Synaptic potentials and effects of amino acid antagonists in the auditory cortex

Neurons of in vitro guinea pig and rat auditory cortex receive a complex synaptic pattern of afferent information. As many as four synaptic responses to a single-stimulus pulse to the gray or white matter can occur; an early-EPSP followed, sequentially, by an early-IPSP, late-EPSP, and late-IPSP. Paired pulse stimulation and pharmacological studies show that the early-IPSP can modify information transmission that occurs by way of the early-EPSP. Each of these four synaptic responses differed in estimated reversal potential, and each was differentially sensitive to antagonism by pharmacological agents. DNQX (6,7-dinitroquinoxaline-2,3-dione), a quisqualate/kainate receptor antagonist, blocked the early-EPSP, and the late-EPSP was blocked by the NMDA receptor antagonist APV (D-2-amino-5-phosphonovalerate). The early-IPSP was blocked by the GABA-a receptor antagonist bicuculline, and the late-IPSP by the GABA-b receptor antagonists 2-OH saclofen or phaclofen. Presentation of stimulus trains, even at relatively low intensities, could produce a long-lasting APV-sensitive membrane depolarization. Also discussed is the possible role of these synaptic potentials in auditory cortical function and plasticity.     

GABAergic Transmission in the Nucleus Accumbens Is Involved in the Termination of Ethanol Self-Administration in Rats

Long-Evans rats ( n = 12) were trained to lever-press on a fixed-ratio 4 schedule of reinforcement with ethanol (10% v/v) presented as the reinforcer. After implantation of bilateral stainless-steel guide cannulae aimed at the nucleus accumbens, site-specific microinjections of muscimol (1–30 ng) and bicuculline (1–10 ng) were tested for effects on ethanol-reinforced responding. Baseline response patterns were characterized by initial high rates that terminated abruptly after ∼20 min. Muscimol administration in the nucleus accumbens decreased the total number of ethanol-reinforced responses and obtained reinforcers. Bicuculline also decreased ethanol-reinforced responses and reinforcers at the highest dose tested. When a dose of bicuculline (1 ng) that was ineffective by itself was coadministered with an effective dose of muscimol (10 ng), the muscimol-induced decreases in responding were blocked. Analysis of response patterns showed that muscimol decreased ethanol self-administration by terminating responding, normally lasting 20 min, after ∼10 min with no changes in local response rate. Bicuculline decreased total responding by producing parallel, but nonsignificant, changes in time course and response rate. These data suggest that GABAergic transmission in the nucleus accumbens is involved in the termination, but not the onset or maintenance of ethanol self-administration. The specificity of this effect gives emphasis to the importance of measuring behavioral parameters, as well as products of behavior (such as intake volume) in the study of ethanol self-administration.     

Genotype-dependent effects of GABAergic agents on sedative properties of ethanol

Two lines of mice, selectively bred for differential sensitivity to the soporific effects of ethanol (ETOH), were administered GABAergic drugs in an effort to evaluate a role for GABA in ETOH sensitivity. ETOH sensitive Long-Sleep mice (LS) showed potentiated ETOH sedation when administered bicuculline, muscimol and aminooxyacetic acid (AOAA). ETOH-insensitive SS mice exhibited reduced ETOH sedation in the presence of the antagonists, bicuculline and picrotoxin, and potentiated sedation in the presence of muscimol and AOAA. These changes in narcosis duration were interpreted as central effects, since blood ethanol levels at waking from ETOH sedation varied with GABAergic drug treatment. Picrotoxin antagonized pentobarbital-induced nacrosis in both lines, but to a greater extent in SS mice. These and other experiments with a genetically heterogeneous stock suggest GABA involvement in genotype-dependent ETOH sensitivity, but do not support a simple role of GABA receptor involvement.     

Potential involvement of a baclofen-sensitive autoreceptor in the modulation of the release of endogenous GABA from rat brain slices in vitro

Summary The effects of the GABA_A agonist, muscimol, and of the enantiomers of the GABA_B agonist, baclofen, on the release of endogenous GABA from slices of the rat cerebral cortex, striatum and hippocampus were measured by means of a HPLC method with electrochemical detection. Moreover, the effect of the GABA_A antagonist, bicuculline, and of the frequency of stimulation were studied in cortical slices. The amount of endogenous GABA released per impulse from cortical slices decreased by about 50% when the frequency was increased from 0.25 Hz to 1 Hz. This might indicate that GABA inhibited its own release. (–)-Baclofen at 1 and 10 M, but not its (+)-enantiomer, markedly inhibited the release of endogenous GABA, to a similar extent in all 3 areas investigated. The effect of (–)-baclofen was dependent on the frequency of stimulation: at lower frequencies (0.25 and 0.5 Hz) it was more marked than at a higher one (4 Hz). This would be expected from the results showing that the release of endogenous GABA decreases with increasing frequency, which suggests that this amino acid inhibits its own release. Muscimol at 10 M, on the other hand, was ineffective in all 3 areas at a stimulation frequency of 0.5 Hz. Bicuculline (10 M) at 4 Hz, at which autosuppression of GABA release is maximal did not enhance the release of endogenous GABA from cortical slices. With cerebellar or nigral slices, no adequate stimulation-induced release of endogenous GABA could be obtained under comparable conditions. These data are compatible with, but do not prove the existence of GABAB-type presynaptic autoreceptors modulating the release of this amino acid. More definite conclusions may possibly be drawn when a GABAB antagonist becomes available, which is expected to enhance GABA release under appropriate conditions.Presented in part at the 3rd Brit. Meeting on Electrochemical detection in Pharmacology and Neurochemistry, Cambridge, March 30–April 1, 1987Send offprint requests to P. C. Waldmeier     

Removal of GABAergic inhibition alters subthreshold input in neurons in forepaw barrel subfield (FBS) in rat first somatosensory cortex (SI) after digit stimulation

Our objective was to test the hypothesis that suppression of GABAergic inhibition results in an enhancement of responses to stimulation of the surround receptive field. Neurons in the forepaw barrel subfield (FBS) in rat first somatosensory cortex (SI) receive short latency suprathreshold input from a principal location on the forepaw and longer latency subthreshold input from surrounding forepaw skin regions. Input from principal and surround receptive field sites was examined before, during, and after administration of the GABA(A) receptor blocker bicuculline methiodide (BMI) (in 165 mM NaCl at pH 3.3-3.5). In vivo extracellular recording was used to first identify the location of the glabrous forepaw digit representation within the FBS. In vivo intracellular recording and labeling techniques were then used to impale single FBS neurons in layer IV as well as neurons in layers III and V, determine the receptive field of the cell, and fill the cell with biocytin for subsequent morphological identification. The intracellular recording electrode was fastened with dental wax to a double-barrel pipette for BMI iontophoresis and current balance. A stimulating probe, placed on the glabrous forepaw skin surface, was used to identify principal and surround components of the receptive field. Once a cell was impaled and a stable recording was obtained, a stimulating probe was placed at a selected site within the surround receptive field. Single-pulse stimulation (1 Hz) was then delivered through the skin probe and the percentage of spikes occurring in 1-min intervals before BMI onset was used as a baseline measure. BMI was then iontophoresed while the periphery was simultaneously stimulated, and spike percentage measured during and after BMI ejection was compared with the pre-BMI baseline. The major findings are: (1) suppression of GABAergic inhibition enhanced evoked responses to firing level from sites in surround receptive fields in 65% of the cells ( n=17); (2) evoked responses were rapidly elevated (within 1 min) to suprathreshold firing in the presence of BMI in 31% of the cells; (3) GABAergic inhibition was reversible [suprathreshold spiking gradually reversed to subthreshold excitatory postsynaptic potentials (EPSPs) in 45% of the cells tested]; (4) BMI altered the stimulus-evoked and non-stimulus-evoked firing pattern in SI neurons from single spikes to burst patterns in all tested cells; and (5) iontophoresis of NaCl (165 mM) without BMI was ineffective in altering evoked responses in control cells ( n=4). The present findings support the notion that subthreshold input from surround receptive fields is one possible mechanism for rapid cortical reorganization in barrel cortex and that GABAergic inhibition may regulate its expression. Possible corticocortical and thalamocortical substrates for subthreshold input to reach barrel neurons are discussed.     

Cross-orientation inhibition in cat is GABA mediated

Summary Visual evoked potentials (VEPs) were recorded from cat cortex (area 17) before, during and after application of the GABA blocker bicuculline (iontophoretic or topical). The stimuli comprised a test sinusoidal grating, and a mask grating oriented either parallel or orthogonal to the test. Both test and mask alternated in contrast at different temporal frequencies. VEPs were averaged in synchrony with the test contrast reversal, so the mask did not contribute directly to the averaged VEP response. Before application of bicuculline, both parallel and orthogonal masks attenuated the amplitude of VEPs and changed the phase response, but in different ways. Orthogonal masks lowered the slope of the contrast response curve without affecting extrapolated threshold, while parallel masks caused the curve to shift to the right. Orthogonal masks increased the phase advance, while parallel masks eliminated it. During application of bicuculline, neither parallel nor the orthogonal masks attenuated VEP amplitudes. The results suggest that although the mechanisms for the action of parallel and orthogonal masks are clearly distinct, both are mediated by the GABA-ergic inhibitory system. Given this evidence, measurement of VEP contrast response curves may provide a simple non-invasive technique for monitoring visual inhibition in humans.     

Water diffusion in the different microenvironments of breast cancer

The parameters that characterize the intricate water diffusion in tumors may serve to reveal their distinct pathology. Specifically, the application of diffusion magnetic resonance imaging (MRI) can aid in characterizing breast cancer, as well as monitoring response to therapy. We present here a non-invasive, quantitative MRI investigation, at high spatial resolution, of water diffusion in hormonal dependent MCF7 breast tumors implanted orthotopically in immunodeficient mice. Distinctive MRI protocols were designed in this study, utilizing a broad range of diffusion times and diffusion gradient strengths. Application of these protocols allowed water diffusion in the tissue extracellular and intracellular compartments to be distinguished, and the effect of restricted diffusion and water exchange on the water diffusion in these compartments to be evaluated. Pixel-by-pixel analysis yielded parametric maps of the estimated volume fraction and apparent diffusion coefficient of each compartment. The diffusion of the water in the extracellular microenvironment was approximately two fold slower than that of free water, and in the intracellular compartment was about one order of magnitude slower than that of free water and demonstrated restriction of water diffusion at long diffusion times. Mapping of the water fraction in each compartment was further employed to monitor changes during tumor progression and to assess tumor response to hormonal manipulation with a new antiestrogenic drug, tamoxifen methiodide (TMI). It was found that, in parallel to the growth arrest by this drug, the volume fraction of the slowly diffusing water increased, suggesting a TMI-induced cell swelling. This study can serve as a basis for extending diffusion breast MRI in the clinical setting.     

A pharmacological study of group I muscle afferent terminals and synaptic excitation in the intermediate nucleus and Clarke's column of the cat spinal cord

Summary When administered microelectrophoretically GABA and piperidine-4-sulphonic acid depolarized the central terminations of muscle group Ia and Ib afferent fibres in the lumbar intermediate nucleus and Clarke's column of cats anaesthetised with pentobarbitone sodium. Both this depolarization, and primary afferent depolarization, generated by impulses in other primary afferent fibres which produce prolonged bicuculline-sensitive inhibition of the firing of group I afferent fibre-excited interneurones in the intermediate nucleus and cells in Clarke's column, are reduced by microelectrophoretic bicuculline methochloride. Systemically administered (±)-baclofen hydrochloride (maximum dose 8 mg kg^–1) depressed the monosynaptic excitation of Clarke's column neurones by impulses in muscle and cutaneous afferent fibres. Microelectrophoretically administered (–)-baclofen reduced the bicuculline-sensitive primary afferent depolarization of group I terminations without, however, reducing the depolarizing action of GABA or piperidine-4-sulphonic acid. The depression by (–)-baclofen of the group I monosynaptic excitation of intermediate nucleus neurones is not reduced by concentrations of bicuculline methochloride adequate to suppress prolonged inhibition of these neurones     

GABA-receptor activation in the globus pallidus and entopeduncular nucleus: opposite effects on reaction time performance in the cat

The possible role of GABAergic mechanisms in the control of the basal ganglia output structures, the globus pallidus (GP) and the entopeduncular nucleus (EP), was studied in cats performing a conditioned flexion movement triggered by an auditory stimulus. The effects of discrete unilateral microinjections of low doses of the GABA_A receptor agonist (muscimol 5–100 ng/ 0.5 l) and antagonist (bicuculline methiodide 25–150 ng/0.5 l) in the GP and the EP were tested on the motor performance of eight animals trained to release a lever in a simple reaction time (RT) schedule after an auditory stimulus. Control injections in neighboring structures did not induce any effect except with five- to tenfold higher doses in the closest injection sites. The dose of 20 ng muscimol injected into the ventral and medial part of the GP produced an arrest of the performance after a few unsuccessful trials (over the RT reinforcement limit of 500 ms), while muscimol injected in sites located in the lateral GP resulted in a dose-dependent lengthening in RTs, with a concomitant increase in the force change latency. In most of the subjects, the force exerted on the lever was higher after muscimol than after vehicle injection. Force change velocity was then significantly increased. In contrast, muscimol injected in the ventral and rostral region of the EP produced a decrease in RTs or a complete cessation of responding after a high number of anticipatory responses (release of the lever before the trigger stimulus). No significant changes in the force change latency could be observed while there was a non-significant tendency for the force levels to be lowered. Bicuculline injections in the EP were found to increase RTs with a concomitant increase in force change latency and a slowness of velocity, while no significant effect was observed following injections in the GP. These results suggest that a balance between GABAergic activity in the two output nuclei of the basal ganglia, the GP and the EP, is crucial for the correct initiation and execution of the conditioned motor task.     

The effects of compounds related to γ-aminobutyrate and benzodiazepine receptors on behavioural responses to anxiogenic stimuli in the rat: Punished barpressing

Rats were trained to press a bar for sucrose reward on a random-interval (RI) schedule and footshock punishment was then introduced for 3-min intrusion periods (signalled by a tone) on an independent RI schedule. Shock intensity was individually adjusted to produce stable intermediate levels of response suppression during the tone for each animal. Groups of animals were then allocated to a number of separate experiments in which they were systemically injected with anxiolytics (chlordiazepoxide HCl or sodium amylobarbitone), GABA antagonists (picrotoxin or bicuculline), the GABA_A agonist muscimol, the GABA_B agonist baclofen, an antagonist (RO 15-1788) at the benzodiazepine receptor and, an inverse agonist (FG 7142) at this receptor. The results showed that the alleviation of punishment-induced suppression of barpressing produced by chlordiazepoxide was blocked or partially blocked by RO 15-1788, picrotoxin and bicuculline but not by FG 7142; that picrotoxin (but not FG 7142) increased the suppression of responding by punishment; that neither muscimol nor baclofen affected responding on their own, but their combination weakly but reliably released punished responding from suppression; and that the anti-punishment effect of amylobarbitone was unaffected by either picrotoxin or bicuculline, though the barbiturate reversed the punishment-enhancing effect of picrotoxin. These results are discussed in the light of the hypothesis that anxiolytic behavioural effects are due to increased GABAergic inhibition.