Diazepam Enhances the Action but Not the Binding of the GABA Analog, THIP
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Brain Research, 297 (1984) 181-186 18 l Elsevier BRE 20108 Diazepam enhances the action but not the binding of the GABA analog, THIP JOHN H. SKERRITT and ROBERT L. MACDONALD Department of Neurology, Universi O' ~/" Michigan Medical Center, Ann Arbor, M148109 ( U. S. A. i (Accepted November 29th, 1983) Key words: GABA receptors -- diazepam -- anticonvulsant -- GABA agonists -- spinal cord cullurcs GABA (4-aminobutyric acid) and its bicyclic analog THIP (4,5,6,7-tetrahydroisoxazolo-[4,5-c]-pyridin-3-ol) produced membrane hyperpolarization and increased chloride ion conductance of mouse spinal cord neurons in cell culture. Above I nM diazepam en- hanced the actions of both GABA and THIP with similar potency and efficacy. Diazepam has been shown to enhance the binding of [~H]GABA to rat brain membranes over similar concentration ranges, with the EC~0 values for enhancement of [;H]GABA binding and increase in membrane conductance being similar. In contrast, binding of [~H]THIP has been shown to be unaltered by diazepam under a variety of conditions. The possible reasons for such a discrepancy between these electrophysiological and neurochemical re- sults with THIP are discussed. The inhibitory neurotransmitter, GABA, has a aptic rather than extrasynaptic localization z.~. widespread distribution in the central nervous sys- Diazepam enhanced the effects of synaptically re- tem, and multiple cellular actions of GABA, likely to leased or exogenous GABA in a wmety of central arise from effects on GABA receptor subclasses, nervous system preparations 9, including cultured have been identified ll. To develop GABA agonists mouse and chick spinal cord neurons '',iT. Diazepam with selective actions, certain GABA analogs of re- also has been shown to enhance [-~H1GABA binding stricted conformation have been designed 12-H. One to rat brain membranes ~,j~,"-2,2:'. In neurochemical key compound is THIP, a bicyclic GABA analog, studies, however, anomalous interactions of diaze- which has potential therapeutic use since it had anti- pare and THIP have been documented. Unlike convulsant and analgesic activity after oral adminis- GABA. THIP failed to enhance [3H]diazepam bind- tration to rodents ~0`~4,j~. ing at 4 °C and instead antagonized GABA-induced Like GABA, TH1P produced bicuculline-sensitive enhancement of benzodiazepine binding4,I.L While inhibition of firing of cat spinal interneurons and dis- diazepam stimulated the binding of [~H]GABA and placed [~H]GABA binding to rat brain membranes [3H]muscimol to rat brain membranes, [3H]THIP with moderate potency 7.1<~5. However, important binding was unaltered by diazepam under a variety of differences between THIP and GABA have been incubation conditions 2~. demonstrated. Although THIP was less potent than Using mouse spinal cord neurons in primary disso- GABA in depolarizing isolated spinal fibers of the ciated cell culture, we have investigated the postsyn- rat, the compound was 20 times stronger than GABA aptic actions of GABA and THIP and the effects of in depression of spinal synaptic activity -~. GABA diazepam upon GABA and THIP responses. We re- usually depolarized apical dendrites of hippocampal port that both amino acids hyperpolarized neurons CA1 pyramidal cells, while THIP has been shown to by increasing chloride ion conductance, the re- elicit hyperpolarizing responses exclusively I. Thus, it sponses were picrotoxin-sensitive and the responses has been suggested that TH1P may selectively acti- to both amino acids were enhanced with similar po- vate a subclass of GABA receptors, possibly of syn- tency and to a similar extent by diazepam. We dem- Correspondence: R. L. Macdonald, University of Michigan, Neuroscience Laboratory Building, 1103 East Huron, Ann Arbor, MI 48109, U.S.A. I)006-8993/84/$03.IlI) © 1984 Elsevier Science Publishers 13, V. 182 A1. KAc B 1. GABA Pre Picro Post ;i ! THIP 2001~M A2. KCI B2. lsec Pre PIcro Post GABA 8pM THIP 200pM Fig. 1. GABA and THIP responses recorded from spinal cord neurons in cell culture. A: miniperfusion (2 s) with 5/~M GABA or 2(10 .uM THIP increased membrane conductance and evoked hyperpolarizing responses during recordings with 4 M KAc-filled intraeellu- lar micropipettes (A 0, and evoked depolarizing responses with 3 M KCl-filled intracellular micropipettes (A2). Resting membrane potentials were --57 mV and --70 mV, respectively. B: picrotoxin (100 F~M) reversibly blocked the effects of iontophoretically applied GABA (8 nA, 400 ms, B 0 and THIP (45 hA, 400 ms, B2) during recordings with KCl-filled micropipettes. (Membrane hyperpolarized to --90 inV.) onstrate that enhancement of GABA responses and recording microplpette: data were recorded on a [3H]GABA binding are similar in dose-dependency 6-channel Gould polygraph. and discuss the possible reasons for difference in di- GABA (0.5 M, pH 3.2) or THIP (0.2 M. pH 3.2~ azepam action on THIP responses and [3H]THIP were applied iontophoretically using 400 ms duration binding. rectangular current pulses ( +0.5--60 hA) at 4-s inter- Cell cultures were prepared from spinal cords with vals. GABA or THIP were also applied at known attached dorsal root ganglia from 12-14-day-old concentrations by miniperfusion using large (2-10 mouse embryos as described earlier 21. Cultures were ~tm) tip diameter micropipettes (0.8 psi ejection pres- maintained for 5-8 weeks prior to electrophysiolog- sure, 2 s duration). ical experiments. Spinal cord neurons were pen- Diazepam or its vehicle (0.01% or less dimethyl- etrated under visual guidance on the modified stage sulfoxide) were applied by miniperfusion (0.2 psi of an inverted phase-contrast microscope, with high ejection pressure, 30 s duration) from a micropipette impedance (25-40 Mf2) glass micropipettes filled positioned 15-100~m from the soma of the cell under with 4 M potassium acetate (KAc) or 3 M potassium study. Such application of medium containing only chloride (KC1). Impalements were accepted only if 0.01% vehicle did not alter responses to iontophoret- membrane potential was greater than 45 mV and ac- ically applied GABA (99.7 +__ 0.7% control. 6 cells) tion potentials could be evoked. or THIP (98.6 -- 2.8% control. 6 cellsj. During as- Cultures were bathed in a phosphate-buffered sa- sessment of diazepam action upon amino acid re- line containing elevated magnesium ions to suppress sponses, cells were impaled with 3 M KCl-containing spontaneous activity (composition in mM: NaCI micropipettes, and GABA or THIP were applied 143.4, KCI 4.2, CaCI 2 0.9, MgCI 2 10.0 and glucose iontophoretically using constant current pulses. 5.6 in 9.5 mM sodium phosphate buffer at pH 7.35- GABA and TH1P responses of 6-9 mV amplitude 7.40) and maintained at 33-34 °C for electrophysio- were evoked following membrane hyperpolarization logical experiments. Using a conventional bridge cir- to --80 to --90 mV and used for assessment of diaze- cuit, simultaneous current injection and membrane pare actions Data were accepted only if the amino potential measurement were possible with a single acid responses returned to control level within 5 rain 183 following removal of the diazepam-containing pi- 35 pette from the vicinity of the cell under study. In most experiments diazepam effects on GABA and THIP 30 / E / responses were studied on the same cell, with 3 cycles v T T/ w 25 /! t ' of diazepam application possible. +,,', Z / When KAc recording micropipettes were used, ap- II T ,,'i GABA },, THIP plication of GABA or THIP to spinal cord neurons w T //. rapidly and reversibly produced membrane hyperpo- 15 ~ /// • larization and an increase in membrane conductance ) , i" 10 (Fig. 1Aj). Use of KCI-containing recording micro- JJ /¢ pipettes allowed chloride ions to enter the cell, chan- i 3 ging the equilibrium potential for chloride ions from about --65 mV to--20 mVl% and both GABA and 2 5 10 20 s'0,00 260 s60 TH1P responses became depolarizing (Fig. IA;). At (/JM) similar amplitudes, responses to THIP were of longer Fig. 2. Concentration-dependency of GABA and THIP re- duration than those to GABA, applied either ionto- sponses. Depolarizing responses were obtained following im- palement of cells with 3 M KCI-containing micropipettes anti phoretically or by miniperfusion for similar durations membrane hyperpolarizatJon to --80 to --90 inV. Responses (Fig. IA). In 21 cells for which GABA and TH1P re- at each concentration are averages of responses on 4-8 cells. sponses of approximately equal size (about 8 mV) Bars indicate _,+ standard error of the mean (S.E.M.). (Portions of the GABA data are from ref. 19.) were obtained following 4{){) ms iontophoretic appli- cations (KCl-filled recording micropipettes), the mean duration at half-maximal amplitude of GABA sponses were studied using miniperfusion and re- responses was 0.47 _+ 0.02 s and of THIP responses cording with 3 M KCl-containing micropipettes was {).66 _+ 0.02 s (I' < 0.001, 2-tailed Student's t- (Fig. 2), Threshold for GABA responses was ap- test). It has been suggested by otherst that the great- proximately 1-2/,M and for THIP 5 !~M, A 10 mV er duration of THIP action may be due to the absence depolarization was produced by approximately 4 ItM of active uptake mechanisms for this amino acid in GABA and 16 !~M THIP, a 20 mV depolarization neuronal tissue. Indeed, TH1P does not inhibit was produced by about 8 uM GABA and 60 ,M 13H]GABA uptake by minislices of rat cerebral cor- THIP and a 30 mV depolarization was produced by text5, and [~H]TH1P is not itself accumulated by such 19/,M GABA and 350!~M THIP. Thus, over the con- slices (Skerritt, unpublished observations). Howev- centration range studied, GABA was 2.5-25 times er, the GABA uptake inhibitor, nipecotic acid (200 more potent than THIP.