Effects of bicuculline and picrotoxin on fast and slow IPSP in pyramidal neurons in hippocampal slices isolated from the rat

The effects of different concentrations of bicuculline and picrotoxin on IPSP of pyramidal neurons with high and low rates of rise ("fast" and "slow" IPSP) were investigated in hippocampal slices isolated from area CA1. These neurons generated mainly fast IPSP in response to antidromic stimulation; orthodromic stimulation produced both fast and slow IPSP, often combining into a single IPSP with two phases. Both these patterns of IPSP were recorded from the apical dendrites as well as the soma and were reversibly inhibited by bicuculline and picrotoxin. Degree of inhibition depended on dose and duration of blocker action, but slow IPSP were more resistant to this action. At the same effective concentration of bicuculline or picrotoxin, slow IPSP were inhibited later and recovered sooner after washout of blocker than fast IPSP. The difference between the inhibitory effect of blockers on fast and slow IPSP persisted under tetanic stimulation, although the progress of reduction in IPSP proceeded far more rapidly than following application of a single stimulus. The reason for this phenomenon is discussed, as well as particular features of -aminobutyric acid (GABA) receptors of pyramidal neurons mediating generation of slow IPSP.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 1, pp. 44–54, January–February, 1990.     

Effects of bicuculline on [3H]SR 95531 binding in discrete regions of rat brains

Effects of bicuculline in vitro, and acute and chronic treatment of a subconvulsive dose of bicuculline on [³H]SR 95531 binding to discrete regions of rat brains were studied in Sprague-Dawley rats. Scatchard analysis of the binding isotherms exhibited two populations of binding sites for [³H]SR 95531 in frontal cortex, cerebellum, striatum and substantia nigra. The apparent K_D for high-affinity sites was significantly increased in the frontal cortex and cerebellum in the presence of bicuculline (1 M) with no change in B_max. In contrast, the apparent affinity for low-affinity sites was not altered in the presence of bicuculline in these regions, whereas the B_max was significantly decreased in the cerebellum. Following acute (2 mg/kg, i.p.) or chronic (2 mg/kg, i.p. for 10 days) bicuculline treatment, [³H]SR 95531 binding was also investigated in various regions of brains. The acute bicuculline treatment did not affect the [³H]SR 95531 binding in any of the regions studied. In contrast, apparent affinity for [³H]SR 95531 was significantly decreased in low-affinity sites of all regions studied in rats treated chronically with bicuculline. The B_max values of high and low-affinity sites were significantly increased in the cerebellum with no change in the frontal cortex, striatum and substantia nigra. The present study demonstrates that chronic bicuculline treatment decreases apparent affinity of [³H]SR 95531 binding whereas the treatment increases apparent affinity of [³H]SR 95531 and [³H]muscimol binding in the cerebellum may be due to true up-regulation of GABA binding sites, involving increased de novo synthesis of receptor protein. These results also suggest that properties of cerebellar GABA_A receptors are different from those in other regions.Abbreviations used GABA -aminobutyric acid - FC frontal cortex - CB cerebellum - ST striatum - SN substantia nigra     

Valproic acid and bicuculline affect the formation of glycerolipid in rat brain

To ascertain the effects of bicuculline and of sodium valproate on the incorporation of glycerol into rat brain lipid, rats were divided into 5 groups: (a) controls; (b) treated with sodium valproate (400 mg/kg body wt); (c) treated with bicuculline (12.5 μmol/kg body wt); (d) treated with sodium valproate as in (b) + bicuculline as in (c); and (e) treated with bicuculline (25 μmol/kg body wt). Only rats of group (c) had seizures, which lasted until the end of the experiment. Each animal received 20 μCi of [2-³H]glycerol by intraventricular route and was sacrificed 12 min afterwards. Hippocampi and cerebella were taken and lipid extracted and separated by chromatography.

The type of treatment influenced very much the fate of injected, labeled glycerol. Indeed, total recovered radioactivity increased following either convulsions or the administration of valproate, whereas both treatments decreased the amount of radioactivity incorporated into lipid. These effects were more evident in cerebella than in hippocampi.

The distribution of radioactivity among lipid classes (diglyceride, triglyceride and total phospholipid) was also affected by seizures, which decreased the labeling ratio phospholipid/neutral lipid. The distribution of radioactivity among phospholipid classes was influenced by bicuculline (both at convulsant and non-convulsant doses) and these effects were sometimes antagonized by valproate. We conclude that some effects of bicuculline are exerted through the systemic modifications due to seizures and that other effects are probably connected to neuronal hyperfiring. The data reported in this paper are consistent with both mechanisms of action proposed for valproate, i.e. increased membrane permeability and modifications of GABAergic systems.     

Effect of trazodone on oxidative metabolism of rat brain in vitro

Oxygen uptake of rat brain homogenate was reduced by 1 mM trazodone, a new atypical antidepressant. Na,K-ATPase activity and the associated oxygen consumption of rat brain slices were also reduced. Oxygen consumption of rat brain slices was enhanced by dopamine and this effect was blocked by 0.0001 mM trazodone. This drug uncoupled oxidative phosphorylation.     

Effects of GABA antagonists,SR 95531 and bicuculline,on GABAA receptor-regulated chloride flux in rat cortical synaptoneurosomes

Synaptoneurosomes isolated from cerebral cortices of male Sprague-Dawley rats were used for studying GABA_A receptor-regulated chloride influx. The in vitro effects of GABA antagonists, SR 95531 (a pyridazinyl GABA derivative) and bicuculline, on pentobarbital-stimulated, muscimol-stimulated or flunitrazepam-enhanced, muscimol-stimulated chloride uptake were studied. The chloride uptake was determined at 30°C, for 5 sec. Pentobarbital and muscimol produced a maximal stimulation of chloride uptake in cortical synaptoneurosomes at 500 M and 50M, respectively. SR 95531 as well as bicuculline had no effect on the basal uptake of chloride. Whereas, SR 95531 (0.3–30 M) and bicuculline (0.1–100 M), when added 5 min before muscimol (50 M), produced a significant concentration-dependent inhibition of muscimol (50 M)-stimulated chloride uptake (IC₅₀ s of 0.89±0.11 M and 13.45±2.10M, respectively). In studies of the inhibitory effects of SR 95531 and bicuculline on pentobarbital (500 M)-stimulated chloride uptake, the IC₅₀ s were 0.81±0.12 M and 3.86±1.14 M, respectively. SR 95531 exhibited a more potent inhibitory effect than bicuculline on flunitrazepam-enhanced, muscimol-stimulated chloride uptake. The results revealed that SR 95531 has a more potent antagonistic effect than bicuculline on GABA_A-regulated chloride flux.     

Effects of chaotropic anions on bicuculline inhibition of high affinity γ-aminobutyric acid binding in bovine retina and rat brain membranes

Chaotropic anions (ions that favour transfer of apolar groups to water) increased bicuculline inhibition of ³H-γ-aminobutyric acid binding to bovine retinal membranes as previously reported for rat forebrain membranes. The increased bicuculline inhibition was reversible when the chaotropic anion was removed thus ruling out the possibility that ‘endogenous regulators’ were being removed by the chaotropic anions. Another possible explanation for the enhanced bicuculline inhibition is an increase in the solubility of bicuculline, an organic compound that is sparingly soluble in water; however, when bicuculline-methiodide, a more water soluble form of bicuculline was used, no difference was noted in this enhancement.Although the chaotropic anions temporarily increase the bicuculline inhibition of γ-aminobutyric acid binding, they do not increase ³H-γ-aminobutyric acid receptor binding as previously suggested. Thiocyanate or perchlorate have no effect on ³H-γ-aminobutyric acid receptor binding to rat forebrain or cerebellar membranes. Although thiocyanate slightly inhibits γ-aminobutyric acid receptor binding to bovine retinal membranes, perchlorate has no effect. The previous observation that sodium perchlorate enhanced γ-aminobutyric acid binding in bovine retina was due to the enhancement of sodium-dependent binding to a nipecotic acid-sensitive binding site (perhaps an uptake site).The mechanism of action of chaotropic anions in vitro on γ-aminobutyric acid binding may be through an alteration in the interaction of the receptor with the antagonists, bicuculline or bicuculline-methiodide, but is not through the removal of a component that blocks the binding site or regulates the receptor's properties, as evidenced by the reversibility of the chaotropic anion effect and the lack of effect on agonist binding.     

Effects of anticonvulsant agents and ethanol on the complexes of epileptic foci created by applications of bicuculline, strychnine, and penicillin in the rat brain cortex

Anticonvulsive effects of direct agonists of the GABA receptor system, medinal and phenazepam, as well as ethanol, on the complexes of epileptic foci created in the rat brain cortex by applications of various convulsant agents (bicuculline, strychnine, or penicillin) were studied. Ethanol, as well as phenazepam and medinal, were shown to possess high anti-epileptic activity in relation to the complexes. It was shown, using an i.v. infusion technique, that threshold doses of strychnine and bicuculline depend, in a linear and a nonlinear fashion, respectively, on the alcohol doses administered. A specific feature was found in the pharmacolgoical effect of ethanol: anticonvulsive activity of ethanol was pronounced within the postinjection interval from 5 min to 4 h, while within the following interval, from 4 to 24 h, the animals turned into a seizure-ready state. The subseizure phase was observed under the conditions when the penicillin complex was induced, and also following i.v. injections of strychnine and bicuculline.     

Laminar differences in bicuculline methiodide's effects on cortical neurons in the rat whisker/barrel system

Extracellular unit recordings were made at various depths within SmI barrel cortex of immobilized, sedated rats, in the presence and absence of titrated amounts of the GABAA receptor antagonist bicuculline methiodide (BMI). Principal and adjacent whiskers were moved singly, or in paired combination in a condition-test paradigm, to assess excitatory and inhibitory receptive field (RF) characteristics. Neurons were classified as regular- or fast-spike units, and divided into three laminar groups: supragranular, granular (barrel), and infragranular. BMI increased response magnitude and duration, but did not affect response latencies. The excitatory RFs of barrel units, which are the most tightly focused on the principal whisker, were the most greatly defocused by BMI; infragranular units were least affected. All three layers had approximately equal amounts of adjacent whisker-evoked, surround inhibition, but BMI counteracted this inhibition substantially in barrel units and less so in infragranular units. The effects of BMI were most consistent in the barrel; more heterogeneity was found in the non-granular layers. These lamina-dependent effects of BMI are consistent with the idea that between-whisker inhibition is generated mostly within individual layer IV barrels as a result of the rapid engagement of strong, local inhibitory circuitry, and is subsequently embedded in layer IV's output to non-layer IV neurons. The latter's surround inhibition is thus relatively resistant to antagonism by locally applied BMI. The greater heterogeneity of non-granular units in terms of RF properties and the effects of BMI is consistent with other findings demonstrating that neighboring neurons in these layers may participate in different local circuits.     

GABA及荷包牡丹碱对金黄地鼠上丘视觉神经...

Visual response properties of single neurons in the superior colliculus of golden hamsters could be altered by iontophoretically applied gamma-aminobutyric acid (GABA) and its antagonist, bicuculline (Bicu). GABA decreased the responses of the superficial cells to stationary flashing stimuli, while Bicu increased the responses and suppressed the inhibition exerted by the surround. The number of spikes evoked by a moving bar/spot decreased after applying GABA in 76.6% of the cells (n = 60) and increased in 90.0% (n = 60) of the cells after Bicu. Similar effects on the spontaneous activities were observed. In addition, 65.0% of the 60 cells recorded have enlarged movement receptive fields after application of Bicu. GABA and Bicu have some effects on the orientation selectivity of the collicular cells too.     

Effects of acute and chronic trazodone administration on serum prolactin levels in adult female rats

Trazodone was tested for its ability to elevate serum prolactin levels in mature female rats. When the drug was administered acutely to female rats at doses up to 80 mg/kg ip, it induced a clear rise in serum prolactin levels, with a minimum effective dose of 20 mg/kg; blood trazodone levels at these doses were between 1.6–2.4 μg/ml. However, trazodone could not be considered to be a potent stimulator of prolactin secretion, since the injection of haloperidol at 2 mg/kg elevated serum prolactin to values twice those seen in animals receiving the 80 mg/kg dose of trazodone. When trazodone was administered chronically in the diet for two or four weeks, at an average daily dose of 80 mg/kg, serum trazodone levels were found to be 100–200 ng/ml when measured at each stage of the estrous cycle. Serum prolactin levels in trazodone-treated animals, however, did $\text{not}$ differ from those in control rats. Moreover, drug-treated animals showed normal proestrus surges in serum prolactin. The results of these studies thus indicate that acutely, at very high doses, trazodone probably can stimulate prolactin secretion modestly in female rats. However, when consumed chronically at 80 mg/kg/day, the drug has no effects on serum prolactin levels. Therefore, if trazodone stimulates prolactin secretion by altering neurotransmission across dopamine and/or serotonin synapses in brain, it is probably not potent in these actions, at least as concerns those dopamine and serotonin neurons that influence the secretion of prolactin.     

Regulation of 35S-TBPS binding by bicuculline is region specific in rat brain

The allosteric regulation of specific 35S-TBPS binding to the convulsant site on the GABAA receptor/chloride (Cl-) ionophore complex was studied in various brain regions in an attempt to characterize regional heterogeneity of the protein subunits forming the complex. Bicuculline methiodide (BIC), a GABAA antagonist, enhanced binding in cortex (CTX), substantia nigra (SN) and cerebellum (CBL), inhibited binding in inferior colliculus (IC) and did not affect binding in superior colliculus (SC). Similar results were found in CBL and IC using SR-95531, another GABAA antagonist. The levels of endogenous GABA in the different tissue samples could not account for the regional differences in binding. When the functional regulation of these receptors was measured using 36Cl- uptake in microsomes, muscimol-stimulated uptake was completely blocked by BIC in CBL and IC but was not affected by BIC in SC. Additionally, picrotoxin completely blocked muscimol-stimulated uptake in CBL but had no effect in IC or SC. These findings provide a functional basis for regional heterogeneity of GABAA receptor.     

Effects of systemic administration of strychnine, L-allylglycine, bicuculline and picrotoxin on the transsynaptic neural destruction in the medullary dorsal horn following transection of the rat inferior alveolar nerve

Adult rats underwent unilateral transection of the inferior alveolar nerve and subsequent intraperitoneal injection of strychnine (1 mg/kg, 3-23 days) at various posttransectional intervals. When they were sacrificed at 18-30 days posttransectionally, many pyknotic neuronal cell bodies were observed in plastic embedded toluidine blue-stained 1 micron-thick sections of the medullary dorsal horn. They were mostly found in the dorsal part of the dorsal horn ipsilateral to the neurotomy and were more abundant in laminae I/II than in laminae III/IV. Similar pyknotic neurons were found after 1 or 2 days of L-allylglycine administration (55.7 mg/kg/day) at a posttransectional interval of 20 days. Unlike those observed after strychnine treatment, the pyknotic neurons after L-allylglycine treatment were evenly distributed throughout laminae I-IV of the dorsal half of the medullary dorsal horn. Twenty-three days of bicuculline (2 mg/kg/day) or picrotoxin (0.5 mg/kg/day) treatment at an interval of 7 days did not yield pyknotic neurons. The results are discussed in the light of intrinsic synaptic circuitry of the dorsal horn.     

Studies on bicuculline binding sites on neuronal membrane using fluorescent antibody technique: Comparative binding of gaba and bicuculline

Highly purified fluorescent labelled anti-bicuculline antibodies were used to mark bicuculline binding sites in cerebral cortex of monkey brain. Specific binding of bicuculline could be demonstrated in the synaptosomal fraction, when bicuculline was added both $\text{in vitro}$ and $\text{in vivo}$. Addition of γ-aminobutyric acid (GABA) to the bicucullinised membrane led to a decrease in fluorescence indicating same receptor loci and establishing GABA-bicuculline antagonism at a molecular level.     

Effects of interleukin-10 on the epileptiform activity development in the hippocampus induced by brief hypoxia, bicuculline and electrical kindling

The comparative effects of antiinflammatory cytokine interleukin-10 on the epileptiform activity development in CA1 hippocampal neurons were studied in different functional models of epileptogenesis that are not accompanied the visible morphological disturbances in the brain cells: --in vitro hypoxic model in the rat hippocampal slices; 2--in vitro disinhibitory model with using GABAA antagonist, bicuculline, in the rat hippocampal slices; 3--partial hippocampal kindling model in freely moving rats. Interleukin-10 (1 ng/ml) depressed the posthypoxic hyperexcitability in CA1 pyramidal neurons of the rat hippocampal slices through a decrease of the effectiveness of hypoxia to depresses the functional neuronal activity in the rat hippocampal slices during hypoxic episode. On the other hand, interleukin-10 (1 ng/ml) did not affect an initiation of epileptiform activity in CA1 pyramidal neurons of the rat hippocampal slices induced by bicuculline. Interleukin-10 (1 ng/5 microl) applied to the dorsal hippocampus in awake rats depressed an initiation of focal seizures ("ictal"-like components of afterdischarges) induced by hippocampal kindling during the first six hours after an application. However, this cytokine did not affect neither the duration of "interictal"-like component of afterdischarges nor motor seizure development. Thus, our findings showed that antiinflammatory cytokine interleukin-10, in addition to its antihypoxic action, exert the neuroprotective effect on the initiation of "ictal"-like, but not "interictal"-like, epileptiform discharges.     

Some observations on habituation of the flexor reflex in the rat: the influence of strychnine, bicuculline, spinal transection, and decerebration

The involvement of inhibition in habituation of the flexor reflex was investigated in intact, spinal, and decerebrate rats. Strychnine and bicuculline were administered in order to determine the contribution of certain forms of central inhibition to the development of habituation. Both strychnine and bicuculline reduced habituation in the intact rat but did not do so in the spinal preparation. Strychnine, in fact, caused a facilitation of habituation in the spinal rat. The impairment of habituation by strychnine was related to the intensity of stimulation used to elicit the reflex. Thus, the effect of strychnine was only demonstrable when relatively high intensities were used. Flexor reflex habituation was shown to be more pronounced in the decerebrate than in the spinal preparation. This difference could be demonstrated only when stimuli of high intensity were given. It is concluded that, in the absence of supraspinal influences, habituation of the flexor reflex does not require inhibitory mechanisms. However, inhibition may play a role in habituation of the component of the reflex that utilizes descending influences.     

The effects of bicuculline and muscimol on glutamate-induced feeding behavior in broiler cockerels

This study was designed to examine the effects of intracerebroventricular (ICV) injection of bicuculline (GABA_A receptor antagonist) and muscimol (GABA_A receptor agonist) on glutamate-induced eating response in 24-h food-deprived (FD24) broiler cockerels. At first, guide cannula was surgically implanted in the right lateral ventricle of chickens. In experiment 1, birds were ICV injected with different doses of glutamate. In experiment 2, birds were administered with effective dose of glutamate after bicuculline. In experiment 3, chickens received muscimol prior to the injection of glutamate, and cumulative food intake was determined at 3-h postinjection. The results of this study showed that glutamate decreases food consumption in FD24 broiler cockerels (P ≤ 0.05), and this reduction occurs in a dose-dependent manner. Moreover, the inhibitory effect of glutamate on food intake was significantly increased with bicuculline pretreatment, and this effect was attenuated with muscimol (P ≤ 0.05). These results suggest that there is an interaction between glutamatergic and GABAergic systems (through GABA_A receptor) on food intake in broiler cockerels.