0306-4522/92 E5.OOf0.00 Pergamon Press plc 0 1992 IBRO

Neuroscience Vol. 46, No. 4, pp. 931-941,1992 Printed in Great Britain

PRIMARY

RECEPTOR IN LAMPREY

FOR INHIBITORY TRANSMITTERS SPINAL CORD NEURONS

K. V. BAEV,K. I. RUSINand B. V. SAFRONOV* Department of Spinal Cord Physiology, A. A. Bogomoletz Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Bogomoletz str. 4, 252601 GSP, Kiev 24, U.S.S.R. Abstract-The action of glycine and GABA on isolated lamprey spinal cord neurons was investigated by means of intracellular perfusion and concentration clamp techniques. These amino acids activated desensitizing chloride ionic conductances. The concentrations of agonists evoking half-maximum effects (EDJ were equal to 16 PM and 1.5 mM for glycine- and GABA-activated currents, respectively. Increase in the transmitter concentration led to a decrease in the time constant of desensitization. Current-voltage relationships of glycine- and GABA-activated currents were strongly dose-dependent. At low agonist concentrations the time constant of activation decreased with membrane hyperpolarization. Glycine- and GABA-activated currents exhibited complete cross-desensitization. The specific glycine antagonist, strychnine, suppressed both glycine- and GABA-activated currents to the same degree. Selective antagonists of GABA receptors, bicuculline and picrotoxin, produced equal blocking effects on glycineas well as GABA-evoked responses. In the cells studied, taurine activated desensitizing ionic conductance. Responses evoked by taurine and glycine applications demonstrated complete cross-desensitization. Taurine-activated currents were sensitive to strychnine, bicuculline and picrotoxin. These results suggest the existence of one receptor-channel complex for the main inhibitory transmitters in lamprey spinal cord neurons. 3,CDioxy-L-B-phenylalanine evoked desensitizing strychnine-sensitive ionic responses which exhibited cross-desensitization with glycine-activated currents.

in the sensitivity to glycine and GABA,‘8*24 while these transmitters are equally potent in mamate molecular structures-receptors controlling conmalian (mouse) spinal cord neurons.5 ductance of the transmembrane ionic channels.‘6*32 Recently, taurine has been considered as a possible Many substances activating either glycine or GABA candidate in inhibitory mediators. However, there are receptors have been found.6,7*33,” Strychnine and some discrepancies in the data on the interaction of bicuculline were described as the specific reversible taurine molecules with receptors. It was found that blockers of glycine- and GABA-evoked responses, taurine’s inhibitory action was similar to that of respectively.9 GABA in frog motoneurons,‘0J5,26 whereas it actiThere are, however, some unusual data about the vated glycine receptors in rat brain neurons*’ and effects induced by these amino acids. In the Mauthner produced a strychnine-sensitive depressant action on cells of goldfish the pretreatment of the preparation spinal neurons.4s” with a solution containing one inhibitory transmitter It was interesting, therefore, to study the influences led to a reduction of the sensitivity to the other in the of some inhibitory transmitters and their agonists on case of short transmitter applications.13 Furthermore, isolated spinal neurons of lower vertebrates, such as the main lifetimes of glycine- and GABA-activated the lamprey, in “concentration clamp”” conditions. channels were equal to 7.15 ms for both amino Preliminary data have been reported in a preceding acids.14 A partial cross-desensitization between glycommunication.3’ tine and GABA has also been described for the cultured spinal neurons of the 13-14-day-old mouse EXPERIMENTAL PROCEDURES embryo,3 which implies the existence of receptors The middle part (about 5-7cm) of the spinal cord was sensitive to both transmitters. It has also been found dissected from 25-30 cm lamprey (Zmnpetrafluuiurilis) after that glycine and GABA activated the same type of 5 min anaesthesia with 5% urethane solution. After the chloride channels which differ only in the structure of preparation of 2-3 mm cross-slices thev were subiected to the recognition site.’ It should be noted that such k&ymatic treatment with a solution contain&g 0.3% pronase for about 2 h at lO-12°C. Dissociation of the cells effects were observed for the central neurons of was produced by passing the slices through a glass pipette vertebrates situated on the lower levels of evolutionwith 0.5mm tip diameter under binocular observation. ary or ontogenetic development. Isolated spinal neurons were of size 50-120 pm and had a characteristic exterior as shown in Fig. 1. The spinal and reticulospinal neurons of lower It was impossible to perform any morphological identifivertebrates (lamprey) demonstrate essential differIt is generally accepted that the main inhibitory transmitters glycine and GABA interact with separ-

*To whom correspondence should be addressed. Abbreviation:

L-DOPA, 3,4-dioxy-L-/3-phenylalanine.

ences

cation of the studied neurons in our experimental conditions. The present study was carried out on 124 neurons. Normal extracellular solution had the composition (in mM): NaCl 120, KC1 4, CaCl, 3, MgCl, 2, glucose 20,

931

932

K. V. BAEVet al.

Fig. 1. Isolated spinal cord neuron of lamprey.

HEPES-NaOH 20 (pH 7.3). Enzymatic treatment was carried out with a solu~on containing (in mM): NaU 120, KC1 4. CaCl, 0.25. EGTA 1. trlucose 20. HEPES-NaOH 20 (PH 7.3): The standard i&&lhtlar perfusion solution contained (in mM): KF 110, gh~~~se 20, Tris-HCl SO(PH 7.3). To determine the ionic nature of the transmitter-activated currents, Cl-, K+-free intracellular solution was used (in mM): NaF 140, gha~~ 20, HEPES-NaOH 10 (PH 7.3). Glycine and GABA were from Sigma. Solutions containing bicuculline and 3,4dioxy-L-Bphenylalanine (L-DGPA) (Reanal, Hungary) were prepared directly before use. All experiments were carried out at 414°C. Voltage clamp and current recordings from the whole

cells were made using glass pipettes.” A 2OOMn resistor was used in the feedback of the current-voltage converter. A Pyrex glass with 2 mm outer diameter was pulled to a tip diameter of about 50 pm in two stages and then !ire polished to an inner diameter of about 5-10 pm. Pipettes f&d with standard intracellular solution had resistances of about 0.3-0.5 MR. Large recording currents in some cases led to a certain shift in clamped potential. However, they did not change the qualitative character of the data obtained. Possible poor space clamp conditions for neurons with long neurites did not make an essential contribution to the present results because similar effects were also observed for relatively small cells (about 50 pm) with one neurite. Liquid junction potentials were corrected as described by Fenwick et al.‘$ The “concentration clamp” method was used for rapid amino acid applicationss” The tip of the pipette containing the perfused cell was positioned into a “J”-shaped glass capillary tube. The suction applied to the upper end of the tube was controlled by an electromagnetic valve. The application was produced by lowering the tube into the dish with agonist-containing solutions and simultaneous opening of the valve. Extra&h&u solutions were exchanged for about SOms. This was estimated by the disappearance of the voltage-activated sodium currents after application of the Na-free solution.

RESULTS Glycine-activated currents

All cells studied (N = 124) responded to glycine application (at holding potential, - 1OOmV) by desensitizing inward ionic currents. Figure 2A demonstrates responses evoked by different amino acid concentrations. An increase in the transmitter dose led to a decrease in the time constant of desensitization. For the currents shown in Fig. 2A this changed from 500 to 250 ms when glycine concentration increased from 10 to 100 PM. Desensitization kinetics were strongly temperature-dependent. In most cells glycine-activated currents exhibited almost complete desensitization, whereas in some A 1

1

10

100

$4

Fig. 2. Glycine-activated currents. (A) Cell responses to application of different glycine concentrations (shown near each trace in PM). Holding potential, - 1OOmV. (B) Dose-response relationship for the experiment shown in A. The smooth curve corresponds to the Hill equation with n =2 and ED~= 16pM.

Glycine- and GABA-gated Cl- current neurons

the responses

component (about 510% of the peak value). Threshold glycine concentrations were of about 5-10pM; 0.2-W mM glycine evoked maximal responses. The concentration evoking half-maximum effect (ED~) for the peak values was found to be 16 Z.IM from dose-response dependence (Fig. 2B). Hill’s coefficients varied from 1.2 to 2.5. For the steady-state currents in our eXperimentS, ED% WaS about three times less than that for the peak values. Neuron responses evoked by 0.1 mM glycine at different holding potentials are shown in Fig. 3A. The currents reversed at -25 to - 30 mV (N = 8), which was in good agreement with the chloride equilibrium potential in the present experimental conditions. To estimate permeability of the channels to other ions (F-, K+,Na+), experiments with perfusion of the neurons by Cl-, K+-free intracellular solution (see Experimental Procedures) were performed. In this case reversal potential shifted to - 75 mV, which gave the ratio of permeabilities of Nat, K+ and F- ions to Cl- as not exceeding 0.05. Current-voltage (Z-V) dependence of the responses evoked by saturating glycine concentrations demonstrated linearity at all voltages studied. Kinetics of the responses were not voltage-dependent. However, at low transmitter doses (not higher than 10 PM) Z-V relationships deflected to the axis of potentials in the voltage range from -60 to - 100 mV and the activation rate of these responses increased with depolarization (Fig. 3B). GABA -activated Each

neuron

currents investigated

A

a small steady-state

revealed

was also

sensitive

to

GABA application, responding with desensitizing chloride ionic currents. GABA responses, as well as A nA

1OOpM-glycine

10

BABA --w-m

cl

nA

5 -100 =+f-

-40.

20 mV -5

Fig. 4. GABA-activated conductances. (A) Neuron responses to rapid applications of different GABA concentrations (indicated near each trace in mM). Holding potential, - 100 mV. Recordings in Figs 2A and 4A were obtained from the same neuron. (B) Dose-response relationship for the experiment shown in A. The data are fitted by the curve of the Hill equation with n = 2 and ED~= 1.5 mM.

(C) Recordings of ionic responses elicited by applications of 1mM GABA at different membrane potentials (in mV). (D) I-V relationship for the recordings shown in C.

glycine-activated responses, sometimes had small (about 510% of the peak values) steady-state component. Examples of the current elicited by different GABA doses are shown in Fig. 4A. Being applied to the same neuron, the saturating glycine and GABA concentrations evoked responses of almost equal amplitude (Figs 2A and 4A). Meanwhile, the kinetics of desensitization for GABA-activated currents were about twice as slow as those for responses evoked by glycine (Fig. 2A). In the traces in Fig. 4A the time constant of desensitization decreased from 1 to 0.5 s when the dose of GABA changed from 1 to 10 mM. The activation threshold for GABA was about 0.1-0.2 mM (Fig. 4B). rmso was found to be 1.5 mM. Hill’s coefficient varied from 1.6 to 2.1. Ionic responses to non-saturating GABA concentrations exhibited strong voltage dependence of the activation kinetics (Fig. 4C) (N = 6). The time constant of activation increased considerably with hyperpolarization. The Z-V relationship exhibited large deflection from linearity at potentials from -60 to - 100 mV (Fig. 4D), as described above for currents evoked by low glycine concentrations. It should be mentioned that when the neuron lost sensitivity to glycine during the experiment, it no longer responded to the application of GABA.

7+ 5

-100

-40

mV

-5

-10

B 2.5 @l-glycine

IPA -100

-LO

4

0 mV

74

A

-4

2nA

750 ms

Fig. 3. Voltage dependence of glycine-activated currents. Recordings and I-V relationshins for ionic currents elicited by appl&ions of high (0.1 mM; A) and low (10 PM; B) concentrations of glycine at different holding potentials (indicated near the corresponding traces in mv).

Cross-desensitization

experiments

Subthreshold glycine doses applied to the neurons desensitized most of the receptors (N = 22). After this, even saturating concentrations of the transmitter could not evoke maximal responses. The desensitiza-

934

K.V. BAEVet al. A

0.1 m M - g t y c i n e

~ |ycine 20

"-

~I

_

0.5 0.0.I

0.5

~J /

J2nA glycine d"

5mM-GABA

0.5S

0.1

1

10

C,pM

Fig. 5. Steady-state desensitization of glycine and GABA receptors by glycine. (A) Recordings of ionic currents evoked by applications of 0.1 mM glycine (upper traces) and 5 mM GABA (lower traces) following 30 s preincubation of the cell in a solution containing different concentrations of glycine (shown near the corresponding traces in #M). Holding potential, -100inV. (B) Dose dependence of the steady-state desensitization of glycine (O) and GABA (O) receptors by glycine for the experiments shown in A. tion was reversible and the cell again acquired initial sensitivity to glycine after 15-30 s of washing in the normal solution. However, the same concentrations of glycine could also desensitize GABA-activated receptors. To determine the interdependence between glycine- and GABA-evoked responses, a quantitative study of cross-desensitization was carried out. The scheme of the experiments was as follows: control doses of glycine or G A B A were applied to the neuron after 30 s preincubation in the solution containing the glycine concentrations studied. Changes in the amplitude of control responses indicated the amount of desensitized receptors. Steady-state desensitization of glycine- (upper traces) and GABA-activated (lower traces) currents by different glycine concentrations is illustrated in Fig. 5A. Dose dependences of the steady-state desensitization are shown in Fig. 5B. Two-fold decreases of control responses were observed at 2 # M glycine for glycine- and 1 # M glycine for GABA-activated currents. The kinetics of the responses did not change with the value of desensitization. Like glycine, pretreatment of the neurons with G A B A also reduced the amplitudes of the responses to application of both transmitters ( N = I 6 ) . Figure 6A demonstrates steady-state desensitization of glycine (upper traces) and GABA-activated (lower traces) currents by GABA. There was little difference between concentration dependences of desensitization for responses evoked by control applications of glycine and G A B A (Fig. 6B). Responses with halfmaximal amplitude were observed after cell preincubation with 0.5 and 0.2 mM G A B A for glycine- and GABA-activated currents, respectively. When the response to one transmitter included the steady-state current, then this was also present in the response to the other transmitter. If application of one amino acid saturated the steady-state component

then it could not be increased by the addition of the other one. The action o f specific blockers

The following experiments were carried out using specific blockers of glycine receptors (strychnine) and GABA receptors (bicuculline and picrotoxin). In these experiments a solution containing control concentration of transmitter with blocker was applied to the neuron after 30s preincubation in a solution containing the same concentration of blocker only. Taking into account the difference in cell sensitivity to both amino acids, the control concentration of GABA was chosen about two orders of magnitude higher than that for glycine. Responses to both transmitters demonstrated sen-

A

B

0.5 mM-gtycine

GABA

i

a1 2 0 GABA

~

n

v

A 0.25 s

&5

5 mM-GABA

. • 0.25nA

0.1

I

C, ml~

0.5s

Fig. 6. Steady-state desensitization of glycine and GABA receptors by GABA. (A) Ionic responses evoked by applications of 0.5 mM glycine (upper traces) and 5 mM GABA (lower traces) following 30 s preincubation of the cell in a solution containing different concentrations of GABA (shown near the corresponding traces in raM). Holding potential, -100 mV. (B) Dose dependence of the steadystate desensitization of glycine (Q) and GABA (O) receptors by GABA for the recordings presented in A.

Glycine- and GABA-gated Cl- current

935

Fig. 7. Blocking effect of strychnine on glycine- and GABA-activated currents. (A) Currents activated by applications of 50 PM glycine (upper traces) and 10 mM GABA (lower traces) in the presence of different strychnine (str) concentrations (indicated near the corresponding recordings in M). Holding potential, - 100 mV. (B) Dose dependence of suppression of currents activated by 50 PM glycine (0) and 10 mM GABA (0) by strychnine (from the experiment in A). (C) Recordings of glycine-elicited currents in the absence and presence of strychnine at membrane potentials of +20 and - 100 mV. (D) Prolongation of the desensitization kinetics of currents activated by 0.5 mM glycine under the action of different strychnine doses (given in M).

sitivity to the presence of strychnine in the extracellular solution (N = 12). Examples of the ionic currents

evoked by applications of 50 PM glycine (upper traces) and 1OmM GABA (lower traces) in the presence of different strychnine concentrations are shown in Fig. 7A. The blocker suppressed the responses to both transmitters to the same degree (Fig. 7B). Strychnine block was reversible and did not exhibit any voltage dependence (Fig. 7C). A considerable prolongation of the activation and desensitization kinetics of ionic responses was a peculiarity of the strychnine blocking action (Fig. 7D). This phenomenon can be clearly seen in the responses to saturating transmitter concentrations at high temperature of solutions, about 14”C, when the desensitization kinetics were most rapid. Increase in the blocker dose enlarged the time constant of activation and desensitization and led to a considerable change in the decay kinetics. Bicuculline, at doses higher than 1 FM, demonstrated a blocking effect on glycine- as well as GABAactivated currents (N = 8). Figure 8A shows the effects of different bicuculline concentrations on the responses evoked by applications of 50 FM glycine (upper traces) and 5 mM GABA (lower traces). The effect of activation and desensitization kinetics pro-

longation, described for strychnine, was also observed, although less pronounced for bicuculline action. Bicuculline produced equal dose-dependent suppression of glycine- and GABA-activated currents (Fig. 8B). The blocking effect depended neither on the holding voltage nor on the direction of ionic current (Fig. 8C, D) for both transmitters. Picrotoxin, like strychnine and bicuculline, also demonstrated a reversible blocking effect on the responses to glycine and GABA (N = 7). Figure 9A shows examples of ionic currents evoked by application of 50 p M glycine (upper traces) and 10 mM GABA (lower traces) in the presence of different picrotoxin concentrations. The blocker did not evoke any changes in the kinetics of response in all the neurons studied. Picrotoxin suppressed responses to both amino acids to the same degree (Fig. 9B). The half-blocking effect was achieved at 10 and 8 PM picrotoxin for glycine- and GABA-activated membrane currents, respectively. The blocking effect was not dependent on the membrane voltage and direction of the ionic current (Fig. 9C). Taurine-activated currents All neurons studied (N = 15) were sensitive to taurine. Its application at doses higher than 5-10 fiM

936

K.V. BAEVet al.

A bic

I

A

10~"~

bic , 100pM- glycine *20

50 0 V bic

20s

tourine

50 I~M-gtycine

~

~

..,J lOO

,oo

O,5s

FF. 2.5 s

5mM-GABA

lOO

I~_

/

1.5 s

500

C [/ Jo.snA (175 s

o

v

-100

0.Ss

0.5

D

B

nA -100 V

-lOO

-40

v

D

-100

Y If:

0.5 s

'

.40m~29

"mV

"

~o

lb

;

1;o

10b0 o,'~M

Fig. 8. Suppression of glycine- and GABA-activated currents by bicuculline. (A) Traces of currents evoked by applications of 50/~M glycine (upper traces) and 5raM GABA (lower traces) in the presence of different bicuculline (bic) concentrations (indicated near the recordings in # M). Holding potential, - 1 0 0 mV. (B) Dose dependence of suppression of currents activated by 50#M glycine ( 0 ) and 5 mM GABA (O) by bicucuUine (from the experiment in A). (C) Recordings of glycine-elicited currents in the absence and presence of bicuculline at membrane potentials of + 20 and -100 mV. (D) i-v relationship of the ionic currents evoked by applications of 0.1 raM glycine in the absence ( 0 ) and presence (@) of 0.1 mM bicuculline.

Fig. 10. Ionic currents evoked by rapid applications of taurine. (A) Cell responses to the applications of different taurine concentrations (indicated by # M). Recordings of the steady-state currents are given at higher resolution. Holding potential, - 100 mV. (B) Dose-response relationships of the peak values (I-q) and the steady-state components (11) for the recordings from A. Hill's coefficient and EDs0 for the steady-state components (n and EBb0) were found to be 1.5 and 12/~M, respectively, by the least squares method. By calculations of Hill's coefficient and EDs0for the peak values (ED~0), 1.5 and 86 #M, three initial points of the dose-effect relationship were not taken into consideration because of slow activation kinetics of the responses at low agonist doses. (C) Recordings of currents activated by applications of 0.5mM taurine at different membrane potentials. (D) l - V relationship for the experiment shown in C.

evoked desensitizing chloride ionic currents (Fig. 10A) similar to those described for glycine and GABA. riDs0 was found to be 86/~M for the peak values and 12/~M for the steady-state currents. Hill's

coefficient was 1.5 for both components (Fig. 10B). At saturating taurine concentrations, I - V dependence was linear at all potentials studied (Fig. 10C, D).

8

J

,

1

10o IJM

10

A

•

B

pic

S0 uM-~I~,cine

pic

10 mM-GABA

50 --~'~ 5 1

C pic

.

~

10 L

is

50 MM-glycine

~ 1

, ~ 10

-J 1001JM 0 V

O.5s

Fig. 9. Effect of picrotoxin on giycine- and GABA-activated currents. (A) Traces of currents evoked by applications of 50 #M glycine (upper traces) and 10 mM GABA (lower traces) in the presence of different picrotoxin (pie) concentrations (indicated near the corresponding recordings in/~M). Holding potential, - 1 0 0 inV. (B) Dose dependence of suppression of currents activated by 50 #M glyeine ( 0 ) and 10 mM GABA (©) by picrotoxin (from the experiment in A). (C) Recordings of glycine-elicited currents in the absence and presence of picrotoxin at membrane potentials of +20 and - 1 0 0 inV.

937

Glycine- and GABA-gated Cl- current

In the study of cross-desensitization (N = 3) different glycine doses reduced, to an equal degree, the responses to control glycine (upper traces) and taurine (lower traces) concentrations (Fig. 11A, B). The responses of cells to taurine applications were sensitive to each of the three specific antagonists (Fig. 11C). Non-speczjIc agonists of inhibitory receptors L-DOPA, when applied to the neurons (N = 4), could also produce direct action on the membrane (Fig. 12A). The threshold for activation by L-DOPA was about 0.1 mM (Fig. 12B). Responses increased with concentration, reaching saturation at doses of about 1OmM. The Hill coefficient was 2.1. I-Y dependence of L-DOPA-activated currents was similar to the responses evoked by GABA (Fig. 12C). L-DOPA- and glycine-activated currents showed complete cross-desensitization. One micromole of strychnine was enough to produce a powerful and reversible blockade of the responses (Fig. 12D). A specific blocker of excitatory N-methyl-n-aspartate receptors, 2-amino-5-phosphonovalerate,‘2 was also a weak agonist of the inhibitory chloride receptors (N = 5) with an activation threshold of about 1 mM. Control experiments Possible contamination of samples by 1% glycine was excluded for the following reason: interaction between the same samples of glycine and GABA was studied in control experiments on isolated spinal

A ,

glycine

100

neurons of chick embryo where these agonists were found to have separate receptorchannel complexes (our and other literature data). Control application of 1 mM glycine evoked maximal response. When applied to the neuron, 10 PM glycine (1% of 1 mM solution) was enough to produce a considerable reduction in the control glycine response. However, preincubation of the cell with GABA at concentrations of 1 or even 5 mM (1% of 5 mM is 50 PM) did not change the maximal glycine response. Thus, our lamprey data on GABA could not be explained by contamination of the samples used.

DISCUS!SION

Common receptor for inhibitory transmitters Experiments studying cross-desensitization and the action of specific blockers indicate that glycine, GABA and taurine activate the same receptorchannel complexes in the membranes of lamprey spinal neurons. Our data about the action of specific blockers, however, contradict the results obtained on lamprey giant interneurons, ‘*but this study was carried out on whole cord preparations and transmitters were applied to perfusing solutions. Investigation of the fast processes of agonist-receptor interaction was impossible under such conditions. Moreover, experiments on whole cord preparations exclude unambiguous understanding of the results.

B

C

uW-plycinc

1 lOOuW-

tourine 1

1 :F= bit

0

1lOO+t- Lourine

lOOpM-

tourine

0.5 \

r!L_

a

Fig. 11. (A) Examples of ionic currents evoked by applications of 0.1 mM glycine (upper traces) and 0.1 mM taurine (lower traces) after 30 s preincubation of the cell with a solution containing different concentrations of glycine (shown near the corresponding traces in FM). Holding potential, - 100 mV. (B) Dose dependence of the steady-state desensitization of glycine- (0) and taurine-activated (0) receptors by glycine (for the experiment shown in A). (C) Traces of responses evoked by 0.1 mM taurine in the absence and presence of 0.5 FM strychnine (upper traces), 100 pM bicuculline (middle traces) and 100 p M picrotoxin (lower traces). Holding potential, - 100 mV.

938

K.W. BAEVet al.

A

B

L-DOPA 0.5 1

0.5 A

2 nA 0.1

1

1'0 mM

5

D C

sir

nA

,-4°,~2°

-100,

Y[;

v

lmM-L-DOPA

1 --

.......... nA 0.Ss

Fig. 12. L-DOPA-activated currents. (A) Traces of neuron responses following rapid applications of different L-DOPA concentrations (indicated near each trace in mM). Holding potential, -100 mV. (B) Dose-response relationship for the experiment shown in A. The data are fitted by the curve of the Hill equation with n = 2 and EDs0= 0.9 mM. (C) 1-V relationship for the responses elicited by application of 1 mM L-DOPA. (D) Oscilloscope traces of responses evoked by applications of 1 mM L-DOPA in the absence and presence of 1/aM strychnine. Holding potential, -100 mV.

How many types o f inhibitory receptors exist?

EDs0 for the steady-state currents was always several times less than that obtained for the peak values for every amino acid studied. However, there are no serious reasons to suggest that each agonist activates two different types of receptors: desensitizing and non-desensitizing. Such behaviour may be rigorously described by the following scheme of agonist (A)-receptor interaction: KA

KD

nA + R ~ A . R ~ A,,D,

(1)

where receptors may exist in initial (R), activated (A,R) and desensitized (A,D) states, n is Hill's coefficient, and Ks and KD are dissociation constants for the first and second transitions, respectively. At equilibrium, the ratio of the steady-state current (I ss) to the maximal current (Ira) can be written as: lss

Im

1 - - X

1+~

K~ [A]"+

1 l+-

K~ 1

=

l+--

Ip I~

Ip I~

[A]° [A]"+ KA

[A]" [A]"+ ED~

(3)

The following relation between the maximal amplitudes and the concentrations evoking half-maximum effects for the fast and steady-state components can be obtained by comparing expressions (2) and (3):

[A]"

l

This gives a dose-response dependence for the steady-state current components. Such a relationship must, however, be different for the peak values. When high agonist concentrations are applied to the cell the first transition in the reaction sequence (1) is much more rapid than the second one (all responses to saturating transmitter concentrations demonstrated fast activation and relatively slow desensitization). We may assume, therefore, that directly after rapid application the "instant" equilibrium between the R and A,R states will be established without any significant transitions of activated receptors into the desensitized state• The scheme of the reaction for this "instant" equilibrium is of classical type, with a Hill dose-response relationship for the peak values of currents (lP):

[A]"

l

K~

X

[A]" + ED~on

(2)

(.oq._ Pm--\E'~/

, l + - -1 KD

(4)

939

Glycine- and GABA-gated Cl- current

Such dose-effect relationships have been found in our experiments for all agonists. For example, for taurine-activated currents (Fig. lOB), lng=2.8 m

and

n xlns=2.8&-0.5. *Cl

ED~,,for

GABA-activated currents, 1.5 mM, is in good agreement with the data obtained for frog spinal neurons, 1 and 3 mM.28*29Meanwhile, in the experiments on lamprey whole-cord preparations it was estimated within 0.246 mM2’ for bath-applied GABA. However, this value should be compared with our ED, calculated for the steady-state component, which was 3-10 times lower (see example with taurine) than that for peak values. Spinal neurons of higher vertebrates are much more sensitive to GABA. The ED= for cultured spinal neurons of chick embryo was found to be 17 PM.‘* Evolutionary development of spinal neuron sensitivity is apparently accompanied by an essential increase in the sensitivity to GABA, whereas glycine is almost equipotent in spinal neurons for all vertebrates. Z-V relationships demonstrated strong deflection from linearity at potentials from - 100 to -60 mV for ionic currents activated by low concentrations of all agonists. In these cases the time constant of activation decreased with membrane depolarization. However, the kinetics of desensitization were not potential-dependent. Such effects may be explained by assuming that the transition of activated receptors from close to open states is performed with an opening rate constant increasing with agonist concentration, whereas a closing one depends only on membrane potential and not on concentration, as has been described for the acetylcholine-activated channels in the frog endplate2sz2 and for GABA-evoked responses in frog sensory neurons.’ Mechanisms

of blocker’s action

In our experimental conditions it was impossible to determine the competitive or non-competitive nature of blocking effects directly. For example, strychnine has been described as a competitive blocker of glycine receptors.23 However, in the present experiments, the dose-response dependence obtained for glycineactivated currents in the presence of strychnine demonstrated a considerable shift in threshold concentrations with simultaneous reduction in the amplitudes of the maximal responses. Such a reduction may, however, be due to a slow dissociation of strychnine molecules from the receptors when the response starts to decay before the establishment of equilibrium in the interaction of agonist and blocker with the receptors. Prolongation of the activation and desensitization obtained in the presence of strychnine and bicuculline may be explained by the competitive nature of blocking actions when binding of the receptor by the blocker molecule preserved it from activation and desensitization by agonist.

To understand the mechanism of this phenomenon, let us consider the scheme of receptor activation in the presence of a competitive blocker (when, for simplicity, the response has no steady-state component): B + nA+R$

A,R -? A,D,

(5)

k, &,

BR where B is blocker, and k, , k_, , kb, k_, and kd are the rate constants. Scheme (5) shows that the response to agonist application in the presence of competitive blocker must differ from that evoked in normal solution. Its amplitude will be diminished because the fraction of receptors given by

is occupied by blocker molecules at the initial moment. However, after the complete desensitization of the ionic response all these receptors will be in the A,D state after passing through the activated state (A,R). Therefore, this fraction of the receptors (0) is opened with a considerable time delay, leading to an increase in the time constant of activation and desensitization. In the present study strychnine was more potent to delay the desensitization than bicuculline which may probably be explained by the difference in the values of k_, for these blockers. Picrotoxin, unlike strychnine and bicuculline, produced neither a shift in the initial part of the dose-effect relations nor any changes in the kinetics of the responses. This may be due to the noncompetitive manner of picrotoxin blocking action when the blocker molecule binds with the receptor, irrespective of its interaction with the agonist. Thus, such effects are indirect evidence of a competitive blocking mechanism for strychnine and bicuculline and a non-competitive one for picrotoxin. This is in agreement with the literature.‘* It is unexpected that L-DOPA is an agonist of inhibitory receptors; 0.5 mM L-DOPA was described as activating rhythmic locomotor activity in the lamprey spinal cord. 3oSuch action was attributed to the influence of L-DOPA on excitatory receptors of acidic amino acids because it was removed by glutamylglycine, the antagonist of these receptors. However, in our experiments 0.5 mM L-DOPA could not activate any acidic amino acid receptors, whereas it produced almost complete desensitization of the inhibitory receptors. According to these facts, the mechanism of the initiation of lamprey locomotor activity by virtue of L-DOPA application appears to be a complicated process including the desensitization

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K. V. BAEVet al

of inhibitory receptors. It can be supposed that L-DOPA, like glycine,” may potentiate responses of N-methyl-D-aspartate receptors in the membranes of lamprey spinal neurons. Thus, the inhibitory receptor-channel complexes of lamprey spinal neurons are activated by a number of substances. Such poor selectivity of these receptors appears to be due to the lower position of Cyclostom-

ata on the evolutionary scale of vertebrates when independent glycine and GABA receptors have not yet been formed and there is only one weakly differentiated primitive type of receptor. It could be suggested that it is a prototype of independent glycine and GABA receptors of the membranes of higher vertebrate spinal neurons as it exhibits a sensitivity to specific blockers of both transmitters.

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