distribution and depression of the GABAB receptor in the spinal dorsal horn of adult rat

γ-Aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in vertebrate nervous system. The metabotropic receptor for GABA, GABA_B receptor, is characterized as a G protein-coupled receptor subtype. In the present study, GABA_B receptor-like immunoreactivity (GABA_BR-LI) in the rat spinal cord and dorsal root ganglion (DRG), as well as GABA_B receptor-mediated depression in the spinal dorsal horn were examined by using immunohistochemistry and whole-cell voltage-clamp recording technique, respectively. Under light microscope, GABA_BR-LI was densely found in laminae I and II of the dorsal horn. DRG cells of various diameters also showed GABA_BR-LI. Electron microscopy further revealed that GABA_BR-LI was also localized in terminals of myelinated, unmyelinated fibers as well as the somatodendritic sites of dorsal horn neurons. Bath application of a GABA_B receptor agonist, baclofen (10 μM, 30 s), induced a slow outward (inhibitory) current in dorsal horn neurons. This slow current was depressed when the postsynaptic G protein-coupled receptor was inhibited, indicating the postsynaptic action of baclofen. Under the condition of postsynaptic GABA_B receptor being inhibited, baclofen (10 μM, 60 s) depressed large (Aβ) and fine (C, Aδ) afferent fiber-evoked monosynaptic excitatory postsynaptic currents, indicating presynaptic inhibition of GABA_B receptor on elicited neurotransmitter release. Taken together, the results suggest that baclofen-sensitive GABA_B receptor is expressed pre- and postsynaptically on primary afferent fibers and neurons in the spinal dorsal horn; activation of GABA_B receptor in the dorsal horn postsynaptically hyperpolarizes dorsal horn neurons and presynaptically inhibits primary afferents.     

Role of GABAB receptors in GABA and baclofen-induced inhibition of adult rat cerebellar interpositus nucleus neurons in vitro

Previous studies suggested that the postsynaptic GABA(B) receptors of deep cerebellar nuclear neurons of adult rats were not activated by selective GABA(B) receptor agonist baclofen or endogenous GABA released by cerebellar cortical Purkinje cells, although the receptors have been demonstrated to exist in the deep cerebellar nuclei. In this study, cerebellar slices of adult rats were prepared for testing effects of GABA, baclofen and muscimol (selective GABA(A) receptor agonist) on cerebellar interpositus nucleus (IN) neurons. Perfusing slices with GABA (10-1000 microM), baclofen (1-30 microM) and muscimol (1-100 microM) respectively produced a dose-dependent inhibitory response on the IN neurons (n = 39, 62 and 50), which was not blocked by low-Ca(2+)/high-Mg(2+) medium (n = 5, 6 and 6), supporting a direct postsynaptic action of these GABAergic agonists. Moreover, both selective GABA(B) receptor antagonist CGP35348 and selective GABA(A) receptor antagonist bicuculline were capable of partially blocking the inhibitory response of IN neurons to GABA (n = 14 and 11), suggesting that the GABA-induced inhibition may contain two components, a GABA(B) receptors-mediated component and a GABA(A) receptors-mediated one. Further experiments revealed that not only muscimol (n = 50) but also baclofen (n = 62) suppressed IN cells' activity. The baclofen-induced inhibition was selectively blocked by CGP35348 (n = 12) but not by bicuculline (n = 8), whereas the muscimol-induced inhibition was selectively antagonized by bicuculline (n = 8) instead of CGP35348 (n = 9). These results indicate that GABA(B) receptors in the IN neurons can be activated not only by GABA but also by baclofen, suggesting that besides GABA(A) receptors, GABA(B) receptors may also be involved in mediating the inhibitory effect of GABA on cerebellar IN neurons of adult rats.     

Severity of atypical absence phenotype in GABAB transgenic mice is subunit specific

Overexpression of GABA_BR1a receptors in mice (R1a⁺) results in an atypical absence seizure phenotype characterized by 3- to 6-Hz slow spike-and-wave discharges (SSWDs), reduced synaptic plasticity, and cognitive impairment. Here we tested the hypothesis that increased R1 expression causes atypical absence epilepsy and is not subunit specific. GABA_BR1b receptors were overexpressed in mouse forebrain (R1b⁺) and confirmed by immunoblot and ³H-CGP54626A autoradiography. The R1b⁺ mice showed a reduction in hippocampal long-term potentiation and GABA_A receptor-mediated inhibitory postsynaptic currents. R1b⁺ mice manifested an electrographic, pharmacological, and behavioral phenotype consistent with atypical absence seizures, though less robust than R1a⁺ in terms of SSWD duration and severity of cognitive impairment. These results suggest that abnormal GABA_BR1b function plays a lesser role in the development of atypical absence epilepsy.     

Structural determinants of activity at the GABAB receptor

Phosphoethanolamine is a phosphomonoester that is reduced in Alzheimer disease brain. Despite its close structural similarity to GABA and the GABA_B partial agonist 3-aminopropylphosphonic acid, phosphoethanolamine binds very poorly to GABA_B receptors (IC₅₀=7.5±0.8 mM). In this study, we examined whether the marked decrease in binding affinity associated with the presence of an ester oxygen in place of the α-CH₂ group of GABAergic compounds also occurred in sulfonates and used high resolution solution NMR and molecular mechanics calculations to determine the structural basis of this decrease in activity. The sulfonate analog of GABA, 3-aminopropylsulfonic acid, became >2500-fold less potent when the α-CH₂ was replaced by an ester oxygen. Structural studies showed that the active α-CH₂ compounds (GABA, 3-aminopropylphosphonic acid, and 3-aminopropylsulfonic acid) prefer a fully extended conformation. The inactive compounds, phosphoethanolamine and ethanolamine-O-sulfate, exist in a gauche conformation around the C_β–C_γ bond. This study, which suggests conformational differences, may explain how PE can be so efficiently excluded from GABA_B receptors, despite being present in millimolar concentrations in brain. Exclusion of phosphoethanolamine from GABA_B receptors may be an important physiologic control, mechanism in the regulation of inhibitory neurotransmission.     

Involvement of GABAB receptor systems in experimental depression: baclofen but not bicuculline exacerbates helplessness in rats

There are two γ-aminobutyric acid (GABA) hypotheses of the antidepressants action: an increase in GABA_A neurotransmission or a decrease in GABA_B neurotransmission may contribute to action of antidepressants. In this study, involvement of GABA_A and GABA_B, receptor systems was examined in the learned helplessness paradigm in rats. Rats were injected with bicuculline or baclofen for 14 days. On day 14, the rats were subjected to 15 inescapable shocks. On day 15, they underwent the 40-trial escape test. Baclofen exacerbated the escape failures in the rats subjected to the inescapable shocks, although baclofen had no effects in the animals without shock pre-treatment. Bicuculline failed to influence the escape failures in the rats with the 15-shock pre-treatment. These results suggest that the long-term increase in GABA_B, neurotransmission but not the long-term attenuation of GABA_A neurotransmission may be related to helplessness in rats.     

GABAB receptors in the dorsomedial hypothalamus and heart rate in anesthetized rats

Previous studies have shown that: (1) activation of neurons in the dorsomedial hypothalamus (DMH) of the rat by blockade of local GABA_A receptors with bicuculline methiodide (BMI) elicits cardiovascular changes resembling those seen in experimental stress, including marked sympathetically-mediated tachycardia, and (2) inhibition of neurons in the same region by local microinjection of the GABA_A receptor agonist muscimol can virtually abolish stress-induced tachycardia. This study examined the possibility that GABA_B receptors exist in the neural circuitry of the DMH, and that stimulation of these receptors might suppress the cardiovascular response to local disinhibition with BMI. Microinjection of BMI 10 pmol into the DMH in urethane-anesthetized rats resulted in marked tachycardia with little or no effect on arterial pressure. Simultaneous injection of the GABA_B receptor agonist baclofen at doses of 2.5, 5.0 and 10 pmol produced dose-related suppression of BMI induced tachycardia. Coinjection of the GABA_B receptor antagonist 2-hydroxysaclofen 100 or 200 pmol had no significant effect on the heart rate response to BMI, but reversed the suppression elicited in the presence of baclofen. These findings indicate that (1) functional GABA_B receptors exist in the DMH, and (2) stimulation of these receptors inhibits the tachycardia resulting from blockade of local GABA_A receptors.     

The GABAB antagonist phaclofen inhibits the late K-dependent IPSP in cat and rat thalamic and hippocampal neurones

Phaclofen (0.5–1 mM) reversibly inhibited the late, bicuculline resistant, K⁺ dependent IPSP recorded in projection cells of the cat and rat dorsal lateral geniculate nucleus and in rat hippocampal CA1 pyramidal neurones. At the same concentrations, phaclofen reversibly blocked the K⁺ dependent, bicuculline insensitive hyperpolarization evoked by GABA and baclofen but had no effect on the GABA_A IPSP. These results represent conclusive evidence that GABA_B receptors mediate the late K⁺ dependent IPSP in cortical and subcortical neurones.     

5-HT1A, GABAB, and pirenzepine-insensitive muscarinic receptors are functionally coupled to distinct pools of the same kind of G proteins in rat hippocampus

In order to probe the interaction between the neurotransmitter receptors and guanine nucleotide-binding regulatory (G) proteins in rat hippocampus, the high-affinity GTPase activity stimulated by 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA), carbachol (CCh), and dopamine (DA) has been investigated, focusing on the additivity among the effects of these agonists at their maximally effective concentrations. There were simple additive relationships among 5-HT, GABA-, and CCh-stimulated activities. As 5-HT, GABA-, and CCh-stimulated high-affinity GTPase activities are mediated by the 5-HT_1A, GABA_B, and pirenzepine-insensitive muscarinic receptors, respectively, the additive effects indicate that these three receptors are independently coupled to distinct pools of G proteins. In contrast, an apparent lack of additivity was seen between 5-HT- and DA-stimulated activities. This phenomenon was likely due to an activation of the common 5-HT_1A receptor-mediated signalling by DA as well as 5-HT, since the DA-sensitive increment of the activity was potently inhibited by the 5-HT₁ receptor antagonist methiothepin, but not by the DA D₂ receptor antagonist raclopride.