GABA receptors and the depressant action of pentobarbital

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I'INS -,lam+ttrv 1981

GABA receptors and the depressant act=on of pentobarbital Jeffery L. Barker and David A. Mathers GABA is considered to be an important neurotransmitter substance mediating inhibitory synaptic events throughout the CNS. Pentobarbital shares some of the cellular actions of GABA and the question of whether its GA BAmimetic effects involve GABA receptors is considered here. T-aminobutyric acid ( G A B A ) is an amino acid synthesized and secreted throughout the mammalian central nervous system (CNS) where it is thought to mediate several important forms of intercellular communication TM. Its role in neuronal function has been characterized primarily in terms of its ability both to depress the electrical excitability of individual nerve cells and to Fig. 1, Stereospecific actions of (+) and ( - ) pentobarbital studied using cultured neurons. A and B are intraeellular recordings made with KAc microelectrodes from two different cultured mouse .spinal neurons. The lower traces in each section show membrane potential and the upper traces indicate the duration o f drug application delivered by pressure from pipettes positioned close to the recorded cells. The pipettes contained I O01.LM o f either (+) or ( - ) pentobarbital (PB). 10 raM MgCl2 was added in both experiments to eliminate ~sFontaneousl'; evoked synaptic activity. (,41) Brief application o f (+) PB (indicated by upward deflection on upper trace) rapidly depolarizes the cell, causing excitation that oudasts the application period. ( - ) PB hyperpohtrizes the tell and increase.s membrane conductance as reflected in the depression of voltage responses to constant current (-0.25nA) stimuli during the hyperpolarizing respon.~e. Resting membrane potential: -51inV. (A2) With the membrane polarized to - 6 1 m V, repeated brief applications of(+) PB cause depolarizing re~po~es which suraraate sufficiently to excite the cell. (+) PB also evokea synaptic activity as reflected in the appearance o f discrete synaptic potentials superimposed on the depolarizing responses. Coincident application o f ( - ) PB blocks both the depolarizing events and the evoked synaptic activity induced by the (+) isomer. ( B I ) Dual effects o f the isomers. Application of lO01zM (+) PB ]or 50 m.~ec is strongly excitatory, while application o] lOOt.tM ( - ) PB for 500 msec n weakly excitatory. Membrane potential: -50inV. ( B 2 ) I sec applications o f the isomer~ to the same cell show that ( - ) PB transiently e~cites and then inhibits spontaneous fring, while (+) PB causes prolonged depolarization that first e.~citea and then depolarizes to a potential which inactivates spike generation. ( B 3 ) 5 sec application of the (+) isomer results in a complex response consisting o f an ininal depolarization followed by a progressive hyperpolarization. After termination o f the drug application, the membrane potential first depolarizes and then repolarizes to certain letels. A 5 sec application o f ( - ) PB leads to a sustained inhibition ofaction potential activity through an increase in membrane potential and conductance as reflected in the decrease in voltage respon,~es (downward deflections) to constant current ( -0.2hA ) stimuli. Membrane potential in B2 and B3: -46inV. I ht ~let/N~rth Holland Biomedical P I c ~ 19Sl

alter secretory processes at specific nerve terminals. The depression of excitability is generated at specialized synaptie junctions between nerve cells and the synaptic signal is brief, lasting less than a second. The alteration in neurosecretory release processes by G A B A has been observed following pharmacological application at peripheral sites. This effect appears to have

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distinctly different structural requirements from those involved in generating both post-synaptic signals and depolarizing responses at dorsal root ganglia. Activity on dorsal root ganglion cell bodies is considered to model GABA-mediated inhibition of transmitter release from primary afferent terminals, although alternative mechanisms k~r "pre-synaptic inhibition' have been proposed. The relationship among these different actions of G A B A and their importance in neuronal function need to be resoh'ed. Pentobarbital is a synthetic drug which has found widespread use in clinical medicine owing to its ability to induce a general anesthetic state. The substance is actually a racemic mixture of two stereoisomers which, when administered separately to animals cause qualitatively different changes in the excitability of the CNS TM. The (+) isomer elevates CNS excitability at low concentrations and depresses excitability at higher concentra-

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lions. The ( - ) isomer is only weakly excitatory, if at all. It produces a steadily progressive anesthetic state as concentration is increased. We will briefly review some recent studies which have closely examined the relationship between some of the cellular effects of pentobarbital on the excitability of CNS neurons and the synaptic actions of GABA.

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Chloride conductance changes One synaptic signal mediated by G A B A on central neuronal membranes involves an increase in conductance to CI- ions5. Since this effectively acts to stabilize membrane potential below the level necessary for action potential activity, the signal is considered to be 'inhibitory' to excitability. The racemic mixture of pentobarbital isomers used clinically can also activate membrane conductance to CI- ions and 30 SEC reduce excitabilitye. The ( - ) isomer is Fig. 2. Membrane current responses to ( - ) pentobarbttal and GABA are both asssociated with an increase in more potent than the ( + ) isomer in evok- membrane current variance. The responses were obtained on a cultured mouse spinal cord cell recorded with two ing this activity8 (Fig. 1), as it is in inducing KCI microelectrodes used in a voltage clamp configuration to hold the cell's membrane potential at - 6 0 m V (V). anesthesia 14. In fact, at low concentrations ( - ) PB was applied by pressure from a pipette containing I O0#M (pressure application period shown in top trace, P). GA BA was iontophoresed from a 1M solution (1). The high-gain, A C-coupled membrane current record (Ira) the (+) isomer of pentobarbital directly is illustrated above the low-gain, DC-coupled record (Ira). Membrane current variance converted to a voltage is excites individual neurons (Fig. 1), while at displayed at the bottom (o'1). Both substances evoke membrane current responses associated with a thickening of higher concentrations it depresses excita- the current tracings and coincident increase in variance. (Copyright, 1980, by the AmericanAssociationfor the bility by increasing the CI- conductance of AdvancementofScience). individual cells. Some structural specificity ion channel when activated by G A B A and drug is estimated to be about five times as for these direct effects is also suggested by ( - ) pentobarbital. Using this assumption long as that opened by G A B A (Fig. 3). At the fact that anticonvulsant barbiturates do we have estimated that the ion channel this level of resolution it appears that the not activate CI- ion conductance over a events for G A B A and ( - ) pentobarbital drug is able to act in a way which mimicks range of concentrations that is clinically are of similar conductance but very differ- the ion channel mechanism utilized by the relevantL ent duration - the channel activated by the amino acid, only the kinetics are different. The elementary events underlying the 4macroscopic changes in agonist-evoked C1- conductances have been studied in detail using cultured mouse spinal neurons because this preparation affords an accessibility to high resolution electrophysiological techniques that is not presently obtaini " v able in vivo. Although all cultured spinal ~_ $(f) cord cells studied thus far appear to be sensitive to GABA, less than half of the cells responded to the ( - ) isomer. The mem~Q brane responses to both G A B A and ( - ) .0(?4. ' ~ pentobarbital are both associated with the appearance of additional membrane current variance 1° (Fig. 2, see also article by .0004, Cull-Candy in this issue, pp. 1-3). Microscopic fluctuations in membrane current -~41 i 10 10o .1 i Io ~o associated with chemically induced FREQUENCY (Hz) responses in muscle cells have been shown to reflect the variation in the number of Fig. 3. Spectral analysis o f membrane current fluctuations induced by ( - ) pentobarbital and GA BA in a cultured elementary events which contribute to the mouse spinal neuron. The spectra were calculated as the fast Fourier transform o f 6144.point samples of response ~1. This elementary event is a membrane-current digitized at 5 msec per point. A. Spectrul analysis of baseline fluctuations in almost all of the cells studied show that in the absence o f agonist, power spectral density is proportional to the inverse o f frequency over step-wize change in conductance which the entire frequency range o f the spectrum and can be approximated by a linear relationship between power spectral occurs in practically an all-or-none man- density and frequency. B. Baseline spectra were subtracted from those obtained during agonist responses and the ner. If we assume that the fluctuations resulting 'difference" spectra were then normalized by dividing each spectral density point by the zero frequency which are observed superimposed on the asymptote o f the spectrum. Least squares analysis o f the normalized spectra show that both are closely approximacroscopic responses shown in Fig. 2 can mated by single Lorentzian equations (solid lines). The cut-off frequency o f such spectrum, fc (marked by downward arrowhead), can be converted into channel lifetime, r, by the fact that r = 112 rrft. Here r = 33.7 msec for be analyzed in the same manner, then we GA BA and 129.1 rasec ]'or ( - ) PB. (Copyright,1980, by the AmericanAssociationfor the Advancementof Scican estimate the properties of single CI- ence).

12

FINS -Janttarv 1981

Possible mechanisms

Presumably G A B A activates C I ion channels by interacting with specific receptor sites in the cell membrane. We have made estimates of single channel properties for a series of synthetic substances structurally related to G A B A and have fl)und that all of the agonists activate channels whose conductance is similar to that of G A B A but whose durations differ significantly ~. The average duration of a single channel event activated by the different agonists correlates significantly (p<.001) with the concentration required to displace G A B A in competitive binding assays on frozen rat brain synaptic membranes. The close correlation suggests that the two assays are measuring a parameter important to G A B A receptor function and imply, but do not prove that the G A B A

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receptor site used in binding assays is related to CI- ion channels. Pentobarbital also activates an ion channel whose apparent conductance is indistinguishable from that of G A B A , but there is no evidence of competitive displacement of G A B A by the drug in binding experiments. Rather, the drug has recently been shown to potentiate G A B A binding by increasing the apparent U .\ affinity of G A B A receptor sites for "\ G A B A TM. Therefore if we interpret the correlation as indicating that all of the synQ\o thetic substances showing competitive displacement of G A B A in binding assays can engage the same G A B A receptor site to \\ activate CI- conductance in neuronal membranes, then ( - ) pentobarbital is unlikely to open CI ion channels by engago /~ ~ ~i~ 11e, ~'.o ~'.~ ing G A B A receptors. However, the "nME(SEC) stereospecific requirements for drug action Fig. 4. ( - ) Pentobarbital potentiates GABA-induced suggest that the interaction of the drug with membrane conductance on a cultured neuron. KAc the m e m b r a n e involves at least one type of recording from a cultured mouse spinal neuron• Memchemical reaction with relatively fixed spabrane potential traces show responses to iontophoresis tial geometry, much in the manner of a o f GA BA in control and during coincident application receptor. It is thus possible that ( - ) peno f lO0~M ( - ) pentobarbitaL Membrane conductance can be calculated from the amplitude o f the tobarbital might engage a relatively downward-going vohuge responses to constant-current specialized receptor-like site in the memstimuli (-O,25nA), which is inversely related to membrane. We have only characterized one brane conductance• Inspection of the figure shows that GABA increases membrane conductance without other type of CI ion channel event associated with the response to a naturally occurchanging membrane potential because the cell is polarized to the inversion potential o f GABA responses • ring ligand on cultured neuronal mem(-60mV), which is the potential where the electrical branes. The ligand is the neutral amino acid and chemical gradients underlying the GA BA response glycine and the elementary event activated are equal and opposite. Membrane conductance by it has electrical properties which are sige~'oked by G A BA has been plotted as a function o f time (open circles). ( - ) Pentobarbital potentiates the nificantly different from those of ( - ) penGA BA-induced conductance change and increases the tobarbital and G A B A both in conductance time constant o f decay (downward arrowheads) withand duration s . Alternatively, the drug out itself having any detectable effect on membrane could interact directly with CI- ion channel properties and without changing the inversion potential for GABA. (J. L. Barker and L. M. Huang, unpub- mechanisms to activate them. Still another possibility stems from the lished obserxations).

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fact that barbiturates are known to potentiate G A B A responses (Fig. 4) 9. The potentiated G A B A responses are associated with ion channel events whose durations are considerably prolonged over control values 4. Thus the action of ( - ) pentobarbital might be due to modulation of CI- ion channels activated by trace amounts of G A B A . But inspection of the baseline spectra obtained just prior to the application of the drug does not reveal the presence of ambient G A B A . If there is G A B A present, it is below the level of detection by this electrophysiological assay. The possibility that the drug causes release of G A B A wi/h coincident modulation of GABA-activated channels cannot be eliminated. However, there is no evidence of any CI- dependent, synaptic-like events released during ( - ) pentobarbital application, so if release does occur, it must be other than quantal in nature. In summary, pentobarbital has a variety of cellular actions which would be expected to contribute to depression of CNS excitability. It appears to be able to activate CI ion channel-like mechanisms in the membrane of CNS neurons possibly through engagement of receptor-like sites. Although the elementary signal evoked by the drug has the same amplitude as that activated by G A B A and although the drug effect can be antagonized by agents which block G A B A responses TM, we cannot yet conclude that the drug activates a G A B A receptor-coupled CI- ion conductance. A second action of the barbiturates involves potentiation of G A B A responses and this modulatory' action also shows some stereospecific requirements. The effect appears

T I N S - J a n u a r y 1981

to be due at least in part to prolongation of channel lifetime. The increase in apparent affinity of membrane binding sites for G A B A in the presence of pentobarbital may be related to the prolongation of the elementary event estimated from fluctuation analysis of G A B A responses potentiated by barbiturates. It remains to be shown whether these transmitter-like and modulatory actions of pentobarbital really do reflect pharmacological activation of m e m b r a n e receptors, and by implication, what physiological roles such receptors might play in neuronal function.

13 8 Huang, L. M. and Barker, J. L. (1980) Science

207, 195-197 9 Macdonald, R.L. andBarker, J.L.(1978)Science

200, 775-777 10 Mathers, D. A. and Barker, J. L. (1980)Science 209, 507-509 11 Neher, E. and Stevens, C. F. (1977)Ann. Rev. Biophys. Bioeng. 6, 345-381

12 Nicoll, R. A. and Wojtowicz, J. M. (1980) Brain Res. 191,225-237 13 Nistri, A. and Constanti, A. (1979) Prog. NeurobioL 13, 117-235 14 Waddell, W. J. and Baggen, B. (1973)Arch. Int. Pharmacodyn. Ther. 205, 40-44 15 Willow,M. and Johnston, G. A. R. (1980) Neurosci. Len. 18, 323-327

Reading list

1 Allan, R. D., Evans, R. H. and Johnston, G. A. R. Biochem. Pharmacol. (in press) 2 Barker, J. L. and Mathers, D. A. Neurosci. Abstr. 10 (in press) 3 Barker, J. L. and McBurney,R. N. (1979) Nature (London) 277, 234-236 4 Barker, J. L. and McBurney, R. N. (1979)Prc~. R. Soc. London B 206, 319-327 5 Barker, J. L. and Ransom, B. R. (1978)./. Physiol. 280, 331-354 6 Baker, J. L. and Ransom, B. R. (1978)J. Physiol. 280, 355-372 7 Bowery, N. G., Doble, A., Hill, D. R., Hudson, A. L., Shaw,J. S. and Turnbull, M. J. (1979) Br. J. Pharmac. 67,444P-445P

Dr Jeffery L. Barker was born in New York City and received a B.A. (magna cum laude) from Harvard Collegeand an M.D. from Boston UniversitySchool of Medicine. After an interns'hipin surgery he came to the National Institutes of Health to do basic research in the neurosciences. Dr Barker is with the Laboratory of Neurophysiology of the National Institute of Neurological and Communicative Disorders and Stroke where he is a Medical Officer in the Public Health Service.

Effects of alcohol on the developing nervous system Salvador Borgesand Paul D. Lewis The s t u d y o f the effects on fetus a n d child o f maternal alcohol intake during p r e g n a n c y is n o t new, f o r descriptions o f clinical findings supported b y laboratory experiment date b a c k to the last century. W h a t is new, however, is the general recognition o f alcohol (ethanol) as an agent which m a y significantly perturb n o r m a l somatic a n d behavioural development. This article examines evidence that alcohol adversely affects the developing brain, and looks at s o m e possible mechanisms and lines o f approach.

In the past, prevailing social and cultural constraints have strongly influenced the acceptance of medical observations, periods o f alarm alternating with those of complacency and often resulting in scepticism or rejection of the published findings22. The crusading and moralistic tone of turnof-the-century temperance writings created an environment in which impartial scientific research on alcohol was very difficult, while enthusiasts of social Darwinism and eugenics were convinced that defective offspring of alcoholic mothers were exclusively the result of 'neuropathic heredity' and not of alcohol teratogenicity. By the 1920s and with the arrival of Prohib-

ition in the U.S., many American researchers came to regard alcoholism as a past problem, and in Britain, despite the absence of national prohibition, research on the subject languished until World War II. During the 1940s, pre-Prohibition literature was derided as being 'unscientific' and 'uncontrolled' and as a result noteworthy research went unacknowledged. 'Fetal Alcohol Syndrome'

Such was the scientific consensus on the effects of alcohol on the developing nervous system that as recently as 1972 it could be authoritatively asserted that the evidence for a link between maternal alcohol-

Dr David A. Mathers is a native of St. Andrews, Scotland. After receiving a B.Sc.(Hons.) in Zoology from Edinburgh University, he undertook doctoral research work at the Universities of Glasgow and Nottingham. He subsequently commenced a postdoctoral studies in the Pharmacology Department of Lund University, Lund, Sweden and the Physiology Institute of the Technical University in Munich, West Germany. He is currentlya Fogarty Visiting Fellow at the National Institutes of Health, Bethesda, Maryland.

ism and abnormal fetal development was largely negative 6. In the same year a clinical report described intrauterine growth failure in most of the offspring of a group of alcoholic mothers 21. This report was soon followed by the description 8 from Seattle of a stereotyped pattern of abnormal development in children born to alcoholic mothers, a pattern which has been termed 'Fetal Alcohol Syndrome' (FAS). Since then, cases have been reported from other countries, including 127 cases in a 'rediscovered' 1968 study from France TM. The most prominent features of FAS are prenatal and postnatal growth deficiency, microcephaly, areas of abnormal nerve cell migration in the brain, craniofacial abnormalities, mental and psychomotor retardation and cardiovascular defects. There is no single feature that is pathognomonic and there is a degree of overlap with several other syndromes, both genetic and due to environmental influences. As a result the FAS remains to some extent controversial and doubt as to its existence has been expressed in some quarters. Its supporters maintain that the complete syndrome, associated with high levels of alcohol consumption during pregnancy, is comparatively rare, but point out that a constellation of minor developmental defects appear to be linked with moderate con(~ Elsevier/North-Holland Biomedicat Press 1981