Neurotransmitter abnormalities in genetically epileptic rodents

A growing body of evidence supports a pathophysiological role for norepinephrine (NE) and serotonin in the regulation of seizures in the genetically epilepsy-prone rat (GEPR). Other evidence indicates that gamma-aminobutyric acid (GABA) and taurine may also participate in the seizure regulation process. Innate deficits in NE and serotonin appear to be causes of the genetically determined seizure-prone states of the GEPR, whereas abnormalities in GABAergic systems and taurine metabolism may represent inadequate attempts of the central nervous system to compensate for the seizure-prone state in these rats. In audiogenic seizure-susceptible (AGS) mice, evidence suggests a role for dopamine as well as GABA and possibly serotonin. NE may contribute to the regulation of seizures in AGS mice, but consistent evidence for a primary role for this monoamine is lacking. It is suggested that there is no single common neurotransmitter abnormality underlying genetic seizure disorders in humans or other animals and that the GEPR and the AGS mouse may both serve as good models for study of the neurochemical abnormalities that underlie the different human epilepsies.     

Noradrenergic and serotonergic determinants of seizure susceptibility and severity in genetically epilepsy-prone rats

Pharmacological studies demonstrate a reciprocal relationship between both noradrenergic and serotonergic transmission and audiogenic seizure severity and susceptibility in the genetically epilepsy-prone rat (GEPR). In contrast, drug-induced changes in the neurochemical indices of dopaminergic activity do not result in alterations in seizure severity. These pharmacological investigations led to the hypothesis that both noradrenergic and serotonergic neurons are capable of regulating seizure severity in the GEPR. Pharmacological investigations also provided evidence that monoaminergic neurons serve as determinants of seizure susceptibility in these epileptic animals. The GEPR is susceptible to environmentally-induced seizures which cannot be precipitated in neurologically normal subjects. Drug studies suggest that monoaminergic decrements serve as one set of susceptibility determinants. However, non-monoaminergic abnormalities also play important roles in the seizure predisposition which characterizes the GEPR. Pathophysiological studies have confirmed and extended the concepts generated by the pharmacological investigations. Noradrenergic and serotonergic deficits do indeed characterize the seizure naive state of the GEPR. These studies have provided a basis for tentative identification of areas of the brain in which monoaminergic abnormalities regulate seizure severity and susceptibility. Monoaminergic defects in some areas such as the thalamus may regulate both susceptibility and severity. In other areas, defects may regulate only severity or susceptibility. In the striatum, noradrenergic defects do not appear to be present and probably are not determinants of the epileptic state of the GEPR.     

Sleep, temperature, activity, and prolactin phenotypes of genetically epilepsy-prone rats

Sleep-wake disturbances are common in epilepsy, yet the potential adverse effect of seizures on sleep is not well characterized. Genetically epilepsy-prone rats (GEPRs) are a well-studied model of genetic susceptibility to audiogenic seizures. To assess their suitability for investigating relationships between seizures and disordered sleep, we characterized the sleep, activity, and tempera ture patterns of 2 GEPR strains (designated 3 and 9) and Sprague-Dawley (SD) rats in the basal state, after forced wakefulness, and after exposure to sound-induced seizures at light onset and dark onset. Because of observed differences in rapid-eye-movement sleep (REMS), we also assessed serum levels of prolactin, which is implicated in REMS regulation. The data reveal that under basal conditions, the GEPR3 strain shows less SWS and REMS, higher core temperatures, and higher serum prolactin concentrations than do GEPR9 and SD strains. All 3 strains respond similarly to enforced sleep loss. Seizures induced at light onset delay the onset of SWS in both GEPR strains. Seizures induced at dark onset do not significantly alter sleep. Genotype assessment indicates that although both GEPR strains are inbred (that is, homozygous at 107 genetic markers), they differ from each other at 74 of 107 loci. Differences in basal sleep, temperature, and prolactin between GEPR3 and GEPR9 strains suggest different homeostatic regulation of these functions. Our detection of concurrent alterations in sleep, temperature, and prolactin in these 2 GEPR strains implicates the hypothalamus as a likely site for anatomic or physiologic variation in the control of these homeostatic processes.     

Anticonvulsant drugs and the genetically epilepsy-prone rat

Anticonvulsant drugs were evaluated in members of two colonies of genetically epilepsy-prone rats (GEPR). Virtually all of the animals in the first colony experience a wild running fit that terminates in a generalized clonic convulsion when they are stimulated by sound. According to our convulsion intensity scoring system, these animals have an audiogenic response score (ARS) of 3 and the colony is designated the GEPR-3 colony. In the second colony, more than 95% of the animals experience a wild running phase terminating in a tonic extensor convulsion when they are stimulated by sound. That is, they have an ARS of 9 and the colony is designated the GEPR-9 colony. All of the established antiepileptic drugs that were tested produced anticonvulsant effects in the GEPR. Three tricyclic antidepressant agents acted as anticonvulsants in doses substantially lower than the toxic doses that produced spontaneous convulsions. Two of the established anticonvulsants, phenobarbital and ethosuximide, produced anticonvulsant effects in very similar doses in members of GEPR-3 and GEPR-9 colonies. Valproic acid produced an anticonvulsant effect in GEPR-3 in significantly lower doses than in GEPR-9. Carbamazepine, phenytoin, imipramine, amitriptyline, and desipramine produced anticonvulsant effects in essentially equimolar doses and in each case the protective dose was significantly lower in GEPR-9 than in GEPR-3 colonies. GEPR did not experience the convulsive effects of imipramine, amitriptyline, and desipramine at lower doses than did control animals. Thus, these epilepsy-prone animals are no more likely to experience convulsions in response to overdose of one of these three drugs than are nonepileptic subjects.     

Anatomical changes of the GABAergic system in the inferior colliculus of the genetically epilepsy-prone rat

The number of GABAergic neurons as determined by GAD immunocytochemistry and total neurons as determined from Nissl preparations were counted and classified at the light microscopic level in the inferior colliculus (IC) of the genetically epilepsy prone rat (GEPR) and the non-epileptic Sprague-Dawley (SD) strain of rat. GAD-positive neurons are abundant in the IC and a significant increase in the number of GAD-positive neurons occurs in the GEPR as compared to the SD in all three subdivisions. However, the most pronounced difference occurs in the ventral lateral portion of the central nucleus, where there is a selective increase in the small (200%) and medium-sized (90%) GABAergic somata (10-15 microns in diameter and 15-25 microns in diameter, respectively). As determined from Nissl preparations an increase in total numbers of neurons also occurs. Thus, a 100% increase in the number of small neurons and a 30% increase in the number of medium-sized neurons occur in the adult GEPR as compared to the SD rat. A statistically significant increase in the numbers of small neurons also occurred in the IC of the young GEPR. At 4 days of age, a 55% increase in the number of small neurons was found, and at 10 days of age this increase was 105%. The numbers of the medium and large neurons were similar in the older group of rats. These data suggest that the increase in cell number observed in the adult GEPR is not compensatory to the seizure activity, but may either be genetically programmed or be a failure of cell death. Based on other studies of genetic models of epilepsy, we propose that the additional GABAergic neurons may disinhibit excitatory projection neurons in the IC.     

The development of kindled seizures is accelerated in the genetically epilepsy-prone rat

The kindling phenomenon was examined in genetically epilepsy-prone (GEPR) and non-epileptic control Sprague-Dawley rats. Kindling stimulations were administered three times a day until each rat had exhibited three Class 5 kindled motor seizures. The mean total number of kindling stimulations required for each experimental group to exhibit three motor seizures of each motor seizure class was determined. The results indicated that the early stage of kindling development was accelerated significantly in both the GEPR-3 and GEPR-9 rats, compared to non-epileptic control rats. Later stages of kindling development were accelerated in GEPR-9 but not GEPR-3 rats. Thus a differential acceleration of kindling development was exhibited by GEPR-3 and GEPR-9 rats. The results suggest the possibility that some brain region(s) involved in the early stages of kindling development may be hyperexcitable in both GEPR-3 and GEPR-9 rats. Other brain region(s) involved with the later stages of kindling development may be more excitable in GEPR-9 rats. These putative alterations may, in part, contribute to the seizure prone state of GEPR rats and the differential seizure responses of GEPR-3 and GEPR-9 rats.     

Cerebral Glutamic Acid Decarboxylase Activity and γ-Aminobutyric Acid Concentrations in Mice Susceptible or Resistant to Audiogenic Seizures

Abstract: DBA/2 mice between 21 and 28 days of age are highly susceptible to sound-induced seizures. Drug studies suggest a possible deficit of γ-Aminobutyric acid (GABA)-mediated neurotransmission may be involved. We have measured the whole brain GABA concentration and glutamic acid decar-boxylase activity in DBA/2 mice at various ages before, during, and after the period of maximal susceptibility to audiogenic seizures. Corresponding determinations were carried out on age-matched TO mice, a strain much less susceptible to audiogenic seizures than DBA/2 mice at all ages. No significant differences in GABA concentration or glutamic acid decarboxylase activity were found between strains at any age. The susceptibility of DBA/2 mice to audiogenic seizures does not result from a gross inability to synthesise or store GABA.     

听原性惊厥易感大鼠下丘GluR2的表达及QR位点编辑水平

听原性惊厥易感大鼠是强直 -阵挛惊厥大发作的一种模型 .一般认为 ,下丘是听原性惊厥发作神经元网络的启动部位 .采用RT PCR、Western印迹、免疫组织化学等方法观察了听原性惊厥易感大鼠 (P77PMC)一次惊厥发作与惊厥点燃状态下AMPA受体亚基GluR2在下丘内表达的改变 ,并采用限制性酶切方法分析了GluR2Q R位点mRNA编辑水平的改变 .研究结果显示 ,一次惊厥发作后下丘内GluR2表达无明显改变 ,惊厥点燃后下丘内GluR2表达降低 ,一次惊厥发作及惊厥点燃状态下GluR2Q R位点处于编辑成熟状态 .提示 ,GluR2表达降低参与了点燃状态下的惊厥发作 ,在听原性惊厥易感大鼠惊厥发作机制中不涉及下丘内GluR2Q R位点编辑水平改变 .     

Neuroanatomical localization of structures responsible for seizures in the GEPR: lesion studies

Identification of the neural substrates subserving audiogenic convulsions in the GEPR is an important task and while it is not yet complete, many laboratories employing various techniques have contributed importantly to our current understanding. The present review focuses on the use of lesions to identify the neural substrates of audiogenic convulsions. Lesions in brain stem nuclei appear to have a much greater ability to attenuate audiogenic convulsions than do forebrain lesions. In fact, some forebrain lesions (dorsal hippocampus, caudate, intralaminar thalamic nuclei) appear to enhance the severity of audiogenic seizures. On the other hand, bilateral lesions in the inferior colliculus (IC) have been shown to completely abolish audiogenic convulsions, while lesions in the pontine reticular formation (PRF nucleus) abolish all aspects except the running episode suggesting that these two brain stem structures are important neural substrates involved in the expression of audiogenic convulsions. Large bilateral lesions of the substantia nigra also appear to attenuate audiogenic convulsions. The effect of lesions on audiogenic convulsions is basically similar to their effect on other generalized seizure models and the data appear to support the hypothesis that there are two anatomical systems involved in the expression of all generalized convulsions: a forebrain system responsible for the expression of face and forelimb clonus; and a brain stem system responsible in the expression of running-bouncing clonus and tonus.     

Cerebral cortical GABA and benzodiazepine binding sites in genetically seizure prone rats

Adult male and female genetically seizure-prone rats were assessed for sound-induced seizures. Heterozygous control groups were compared with mild seizure (designated GEPR 3) and severe seizure animals (GEPR 9). Groups of animals were killed and crude synaptosome fractions (P2) prepared from freshly dissected cerebral cortices. Binding sites for gamma-aminobutyric acid (GABA) were assessed by [3H]-muscimol in the absence or presence of excess GABA and/or pentobarbital. Binding sites for benzodiazepines were assessed by [3H]-flunitrazepam in the presence or absence of clonazepam. Compared to controls, GEPR 3 animals had a modest increase and GEPR 9 animals a larger increase in Bmax for both high and low affinity GABA sites, with no change in Kd. Chloride-dependent, barbiturate-enhanced GABA binding (increased Bmax) was observed in all conditions and groups. Likewise benzodiazepine binding (Bmax) increased slightly in GEPR 9 animals. There were no observed changes in binding sites for a survey of biogenic amines. Seizure-prone animals appear to have compensatory denervation-like supersensitivity for their most prominent inhibitory receptor, which may or may not be linked to the seizure event.     

Long lasting effect of a single audiogenic seizure on GABA turnover rates and steady-state levels

GABA turnover rates (TOR) and steady-state levels (SSL) were determined, 16–18 h after a single acoustic stimulation, in 15 brain areas of 3 mouse sublines. Each subline differs in its response to an acoustic stimulation (Rb1 mice are clonic-tonic seizure-prone, Rb2: clonic seizure-prone, Rb3: seizure-resistant). TOR and SSL were compared to those of unstimulated control mice and to those of repeatedly stimulated mice of the same subline. Following a single acoustic stimulation long-lasting alterations of GABA metabolism, mainly large alterations of GABA TOR, are observed. Most of the effects elicited after repeated stimulations, either on SSL or TOR, are not those of the last stimulation and repeated seizures (and/or stimulations) strengthen the effect of a single one. It appears that, for each of the Rb sublines, a specific and quite simple profile of the alterations of GABA metabolism in response to a single or repeated audiogenic seizures (and/or stimulations) can be given. The global analysis through the correlation of GABA TOR and SSL gives an indication that the alterations of the parameters of the correlation observed are to be allocated to the audiogenic seizures. Furthermore the tonic and clonic components of the audiogenic seizures can be distinguished.     

Long lasting effects of audiogenic seizures on synaptosomal neurotransmitter amino acids in Rb mice

Long lasting alterations of synaptosomal amino acid neurotransmitters following a single or several audiogenic seizures and/or acoustic stimulations were investigated in six brain areas-olfactory bulbs (OB), amygdala (A), hippocampus (Hi), cerebellum (C), inferior colliculus (IC), ponsmedulla (P)- of three sublines of Rb mice: audiogenic seizure-prone Rb1 and Rb2, seizure-resistant Rb3. Changes in the synaptosomal levels of aspartate (Asp), glutamate (Glu), taurine (Tau), 4-amino butyrate (GABA), glycine (Gly) and some closely related precursors, serine (Ser) and glutamine (Gln), were recorded 15–18 hours after a single or multiple acoustic stimulations. Changes were more frequent, or larger, after polystimulation. Some alterations appeared to be attributable to an effect of the acoustic stress.In both seizure-prone sublines, after a single or repeated seizures, an increase in synaptosomal Asp was observed in IC. Decreases in Asp and Tau in OB and Ser in A, an increase in Gln in IC were only observed after repeated seizures, in Rb1 and Rb2 mice.Abbreviations used GABA 4-aminobutyrate - Tau taurine - Gly glycine - Ser serine - Asp aspartate - Glu glutamate - Gln glutamine - OB olfactory bulbs - A amygdala - Hi hippocampus - C cerebellum - IC interior colliculus - P pons Professeur Paul Mandel passed away on 6th October, 1992Special issue dedicated to Dr. Bernard W. Agranoff.     

Gabuculine and isogabaculine: in vivo biochemistry and pharmacology in mice.

Two irreversible enzyme-activated GABA-transaminase inhibitors, gabaculine (5-amino cyclohex-1, 3-dienyl carboxylic acid) and an isomer, isogabaculine (3-amino cyclohex-1, 5-dienyl carboxylic acid), were investigated in mice for their effects on brain GABA metabolism and on seizures induced by a variety of stimuli. Biochemical and pharmacological activities of the two inhibitors were very similar. Both produce dose- and time-related, sustained inhibition of GABA-T activity and, to a lesser extent, of GAD activity and long-lasting increases in brain GABA concentrations. Both protect mice against audiogenic seizures and significantly decrease the frequency of seizures induced by isoniazid, thiosemicarbazide and pentylenetetrazol. They do not affect the frequency of seizures induced by strychnine, bicuculline or picrotoxin and do not alter the threshold to electroconvulsive shock. Although the effects of gabaculine and isogabaculine on brain GABA metabolism resemble those of other GABA-T inhibitors, important differences in pharmacological activities exist.     

Evidence for a GABAergic Projection from the Dorsal Nucleus of the Lateral Lemniscus to the Inferior Colliculus

Abstract: This study attempts to determine whether the pathways from the guinea pig dorsal nucleus of the lateral lemniscus (DNLL) to the inferior colliculus (IC) use γ-aminobutyric acid (GABA) as a transmitter. Injections of kainic acid (KA) were used to destroy neurons in the left DNLL. Two to 4 days after the injection, Nissl-stained sections through the lesion site showed destruction of the DNLL neurons. The lesions varied in size; 12–100% of the DNLL neurons were destroyed on the injected side without damage to the ipsilateral IC. Two to 4 days after the injection, the electrically evoked, Ca²⁺-dependent release and high-affinity uptake of [³H]GABA were measured in dissected pieces of the left and right IC. These activities were compared with those in the IC taken from unlesioned controls and from sham controls, which received injections of saline instead of KA. Each IC was divided into a dorsal piece, which contained the dorsal cortex and dorsomedial nucleus, and a ventral piece, which contained the central and lateral nuclei. Lesions of the left DNLL depressed the release and uptake of [³H]GABA in the ventral pieces of the IC, but there was a greater depression in the ventral IC contralateral to the lesioned DNLL. There were good correlations between the percentage of neuronal loss in the left DNLL and deficits in [³H]GABA release and uptake activities in the ipsi- and contralateral ventral IC. By contrast, there was no depression of [³H]GABA release and uptake in the dorsal pieces of the IC. The localization of the deficits in release and uptake appears to match the distribution of the synaptic endings of the DNLL pathways in the IC. This correspondence associates GABA release and uptake activities with the DNLL projections to the IC and, therefore, suggests that GABA may be a transmitter of these pathways. The release and uptake of [¹⁴C]glycine was also measured to determine whether glycine might be a transmitter of the DNLL pathways to the IC. Lesions of the left DNLL failed to alter the Ca²⁺-dependent release or the uptake of [¹⁴C]glycine, suggesting that DNLL neurons are unlikely to use this compound as a transmitter.     

GABA介导杏仁外侧核对皮层A Ⅰ区神经元声反应的抑制:在体微电泳研究

在SD大鼠上应用多顺利完成微电极方法,观察微电泳CABA及其受体的拮抗剂或激动剂对杏仁外侧核（LA）抑制皮层AⅠ神经元声反应效应的影响。结果显示,电泳GABA能抑制皮层AⅠ区神经元的电活动,电泳GABAA受体拮抗剂bicuculline（BIC）则能易化其反应;电刺激LA能抑制皮层AⅠ区听神经元声反应,电泳GABA产生类拟于刺激LA的抑制效应;LA对皮层AⅠ区神经的抑制效应能被BIC所翻转,而不能被什氨酸受体拮抗剂strychnine所翻转,电泳GABAB型受体例激动剂baclofen对神经元声反应无影响。上术结果表明,GABA可能是介民LA抑制皮层AⅠ区神经元声反应的最终递质,并且是通过GABAA受体作用的。     

Elevated levels of group-III metabotropic glutamate receptors in the inferior colliculus of genetically epilepsy-prone rats following intracollicular administration of l-serine-O-phosphate

The selective group-III metabotropic glutamate receptor agonist, L-serine-O-phosphate (L-SOP), when injected bilaterally into the inferior colliculus of the sound sensitive genetically epilepsy-prone (GEP) rats produces a short proconvulsant excitation followed by a long phase of protection against sound-induced seizures lasting for 2-4 days. We have studied this prolonged suppression of audiogenic seizures using pharmacological and molecular biological approaches including semiquantitative RT-PCR and western blotting. The intracerebroventricular injection of the protein synthesis inhibitor cycloheximide (120 microg) 30 min beforehand significantly reduces the proconvulsant seizure activity and the prolonged anticonvulsant effect of intracollicular L-SOP (500 nmol/side). The sensitive semiquantitative RT-PCR revealed a significant up-regulation in mGlu(4) and mGlu(7) mRNA levels in the inferior colliculus at 2 days (maximum suppression of audiogenic seizures) after intracollicular L-SOP injection compared with the non-injected, 2-day post-vehicle treated and 7-day (return to expressing audiogenic seizures) post-drug or vehicle-treated groups. No significant changes were observed in mGlu(6) or mGlu(8) mRNA expression levels in drug-treated compared with control groups. Examination of mGlu(4a) and mGlu(7a) protein levels using western blotting showed a significant increase in mGlu(7a) but no significant change in mGlu(4a) protein levels 2 days after L-SOP treatment compared with the control groups (non-injected and 2-day vehicle-injected group). These results suggest that up-regulation of mGlu(7) receptors is involved in the prolonged anticonvulsant effect of L-SOP against sound-induced seizures in GEP rats. The potential use of mGlu(7) agonists as novel anti-epileptic agents merits investigation.     

Epilepsy and electromagnetic fields: effects of simulated atmospherics and 100-Hz magnetic fields on audiogenic seizure in rats

In order to study the possible association between epileptic seizures and natural electromagnetic fields, 32 female audiogenic seizure (AGS)-susceptible rats were exposed to simulated 10 kHz and 28 kHz atmospherics and to a sinusoidally oscillating magnetic field with a frequency of 100 Hz and field strength of 1 A/m. After the electromagnetic exposure, seizures were induced in the rats with a sound stimulus. The severity of the seizure was determined on an ordinal scale, the audiogenic response score (ARS). The time from the beginning of the sound stimulus to the onset of the seizure (seizure latency) and the duration of the convulsion was measured. No differences from the control experiments were found in the experiments with simulated atmospherics, but the 100 Hz magnetic field increased the seizure latency by about 13% (P<0.02). The results do not support the hypothesis that natural atmospheric electromagnetic signals could affect the onset of epileptic seizures, but they suggest that AGS-susceptible rats may be a useful model for studying the biological effects of electromagnetic fields.     

Effects of cimetidine-like drugs on recombinant GABAA receptors

Even though conventional systemic doses of cimetidine and other histamine H(2) antagonists display minimal brain penetration, central nervous system (CNS) effects (including seizures and analgesia) have been reported after administration of these drugs in animals and man. To test the hypothesis that cimetidine-like drugs produce these CNS effects via inhibition of GABA(A) receptors, the actions of these drugs were studied on seven different, precisely-defined rat recombinant GABA(A) receptors using whole-cell patch clamp recordings. The H(2) antagonists famotidine and tiotidine produced competitive and reversible inhibition of GABA-evoked currents in HEK293 cells transfected with various GABA(A) receptor subunits (IC(50) values were between 10-50 microM). In contrast, the H(2) antagonist ranitidine and the cimetidine congener improgan had very weak (if any) effects (IC(50) > 50 microM). Since the concentrations of cimetidine-like drugs required to inhibit GABA(A) receptors in vitro (greater than 50 microM) are considerably higher than those found during analgesia and/or seizures (1-2 microM), the present results suggest that cimetidine-like drugs do not appear to produce seizures or analgesia by directly inhibiting GABA(A) receptors.     

Interaction of 3beta, 5alpha-tetrahydrodeoxycorticosterone in rat and guinea-pig neurons: a comparison of Ca2+ - and GABA(A)-CI- -channel current modulation.

A comparison of the interaction of 3beta, 5alpha-tetrahydrodeoxycorticosterone (TDOC) on voltage-gated Ca2+ -and the gamma-aminobutyric receptor (GABA(A)) gated-Cl- -channels was examined in freshly dissociated guinea-pig (GP) and rat hippocampal CA1 neurons and rat hypothalamic ventromedial nucleus (VMN) neurons. The steady-state inhibition of the peak Ca2+ channel current evoked by depolarized steps from -80 to -10 mV by TDOC increased in concentration-dependent manner with IC50 values of 1 and 6 pM for rat and GP CA1 neurons, respectively and 3 nM for rat VMN neurons. TDOC rapidly and reversibly inhibited a fraction (up to 26%) of the total Ca2+ channel current in all neurons. Intracellular dialysis with GDP-beta-S (500 microM) significantly diminished the TDOC inhibition of the Ca2+ channel current, suggesting a G-protein involvement. In neurons isolated from pertussis-toxin-treated animals by chronic intracerebroventricular (1000 ng/24/48 h) infusion, the TDOC inhibition was also significantly diminished, suggesting modulation by the Galphai and/or Galphao G-protein subunits. The peak GABA-gated inward Cl- current was enhanced in both species from 0.1 to 10 microM with the greatest increase (48% at 10 microM) seen in the VMN. There was no difference in the enhancement of the GABA current in the CA1 region of both species. The results show that in contrast to the 3a-series, the 3beta-series weakly enhance the GABA-evoked Cl- current but potently inhibit the Ca2+ channel current. In addition, these results also suggest a common mode of action and a lack of interspecies difference for this steroid.     

A method for detection and characterization of GABA(A) receptor ligands using calcium-sensitive fluorescent probes

A method for detecting and characterizing possible ligands of neuronal GABA(A) receptors has been developed, which is based on measuring the calcium response to GABA by the fluorescence of a two-wavelength Ca-sensitive probe Fura-2. In a young (2–4 days) rat hippocampal cell culture, GABA induced depolarization and a transient increase in Ca²⁺ concentration in the cytosol of neurons due to activation of voltage-dependent calcium channels. A brief application of GABA could attenuate the calcium response to a subsequent addition of depolarizing agents (GABA or KCl). However, at modest amplitudes of calcium response to GABA, the reduction of the subsequent effect of KCl was insignificant, and the amplitudes of responses to KCl and to GABA proved to be linearly correlated, with a slope of ∼3.4. Therefore, the GABA calcium signals could be normalized in order to compare experiments performed on different days and different cultures. With such normalization, we estimated the EC₅₀ for GABA in neurons at ∼2.23 μM and the Hill coefficient at ∼1.9. A blocker of voltage-dependent calcium channels nifedipine suppressed the calcium responses both to KCl and to GABA, so that the linear relationship between their amplitudes was retained. To further validate the method, the IC50 and the type of inhibition were verified for known noncompetitive and competitive antagonists of GABA(A) receptors.