GABA and muscimol open ion channels of different lifetimes on cultured mouse spinal cord cells

242

Brain Research, 204 (1981) 242-247 © Elsevier/North-Holland Biomedical Press

GABA and muscimol open ion channels of different lifetimes on cultured mouse spinal cord cells

DAVID A. MATHERS and JEFFREY L. BARKER Laboratory o f Neurophysiology, National Institute of Neurological and Communicative Disorders and Stroke, Bldg. 36, Rm. 2C-02, Bethesda, Md. 20205 (U.S.A.}

(Accepted July 31st, 1980) Key words: GABA - - muscimol - - fluctuation analysis - - inhibition - - spinal cord neurons

Intracellular recordings of cultured mouse spinal neurons were used to examine the effects of yaminobutyric acid (GABA) and the GABA analog muscimol on neuronal excitability, Museimol was about twice as effective as GABA in increasing membrane conductance to Cl-ions. Fluctuation analysis of membrane current responses showed that muscimol activated ion channels with the same conductance as GABA but with about twice the average duration. The results provide an economical explanation for the greater potency of muscimol in depressing excitability. ~-Aminobutyric acid (GABA) is an amino acid endogenous to both invertebrate and vertebrate nervous systems where it is thought to function as a neurotransmitter, inhibiting excitability through an increase in C1- ion conductance 6,11,17. This inhibitory action can be readily mimicked by the plant alkaloid muscimol (3-hydroxy-5aminomethylisoxazole) whose structure resembles G A B A 7,9,1a,15. When compared to GABA, muscimol has always proved to be a more effective agonist, having equipotent inhibitory effects at a concentration several times lower than that required for G A B A 7,a,~a,15. Furthermore, the drug competes strongly with G A B A for receptor sites in binding assaysS,~, is, leading to the suggestion that muscimol can activate G A B A receptorsS,7,9,12,13,15, is. The advent of fluctuation analysis and single-channel recordings has shown that the elementary events associated with the actions of G A B A and other neurotransmitters may be equated with the opening of many individual ion channels4,14,16. The electrical properties of these channels depend on the nature of the neurotransmitter. The channel properties can therefore be used as an estimate of the molecular effectiveness of an agonist in terms of its ability to induce charge transfer across the cell membrane 18. We have applied fluctuation analysis to G A B A and muscimol responses on cultured mouse spinal neurons and have found an explanation for the greater protency of muscimol previously noted. Embryonic mouse spinal cord cells were grown in dissociated cell culture according to methods previously reported ~a. After 6 weeks in culture the ceils were large enough (about20--25/~m in diameter) to permit stable intracellular recordings on the modified stage o f an inverted phase microscope. The neurons were bathed in buffered

243

A

40pMGABA P

L

20juMMUSCIMOL

r

V

10PSI I 10mV 4nA 30SEC

I Slllllffilffil B

~

P 10pMGA

.~uMMUS

20/uMGABA

10pMMUS IIU'

V

C V

MUSCIMOL

Vc=-70mV

GABA

5mVI

M Im~

J~

-

Imll

X

O2 Fig. 1. GABA and muscimoi responses on cultured mouse spinal neurons. Membrane potential recordings in A were made using KAc microelectrodes, while those in B and C were made with KC1 microelectrodes. P is the trace monitoring positive pressure applied to micropipettes containing known concentrations of the agonists. The indicated polarity of the pressure pulses is arbitrary. I shows the current passed through a bridge circuit to sample membrane conductance during agonist action. The conductance evoked in each pair of agonist responses is approximately equal. C: with the cell's membrane potential clamped to --70 mV muscimol and GABA evoke membrane current responses, Im (upper trace) which are associated both with an increase in the thickening of the high gain, AC-coupled current recording I~n (lower trace) and with an increase in the membrane current variance, a2 (which was converted to a voltage for display). The bandwidth of the AC-coupled recording was 0.1-1 kHz and that for the variance 0.1-125 Hz. M is the event marker trace indicating when the 30-sec samples were taken for spectral analysis (see Fig. 2).

244 Hanks Medium containing 1 mM CaClz, 10 mM MgCI~ and 0.5 ,uM tetrodotoxin to eliminate all evoked synaptic activity and allow better examination o f agonist effects on membrane properties. Spinal cord (SC) neurons were impaled with either one or two microelectrodes filled with 4 M potassium acetate (KAc) or 3 M KCI (50-80 Mf~ resistance) and membrane properties recorded using conventional or voltage-clamp techniques. GABA and muscimol (Sigma, St. Louis) were applied by pressure to the cell body from 5/~m tip diameter pipettes filled with bathing medium containing agonists at known concentrations. On occasion G A B A was iontophoresed from a pipette containing 1 M agonist. Analysis of current fluctuations was carried out with the aid of PDP 11/10 and 11/40 computers. Both GABA and muscimol increased membrane conductance in a dose-dependent, reversible manner in all SC cells tested (Fig. 1). The polarity of the associated voltage response was hyperpolarizing with KAc recordings (Fig. 1A) and depolarizing with KCI recordings (Fig. IB). Responses to GABA and muscimol had identical inversion potentials, suggesting that muscimol, like GABA a, activates a C1 - ion conductance. Since pressure pipettes were not uniform we routinely increased the pressure applied until a maximum conductance response was obtained. On 10 cells where dose-response curves were performed, muscimol was typically about twice as potent as GABA in increasing membrane conductance over the linear part of the dose-response curve (Fig. 1A and B). Fluctuation analysis of agonist-induced current responses was performed on SC cells voltage-clamped with two KC 1 microelectrodes. The use of the KC 1-filled microelectrodes permits a relatively stable, sizable driving force 3. Results were obtained from 16 cells studied with G A B A alone, 6 studied with muscimol alone and 5 tested with both agonists. Both agonists induced an increase in membrane current accompanied by the appearance of additional membrane current variance (Fig. 1C). Variances of membrane current fluctuations during baseline records and agonist responses were averaged over 30-see samples. Agonist-induced variance, obtained by subtracting the former from the latter was proportional to the mean current change for responses up to 3 nA in amplitude. The agonist-induced fluctuations were analyzed on the assumption that they derive from the statistical variation in the number of agonistactivated open ion channels about a mean level of open channels. The average conductance, ),, of a single channel can be estimated from the relation 7 -- ~ / ( A I ( V e - - E R ) ) where tr2 is the variance associated with agonist-induced change in membrane current, &I, ER is the reversal potential of the agonist response and Ve is the holding TABLE I Properties o f channels activated by GABA and muscimol on cultured mouse spinal neurons

Conductance (pS) Duration (msec)

GABA (n = 21 cells)

?duscimol (n = 11 cells)

14.5 + 7 (mean 4- S.D.) 30.7 ± 7

12.7 4- 4.0* 67.2 4- 16"*

Student's t-test: *NS (P > 0.40);

** P < 0.001.

245

MUSCIMOL

S(f) ' s(o).l:

.01r

.f ~

" .... i

........ FREQUENCY

I0

.....

I--~

(Hz)

Fig. 2. Power spectral density of GABA and muscimol fluctuations on a single cultured mouse spinal neuron. Same cell as shown in Fig. IC. The spectra were obtained as the difference between the spectra derived from baseline fluctuations and those calculated during agonist applications. Those illustrated are the average of 17 'difference' spectra calculated for each of the agonists. The spectra have been normalized by dividing each spectral density point S(f), by the zero frequency asymptote of the spectrum, S(0). Least-squares analysis of the data revealed that both spectra were closely approximated by single Lorentzians (solid lines) described by the equation Sff)/S(0) = 1/[1 + (f/fc)2] whose half-frequency, fe is 5.05 Hz for GABA and 3.12 Hz for muscimol. Since mean channel lifetime, r = 1/2nfc, " ~ G A B A = 31.5 msec and t~tuse = 50.9 msec. potential under voltage-clamp. Estimates o f 7 for muscimol and G A B A were not significantly different (Table I), indicating that b o t h agonists open channels o f similar conductance. Estimates o f the average duration o f the elementary events were obtained f r o m the power spectral density o f current fluctuations in the presence and absence o f the agonists. The spectra were calculated as the Fast Fourier Transform s o f 6144-point samples o f m e m b r a n e current digitized at 5 msec per point. Spectra o f baseline fluctuations were subtracted f r o m spectra obtained during agonist responses to give difference spectra o f agonist-induced fluctuations. The results to be described were derived f r o m 139 observations with G A B A on 21 cells and f r o m 63 observations on 11 cells with muscimol. F o r holding potentials in the - - 5 0 to - - 9 0 m V range, agonist spectra could be described by a single Lorentzian term, S(f)/S(0) = 1/[1 q- (f/f e) 2] where S(f) denotes spectral density as a function o f frequency, f, and fc is the half-power frequency o f the spectrum 16 (Fig. 2). In terms o f a simple model o f channel operation, the nature o f the spectra suggests that b o t h agonists open a population o f channels with lifetimes exponentially distributed a r o u n d an average duration, T, given

246 by the equation z = 1/2~rfcl 6. As shown in Table I the mean lifetime of muscimolinduced channels, zMusc = 67.2 msec, is slightly more than double that of GABAinduced channels, ZGABA ---- 30.7 msec, the ratio VMrJSC/VGABA being 2.19. The lifetimes of both sets of channels appeared to vary little over the - - 5 0 to - - 9 0 mV range of membrane potential. The present results show that muscimol opens ion channels of about the same conductance and twice the average lifetime as those activated by GABA. Since the macroscopic agonist-induced current is proportional to the mean channel lifetime 1, this observation provides an economical explanation for the greater potency of muscimol on cultured neurons. However, the possibility that other factors, e.g. agonist affinity and available receptor number, may also contribute to the different potencies of GABA and muscimol cannot at present be discounted. The present results also show that an exogenous plant alkaloid can operate channels much in the manner of a natural ligand. This GABA-mimetic action of muscimol at the single channel level does not, however, prove that muscimol is interacting with G A B A receptors to open the same ion channels. Further, since several functionally distinct types of G A B A receptor have been identified 2,4,1°, competition for a site in a binding assay does not establish that muscimol is interacting with G A B A receptors coupled to C1- ion channels. If we, however, tentatively conclude that muscimol interacts with GABA receptors to open ion channels, then muscimol is a more effective agonist in causing charge transfer at these channels. It seems probable that extension of the present study to other structural analogues of G A B A will provide insights into the molecular determinants for ion channel gating at the G A B A receptor o f mammalian neurones. 1 Adams, P. R., Relaxation experiments using bath applied suberyldicholine, J. PhysioL (Lend.), 268 (1977) 271-289. 2 Barker, J. L., MacDonald, J. F. and Mathers, D. A., Three GABA receptor functions on cultured mouse spinal neurons, Brain Res. Bull., in press. 3 Barker, J. L. and Ransom, B. R., Pentobarbitone pharmacology of mammalian central neurones grown in tissue culture, J. Physiol. (Lend.), 280 (1978) 355-372. 4 Dudel, J., Finger, W. and Stettmeier, H., GABA-induced membrane current noise and the time course of the inhibitory synaptic current in crayfish muscle, Neurosci. Lett., 6 (1977) 203-208. 5 Enna, S. and Snyder, S. H., Influencesof ions, enzymesand detergents on GABA receptor binding in synaptic membranes of rat brain, Molec. PharmacoL, 13 (1977) 442-453. 6 Gershenfeld, H. M., Chemical transmission in invertebrate central nervous systems and neuromuscular junctions, PhysioL Rev., 53 (1973) 1-119. 7 Horri, N., Ikeda, K. and Roberts, E., Muscimol, GABA and picrotoxin: effects on membrane conductance of a crustacean neuron, Brain Research, 141 (1978) 364-370. 8 Jenkins, G. M. and Watts, D. G., Spectral Analysis and its Applications, Holden-Day, San Francisco, 1968. 9 Johnston, G. A. R. et al., Central actions of ibotenic acid and muscimol, Biochem. Pharmacol., 17 (1968) 2488-2489. 10 Kate, E. and Kuba, K., Inhibition of transmitter release in bullfrog synoptic ganglia induced by y-aminobutyric acid, J. Physiol. (Lend.), 298 (1980) 271-283. 11 Krnjevic, K., Chemical nature of synaptic transmission in vertebrates, Physiol. Rev., 54 (1974) 418-540. 12 Krogsgaard-Larsen, P., In F. Fonnum (Ed.), Amino Acids as Chemical Transmitters, Plenum, New York, 1979, pp. 305-322. 13 Krogsgaard-Larsen, P. et al., Structure and biological activity of a series of conformationally restricted analogues of GABA, J. Neurochem., 25 (1975) 803-809.

247 14 McBurney, R. N. and Barker, J. L., GABA-induced conductance fluctuations in cultured spinal neurons, Nature (Lond.), 274 (1978) 596-597. 15 Naik, S. R., Guidotti, A. and Costa, E., Central GABA receptor agonists: comparison of mnscimol and baclofen, Neuropharmacology, 15 (1976) 479-484. 16 Neher, E. and Stevens, C. F., Conductance fluctuations and ionic pores in membranes, Ann. Reo. Biophys. Bioengng., 6 (1977) 345-381. 17 Nistri, A. and Constanti, A., GABA and glutamate receptors in vertebrate and invertebrate nerve and muscle tissue, Progr. Neurobioi., 13 (1979) 117-235. 18 Olsen, F. W., Greenlee, D., VanNess, P. and Ticku, M. K., In F. Fonnum (Ed.) Amino Acids as Chemical Transmitters, Plenum, New York, 1978, pp. 467-486.