121

J. Phyaiol. (1978), 283, pp. 121-132 With 8 text-ftgure8 Printed in Great Britain

THE BLOCKADE OF GABA MEDIATED RESPONSES IN THE FROG SPINAL CORD BY AMMONIUM IONS AND FUROSEMIDE

BY R. A. NICOLL From the Departments of Pharmacology and Physiology, University of California, San Francisco, U.S.A. (Received 24 October 1977) SUMMARY

1. A variety of compounds which are known to block chloride transport in a variety of systems have been examined for their effects on amino acid and synaptic responses in the frog spinal cord in vitro. 2. A number of monocarboxylic aromatic acids, copper sulphate, and acetazolamide had no effect on any of the responses. 3. Ammonium ions blocked the motoneurone hyperpolarizing responses to all the neutral amino acids tested, but had little effect on the primary afferent depolarizing responses. 4. Furosemide also blocked the motoneurone hyperpolarizing responses to all the neutral amino acids. In addition it selectively blocked dorsal root potentials and the action of GABA and /?-alanine on primary afferents. 5. Intracellular recording from dorsal root ganglion cells demonstrated that furosemide had little effect on the reversal potential for the GABA response. These results suggest that furosemide acts primarily by blocking the conductance increase elicited by GABA. 6. The results with furosemide provide indirect evidence that chloride ions are involved in generating the GABA depolarizations of primary afferent terminals and dorsal root potentials. INTRODUCTION

It has long been known that chloride ions are involved in the generation of inhibitory post-synaptic potentials (i.p.s.p.s) in the vertebrate (Eccles, 1964). It was originally assumed that chloride ions were passively distributed across the motoneurone cell membrane, but recent evidence, based on the action of ammonium ions, strongly suggests that motoneurones possess an outward chloride pump (Lux, Loracher & Neher, 1970; Lux, 1971; Meyer & Lux, 1974; Llinas, Baker & Precht, 1974). From these studies it was concluded that the inhibitory transmitter selectively increases permeability only to chloride ions. Considerable evidence indicates that y-aminobutyric acid (GABA) and glycine are transmitters mediating i.p.s.p.s in the C.N.S. (Curtis & Johnston, 1974; Krnjevic, 1974). However, it is also established that GABA has a strong depolarizing action on primary afferents and is the putative transmitter mediating dorsal root potentials (Davidoff, 1972; Barker & Nicoll, 1973; Barker, Nicoll & Padjen, 1975a, b). The ionic mechanism of this depolarization on the cell body of dorsal root ganglion cells

R. A. NICOLL appears to result from a selective increase in chloride permeability (Nishi, Minota & Karczmar, 1974; Deschenes, Feltz & Lamont, 1976; Gallagher, Higashi & Nishi, 1978) and thus the cell body membrane must possess an inward chloride pump. Although initial results based on extracellular recording of the GABA response from the intramedullary portion of primary afferents suggested that sodium ions were involved in the response (Barker & Nicoll, 1973) a number of technical limitations inherent in extracellular recording have prevented a clear understanding of the ionic mechanisms of the GABA depolarization at this site. In addition, the small size of the primary afferent terminals precludes an analysis with intracellular microelectrodes. If a substance could be shown to block chloride conductance and/or the presumed inward chloride pump in dorsal root ganglion cells, it would provide a useful tool for determining if a chloride mechanism were also involved in the dorsal root potentials and the GABA response of primary afferent terminals. Therefore, the actions of a number of substances proposed as blockers of chloride transport in other systems have been tested on amino acid and synaptic responses in the frog spinal cord. These substances include ammonium salts (Lux et al. 1970; Lux, 1971; Llinas et al. 1974; Meyer & Lux, 1974; Raabe & Gummit, 1975), furosemide (Burg, Stoner, Cardinal & Green, 1973; Candia, 1973; Lote, 1974; Montoreano, Rabito & Villamil, 1975; Brazy & Gunn, 1976), acetazolamide (Alvarado, Dietz & Mullen, 1975; Nellans, Frizzell & Shutz, 1975), copper (Chiarandini, Stefani & Gerschenfeld, 1967; Lux & Globus, 1968; Dreifuss, Kelly & Krnjevic, 1969), and a number of monocarboxylic aromatic acids (Bryant & Morales-Aguilera, 1971; Palade & Barchi, 1977). A brief account of some of these results has appeared (Nicoll, 1975). 122

METHODS The dissection, chamber, and techniques of sucrose gap recording have recently been described (Barker et al. 1975a, b). The Ringer solution was bubbled with 100 % oxygen and consisted of 115 mM-NaCl, 2 mM-CaCl2, 2 mi-KCI, glucose 1 g/l. and 10 mM-Tris (hydroxymethyl) aminomethane buffered to pH 7*3 with HCl. The Ringer solution was passed through a cooling block attached to a thermoelectric cooling device (Cambion) just before entering the chamber. The preparation was maintained at 13-15 'C which noticeably improved the size of the motoneurone hyperpolarizing responses to the neutral amino acids. All of the drug responses were obtained in a Ringer solution containing 10 mM-MgSO4, which blocked indirect synaptic effects. Drugs were administered either by manually turning a stopcock orby applying a voltage gate from a Digitimer stimulator to a solenoid valve (General Valve). The latter procedure permitted very short and reproducible drug applications. For intracellular recording, micro-electrodes filled with 2 M-K methylsulphate and having resistances of 15-25 MCI were used. The signal was passed through a WPI amplifier (M-701) which also permitted passing current through the micro-electrode to vary the neuronal membrane potential. To balance the bridge during current injection the negative capacitance feedback was maximally compensated so as to eliminate the transients at the beginning and end of the current pulse. Only those electrodes were used which showed minimal separation of the trace during the passage of long current pulses, indicating that the electrode characteristics had not changed during the current pulse. In some experiments GABA was applied ionophoretically from pipettes filled with 1 M-GABA (pH adjusted to 3-5 with HCl). The ionophoretic pipette was independently positioned over the impaled neurone. All responses in this study were recorded on a Brush 280 pen recorder. The following substances were used: ammonium acetate (Sigma), ammonium chloride (Fisher), Na acetate (Fisher), furosemide (Hoechst), acetazolamide (Sigma), copper sulphate (Sigma),

GABA AND CHLORIDE TRANSPORT BLOCKERS

123

3-chloro-2,5,6-trimethyl-benzoic acid (U-23, 223) (Upjohn), 5,6-dihydro-5,5-dimethyl-7-carboxybenz (c) acridine (Lilly-29358) and phenanthrene-9-carboxylic acid (Lilly-65865) (both from Eli Lilly) and anthracene-9-carboxylic acid (Aldrich). All substances were added directly to Ringer at the beginning of each experiment, and the pH was adjusted when necessary with dilute HCl or NaOH. At least three preparations were used to examine the effects of a substance on each type of response.

NH4

A

acetate

(2 mM) 27 min

GABA GLU

25 mVm

ibexm

2-5 min

B

NH4

acetate

(4mM)

90 min

Fig. 1. Effect of ammonium acetate on frog motoneurone alternating GABA and glutamate responses. A, 2 mM-ammonium acetate reduces the GABA hyperpolarization (downward response) without affecting the glutamate (GLU) response (upward response). B, 4 mM-ammonium acetate reverses the GABA hyperpolarization into a depolarization. also transiently depolarizes the motoneurones and increases the size of the glutamate response. Both amino acids (0.2 mM) were applied for 14 sec. The calibration in A also applies to B. RESULTS

The effect of ammonium ions and furosemide on amino acid induced hyperpolarizing responses of motoneurones Ammonium acetate readily blocked the hyperpolarizing responses elicited in motoneurones by the neutral amino acids but had little effect on the depolarizing responses to glutamate (Fig. 1A). The threshold concentration for this antagonism was 0 1 mm and complete block of the hyperpolarizations could usually be obtained with a concentration of 0.5 mM (Fig. 2A). At this concentration ammonium acetate had no effect on the membrane potential of motoneurones and block of the responses generally was complete in 15 min. An important observation was that all of the amino acid responses were equally sensitive to ammonium acetate (Fig. 2A and B) even though they elicited hyperpolarizations through different receptor mechanisms (Curtis & Johnston, 1974; Nicoll, Padjen & Barker, 1976). At a concentration of

R. A. NICOLL 124 greater than 2 mM, ammonium acetate depolarized the motoneurones (Fig. 1 B). At these high concentrations the hyperpolarizations were blocked in a few minutes and were replaced by depolarizing responses. In addition, the glutamate response was usually larger. This reversal of the hyperpolarizing response was also seen with glycine and fl-alanine responses (Fig. 2B). The effect of ammonium acetate was completely reversible with prolonged washing (Figs. 1 and 2). Ammonium chloride was just as effective as amnmonium acetate in blocking hyperpolarizing responses, whereas Na acetate (10 mM) was entirely ineffective. Intracellular recording from motoneurones in the presence of ammonium acetate demonstrated that GABA was

A

BALA GABA GLY

B BALA GLY GABA

C

GABA BALA

GLY

GLU

NH4 acetate (0-6 mm) NH4 acetate (2

m

mM)

GV

2-5 min

Furosemide

Wash

(2 mm) -Wash D

GABA

Wash

-100 Fig. 2. Ammonium acetate and furosemide blockade of amino acid hyperpolarizing responses from frog motoneurones. A, 10 min exposure to 0-6 mM-ammonium acetate blocks all of the hyperpolarizations which are restored after 20 min of washing in a drug-free Ringer solution. Concentration of all amino acids was 0 5 mm. B, higher concentrations of ammonium acetate (2 mM) reverse the hyperpolarizations into large depolarizations. The responses were obtained 40 min after initiating the test solution. Washing for 2 hr completely restores the hyperpolarizing responses. Amino acids were applied in a concentration of 1 mm. C, 2 mM-furosemide applied for 6 min blocks the hyperpolarizing responses but has no effect on the glutamate depolarization. The hyperpolarizations are restored with 2 hr of washing in a drug-free Ringer. The amino acids were all at a concentration of 0-2 mm. The calibration in C also applied to the record in A and B. D, GABA (1 mM) response from an antidromically identified motoneurone in the presence of 4 mM-ammonitim acetate. The hyperpolarizations to constant current pulses are fused together. The time calibration in C also applies to D.

125 GABA AND CHLORIDE TRANSPORT BLOCKERS still capable of eliciting a large increase in membrane conductance (Fig. 2D), although hyperpolarizing responses (six cells) were never observed Furosemide also selectively antagonized the amino acid induced hyperpolarizations and the responses to the different neutral amino acids were equally sensitive to the action of furosemide (Fig. 2 C). The minimally effective concentration for furosemide was 0-2 mm. The only differences between the action of furosemide and that of ammonium were that furosemide, even in high concentrations (about 4 mM), rarely reversed the hyperpolarizations into depolarizations and furosemide had no direct effect on the membrane potential. A, Control

A2 NH4 acetate

(1 mM)

A3 NH4 acetate (3

B,

mM)

GLU GABA BALA

GLY

IVm

DR-DRP

V

d~~~~mV ~~~25mV 2 sec

B2

NH4 acetate (2 mM)

DR-VRP

VR-DRP

mV 1 05se

Fig. 3. The effect of ammonium acetate on responses from primary afferents. A, shows the control root potential responses. The response on the top is the dorsal root potential generated by stimulating an adjacent dorsal root (DR-DRP), the middle response is the ventral root potential elicited by dorsal root stimulation (DRVRP) and the bottom record is the dorsal root potential elicited by ventral root stimulation (VR-DRP). In A2 the preparation had been perfused for 15 min in 1 mM-ammonium acetate and in A3 the concentration was increased to 3 mm for an additional 10 min. The calibration for the DR-DRP also applies to the DR-VRP. B, 20 min exposure to 2 mi-ammonium acetate slightly increased the size of the amino acid responses. Amino acids were all applied at a concentration of 1 mM.

The effect of ammonium acetate on root potentials and amino acid induced depolarization of primary afferents Ammonium acetate in concentrations which entirely blocked amino acid induced hyperpolarizations of motoneurones had surprisingly little effect on the root potentials. At a concentration of 0*5 mm there was often a slight selective depression of the dorsal root potentials (Fig. 3A2), but at higher concentrations all of the

126 R. A. NICOLL potentials were reduced in a rather indiscriminate fashion (Fig. 3A3). These effects were partially reversible although the VR-DRP rarely returned to control amplitude. At no concentrations were the root potentials prolonged, as is seen with many convulsants which block post-synaptic inhibition (Davidoff, 1972; Barker & Nicoll, 1973). In fact, ammonium acetate usually reduced the duration of the root potentials. The non-selective depression of all root potentials suggests that ammonium acetate is not interfering with the depolarizing action of GABA, the presumed transmitter of primary afferent depolarization. This was tested directly by observing the effects of ammonium acetate on the amino acid induced depolarizations of primary afferents. At

Control

A2 NH4 acetate (4 mM)

A3 20 min ^

"

-

7~~~~~~~~~~~~~-0

100 sec

10 sec

90 mV

30 min mV

_

B

A4

Control 70. mV

D i 61 mV

El15

Control (RMP=61 mV) o NH4 acetate (4 mM) (RMP=64mV) *

0

c

NH4

1 j5 mV < < 5 (4 mM) 3s 1 64 mV 76 mV

acetate

84 mV

_IY ?

/

The blockade of GABA mediated responses in the frog spinal cord by ammonium ions and furosemide.

121 J. Phyaiol. (1978), 283, pp. 121-132 With 8 text-ftgure8 Printed in Great Britain THE BLOCKADE OF GABA MEDIATED RESPONSES IN THE FROG SPINAL COR...
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