J. Phy.iol. (1978), 275, pp. 135-148 With 9 text-filgur&s Printed in Great Britain
135
FIELD POTENTIALS, INHIBITION AND THE EFFECT OF PENTOBARBITONE IN THE RAT OLFACTORY CORTEX SLICE
BY HILARY G. PICKLES AND M. A. SIMMONDS From the Department of Pharmacology, The School of Pharmacy, University of London, 29/39 Brunswick Square, London, WC1N lAX
(Received 16 August 1977) SUMMARY
1. Field potentials were evoked in slices of rat olfactory cortex by stimulating the lateral olfactory tract. In addition to previously described components of the waveform, a further distinct surface-negative potential of low amplitude and long duration (I-wave) has been described. 2. Pentobarbitone, at concentrations of 10-5 M and above, markedly enhanced the amplitude and duration of the I-wave with only minimal effect on other components of the field potential. 3. The I-wave was reversibly reduced by the GABA antagonists bicuculline and picrotoxin and was also attenuated at rapid rates of stimulation. Low chloride medium usually caused a transient increase in amplitude of the I-wave followed by a gradual reduction, suggesting that a chloride-mediated depolarization was involved. 4. Evoked inhibition, which was most probably post-synaptic, occurred in parallel with the I-wave. This was monitored as a suppression of, or increase in latency of the population spike evoked by a second stimulus at appropriate intervals after the first. Pentobarbitone substantially increased the duration of the post-synaptic inhibition, without obvious changes in the presynaptic inhibitory phenomenon associated with antidromic firing in the lateral olfactory tract. 5. It is proposed that the I-wave is the field potential representation of a population depolarizing i.p.s.p. and that the main action of pentobarbitone is to enhance this inhibition. INTRODUCTION
In many preparations, barbiturates are now thought to enhance inhibition. For example, presynaptic inhibition is affected in the spinal cord (Eccles, Schmidt & Willis, 1963) and cuneate nucleus (Banna & Jabbur, 1969) and post-synaptic inhibition in the olfactory bulb (Nicoll, 1972) and hippocampus (Nicoll, Eccles, Oshima & Rubia, 1975). In the olfactory cortex slice, however, it has been suggested that the main action of pentobarbitone might be a more direct reduction in excitatory transmission (Richards, 1972a),- as previously proposed for the action on spinal motoneurones (Weakly, 1969). Nevertheless, an enhancement of inhibition by pentobarbitone in the olfactory cortex slice could not be completely excluded since no convincing field potential correlate of post-synaptic inhibition had been demonstrated in this preparation.
1I. G. PICKLES AND M. A. SIMMONDS 136 In vivo studies in the olfactory cortex (Legge, Randic & Straughan, 1966; Biedenbach & Stevens, 1969b; Haberly, 1973a) have shown the presence of prolonged inhibition (for up to 400 msec) following lateral olfactory tract stimulation and this inhibition is generally assumed to be post-synaptic. At one time it was thought that the surface-positive waves recorded in vivo (Biedenbach & Stevens, 1969a; Haberly, 1973b) and in vitro by some workers (Harvey, Scholfield & Brown, 1974; Richards & Sercombe, 1968) were inhibitory field potentials representing hyperpolarizing i.p.s.p.s of the pyramidal cell bodies. However, it has now been suggested that the P-wave reflects a population e.p.s.p. of deeper cells (Halliwell, 1976a, b). Recently, we have demonstrated that a long latency, surface-negative field potential associated with a form of presynaptic inhibition could be elicited in the rat olfactory cortex slice, provided that the lateral olfactory tract was stimulated at very low frequencies (Pickles & Simmonds, 1976a). This prompted us to re-examine the effects of pentobarbitone on this tissue. Our preliminary experiments have shown a striking prolongation by pentobarbitone of the long latency negative field potential (Pickles & Simmonds, 1976b). In the present paper, we characterize this phenomenon further and suggest that it could be due to the enhancement of a component of the field potential representing post-synaptic inhibition. This interpretation is strongly supported by intracellular recordings from superficial pyramidal cells in the olfactory cortex slice (Scholfield, 1976, 1977, 1978 a, b) which suggest that evoked i.p.s.p.s are normally in the depolarizing direction in this preparation and that the post-synaptic inhibition is markedly prolonged by pentobarbitone. METHODS
Slices of rat olfactory cortex approx. 0 5 mm thick were prepared as previously described (Pickles & Simmonds, 1976a). A preincubated slice was placed in a chamber and perfused at room temperature with oxygenated medium with the following composition: NaCl 118.1 mm, KCl 241 mM, CaCl2 2-5 mM, KH2PO4 0 93 mm, MgSO4 2-2 mm, NaHCO3 25 mm, glucose 11 1 mm. The lateral olfactory tract was stimulated with 50 #usec square pulses of amplitude supramaximal for the N-wave at 0-011 Hz (every 90 sec) unless otherwise specified. Field potential responses were recorded from the prepiriform cortex with a glass micro-electrode of 2-3 MQ resistance filled with 3 m-NaCl. Antidromic activity was monitored via a portion of the tract rostral to the stimulating electrode drawn into a suction electrode. Both recordings were made via DC preamplifiers and either a Medelec U.V. recorder or a Datalab DL 901 transient recorder connected to a chart recorder. An indifferent electrode of Ag/AgCl was in the bottom of the chamber. The olfactory cortex slice does not exhibit spontaneous activity but, following stimulation of the lateral olfactory tract, there can be recorded from the prepiriform cortex surface (i) a presynaptic mass action potential, (ii) a population e.p.s.p. (N-wave) (Yamamoto & Mcllwain, 1966) with a superimposed population spike which represents firing of the cells (Richards & Sercombe, 1968) and (iii) another series of population e.p.s.p.s. initiated indirectly via presynaptic terminal depolarization (late N-wave) (Pickles & Simmonds, 1976a). There is also (iv) a long duration negative tail following the late N-wave, not previously characterized. In view of the results which follow, we have called this the I-wave (inhibitory wave). All these components are shown in Fig. 1. All measurements of the evoked field potential were made from the recording baseline. The N-wave amplitude was measured just prior to the peak of negativity, at a latency which was fixed for each slice and varied between 5 and 10 msec from the stimulus; the late N-wave was measured at its first peak irrespective of the latency, which was dependent on stimulation frequency; the I-wave was measured at the latency specified in each case. Latency measurements were made on the population spike from the stimulus artifact to the peak of positivity.
PENTOBARBITONE AND INHIBITION
137
Sodium pentobarbitone was dissolved in oxygenated Krebs solution to give a final concentration from 3 x 10-6 M to 3 x 104 M, i.e. concentrations not exceeding the minimum calculated for extracellular fluid during anaesthesia (see Richards, 1972a), and after recording for at least i hr in normal solution each slice was exposed to pentobarbitone solution. Measurements for the dose-response curves were made after 1 hr in pentobarbitone solution. Because recovery was so prolonged, each slice was exposed to only one dose of the drug. N late N Tract action
I
potential
|1 mV
A 0-011 Hz
Z
0-2 mV
N
B 1 0 Hz
#(ii)
1 00 msec Fig. 1. Field potential (top record of each pair) and simultaneous recording from a small section of the rostral end of the lateral olfactory tract (bottom record) upon stimulation of the tract; A, at a slow rate (0 011 Hz) and B, at a fast rate (1 Hz). At slow stimulation rates the action potential and N-wave are followed by a late N-wave which merges into the I-wave. At faster rates the late N-wave and the associated antidromic activity in the tract are no longer apparent, whereas a reduced I-wave is still present. In this figure, as in all subsequent figures, negativity is shown upwards. Our interpretation of the components of the tract recording is as follows: (i) initial mass action potential (off scale); (ii) afterhyperpolarization resulting from this action potential; (iii) evoked antidromic firing - perhaps equivalent to the dorsal root reflex in spinal cord; (iv) afterhyperpolarization resulting from this; (v) depolarization without evoked firing, perhaps equivalent to the dorsal root potential in spinal cord. In recordings where the evoked firing (iii) is less marked, this shallow depolarization is more obvious and can be seen throughout the period occupied by (iii) and (iv) in this recording.
RESULTS
The field potential
Effect of pentobarbitone On addition of pentobarbitone to the solution perfusing the slice, there were marked changes in the evoked field potential (Fig. 2). At a stimulation frequency of
138 H. G. PICKLES AND M. A. SIMMONDS 0.011 Hz, the most striking change was an enhancement in both amplitude and duration of the I-wave. For example, after 1 hr in the presence of 10-4 m-pentobarbitone, the I-wave had increased 9-10 fold at post-stimulus latencies of 400700 msec and the duration was sometimes increased in excess of 10 sec. At all of the doses used, the effect of pentobarbitone on the I-wave increased progressively throughout the period of contact (up to 12 hr) and was subsequently slowly reversible over several hours (Fig. 3). The effect was dose dependent (Fig. 4) but was still less than maximal at the highest concentrations used.
(1) (2) Control
(
>-
11 mV
5 mV | ~~~~~~~0
(3)
Pentobarbitone 10-4 M
Partial recovery
500 msec Fig. 2. The effect of 104 m-pentobarbitone on typical recordings. The cortical field potential (top) and the recording from the cut end of the lateral olfactory tract (bottom) are shown following stimulation at 0.011 Hz. While the amplitude of the N- (1) and late N- (2) waves and of the tract afterhyperpolarization and antidromic firing (3) showed the usual progressive increase with time, little affected by 1*25 hr in pentobarbitone, the substantial increase in the I-wave (4) during exposure to pentobarbitone was at least partially reversible. Note that the duration of the tract events was not affected by pentobarbitone, although the I-wave duration was increased to about 4 sec in this slice.
139 PENTOBARBITONE AND INHIBITION The amplitude of the late N-wave was little affected by the presence of pentobarbitone, sometimes showing a small decrease in spite of the presumably underlying I-wave being increased in amplitude. This lack of a clear change in the late N-wave was matched by the lack of any consistent change in a correlate, the antidromic
6r
10-4 M pentobarbitone
5_ ,
4.1
E
P..
O."^
*
....
....
Late N-wave
o3 I/-wave
0**
E
135
FIELD POTENTIALS, INHIBITION AND THE EFFECT OF PENTOBARBITONE IN THE RAT OLFACTORY CORTEX SLICE
BY HILARY G. PICKLES AND M. A. SIMMONDS From the Department of Pharmacology, The School of Pharmacy, University of London, 29/39 Brunswick Square, London, WC1N lAX
(Received 16 August 1977) SUMMARY
1. Field potentials were evoked in slices of rat olfactory cortex by stimulating the lateral olfactory tract. In addition to previously described components of the waveform, a further distinct surface-negative potential of low amplitude and long duration (I-wave) has been described. 2. Pentobarbitone, at concentrations of 10-5 M and above, markedly enhanced the amplitude and duration of the I-wave with only minimal effect on other components of the field potential. 3. The I-wave was reversibly reduced by the GABA antagonists bicuculline and picrotoxin and was also attenuated at rapid rates of stimulation. Low chloride medium usually caused a transient increase in amplitude of the I-wave followed by a gradual reduction, suggesting that a chloride-mediated depolarization was involved. 4. Evoked inhibition, which was most probably post-synaptic, occurred in parallel with the I-wave. This was monitored as a suppression of, or increase in latency of the population spike evoked by a second stimulus at appropriate intervals after the first. Pentobarbitone substantially increased the duration of the post-synaptic inhibition, without obvious changes in the presynaptic inhibitory phenomenon associated with antidromic firing in the lateral olfactory tract. 5. It is proposed that the I-wave is the field potential representation of a population depolarizing i.p.s.p. and that the main action of pentobarbitone is to enhance this inhibition. INTRODUCTION
In many preparations, barbiturates are now thought to enhance inhibition. For example, presynaptic inhibition is affected in the spinal cord (Eccles, Schmidt & Willis, 1963) and cuneate nucleus (Banna & Jabbur, 1969) and post-synaptic inhibition in the olfactory bulb (Nicoll, 1972) and hippocampus (Nicoll, Eccles, Oshima & Rubia, 1975). In the olfactory cortex slice, however, it has been suggested that the main action of pentobarbitone might be a more direct reduction in excitatory transmission (Richards, 1972a),- as previously proposed for the action on spinal motoneurones (Weakly, 1969). Nevertheless, an enhancement of inhibition by pentobarbitone in the olfactory cortex slice could not be completely excluded since no convincing field potential correlate of post-synaptic inhibition had been demonstrated in this preparation.
1I. G. PICKLES AND M. A. SIMMONDS 136 In vivo studies in the olfactory cortex (Legge, Randic & Straughan, 1966; Biedenbach & Stevens, 1969b; Haberly, 1973a) have shown the presence of prolonged inhibition (for up to 400 msec) following lateral olfactory tract stimulation and this inhibition is generally assumed to be post-synaptic. At one time it was thought that the surface-positive waves recorded in vivo (Biedenbach & Stevens, 1969a; Haberly, 1973b) and in vitro by some workers (Harvey, Scholfield & Brown, 1974; Richards & Sercombe, 1968) were inhibitory field potentials representing hyperpolarizing i.p.s.p.s of the pyramidal cell bodies. However, it has now been suggested that the P-wave reflects a population e.p.s.p. of deeper cells (Halliwell, 1976a, b). Recently, we have demonstrated that a long latency, surface-negative field potential associated with a form of presynaptic inhibition could be elicited in the rat olfactory cortex slice, provided that the lateral olfactory tract was stimulated at very low frequencies (Pickles & Simmonds, 1976a). This prompted us to re-examine the effects of pentobarbitone on this tissue. Our preliminary experiments have shown a striking prolongation by pentobarbitone of the long latency negative field potential (Pickles & Simmonds, 1976b). In the present paper, we characterize this phenomenon further and suggest that it could be due to the enhancement of a component of the field potential representing post-synaptic inhibition. This interpretation is strongly supported by intracellular recordings from superficial pyramidal cells in the olfactory cortex slice (Scholfield, 1976, 1977, 1978 a, b) which suggest that evoked i.p.s.p.s are normally in the depolarizing direction in this preparation and that the post-synaptic inhibition is markedly prolonged by pentobarbitone. METHODS
Slices of rat olfactory cortex approx. 0 5 mm thick were prepared as previously described (Pickles & Simmonds, 1976a). A preincubated slice was placed in a chamber and perfused at room temperature with oxygenated medium with the following composition: NaCl 118.1 mm, KCl 241 mM, CaCl2 2-5 mM, KH2PO4 0 93 mm, MgSO4 2-2 mm, NaHCO3 25 mm, glucose 11 1 mm. The lateral olfactory tract was stimulated with 50 #usec square pulses of amplitude supramaximal for the N-wave at 0-011 Hz (every 90 sec) unless otherwise specified. Field potential responses were recorded from the prepiriform cortex with a glass micro-electrode of 2-3 MQ resistance filled with 3 m-NaCl. Antidromic activity was monitored via a portion of the tract rostral to the stimulating electrode drawn into a suction electrode. Both recordings were made via DC preamplifiers and either a Medelec U.V. recorder or a Datalab DL 901 transient recorder connected to a chart recorder. An indifferent electrode of Ag/AgCl was in the bottom of the chamber. The olfactory cortex slice does not exhibit spontaneous activity but, following stimulation of the lateral olfactory tract, there can be recorded from the prepiriform cortex surface (i) a presynaptic mass action potential, (ii) a population e.p.s.p. (N-wave) (Yamamoto & Mcllwain, 1966) with a superimposed population spike which represents firing of the cells (Richards & Sercombe, 1968) and (iii) another series of population e.p.s.p.s. initiated indirectly via presynaptic terminal depolarization (late N-wave) (Pickles & Simmonds, 1976a). There is also (iv) a long duration negative tail following the late N-wave, not previously characterized. In view of the results which follow, we have called this the I-wave (inhibitory wave). All these components are shown in Fig. 1. All measurements of the evoked field potential were made from the recording baseline. The N-wave amplitude was measured just prior to the peak of negativity, at a latency which was fixed for each slice and varied between 5 and 10 msec from the stimulus; the late N-wave was measured at its first peak irrespective of the latency, which was dependent on stimulation frequency; the I-wave was measured at the latency specified in each case. Latency measurements were made on the population spike from the stimulus artifact to the peak of positivity.
PENTOBARBITONE AND INHIBITION
137
Sodium pentobarbitone was dissolved in oxygenated Krebs solution to give a final concentration from 3 x 10-6 M to 3 x 104 M, i.e. concentrations not exceeding the minimum calculated for extracellular fluid during anaesthesia (see Richards, 1972a), and after recording for at least i hr in normal solution each slice was exposed to pentobarbitone solution. Measurements for the dose-response curves were made after 1 hr in pentobarbitone solution. Because recovery was so prolonged, each slice was exposed to only one dose of the drug. N late N Tract action
I
potential
|1 mV
A 0-011 Hz
Z
0-2 mV
N
B 1 0 Hz
#(ii)
1 00 msec Fig. 1. Field potential (top record of each pair) and simultaneous recording from a small section of the rostral end of the lateral olfactory tract (bottom record) upon stimulation of the tract; A, at a slow rate (0 011 Hz) and B, at a fast rate (1 Hz). At slow stimulation rates the action potential and N-wave are followed by a late N-wave which merges into the I-wave. At faster rates the late N-wave and the associated antidromic activity in the tract are no longer apparent, whereas a reduced I-wave is still present. In this figure, as in all subsequent figures, negativity is shown upwards. Our interpretation of the components of the tract recording is as follows: (i) initial mass action potential (off scale); (ii) afterhyperpolarization resulting from this action potential; (iii) evoked antidromic firing - perhaps equivalent to the dorsal root reflex in spinal cord; (iv) afterhyperpolarization resulting from this; (v) depolarization without evoked firing, perhaps equivalent to the dorsal root potential in spinal cord. In recordings where the evoked firing (iii) is less marked, this shallow depolarization is more obvious and can be seen throughout the period occupied by (iii) and (iv) in this recording.
RESULTS
The field potential
Effect of pentobarbitone On addition of pentobarbitone to the solution perfusing the slice, there were marked changes in the evoked field potential (Fig. 2). At a stimulation frequency of
138 H. G. PICKLES AND M. A. SIMMONDS 0.011 Hz, the most striking change was an enhancement in both amplitude and duration of the I-wave. For example, after 1 hr in the presence of 10-4 m-pentobarbitone, the I-wave had increased 9-10 fold at post-stimulus latencies of 400700 msec and the duration was sometimes increased in excess of 10 sec. At all of the doses used, the effect of pentobarbitone on the I-wave increased progressively throughout the period of contact (up to 12 hr) and was subsequently slowly reversible over several hours (Fig. 3). The effect was dose dependent (Fig. 4) but was still less than maximal at the highest concentrations used.
(1) (2) Control
(
>-
11 mV
5 mV | ~~~~~~~0
(3)
Pentobarbitone 10-4 M
Partial recovery
500 msec Fig. 2. The effect of 104 m-pentobarbitone on typical recordings. The cortical field potential (top) and the recording from the cut end of the lateral olfactory tract (bottom) are shown following stimulation at 0.011 Hz. While the amplitude of the N- (1) and late N- (2) waves and of the tract afterhyperpolarization and antidromic firing (3) showed the usual progressive increase with time, little affected by 1*25 hr in pentobarbitone, the substantial increase in the I-wave (4) during exposure to pentobarbitone was at least partially reversible. Note that the duration of the tract events was not affected by pentobarbitone, although the I-wave duration was increased to about 4 sec in this slice.
139 PENTOBARBITONE AND INHIBITION The amplitude of the late N-wave was little affected by the presence of pentobarbitone, sometimes showing a small decrease in spite of the presumably underlying I-wave being increased in amplitude. This lack of a clear change in the late N-wave was matched by the lack of any consistent change in a correlate, the antidromic
6r
10-4 M pentobarbitone
5_ ,
4.1
E
P..
O."^
*
....
....
Late N-wave
o3 I/-wave
0**
E
