67
Journal of Physiology (1991), 440, pp. 67-83 With 10 figures Printed in Great Britain
STRYCHNINE-SENSITIVE GLYCINE RESPONSES OF NEONATAL RAT HIPPOCAMPAL NEURONES
BY SUSUMU ITO* AND ENRICO CHERUBINIt From the Unite 029, INSERM, 123 Boulevard de Port-Royal, 75014 Paris, France
(Received 5 November 1990) SUMMARY
1. Intracellular recordings employing current and voltage clamp techniques were used to study the effects of glycine on rat CA3 hippocampal neurones during the first 3 weeks of postnatal (P) life. 2. Glycine (0 3-1 mM) depolarized neurones from rats less than 4 days old (P4). Neurones from older neonates (P5-P7) were hyperpolarized by glycine, whereas adult neurones were unaffected. 3. Both depolarizing and hyperpolarizing responses were associated with large conductance increases; they reversed polarity at a potential which changed with the extracellular chloride concentration. The responses persisted in tetrodotoxin (1 gM) or in a solution with a much reduced calcium concentration. 4. Strychnine (1 #M) but not bicuculline (10-50 JIM) antagonized the effects of glycine. The action of strychnine was apparently competitive with a dissociation constant of 350 nm. 5. In voltage clamp experiments, glycine elicited a non-desensitizing outward current at -60 mV. When a maximal concentration of glycine was applied at the same time as y-aminobutyric acid (GABA), the conductance increase induced by the two agonists was additive, suggesting the activation of different populations of channels. 6. Concentrations of glycine lower than 100 JM did not affect membrane potential. However, at 30-50 #M glycine increased the frequency of spontaneous GABAmediated synaptic responses; this action was not blocked by strychnine. 7. It is concluded that during the first 2 weeks of life glycine acts at strychninesensitive receptors to open chloride channels. INTRODUCTION
Glycine is the main inhibitory transmitter in hindbrain areas (Aprison & Daly, 1978). Its effect is mediated through the activation of high-affinity receptors localized on neurones of the spinal cord, medulla and pons (Young & Snyder, 1973). The interaction of glycine with its receptor results in an increase in chloride * Present address: Department of Neurophysiology, Medical Research Institute, Tokyo Medical and Dental University, Kanda-Surugadai Chiyoda-ku, Tokyo, Japan. t Present address and to whom correspondence should be sent: International School for Advanced Studies, Strada Costiera 11, 34014 Trieste, Italy.
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S. ITO AND E. CHERUBINI
conductance (Coombs, Eccles & Fatt, 1955; Araki, Ito & Oscarsson, 1961; Eccles, 1964) an effect specifically blocked by the convulsant alkaloid strychnine (Curtis, Hosli & Johnston, 1968). The neurodepressant potency of glycine is related to the uneven distribution of this endogenous amino acid, being greatest in the spinal cord and brain stem level and progressively decreasing in more rostral areas (Kishimoto, Simon & Aprison, 1981). Electrophysiological experiments on cat and rat cortical neurones in vivo have shown that on these cells glycine does not play a major role in synaptic inhibition (Kelly & Krnjevic, 1969; Levi, Bernardi, Cherubini, Gallo, Marciani & Stanzione, 1982). Furthermore, glycine fails to change membrane potential or conductance of adult rat hippocampal neurones in vivo (Ben-Ari, Krnjevic, Reiffenstein & Reinhardt, 1981). In addition to its classical inhibitory action, glycine serves also as an allosteric regulator of excitatory neurotransmission mediated by the N-methyl-D-aspartate (NMDA) receptors (Johnson & Ascher, 1987). This action involves distinct glycine binding sites, which are insensitive to strychnine (Bristow, Bowery & Woodruff, 1986) and are mainly localized to rostral brain areas, including cortex and hippocampus (Kessler, Terramani, Lynch & Baudry, 1989). We have recently reported that, in hippocampal neurones from neonatal (but not adult) rats, micromolar concentrations of glycine enhance the frequency of y-aminobutyric acid (GABA)-mediated synaptic potentials and potentiate NMDA responses (Gaiarsa, Corradetti, Cherubini & Ben-Ari, 1990). This effect, which is strychnine insensitive, is clearly mediated by the allosteric site of the NMDA receptor located on GABAergic interneurones (Ben-Ari, Cherubini, Corradetti & Gaiarsa, 1989) since it can be prevented by the specific NMDA receptor antagonist D-(-)-2-amino-5phosphonovalerate (AP-5; Gaiarsa et al. 1990). We now report that, in addition to its facilitatory action on NMDA-mediated events, glycine induced strychnine-sensitive, chloride-dependent responses detectable only during the first 2 weeks of postnatal life. Part of this work has been reported in a preliminary form (Ito & Cherubini, 1990). METHODS
Experiments were performed on CA3 hippocampal neurones from slices obtained from 2- to 21day-old (P2-P21 days; 0 is taken as the day of birth) and adult Wistar rats. The methods for preparing and maintaining the slices have been extensively reported (Ben-Ari et al. 1989). Rats were decapitated under ether anaesthesia and their brains quickly removed from the skulls. Hippocampi were dissected free; transverse 600 ,am thick slices were cut and immediately incubated at room temperature (20-22 °C) in artificial cerebrospinal fluid (ACSF) of the following composition (mm): NaCl, 126; KCl, 3-5; CaCl2, 2; NaH2PO4, 1-2; MgCl2, 13; NaHCO3, 25; glucose, 11. Equilibrating the ACSF with 95% 02 and 5 % CO2 gave a pH of 7 3-7A4. The slices were allowed to recover for at least 1 h before being transferred to a recording chamber in which they were continuously superfused at 33-34 °C with oxygenated ACSF at a rate of 25-3 ml min-'. Intracellular recordings were made with microelectrodes filled with 2 M-K-methylsulphate (resistance 50-100 MQ). In a few experiments CA3 neurones were impaled with microelectrodes containing 3 M-KCl or 3 M-CsCl (resistance 40-60 MQ). Current was injected through the recording electrode by means of an Axoclamp 2A amplifier. Bridge balance was checked repeatedly during the experiments and capacitative transients (with the electrode tip outside the neurone) were reduced to a minimum by negative capacity compensation. Stabilization of intracellular recording was helped by the injection of small hyperpolarizing currents (0 1-0 3 nA) but in cells from slices obtained from the youngest animals it often was necessary to maintain such a small current
GLYCINE ON NEONATAL HIPPOCAMPAL NEURONES
69
indefinitely (Schwartzkroin & Kunkel, 1982). Membrane potential was estimated from the potential observed upon withdrawal of the electrode from the cell. Membrane input resistance was measured from the amplitude of small hyperpolarizations (200-300 ms duration) evoked by passing known currents across the cell membrane. The conductance increment induced by glycine (fractional conductance increase, Ag, Morita, North & Tokimasa, 1982) was calculated from (R/R') -1, where R is the input resistance at the resting potential and R' is the input resistance during glycine response. In voltage clamp experiments, membrane currents were recorded via a single-electrode voltaae clamp amplifier (Axoclamp 2A, Axon Instruments, Inc.), switching between voltage recording and current injection at 3-4 kHz (30 % duty cycle). The voltage signal at the head stage amplifier was continuously monitored on a separate oscilloscope to ensure optimal performance of the voltage clamp system. Responses were digitized and displayed on a Nicolet digital oscilloscope and on a computer-driven chart-recorder. Some experiments were performed in a low-calcium-highmagnesium ACSF (0-2 mM-Ca2+ and 6 mM-Mg2+ instead of 2 and 1-3 mM) or in a low-chloride ACSF where 126 mM-NaCl was substituted by 126 mM-sodium isethionate, corresponding to 92-6% chloride substitution. Drugs were dissolved in ACSF and applied through a three-way tap system by changing the superfusing solution to one which differed only in its content of drug(s). There was a delay of 20-30 s between turning the tap and the first arrival at the tissue of the changed solution. The ratio of flow rate to bath volume ensured complete exchange within 1 min. Drugs used were: glycine, GABA, strychnine, bicuculline, all purchased from Sigma; D-(-)-2-amino-5-phosphonovalerate (AP-5) and (± )3-(2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid (CPP) purchased from CRB or Tocris. Unless otherwise stated, all data are expressed as means+ S.D. RESULTS
Stable intracellular recordings were obtained from sixty-four pyramidal neurones in slices from 2- to 21-day-old rats or from three adult rats. In agreement with a previous study (Ben-Ari et al. 1989), all neurones recorded during the first postnatal week with potassium methylsulphate electrodes (n = 42) had a resting membrane potential of - 65-2 + 78 mV (ranging from -55 to -75 mV, see Fig. 5A), resting input resistance ranging from 80 to 130 MQ and action potentials greater than 55 mV. Membrane potential changes induced by glycine Days PO-P4 Superfusion with glycine (0-3 mm), caused at resting membrane potential a membrane depolarization (4 ± P17 mV, n = 7), which was associated with an increase in membrane input conductance. The depolarization had a latency of 20-30 s after glycine reached the tissue and achieved its peak amplitude at 1 min. This effect washed out in a few minutes. A typical example from a 3-day-old rat is shown in Fig. 1A. The sensitivity to glycine varied from cell to cell, but in a given neurone the response varied with the concentration applied. The minimum dose of glycine required to produce membrane potential changes was 100 ,LM. Increasing the concentration of glycine increased the peak amplitude, particularly the rate of rise of the depolarization. As already reported (Ben-Ari et al. 1989), > 90 % of the neurones at P0-P4 exhibit spontaneous GABA-mediated giant depolarizing potentials (GDPs). These consisted of brief, high-frequency bursts of spikes riding on large depolarizing potentials. Concentrations of glycine lower than those required to cause membrane potential changes (30-50 JtM) increased the frequency of GDPs. This effect, which was insensitive to strychnine (1 /M), was blocked by the NMDA receptor antagonists AP-
S. ITO AND E CHER UBINI
70
5 or CPP (50 JLM), suggesting that it was mediated by the allosteric modulatory site of the NMDA receptors located on GABAergic interneurones (Gaiarsa et al. 1990). A similar effect was seen with application of the higher glycine concentrations, but it occurred transiently and before the membrane potential changed, presumably a A
Gly (300 .=.
-61
=
gM, P3)
mV'
Gly (1 mM, P6)
-64
MV
II
~I10 mV t
tt
~~~1min
B
Control (P7)
-63 mV
4p
TTX (1 gM)
Low calcium, high magnesium
ft4V 10 mV 1 min
Fig. 1. Glycine depolarized or hyperpolarized neonatal neurones. A, at P3, bath application of glycine (Gly, open bars) induced a membrane depolarization associated with an increase in input conductance. Note that glycine first increased and then suppressed (for 5 min) spontaneously occurring GABA-mediated giant depolarizing potentials. At P6, glycine induced a membrane hyperpolarization and an increase in input conductance. The effect of glycine was associated with an increase in frequency of spontaneous GABA-mediated hyperpolarizing potentials (arrows) followed by their suppression. Upward deflections on the left panel are action potentials riding on slow depolarizations; on the right panel are single spikes. B, the responses to glycine (1 mM, open bars) were unchanged in the presence of TTX or in a low-calcium (0-2 mM), highmagnesium (6 mM) solution. Downward deflections in this and following figures are electrotonic potentials resulting from injection of a fixed current pulse through the recording electrode.
reflection of the slowly increasing concentration of glycine in the tissue slice when it was applied by superfusion. Days P4-P7 Towards the end of the first postnatal week, during a transitional period characterized by the progressive disappearance of GDPs and their replacement by large hyperpolarizing potentials (responses to GABA also changed from the depolarizing to the hyperpolarizing ones; Ben-Ari et al. 1989), glycine induced membrane hyperpolarization and increased input conductance (Fig. 1A). Like the depolarizing response, the hyperpolarizing response was also dose dependent. Increasing the concentration of glycine caused an increase in the peak
GLYCINE ON NEONATAL HIPPOCAMPAL NEURONES
71
amplitude of the membrane hyperpolarization, particularly in the rise of the hyperpolarization. At P7, concentrations of glycine of 0 3 and 1 mm caused (at resting membrane potential) a hyperpolarization of 2-2 + 0-6 (n = 9) and 4-3 + 1-5 mV (n = 7) respectively. The depolarizing or hyperpolarizing response to glycine was unchanged by tetrodotoxin (TTX, 1 ,tM) or by a low-calcium (0-2 mM), high-magnesium (6 mm) solution, implying that it probably did not result from action potential generation and release of transmitter(s) from surrounding cells (Fig. 1B). As for GDPs, the hyperpolarizing effect of glycine was associated with a transient increase in frequency of spontaneous GABA-mediated large hyperpolarizing potentials which, at this stage of development, replace GDPs (Gaiarsa et al. 1990). The spontaneous firing was also completely and reversibly blocked by glycine (Fig. 1A). Days P7-adult After the first postnatal week, the sensitivity to glycine decreased gradually and completely disappeared after P14. At the end of the second postnatal week and in adult neurones, glycine at concentrations of 3-10 mm failed to induce any change in membrane potential, input conductance or synaptic noise.
Conductance changes elicited by glycine Days PO-P4 Glycine-induced depolarizations were accompanied by an increase in cell input conductance, measured from the amplitude of the hyperpolarizing electrotonic potentials (Fig. 2A). The conductance increase persisted after repolarizing the membrane potential to its initial value at rest. The conductance increase reached its maximum within 20-30 s after the beginning of glycine application. Applications of 1-2 min were usually enough to obtain steady-state responses; prolongation of the drug application did not show any further significant change in conductance. Although the sensitivity to glycine varied from cell to cell, the conductance change became detectable at a concentration of 100 #M (Fig. 2A). The conductance changes increased with higher concentrations and reached the maximum at concentrations of 3-10 mm. With these glycine concentrations, the amplitude of the response and associated conductance increase, after an initial peak, declined to reach a steadystate value; this was also seen when the application of glycine was maintained for several minutes.
Days P4-P7
Glycine-induced hyperpolarizations were associated with an increase in cell input conductance. Figure 2B shows the mean fractional conductance increase induced by increasing concentration of glycine during this period of postnatal life. The halfmaximal conductance change was obtained with a glycine concentration of 1H1 mm. The Hill coefficient, calculated in two neurones from the conductance changes induced by increasing concentrations of glycine, was 1-6 and 1-8 respectively.
S. ITO AND E. CHER UBINI
72
Days P7-adult After the first postnatal week, the conductance increase induced by glycine gradually decreased. At P14 and in adult neurones, glycine at concentrations up to 3 mm, applied for a longer period of time, failed to induce any change in membrane A
Gly (100
kiM, P7)
Gly (300 PM)
Gly (1 mM)
Ti
-59 mV
I
10m
W
1 min
B u)
(10)
(2)
(24) (7)
o o 0-
~~~~~(4)/ -4
-3
-2
log lglycinel
Fig. 2. Glycine induced a dose-dependent increase in input conductance. A, increasing concentrations of glycine (open bars) induced membrane hyperpolarization associated with an increase in input conductance. The graph in B represents the fractional conductance increase produced in CA3 neurones during the first postnatal week by increasing concentrations of glycine. Each point represents the mean and S.E.M. of the samples (numbers in parentheses).
potential or input conductance. These results are summarized in Fig. 3, where the fractional conductance increase induced by glycine (1 mM) is plotted as a function of age (postnatal days). During the first postnatal week, every neurone tested responded to glycine with a mean fractional conductance change of 0-85 + 0-38 (n = 24). During the second postnatal week, some neurones were insensitive to glycine and the mean fractional conductance change fell to 0 4 at PIO and to 0-2 at P14. At P21 no neurones (n = 6) responded to glycine. Reversal potential of the responses to glycine The amplitude of glycine-induced depolarizations and hyperpolarizations was a linear function of the membrane potential in the range -90 to -40 mV. The depolarizing response to glycine increased with membrane hyperpolarization and decreased with depolarization. At P2-P4, its reversal potential was - 53-5 + 9-6 mV
GLYCINE ON NEONATAL HIPPOCAMPAL NEURONES 73 (Fig. 4, n = 10) with potassium methylsulphate-containing electrodes and -23 + 7-2 mV (n = 5) with KCl- or CsCl-filled electrodes. After P4, the responses to glycine shifted from the depolarizing to the hyperpolarizing direction. Thus the amplitude of glycine-induced hyperpolarization increased with depolarization and A
Gly (300 pM, P6)
Gly (300 pm, P3) -60
-63 mV
Gly (3 mM, P14) -68 mV
....
mV
jiQ mV B
1
°
min
a, CD)
1
(9)
C4
(9)
(6)
()
(6 )
o
0CC.) .L
5_
(; 1
7
)
(6) 6
14
21
Age (days) Fig. 3. Glycine induced a conductance increase only during the first 2 weeks of postnatal life. A, glycine (open bars) caused membrane depolarization or hyperpolarization associated with an increase in input conductance at P3 and P6 respectively. At P14, a higher concentration of glycine, applied for 2 min, failed to induce any change in membrane potential or input conductance. B, fractional conductance increase induced by glycine (1 mM) as a function of age (postnatal days). Each point represents the mean and S.E.M. (numbers in parentheses). TTX (1 /iM) present throughout the experiment.
decreased with hyperpolarization. The reversal potential recorded between P5 and P7 was - 63-2 + 11-6 mV with potassium methylsulphate-containing electrodes (Fig. 4, n = 20) and -40X2 + 10-6 mV with KCl-filled electrodes (n = 5). Figure 5 shows the distribution values of the resting membrane potential and the reversal potential of the responses to glycine in neurones recorded at P2-P7 with potassium methylsulphate-containing electrodes. In agreement with a previous report (Ben-Ari et al. 1989), we observed that, in contrast to the reversal potential of the responses to glycine, which showed a significant difference (P < 0-01, rank-sum test) between the P2-P4 and P5-P7 groups, the resting membrane potential of cells at P2-P4 was not significantly different from that recorded at P5-P7. The mean resting membrane potential at P2-P4 was - 66-6 + 7 mV (n = 11) and at P5-P7 was - 64-5 + 8 mV (n = 31). The regression line for reversal potentials crossed that of resting membrane potentials between P5 and P6.
S. ITO AND E. CHER UBINI
74 A
Gly (300 piM, P7, 0)
Gly(1 mM, P2,A) lZZ=
-38 mV
i
E
B
-68 mv
-10 CDv
O.
0
A -48
-78
.p
I,!
-88 r8 " .~~ ~~~~~~~~~
-68 ..;.
:,
10
.,...
mv
'; il,
ii, "!
CD 0 A -4 90 -80 -70 A -40 -50 0 . ° > -6 60 'A ' -5 0) °O 0 0 V -1 0 Membrane potential (rmV)
lJ jI10 m\
Q ~~~~~~~E
v
1 min
1 min
Fig. 4. Reversal of glycine responses. A, changing the membrane potential by passing a steady current through the recording electrode changed the amplitude of the depolarization or hyperpolarization induced by glycine (open bars) at P2 (A) and P7 (0) respectively. The resting membrane potential of both cells was -68 mV. B, relationship between the membrane potential and the amplitude of glycine responses for the cells shown in A. Note the shift in the reversal potential from -55 mV (at P2) to -80 mV (at P7). TTX (1 /sM) present throughout the experiment. A
g
B
-30-
-30
-
-50
-
-70
-
4-
C
2
0 a 0
E
-50-
E
-70-
a) 0)
t
.
s
.
0~~~
._
M
0 *
9.
C
.
-zo
0
0)
. a
E
U)
cc
C
-
0) a -inn c
-
I
I
1
2
I
-I 3
4
5
_1nn
I
I
I
I
6
-
7
8
1
2
3
4
5
6
7
Age (days) Age (days) Fig. 5. Changes in reversal potential for glycine are independent from the resting membrane potential. Plot of the distribution values of the resting membrane potentials (A) and the reversal potentials of glycine responses (B) as a function of age (postnatal days). The regression line for reversal potentials crossed that of resting membrane potentials between P5 and P6.
18
Ionic mechanisms Changing the extracellular chloride concentrations [CI-]0 from 136-1 to 101 mm (isethionate substitution) increased the amplitude of the depolarizing response to glycine. In a low-chloride medium the hyperpolarizing response to glycine shifted to
GLYCINE ON NEONATAL HIPPOCAMPAL NEURONES
75
the depolarizing direction. Once it reached the threshold, the depolarization gave rise to action potentials (Fig. 6). A 13-5-fold change in [Cl-]0 yielded a change in glycine reversal potential, Egly, of 40 +3-6 mV (n = 4). As shown in Fig. 6B, the slight deviation of Egly from chloride reversal potential (Ec1), calculated from the Nernst A
-70~mV1'/
Low Cl-, Gly (1 mM)
Control, Gly (1 mM, P6)
|tX
Wash, Gly (1 mM)
|
0 T
-25 mV 1 min
B
Eci
0
E-50
)
-
_(
(3)
(7)
-100- r 10
30
100
[CI-]o (mM) Fig. 6. Chloride dependency of the response to glycine. A, the response to glycine (open bar) recorded in the presence of TTX (1 uM) shifted to the depolarizing direction and increased in amplitude during superfusion with low-chloride (10 mM) medium (isethionate substitution). The depolarization gave rise to calcium action potentials. B, relation between the reversal potential for glycine responses obtained at P6-P7 (Eg,y) and the extracellular chloride concentration [Cl-]0. Each point represents the mean and S.D. for the samples (numbers in parentheses). The dashed line represents the change in 67-8 mV for 13-5-fold change of [Cl-]0, predicted from the Nernst equation.
equation, might reflect either a small permeability of isethionate through chloride channels or slow changes in driving forces concomitant with movements of chloride ions in and out of the cells (Adams, Constanti & Banks, 1981). Reduction of calcium concentration from 2 to 0-2 mm changed neither the amplitude (Fig. IB) nor the reversal potential of the responses to glycine. The role of potassium ions in glycine response was assessed by recording with CsClfilled electrodes. Glycine depolarizations were similar whether Cs+ or K+ was used in the recording electrode. In three cells recorded with electrodes containing CsCl, at resting membrane potential, glycine (0 3 ,tM) caused a membrane depolarization of 5-2 + 2 mV.
S. ITO AND E. CHER UBINI
76
Strychnine selectively antagonizes glycine responses Bath application of strychnine (0 3-1 ,tM) reversibly depressed or abolished glycine responses. Usually strychnine (1 ,UM) completely antagonized the responses to glycine (300-500 /tM). The depression of glycine response was maximal within 3-5 min from Control
A
Gly (1 mM)
Gly (3 mM)
B
-59 mV
(
m -59
-l ' 4
Strychnine (1 pM) X1I
V _ TT u5 Control
X ~~~Strychnine S ry pM X
03
13pU,Mg3 M
/
0
Strychnine (3pM) 11,1
, 1 0
'""jJlOmV 1 min
i. 00
PM
300
PM
1 mMM
3
10 M
30
MMm
[Glycine]
C
2-
0) 0
1pm 3pm 10 pm Strychnine Fig. 7. Strychnine antagonizes the responses to glycine in an apparently competitive manner. A, responses obtained at P7 to two different concentrations of glycine (open bars) in control conditions and in the presence of 1 or 3 /tM-strychnine (in the presence of TTX, 1 /IM). B, dose-response curves for glycine before and during superfusion with strychnine in the concentrations indicated (/uM). Strychnine caused a parallel shift of the glycine concentration-response curve. C, Schild plot of results shown in B. Linear regression line (slope 1) yielded an estimated pA2 (-log Kd) value of 6-57 for strychnine. DR, dose ratio. 1
Onm
the beginning of strychnine superfusion and a complete recovery was obtained 20-30 min after wash. Figure 7 shows the parallel shift to the right of the glycine concentration-response curve by increasing doses of strychnine. The dose ratio was 30 + 6 at 10 /tM-strychnine and the apparent dissociation constant value (apparent Kd) for strychnine was 350+ 76 nM (n = 4). To see whether the depressant effect of strychnine was selective for glycine, both strychnine and bicuculline were tested on the responses induced in the same neurones by superfusion of glycine and GABA (n = 6). Strychnine (1 ,UM) antagonized the response to glycine but had little effect on the GABA response. In contrast
GLYCINE ON NEONATAL HIPPOCAMPAL NEURONES
77
bicuculline (10-50 /uM) depressed the GABA response without affecting the response to glycine (Fig. 8). Comparison of the responses to glycine and GABA In neonatal hippocampal neurones at PO-P4, GABA, acting via GABAA receptors, causes a bicuculline-sensitive depolarization and an increase in chloride conductance Control GABA (300 AiM)
Gly (1 mM)
PII
-62
I
Journal of Physiology (1991), 440, pp. 67-83 With 10 figures Printed in Great Britain
STRYCHNINE-SENSITIVE GLYCINE RESPONSES OF NEONATAL RAT HIPPOCAMPAL NEURONES
BY SUSUMU ITO* AND ENRICO CHERUBINIt From the Unite 029, INSERM, 123 Boulevard de Port-Royal, 75014 Paris, France
(Received 5 November 1990) SUMMARY
1. Intracellular recordings employing current and voltage clamp techniques were used to study the effects of glycine on rat CA3 hippocampal neurones during the first 3 weeks of postnatal (P) life. 2. Glycine (0 3-1 mM) depolarized neurones from rats less than 4 days old (P4). Neurones from older neonates (P5-P7) were hyperpolarized by glycine, whereas adult neurones were unaffected. 3. Both depolarizing and hyperpolarizing responses were associated with large conductance increases; they reversed polarity at a potential which changed with the extracellular chloride concentration. The responses persisted in tetrodotoxin (1 gM) or in a solution with a much reduced calcium concentration. 4. Strychnine (1 #M) but not bicuculline (10-50 JIM) antagonized the effects of glycine. The action of strychnine was apparently competitive with a dissociation constant of 350 nm. 5. In voltage clamp experiments, glycine elicited a non-desensitizing outward current at -60 mV. When a maximal concentration of glycine was applied at the same time as y-aminobutyric acid (GABA), the conductance increase induced by the two agonists was additive, suggesting the activation of different populations of channels. 6. Concentrations of glycine lower than 100 JM did not affect membrane potential. However, at 30-50 #M glycine increased the frequency of spontaneous GABAmediated synaptic responses; this action was not blocked by strychnine. 7. It is concluded that during the first 2 weeks of life glycine acts at strychninesensitive receptors to open chloride channels. INTRODUCTION
Glycine is the main inhibitory transmitter in hindbrain areas (Aprison & Daly, 1978). Its effect is mediated through the activation of high-affinity receptors localized on neurones of the spinal cord, medulla and pons (Young & Snyder, 1973). The interaction of glycine with its receptor results in an increase in chloride * Present address: Department of Neurophysiology, Medical Research Institute, Tokyo Medical and Dental University, Kanda-Surugadai Chiyoda-ku, Tokyo, Japan. t Present address and to whom correspondence should be sent: International School for Advanced Studies, Strada Costiera 11, 34014 Trieste, Italy.
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S. ITO AND E. CHERUBINI
conductance (Coombs, Eccles & Fatt, 1955; Araki, Ito & Oscarsson, 1961; Eccles, 1964) an effect specifically blocked by the convulsant alkaloid strychnine (Curtis, Hosli & Johnston, 1968). The neurodepressant potency of glycine is related to the uneven distribution of this endogenous amino acid, being greatest in the spinal cord and brain stem level and progressively decreasing in more rostral areas (Kishimoto, Simon & Aprison, 1981). Electrophysiological experiments on cat and rat cortical neurones in vivo have shown that on these cells glycine does not play a major role in synaptic inhibition (Kelly & Krnjevic, 1969; Levi, Bernardi, Cherubini, Gallo, Marciani & Stanzione, 1982). Furthermore, glycine fails to change membrane potential or conductance of adult rat hippocampal neurones in vivo (Ben-Ari, Krnjevic, Reiffenstein & Reinhardt, 1981). In addition to its classical inhibitory action, glycine serves also as an allosteric regulator of excitatory neurotransmission mediated by the N-methyl-D-aspartate (NMDA) receptors (Johnson & Ascher, 1987). This action involves distinct glycine binding sites, which are insensitive to strychnine (Bristow, Bowery & Woodruff, 1986) and are mainly localized to rostral brain areas, including cortex and hippocampus (Kessler, Terramani, Lynch & Baudry, 1989). We have recently reported that, in hippocampal neurones from neonatal (but not adult) rats, micromolar concentrations of glycine enhance the frequency of y-aminobutyric acid (GABA)-mediated synaptic potentials and potentiate NMDA responses (Gaiarsa, Corradetti, Cherubini & Ben-Ari, 1990). This effect, which is strychnine insensitive, is clearly mediated by the allosteric site of the NMDA receptor located on GABAergic interneurones (Ben-Ari, Cherubini, Corradetti & Gaiarsa, 1989) since it can be prevented by the specific NMDA receptor antagonist D-(-)-2-amino-5phosphonovalerate (AP-5; Gaiarsa et al. 1990). We now report that, in addition to its facilitatory action on NMDA-mediated events, glycine induced strychnine-sensitive, chloride-dependent responses detectable only during the first 2 weeks of postnatal life. Part of this work has been reported in a preliminary form (Ito & Cherubini, 1990). METHODS
Experiments were performed on CA3 hippocampal neurones from slices obtained from 2- to 21day-old (P2-P21 days; 0 is taken as the day of birth) and adult Wistar rats. The methods for preparing and maintaining the slices have been extensively reported (Ben-Ari et al. 1989). Rats were decapitated under ether anaesthesia and their brains quickly removed from the skulls. Hippocampi were dissected free; transverse 600 ,am thick slices were cut and immediately incubated at room temperature (20-22 °C) in artificial cerebrospinal fluid (ACSF) of the following composition (mm): NaCl, 126; KCl, 3-5; CaCl2, 2; NaH2PO4, 1-2; MgCl2, 13; NaHCO3, 25; glucose, 11. Equilibrating the ACSF with 95% 02 and 5 % CO2 gave a pH of 7 3-7A4. The slices were allowed to recover for at least 1 h before being transferred to a recording chamber in which they were continuously superfused at 33-34 °C with oxygenated ACSF at a rate of 25-3 ml min-'. Intracellular recordings were made with microelectrodes filled with 2 M-K-methylsulphate (resistance 50-100 MQ). In a few experiments CA3 neurones were impaled with microelectrodes containing 3 M-KCl or 3 M-CsCl (resistance 40-60 MQ). Current was injected through the recording electrode by means of an Axoclamp 2A amplifier. Bridge balance was checked repeatedly during the experiments and capacitative transients (with the electrode tip outside the neurone) were reduced to a minimum by negative capacity compensation. Stabilization of intracellular recording was helped by the injection of small hyperpolarizing currents (0 1-0 3 nA) but in cells from slices obtained from the youngest animals it often was necessary to maintain such a small current
GLYCINE ON NEONATAL HIPPOCAMPAL NEURONES
69
indefinitely (Schwartzkroin & Kunkel, 1982). Membrane potential was estimated from the potential observed upon withdrawal of the electrode from the cell. Membrane input resistance was measured from the amplitude of small hyperpolarizations (200-300 ms duration) evoked by passing known currents across the cell membrane. The conductance increment induced by glycine (fractional conductance increase, Ag, Morita, North & Tokimasa, 1982) was calculated from (R/R') -1, where R is the input resistance at the resting potential and R' is the input resistance during glycine response. In voltage clamp experiments, membrane currents were recorded via a single-electrode voltaae clamp amplifier (Axoclamp 2A, Axon Instruments, Inc.), switching between voltage recording and current injection at 3-4 kHz (30 % duty cycle). The voltage signal at the head stage amplifier was continuously monitored on a separate oscilloscope to ensure optimal performance of the voltage clamp system. Responses were digitized and displayed on a Nicolet digital oscilloscope and on a computer-driven chart-recorder. Some experiments were performed in a low-calcium-highmagnesium ACSF (0-2 mM-Ca2+ and 6 mM-Mg2+ instead of 2 and 1-3 mM) or in a low-chloride ACSF where 126 mM-NaCl was substituted by 126 mM-sodium isethionate, corresponding to 92-6% chloride substitution. Drugs were dissolved in ACSF and applied through a three-way tap system by changing the superfusing solution to one which differed only in its content of drug(s). There was a delay of 20-30 s between turning the tap and the first arrival at the tissue of the changed solution. The ratio of flow rate to bath volume ensured complete exchange within 1 min. Drugs used were: glycine, GABA, strychnine, bicuculline, all purchased from Sigma; D-(-)-2-amino-5-phosphonovalerate (AP-5) and (± )3-(2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid (CPP) purchased from CRB or Tocris. Unless otherwise stated, all data are expressed as means+ S.D. RESULTS
Stable intracellular recordings were obtained from sixty-four pyramidal neurones in slices from 2- to 21-day-old rats or from three adult rats. In agreement with a previous study (Ben-Ari et al. 1989), all neurones recorded during the first postnatal week with potassium methylsulphate electrodes (n = 42) had a resting membrane potential of - 65-2 + 78 mV (ranging from -55 to -75 mV, see Fig. 5A), resting input resistance ranging from 80 to 130 MQ and action potentials greater than 55 mV. Membrane potential changes induced by glycine Days PO-P4 Superfusion with glycine (0-3 mm), caused at resting membrane potential a membrane depolarization (4 ± P17 mV, n = 7), which was associated with an increase in membrane input conductance. The depolarization had a latency of 20-30 s after glycine reached the tissue and achieved its peak amplitude at 1 min. This effect washed out in a few minutes. A typical example from a 3-day-old rat is shown in Fig. 1A. The sensitivity to glycine varied from cell to cell, but in a given neurone the response varied with the concentration applied. The minimum dose of glycine required to produce membrane potential changes was 100 ,LM. Increasing the concentration of glycine increased the peak amplitude, particularly the rate of rise of the depolarization. As already reported (Ben-Ari et al. 1989), > 90 % of the neurones at P0-P4 exhibit spontaneous GABA-mediated giant depolarizing potentials (GDPs). These consisted of brief, high-frequency bursts of spikes riding on large depolarizing potentials. Concentrations of glycine lower than those required to cause membrane potential changes (30-50 JtM) increased the frequency of GDPs. This effect, which was insensitive to strychnine (1 /M), was blocked by the NMDA receptor antagonists AP-
S. ITO AND E CHER UBINI
70
5 or CPP (50 JLM), suggesting that it was mediated by the allosteric modulatory site of the NMDA receptors located on GABAergic interneurones (Gaiarsa et al. 1990). A similar effect was seen with application of the higher glycine concentrations, but it occurred transiently and before the membrane potential changed, presumably a A
Gly (300 .=.
-61
=
gM, P3)
mV'
Gly (1 mM, P6)
-64
MV
II
~I10 mV t
tt
~~~1min
B
Control (P7)
-63 mV
4p
TTX (1 gM)
Low calcium, high magnesium
ft4V 10 mV 1 min
Fig. 1. Glycine depolarized or hyperpolarized neonatal neurones. A, at P3, bath application of glycine (Gly, open bars) induced a membrane depolarization associated with an increase in input conductance. Note that glycine first increased and then suppressed (for 5 min) spontaneously occurring GABA-mediated giant depolarizing potentials. At P6, glycine induced a membrane hyperpolarization and an increase in input conductance. The effect of glycine was associated with an increase in frequency of spontaneous GABA-mediated hyperpolarizing potentials (arrows) followed by their suppression. Upward deflections on the left panel are action potentials riding on slow depolarizations; on the right panel are single spikes. B, the responses to glycine (1 mM, open bars) were unchanged in the presence of TTX or in a low-calcium (0-2 mM), highmagnesium (6 mM) solution. Downward deflections in this and following figures are electrotonic potentials resulting from injection of a fixed current pulse through the recording electrode.
reflection of the slowly increasing concentration of glycine in the tissue slice when it was applied by superfusion. Days P4-P7 Towards the end of the first postnatal week, during a transitional period characterized by the progressive disappearance of GDPs and their replacement by large hyperpolarizing potentials (responses to GABA also changed from the depolarizing to the hyperpolarizing ones; Ben-Ari et al. 1989), glycine induced membrane hyperpolarization and increased input conductance (Fig. 1A). Like the depolarizing response, the hyperpolarizing response was also dose dependent. Increasing the concentration of glycine caused an increase in the peak
GLYCINE ON NEONATAL HIPPOCAMPAL NEURONES
71
amplitude of the membrane hyperpolarization, particularly in the rise of the hyperpolarization. At P7, concentrations of glycine of 0 3 and 1 mm caused (at resting membrane potential) a hyperpolarization of 2-2 + 0-6 (n = 9) and 4-3 + 1-5 mV (n = 7) respectively. The depolarizing or hyperpolarizing response to glycine was unchanged by tetrodotoxin (TTX, 1 ,tM) or by a low-calcium (0-2 mM), high-magnesium (6 mm) solution, implying that it probably did not result from action potential generation and release of transmitter(s) from surrounding cells (Fig. 1B). As for GDPs, the hyperpolarizing effect of glycine was associated with a transient increase in frequency of spontaneous GABA-mediated large hyperpolarizing potentials which, at this stage of development, replace GDPs (Gaiarsa et al. 1990). The spontaneous firing was also completely and reversibly blocked by glycine (Fig. 1A). Days P7-adult After the first postnatal week, the sensitivity to glycine decreased gradually and completely disappeared after P14. At the end of the second postnatal week and in adult neurones, glycine at concentrations of 3-10 mm failed to induce any change in membrane potential, input conductance or synaptic noise.
Conductance changes elicited by glycine Days PO-P4 Glycine-induced depolarizations were accompanied by an increase in cell input conductance, measured from the amplitude of the hyperpolarizing electrotonic potentials (Fig. 2A). The conductance increase persisted after repolarizing the membrane potential to its initial value at rest. The conductance increase reached its maximum within 20-30 s after the beginning of glycine application. Applications of 1-2 min were usually enough to obtain steady-state responses; prolongation of the drug application did not show any further significant change in conductance. Although the sensitivity to glycine varied from cell to cell, the conductance change became detectable at a concentration of 100 #M (Fig. 2A). The conductance changes increased with higher concentrations and reached the maximum at concentrations of 3-10 mm. With these glycine concentrations, the amplitude of the response and associated conductance increase, after an initial peak, declined to reach a steadystate value; this was also seen when the application of glycine was maintained for several minutes.
Days P4-P7
Glycine-induced hyperpolarizations were associated with an increase in cell input conductance. Figure 2B shows the mean fractional conductance increase induced by increasing concentration of glycine during this period of postnatal life. The halfmaximal conductance change was obtained with a glycine concentration of 1H1 mm. The Hill coefficient, calculated in two neurones from the conductance changes induced by increasing concentrations of glycine, was 1-6 and 1-8 respectively.
S. ITO AND E. CHER UBINI
72
Days P7-adult After the first postnatal week, the conductance increase induced by glycine gradually decreased. At P14 and in adult neurones, glycine at concentrations up to 3 mm, applied for a longer period of time, failed to induce any change in membrane A
Gly (100
kiM, P7)
Gly (300 PM)
Gly (1 mM)
Ti
-59 mV
I
10m
W
1 min
B u)
(10)
(2)
(24) (7)
o o 0-
~~~~~(4)/ -4
-3
-2
log lglycinel
Fig. 2. Glycine induced a dose-dependent increase in input conductance. A, increasing concentrations of glycine (open bars) induced membrane hyperpolarization associated with an increase in input conductance. The graph in B represents the fractional conductance increase produced in CA3 neurones during the first postnatal week by increasing concentrations of glycine. Each point represents the mean and S.E.M. of the samples (numbers in parentheses).
potential or input conductance. These results are summarized in Fig. 3, where the fractional conductance increase induced by glycine (1 mM) is plotted as a function of age (postnatal days). During the first postnatal week, every neurone tested responded to glycine with a mean fractional conductance change of 0-85 + 0-38 (n = 24). During the second postnatal week, some neurones were insensitive to glycine and the mean fractional conductance change fell to 0 4 at PIO and to 0-2 at P14. At P21 no neurones (n = 6) responded to glycine. Reversal potential of the responses to glycine The amplitude of glycine-induced depolarizations and hyperpolarizations was a linear function of the membrane potential in the range -90 to -40 mV. The depolarizing response to glycine increased with membrane hyperpolarization and decreased with depolarization. At P2-P4, its reversal potential was - 53-5 + 9-6 mV
GLYCINE ON NEONATAL HIPPOCAMPAL NEURONES 73 (Fig. 4, n = 10) with potassium methylsulphate-containing electrodes and -23 + 7-2 mV (n = 5) with KCl- or CsCl-filled electrodes. After P4, the responses to glycine shifted from the depolarizing to the hyperpolarizing direction. Thus the amplitude of glycine-induced hyperpolarization increased with depolarization and A
Gly (300 pM, P6)
Gly (300 pm, P3) -60
-63 mV
Gly (3 mM, P14) -68 mV
....
mV
jiQ mV B
1
°
min
a, CD)
1
(9)
C4
(9)
(6)
()
(6 )
o
0CC.) .L
5_
(; 1
7
)
(6) 6
14
21
Age (days) Fig. 3. Glycine induced a conductance increase only during the first 2 weeks of postnatal life. A, glycine (open bars) caused membrane depolarization or hyperpolarization associated with an increase in input conductance at P3 and P6 respectively. At P14, a higher concentration of glycine, applied for 2 min, failed to induce any change in membrane potential or input conductance. B, fractional conductance increase induced by glycine (1 mM) as a function of age (postnatal days). Each point represents the mean and S.E.M. (numbers in parentheses). TTX (1 /iM) present throughout the experiment.
decreased with hyperpolarization. The reversal potential recorded between P5 and P7 was - 63-2 + 11-6 mV with potassium methylsulphate-containing electrodes (Fig. 4, n = 20) and -40X2 + 10-6 mV with KCl-filled electrodes (n = 5). Figure 5 shows the distribution values of the resting membrane potential and the reversal potential of the responses to glycine in neurones recorded at P2-P7 with potassium methylsulphate-containing electrodes. In agreement with a previous report (Ben-Ari et al. 1989), we observed that, in contrast to the reversal potential of the responses to glycine, which showed a significant difference (P < 0-01, rank-sum test) between the P2-P4 and P5-P7 groups, the resting membrane potential of cells at P2-P4 was not significantly different from that recorded at P5-P7. The mean resting membrane potential at P2-P4 was - 66-6 + 7 mV (n = 11) and at P5-P7 was - 64-5 + 8 mV (n = 31). The regression line for reversal potentials crossed that of resting membrane potentials between P5 and P6.
S. ITO AND E. CHER UBINI
74 A
Gly (300 piM, P7, 0)
Gly(1 mM, P2,A) lZZ=
-38 mV
i
E
B
-68 mv
-10 CDv
O.
0
A -48
-78
.p
I,!
-88 r8 " .~~ ~~~~~~~~~
-68 ..;.
:,
10
.,...
mv
'; il,
ii, "!
CD 0 A -4 90 -80 -70 A -40 -50 0 . ° > -6 60 'A ' -5 0) °O 0 0 V -1 0 Membrane potential (rmV)
lJ jI10 m\
Q ~~~~~~~E
v
1 min
1 min
Fig. 4. Reversal of glycine responses. A, changing the membrane potential by passing a steady current through the recording electrode changed the amplitude of the depolarization or hyperpolarization induced by glycine (open bars) at P2 (A) and P7 (0) respectively. The resting membrane potential of both cells was -68 mV. B, relationship between the membrane potential and the amplitude of glycine responses for the cells shown in A. Note the shift in the reversal potential from -55 mV (at P2) to -80 mV (at P7). TTX (1 /sM) present throughout the experiment. A
g
B
-30-
-30
-
-50
-
-70
-
4-
C
2
0 a 0
E
-50-
E
-70-
a) 0)
t
.
s
.
0~~~
._
M
0 *
9.
C
.
-zo
0
0)
. a
E
U)
cc
C
-
0) a -inn c
-
I
I
1
2
I
-I 3
4
5
_1nn
I
I
I
I
6
-
7
8
1
2
3
4
5
6
7
Age (days) Age (days) Fig. 5. Changes in reversal potential for glycine are independent from the resting membrane potential. Plot of the distribution values of the resting membrane potentials (A) and the reversal potentials of glycine responses (B) as a function of age (postnatal days). The regression line for reversal potentials crossed that of resting membrane potentials between P5 and P6.
18
Ionic mechanisms Changing the extracellular chloride concentrations [CI-]0 from 136-1 to 101 mm (isethionate substitution) increased the amplitude of the depolarizing response to glycine. In a low-chloride medium the hyperpolarizing response to glycine shifted to
GLYCINE ON NEONATAL HIPPOCAMPAL NEURONES
75
the depolarizing direction. Once it reached the threshold, the depolarization gave rise to action potentials (Fig. 6). A 13-5-fold change in [Cl-]0 yielded a change in glycine reversal potential, Egly, of 40 +3-6 mV (n = 4). As shown in Fig. 6B, the slight deviation of Egly from chloride reversal potential (Ec1), calculated from the Nernst A
-70~mV1'/
Low Cl-, Gly (1 mM)
Control, Gly (1 mM, P6)
|tX
Wash, Gly (1 mM)
|
0 T
-25 mV 1 min
B
Eci
0
E-50
)
-
_(
(3)
(7)
-100- r 10
30
100
[CI-]o (mM) Fig. 6. Chloride dependency of the response to glycine. A, the response to glycine (open bar) recorded in the presence of TTX (1 uM) shifted to the depolarizing direction and increased in amplitude during superfusion with low-chloride (10 mM) medium (isethionate substitution). The depolarization gave rise to calcium action potentials. B, relation between the reversal potential for glycine responses obtained at P6-P7 (Eg,y) and the extracellular chloride concentration [Cl-]0. Each point represents the mean and S.D. for the samples (numbers in parentheses). The dashed line represents the change in 67-8 mV for 13-5-fold change of [Cl-]0, predicted from the Nernst equation.
equation, might reflect either a small permeability of isethionate through chloride channels or slow changes in driving forces concomitant with movements of chloride ions in and out of the cells (Adams, Constanti & Banks, 1981). Reduction of calcium concentration from 2 to 0-2 mm changed neither the amplitude (Fig. IB) nor the reversal potential of the responses to glycine. The role of potassium ions in glycine response was assessed by recording with CsClfilled electrodes. Glycine depolarizations were similar whether Cs+ or K+ was used in the recording electrode. In three cells recorded with electrodes containing CsCl, at resting membrane potential, glycine (0 3 ,tM) caused a membrane depolarization of 5-2 + 2 mV.
S. ITO AND E. CHER UBINI
76
Strychnine selectively antagonizes glycine responses Bath application of strychnine (0 3-1 ,tM) reversibly depressed or abolished glycine responses. Usually strychnine (1 ,UM) completely antagonized the responses to glycine (300-500 /tM). The depression of glycine response was maximal within 3-5 min from Control
A
Gly (1 mM)
Gly (3 mM)
B
-59 mV
(
m -59
-l ' 4
Strychnine (1 pM) X1I
V _ TT u5 Control
X ~~~Strychnine S ry pM X
03
13pU,Mg3 M
/
0
Strychnine (3pM) 11,1
, 1 0
'""jJlOmV 1 min
i. 00
PM
300
PM
1 mMM
3
10 M
30
MMm
[Glycine]
C
2-
0) 0
1pm 3pm 10 pm Strychnine Fig. 7. Strychnine antagonizes the responses to glycine in an apparently competitive manner. A, responses obtained at P7 to two different concentrations of glycine (open bars) in control conditions and in the presence of 1 or 3 /tM-strychnine (in the presence of TTX, 1 /IM). B, dose-response curves for glycine before and during superfusion with strychnine in the concentrations indicated (/uM). Strychnine caused a parallel shift of the glycine concentration-response curve. C, Schild plot of results shown in B. Linear regression line (slope 1) yielded an estimated pA2 (-log Kd) value of 6-57 for strychnine. DR, dose ratio. 1
Onm
the beginning of strychnine superfusion and a complete recovery was obtained 20-30 min after wash. Figure 7 shows the parallel shift to the right of the glycine concentration-response curve by increasing doses of strychnine. The dose ratio was 30 + 6 at 10 /tM-strychnine and the apparent dissociation constant value (apparent Kd) for strychnine was 350+ 76 nM (n = 4). To see whether the depressant effect of strychnine was selective for glycine, both strychnine and bicuculline were tested on the responses induced in the same neurones by superfusion of glycine and GABA (n = 6). Strychnine (1 ,UM) antagonized the response to glycine but had little effect on the GABA response. In contrast
GLYCINE ON NEONATAL HIPPOCAMPAL NEURONES
77
bicuculline (10-50 /uM) depressed the GABA response without affecting the response to glycine (Fig. 8). Comparison of the responses to glycine and GABA In neonatal hippocampal neurones at PO-P4, GABA, acting via GABAA receptors, causes a bicuculline-sensitive depolarization and an increase in chloride conductance Control GABA (300 AiM)
Gly (1 mM)
PII
-62
I
