Brain Research, 542 (1991) 273-279 Elsevier
273
BRES 16365
G A B A A receptors modulate axonal conduction in dorsal columns of neonatal rat spinal cord K. Sakatani, M. Chesler and A.Z. Hassan Department of Neurosurgery, New York University Medical Center, New York, NY 10016 (U.S.A.) (Accepted 18 September 1990)
Key words: y-Aminobutyric acid; Axon; Conduction; Dorsal column; Myelination; Development
y-Aminobutyric acid (GABA) can influence conduction in a number of axonal preparations from the peripheral and central nervous system. In the spinal cord, the excitability of primary afferent terminals has long been known to be affected by GABA. Whether conduction in the long fiber tracts of the spinal cord can be similarly modulated is unknown. Since GABA causes a pronounced depression of excitability in preparations of unmyelinated axons, and myelination is incomplete in the neonatal rat, we tested whether GABA can modulate conduction in the dorsal columns of 10-17-day-old rats. Experiments were performed in vitro, on isolated dorsal column segments (n = 18). The extracellular compound action potential evoked by submaximal stimuli was recorded with a glass micropipette positioned 0.5-2.0 mm from a stimulating electrode. At concentrations of 10-4-10 -3 M, GABA decreased excitability, reversibly depressing the compound action potential amplitude, and increasing the latency by 47 + 11% and 22 + 9% (mean + S.E.M., n = 5, 10-3 M), respectively. These effects were blocked by picrotoxin and mimicked by isoguvacine (10-4 M), which decreased the compound action potential amplitude by 44 + 10% and increased the latency by 9 + 4% (n = 5). Lower concentrations of these agents caused a modest increase in excitability. At 10-5 M, GABA increased the compound action potential amplitude by 14 + 2% and decreased the latency by 3 + 2% (n = 5). Our results demonstrate that functional GABA^ receptors are present in neonatal dorsal columns. While the physiologic role of these receptors is not yet clear, these data suggest that axonal conduction in long tracts of the spinal cord may be subject to modulation by extrasynaptic mechanisms. INTRODUCTION
similar p r o n o u n c e d effects on excitability in neonatal dorsal columns.
y - A m i n o b u t y r i c acid ( G A B A ) has long been known to depolarize p r i m a r y afferent terminals in the dorsal horn o f the spinal cord, where it plays a m a j o r role in presynaptic inhibition 4,5. A t the distal terminals of these fibers, in the cuneate a n d gracilis nuclei, G A B A also has a depolarizing action 29,44,51,52. B e t w e e n these sites, pri-
We have addressed this issue by studying the effect of G A B A on the c o m p o u n d action potential in isolated neonatal dorsal columns in vitro. O u r results d e m o n s t r a t e that the activation of G A B A A receptors can profoundly influence axonal conduction in the dorsal columns. While the physiological role of these receptors is not yet clear, our data indicate that axonal conduction in the long spinal tracts may be subject to extrasynaptic modulation. A portion of these results has appeared in abstract form 49.
m a r y afferent axons run in the dorsal columns of the spinal cord. W h e t h e r these long dorsal column axons are subject to similar G A B A e r g i c effects is not known. It is well established that G A B A can m o d u l a t e excitability in a n u m b e r of axonal preparations. These include several p e r i p h e r a l nerves s,16,4°-42 and a few fiber p r e p a r a t i o n s of the central nervous system (CNS), including olfactory tract 17, optic nerve 46'52, and hypothal a m o - n e u r o h y p o p h y s i a l axons 6°. A m o n g the axonal preparations affected by G A B A , unmyelinated fibers a p p e a r to be particularly sensitve. Thus, in frog optic nerve 46, rat olfactory nerve 17 and rat vagus 16, G A B A caused a m a r k e d depression of the c o m p o u n d action potential. Since myelination of rat central axons is not complete until after m a t u r a t i o n zs, G A B A m a y be expected to have
MATERIALS AND METHODS Neonatal Long-Evan's hooded rats (n = 18) of either sex ranging in age from 10 to 17 days old were anesthetized with pentobarbital (i.p., 40 mg/kg) and decapitated. The spinal cord was rapidly removed and placed in oxygenated cold (5-10 *C) Ringer's solution. A length of the thoracic spinal cord was dissected, then hemisected. A 5-8 mm section of dorsal column was isolated from the hemisected cord with mieroseissors at 40x magnification (Fig. 1A). Care was taken to avoid inclusion of the dorsal gray matter in the segment. The absence of gray matter was confirmed histologically at the end of experiments, using hematoxylin-eosin and KIQverBarrera stains. The isolated dorsal column segment was secured in a recording
Correspondence: K. Sakatani, Department of Neurosurgery, New York University Medical Center, 550 First Avenue, New York, NY 10016, U.S.A. 0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
274
A
C DF
VR
l
B N1
P2
11
,
_~i.5 mV
l
Dorsal Column
1 msec
Fig. 1. A: isolation of dorsal column segment from the dissected hemicord. B: schematic of stimulation-recording arrangement with isolated dorsal column segment. C: top trace, single triphasic compound action potential recorded from the isolated dorsal column; bottom trace, averaged compound action potentials (n = 10) from the same preparation. Arrows indicate the onset of stimulation. Calibration: 0.5 mV (positivity downward), 1 ms.
chamber and superfused with Ringer's solution at room temperature (20-24 °C). For 1-2 h after dissection, the preparation was superfused with Ringer to allow washout of pentobarbital and stabilization of responses. In previous reports 14"15"45, 15-30 min
have been required to wash out barbiturate effects on GABA receptors in in vitro preparations. It was notable that using a different anesthesia (e.g. methoxyflurane), comparable results were found. During the 1-2 h superfusion, the compound action potential
A Control
GABA (1 mM)
Wash
Control
1.0 m ~ 1.3 m ~
B
C
,oo 110' 100'
GAB.~, 2b
._1 90
,~o
(mi,,)
6
20
40
(min)
Fig. 2. Effects of GABA on the dorsal column compound action potential. A: traces of an averaged dorsal column compound action potential recorded in normal Ringer's solution, ater 12 min in 1 mM G A B A and after 30 rain of wash. Fourth trace shows control and G A B A sweeps superimposed. B: reversible effect of 1 mM GABA on the amplitude of individual compound action potentials. C'. reversible effect of 1 mM G A B A on the latency of individual compound action potentials. Calibration: 1 mV, all traces; 1.3 ms, first 3 traces; 1.0 ms, fourth trace.
275 amplitude increased and the latency decreased to stable values, at which time experiments were begun. The composition of the Ringer's solution was (in mM): N a O 124, KCI 3, Na2HPO 4 1, NaHCO 3 26, MgCI2 1.5, CaCl 2 1.5, glucose 10. The Ringer was saturated with 5% CO 2 in 95% 0 2, pH 7.4. GABA, picrotoxin (Sigma) and isoguvacine (Research Biochemicals) were added directly to solutions. Bipolar platinum electrodes were used to deliver constantcurrent, submaximal stimuli (0.1-0.2 ms, 2-3 mA, 0.2 Hz). The elicited compound action potential of the dorsal column was recorded with a glass microelectrode (1 M NaCI, 1-2 Mr2) inserted into the dorsal column 0.5-2 mm from the stimulating electrode (Fig. 1B). In several cases, we recorded the compound action potential at two different conduction distances simultaneously. Responses were digitized at 44 kHz (Neurocorder, DR-484) and stored on video tape. Response amplitudes and latencies of individual compound action potentials were analyzed with a microcomputer (NEC, 9801) and then displayed graphically (Microsoft, Excel) on an Apple Macintosh II computer. Responses were also averaged (10-40 times) and displayed to assess changes in waveforms. Statistics are expressed as mean + S.E.M.
RESULTS
Fig. 1C illustrates a typical triphasic (positive-negative-positive) compound action potential recorded from an isolated dorsal column. The compound action potential amplitudes varied from animal to animal, but within a given preparation, remained constant for more than 6 h in vitro, The average peak-to-peak amplitude (P1-N1, Fig. 1C) of the compound action potential was 0.56 + 0.1 mV (n = 12 preparations). The latency of the negative peak was 1.85 + 0.23 ms, at a mean recording distance of 1.1 + 0.1 mm. Superfusion of GABA (10-4-10 -3 M) reversibly depressed the compound action potential amplitude and prolonged the latency. Fig. 2A shows traces of averaged compound action potentials recorded in control solution, after 12 min in 10-3 M GABA, and after 30 min of wash.
A A
GABA (0.1 mM)
1:30 A
~
e-
+PTX
A
8
m
M
~ ~ t .
100
100, "10
" PTX~
Q.
I
E
50
GABA ~ 50
20
0
4o (min) f
J 0.5mV
0
GABA
4'0 (min)
0.5 m V
B lmN¢
120
B
~.
Isoguvacine(0.1raM)
.130 ~Aml~itude (1)
100
~
Control
A
loo
"10
o~
80,
E
60
//
110 8
.= ~.
8 @ 0.5 m V
=.=. _....i
I00 a
-T
273
BRES 16365
G A B A A receptors modulate axonal conduction in dorsal columns of neonatal rat spinal cord K. Sakatani, M. Chesler and A.Z. Hassan Department of Neurosurgery, New York University Medical Center, New York, NY 10016 (U.S.A.) (Accepted 18 September 1990)
Key words: y-Aminobutyric acid; Axon; Conduction; Dorsal column; Myelination; Development
y-Aminobutyric acid (GABA) can influence conduction in a number of axonal preparations from the peripheral and central nervous system. In the spinal cord, the excitability of primary afferent terminals has long been known to be affected by GABA. Whether conduction in the long fiber tracts of the spinal cord can be similarly modulated is unknown. Since GABA causes a pronounced depression of excitability in preparations of unmyelinated axons, and myelination is incomplete in the neonatal rat, we tested whether GABA can modulate conduction in the dorsal columns of 10-17-day-old rats. Experiments were performed in vitro, on isolated dorsal column segments (n = 18). The extracellular compound action potential evoked by submaximal stimuli was recorded with a glass micropipette positioned 0.5-2.0 mm from a stimulating electrode. At concentrations of 10-4-10 -3 M, GABA decreased excitability, reversibly depressing the compound action potential amplitude, and increasing the latency by 47 + 11% and 22 + 9% (mean + S.E.M., n = 5, 10-3 M), respectively. These effects were blocked by picrotoxin and mimicked by isoguvacine (10-4 M), which decreased the compound action potential amplitude by 44 + 10% and increased the latency by 9 + 4% (n = 5). Lower concentrations of these agents caused a modest increase in excitability. At 10-5 M, GABA increased the compound action potential amplitude by 14 + 2% and decreased the latency by 3 + 2% (n = 5). Our results demonstrate that functional GABA^ receptors are present in neonatal dorsal columns. While the physiologic role of these receptors is not yet clear, these data suggest that axonal conduction in long tracts of the spinal cord may be subject to modulation by extrasynaptic mechanisms. INTRODUCTION
similar p r o n o u n c e d effects on excitability in neonatal dorsal columns.
y - A m i n o b u t y r i c acid ( G A B A ) has long been known to depolarize p r i m a r y afferent terminals in the dorsal horn o f the spinal cord, where it plays a m a j o r role in presynaptic inhibition 4,5. A t the distal terminals of these fibers, in the cuneate a n d gracilis nuclei, G A B A also has a depolarizing action 29,44,51,52. B e t w e e n these sites, pri-
We have addressed this issue by studying the effect of G A B A on the c o m p o u n d action potential in isolated neonatal dorsal columns in vitro. O u r results d e m o n s t r a t e that the activation of G A B A A receptors can profoundly influence axonal conduction in the dorsal columns. While the physiological role of these receptors is not yet clear, our data indicate that axonal conduction in the long spinal tracts may be subject to extrasynaptic modulation. A portion of these results has appeared in abstract form 49.
m a r y afferent axons run in the dorsal columns of the spinal cord. W h e t h e r these long dorsal column axons are subject to similar G A B A e r g i c effects is not known. It is well established that G A B A can m o d u l a t e excitability in a n u m b e r of axonal preparations. These include several p e r i p h e r a l nerves s,16,4°-42 and a few fiber p r e p a r a t i o n s of the central nervous system (CNS), including olfactory tract 17, optic nerve 46'52, and hypothal a m o - n e u r o h y p o p h y s i a l axons 6°. A m o n g the axonal preparations affected by G A B A , unmyelinated fibers a p p e a r to be particularly sensitve. Thus, in frog optic nerve 46, rat olfactory nerve 17 and rat vagus 16, G A B A caused a m a r k e d depression of the c o m p o u n d action potential. Since myelination of rat central axons is not complete until after m a t u r a t i o n zs, G A B A m a y be expected to have
MATERIALS AND METHODS Neonatal Long-Evan's hooded rats (n = 18) of either sex ranging in age from 10 to 17 days old were anesthetized with pentobarbital (i.p., 40 mg/kg) and decapitated. The spinal cord was rapidly removed and placed in oxygenated cold (5-10 *C) Ringer's solution. A length of the thoracic spinal cord was dissected, then hemisected. A 5-8 mm section of dorsal column was isolated from the hemisected cord with mieroseissors at 40x magnification (Fig. 1A). Care was taken to avoid inclusion of the dorsal gray matter in the segment. The absence of gray matter was confirmed histologically at the end of experiments, using hematoxylin-eosin and KIQverBarrera stains. The isolated dorsal column segment was secured in a recording
Correspondence: K. Sakatani, Department of Neurosurgery, New York University Medical Center, 550 First Avenue, New York, NY 10016, U.S.A. 0006-8993/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
274
A
C DF
VR
l
B N1
P2
11
,
_~i.5 mV
l
Dorsal Column
1 msec
Fig. 1. A: isolation of dorsal column segment from the dissected hemicord. B: schematic of stimulation-recording arrangement with isolated dorsal column segment. C: top trace, single triphasic compound action potential recorded from the isolated dorsal column; bottom trace, averaged compound action potentials (n = 10) from the same preparation. Arrows indicate the onset of stimulation. Calibration: 0.5 mV (positivity downward), 1 ms.
chamber and superfused with Ringer's solution at room temperature (20-24 °C). For 1-2 h after dissection, the preparation was superfused with Ringer to allow washout of pentobarbital and stabilization of responses. In previous reports 14"15"45, 15-30 min
have been required to wash out barbiturate effects on GABA receptors in in vitro preparations. It was notable that using a different anesthesia (e.g. methoxyflurane), comparable results were found. During the 1-2 h superfusion, the compound action potential
A Control
GABA (1 mM)
Wash
Control
1.0 m ~ 1.3 m ~
B
C
,oo 110' 100'
GAB.~, 2b
._1 90
,~o
(mi,,)
6
20
40
(min)
Fig. 2. Effects of GABA on the dorsal column compound action potential. A: traces of an averaged dorsal column compound action potential recorded in normal Ringer's solution, ater 12 min in 1 mM G A B A and after 30 rain of wash. Fourth trace shows control and G A B A sweeps superimposed. B: reversible effect of 1 mM GABA on the amplitude of individual compound action potentials. C'. reversible effect of 1 mM G A B A on the latency of individual compound action potentials. Calibration: 1 mV, all traces; 1.3 ms, first 3 traces; 1.0 ms, fourth trace.
275 amplitude increased and the latency decreased to stable values, at which time experiments were begun. The composition of the Ringer's solution was (in mM): N a O 124, KCI 3, Na2HPO 4 1, NaHCO 3 26, MgCI2 1.5, CaCl 2 1.5, glucose 10. The Ringer was saturated with 5% CO 2 in 95% 0 2, pH 7.4. GABA, picrotoxin (Sigma) and isoguvacine (Research Biochemicals) were added directly to solutions. Bipolar platinum electrodes were used to deliver constantcurrent, submaximal stimuli (0.1-0.2 ms, 2-3 mA, 0.2 Hz). The elicited compound action potential of the dorsal column was recorded with a glass microelectrode (1 M NaCI, 1-2 Mr2) inserted into the dorsal column 0.5-2 mm from the stimulating electrode (Fig. 1B). In several cases, we recorded the compound action potential at two different conduction distances simultaneously. Responses were digitized at 44 kHz (Neurocorder, DR-484) and stored on video tape. Response amplitudes and latencies of individual compound action potentials were analyzed with a microcomputer (NEC, 9801) and then displayed graphically (Microsoft, Excel) on an Apple Macintosh II computer. Responses were also averaged (10-40 times) and displayed to assess changes in waveforms. Statistics are expressed as mean + S.E.M.
RESULTS
Fig. 1C illustrates a typical triphasic (positive-negative-positive) compound action potential recorded from an isolated dorsal column. The compound action potential amplitudes varied from animal to animal, but within a given preparation, remained constant for more than 6 h in vitro, The average peak-to-peak amplitude (P1-N1, Fig. 1C) of the compound action potential was 0.56 + 0.1 mV (n = 12 preparations). The latency of the negative peak was 1.85 + 0.23 ms, at a mean recording distance of 1.1 + 0.1 mm. Superfusion of GABA (10-4-10 -3 M) reversibly depressed the compound action potential amplitude and prolonged the latency. Fig. 2A shows traces of averaged compound action potentials recorded in control solution, after 12 min in 10-3 M GABA, and after 30 min of wash.
A A
GABA (0.1 mM)
1:30 A
~
e-
+PTX
A
8
m
M
~ ~ t .
100
100, "10
" PTX~
Q.
I
E
50
GABA ~ 50
20
0
4o (min) f
J 0.5mV
0
GABA
4'0 (min)
0.5 m V
B lmN¢
120
B
~.
Isoguvacine(0.1raM)
.130 ~Aml~itude (1)
100
~
Control
A
loo
"10
o~
80,
E
60
//
110 8
.= ~.
8 @ 0.5 m V
=.=. _....i
I00 a
-T
