575
Journal of Physiology (1992), 457, pp. 575-590 With 8 figures Printed in Great Britain
INACTIVATION CHARACTERISTICS OF A SUSTAINED, Ca2+INDEPENDENT K+ CURRENT OF RAT HIPPOCAMPAL NEURONES IN VITRO BY ANDREA NISTRI AND ENRICO CHERUBINI From INSERM U. 29, 123 Boulevard de Port Royal, 75014 Paris, France and the Laboratory of Biophysics, International School for Advanced Studies (SISSA), 34014 Trieste, Italy
(Received 24 July 1991) SUMMARY
1. Current or voltage clamp recordings from CA3 neurones of the adult rat hippocampal slice were performed to study the inactivation properties of a slow outward K+ current identified as the delayed rectifier (IK). 2. In current clamp experiments, burst firing evoked from resting membrane potential by intracellular current injection was reduced or blocked by conditioning hyperpolarizing pre-pulses of 20-40 mV amplitude. This effect was inhibited by tetraethylammonium (TEA; 20 mM) but was unaffected by Cs' (3 mM), 4aminopyridine (4-AP; 2 mm), carbachol (30-50 ,rm), mast cell degranulating peptide (MCDP; 300 nM), thyrotrophin releasing hormone (TRH; 1 /LM) or by a Ca2"-free solution containing Mn2+ or Co2+ (2 mM). 3. Single-electrode voltage clamp experiments were carried out on neurones superfused with Ca2+-free solution, containing tetrodotoxin (TTX; 1 gM), Mn2+ or Co2+ (2 mM), 4-AP (2 mM), Cs' (3 mM) and carbachol (30 ,M). Step depolarizations from a holding potential of -55 mV activated an outward current which reached a plateau after 200 ms, followed by an outward tail current. Such an outward current had the characteristics of IK. 4. The outward currents were significantly potentiated by conditioning hyperpolarizing pre-pulses suggesting the IK was reduced by a voltage-dependent inactivation process. Removal of inactivation was a function of the amplitude of the conditioning hyperpolarizing pre-pulse. At a holding potential of -55 mV removal of inactivation was time dependent with a time constant of 211 ms. High K+ (12'5 or 21-5 mM) solutions did not affect the inactivation characteristics of IK. 5. Tetraethylammonium (20 mM) or low concentrations of Ba21 (0'1 mM) readily depressed the outward current without significantly affecting the inactivation process. Dendrotoxin (200 nM) also depressed such a slow current but, in addition, increased the inactivation process of IK. 6. It is suggested that removal of inactivation of IK by hyperpolarization can modulate cell excitability by fully restoring the ability of IK to inhibit burst firing of CA3 hippocampal neurones. MS 9583
576
A. NISTRI AND E. CHER UBINI INTRODUCTION
Potassium channels play an important role in regulating neuronal excitability. On the basis of their kinetics and pharmacological properties several voltage-dependent K+ currents, activated by membrane depolarization, have been identified in central neurones (for reviews see Rudy, 1988; Storm. 1990). One of them, the delayed rectifier (IK), was originally described by Hodgkin & Huxley (1952) to contribute to repolarization of the action potential of the squid giant axon. Other functional properties of the delayed rectifier in peripheral nerve fibres have been investigated in a number of studies (Rudy, 1988). Recently, in bullfrog sympathetic ganglion cells, pharmacological inhibition of the delayed rectifier had been found to prolong action potential duration, to reduce the fast component of the spike afterhyperpolarization and to enhance spike frequency adaptation (Goh, Kelly & Pennefather, 1989). Furthermore, in bullfrog sensory or autonomic ganglion cells a slowly inactivating delayed K+ current (which is partly active at resting membrane potential) can contribute to the resting membrane conductance (Tokimasa, Tsurusaki & Akasu, 1991). In the case of mammalian brain cells, two voltage-activated outward K+ currents (a fast and a slow one) are observed as 'window' currents already active below threshold for action potential generation by cat cortical neurones (Spain, Schwindt & Crill, 1991 a). The slow current regulates the repetitive firing pattern of these neurones but it also displays voltage-dependent inactivation as suggested by the fact that the accommodation of spike frequency can be enhanced by removing inactivation of the slow K+ current with a hyperpolarizing pre-pulse or can be reduced by pharmacological block of the same current (Spain, Schwindt & Crill, 1991 b). In the hippocampus, the interplay between various ionic conductances of CA3 pyramidal cells is thought to generate 'endogenous' bursts in this region (Hablitz & Johnston, 1981; Wong & Prince, 1981). The bursting behaviour of these neurones is in turn involved in the initiation of synchronized population discharges (Traub & Wong, 1982; Miles & Wong, 1983, 1986). These phenomena are usually prevented by the activation of several K+ currents whose pharmacological block induces intense and sustained bursting activity (Gho, King, Ben Ari & Cherubini, 1986; Cherubini, Neuman, Rovira & Ben Ari, 1988). In the present study on CA3 neurones of the rat hippocampal slice preparation we report a new property of the slowly inactivating, voltage-dependent K+ current, which resembles 'K described in hippocampal neurones either grown in culture (Segal & Barker, 1984) or acutely dissociated (Numann, Wadman & Wong, 1987; Sah, Gibb & Gage, 1988). We have found that, in analogy to the experiments on feline sensorimotor neurones (Spain et al. 1991 b), removal of inactivation of this current by a conditioning hyperpolarizing pre-pulse, largely increased the steady-state amplitude of 'K and reduced burst activity electrically evoked in CA3 neurones. Part of this work has been presented as an abstract (Nistri & Cherubini, 1992). METHODS
Experiments were performed on hippocampal slices obtained from adult Wistar rats (both sexes). The brain was quickly removed from rats under ether anaesthesia and both hippocampi were dissected free. The methods for preparing and maintaining the slices have been extensively
DELA YED K+ CURRENT IN HIPPOCAP1UPLS
577
reported (Nistri & Cherubini, 1991). Briefly, transverse 450 Iam-thick slices were cut and immediately incubated at room temperature (20-22 'C) in artificial cerebrospinal fluid (ACSF) containing (mM): NaCl, 126; KCl, 35; Ca"l2, 2; NaH2PO4, 1-2; MgCl26H2O, 1-3; NaHCO3, 25; glucose, 1 1. Equilibrating the ACSF with 95 %02 and 5 % CO2 gave a pH of 7 3-7 4. The slices were allowed to recover for 1 h before being transferred to a recording chamber in which they were .continuously superfused at 34 'C with oxygenated ACSF at a rate of 25-3 ml min-. Intracellular recordings were obtained from hippocampal CA3 neurones using microelectrodes filled, in most cases, with 3 M KCl (resistance 40-50 MQ). In a few cases CA3 neurones were impaled with microelectrodes containing 2 M potassium methylsulphate (resistance 60-80 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 were reduced to a minimum by negative capacity compensation. In voltage clamp experiments, membrane currents originated mainly from the neuronal soma were recorded via a single-electrode voltage clamp amplifier (Axoclamp 2A), switching between voltage recording and current injection at 3-5 kHz (300% duty cycle). The voltage signal at the head-stage amplifier was continuously monitored on a separate oscilloscope to ensure correct operation of the voltage clamp system. Responses were digitized and displayed on a Nicolet digital oscilloscope and on a chart recorder. The slowly inactivating voltage-dependent K+ current, which we refer to as IK' was studied in isolation in a nominally Ca2+-free solution, containing tetrodotoxin (TTX, 1 ,UM), Co2+ or Mn2+ (2 mM), Cs' (3 mM), 4-aminopyridine (4-AP, 2 mm) and carbachol (30-50 AtM). TTX was used to eliminate fast sodium currents. Co21 or Mn2' replaced Ca2+ in order to eliminate inward Ca21 currents and Ca2+activated K+ currents (Storm, 1990). Cs' was used to suppress the hyperpolarization-activated inward rectification (Halliwell & Adams, 1982) while 4-AP was employed to block fast K+ currents such as 'A (Rogawski, 1985) and ID (Storm, 1988). Carbachol was used to reduce the M-current as well as other K+ currents, including the 'leak' current and the slow Ca2+-dependent K+ current (VAHP; Storm 1990). Such concentrations of carbachol are approximately 10 and 100 times larger than those needed to block by 50 % the M-current and IAHP of hippocampal neurones (Storm, 1990). Under voltage clamp, current-voltage (I-V) plots were constructed by stepping the voltage to various potentials and measuring the current when it had reached a steady-state value (usually at 200 ms). The I-V relation appeared to be linear between -50 and -70 mV (this linearity was maintained when potassium methylsulphate electrodes were used). A least-squares routine was fitted to the linear part of the I-V curve, the slope of which was taken to calculate passive 'leak' conductance. Assuming that the leak conductance was time- and voltage-independent, the I-V relation of IK was plotted after subtracting the observed currents from the extrapolated 'leak' currents at the same level of test potential (original current tracings shown in the figures are not leak subtracted). Drugs were dissolved in ACSF to give the final concentration stated in the text and applied via a three-way tap system. TTX, carbachol, 4-AP, and tetraethylammonium chloride (TEA) were purchased from Sigma; thyrotrophin-releasing hormone (TRH) was kindly supplied by CyanamidTakeda (100% pure compound) while mast cell degranulating pepetide (MCDP) was a gift by Alomone Labs, Jerusalem. Pure dendrotoxin (toxin I) was kindly donated by Dr D. G. Owen (Wyeth Labs). Data are expressed as means+S.E.M. Statistical analysis was performed using Student's paired t test. RESULTS
Stable intracellular recordings were obtained from thirty-eight CA3 pyramidal cells. In keeping with previous studies (Sim & Cherubini, 1990; Neuman, Ben Ari & Cherubini, 1991), all neurones had initial resting membrane potentials more negative than -55 mV, spike overshoots of 15-20 mV and resting input resistances ranging from 25 to 68 MQ. Current clamp experiments CA3 pyramidal neurones, when depolarized from rest by a 50-80 ms depolarizing current pulse, exhibited a burst of action potentials, followed by a slow afterhyperpolarization. Such a burst comprised a slowly depolarizing potential with
578
A. NISTRI AND K CHER UBINI
superimposed spikes. Spike frequency adaptation was also observed as a decline in firing rate from a fast to a slower rate at the end of the pulse. When a conditioning pre-pulse of variable amplitude (ranging from -10 to -60 mV) and/or duration (ranging from 20 to 500 ms) preceded the depolarizing test pulse, a clear reduction A
B
120 mV
-56 mV
!_ _ _ _
_
-
12 nA 100 ms
Fig. 1. Inhibition of cell firing by preceding hyperpolarizing potential. A, two superimposed oscilloscope tracings showing membrane potential (top) and injected current pulses (bottom). Cell membrane potential was -56 mV. Note suppression of firing and reduction in the amplitude of depolarizing electrotonic potential after a hyperpolarizing current pulse. B, superimposed oscilloscope tracings of spike activity evoked at increasing intervals from hyperpolarizing current pulse. The last response on the right was elicited without preceding the hyperpolarization and comprised four action potentials. The response immediately after the hyperpolarizing pre-pulse generated only one spike, while the second and third ones produced two spikes with progressively smaller interspike intervals. A different cell from A held at the same level of membrane potential.
or complete block in firing rate was seen. An example is shown in Fig. IA in which a depolarizing pulse from a membrane potential of -56 mV elicited a burst of action potentials blocked by a conditioning hyperpolarizing pre-pulse to -116 mV. Similar results were obtained in six cells. The inhibiting action of a hyperpolarization on subsequent firing was persistent as shown by the example of Fig. 1 b: as the interval between the end of the conditioning pre-pulse and the depolarizing pulse was decreased from 300 ms to zero, a burst of four closely spaced action potentials (unaffected by the pre-pulse) was replaced first by two spikes and then by just one spike. The inhibition of the burst was also a function of the amplitude and duration of the conditioning hyperpolarizing pre-pulse. Thus, a conditioning hyperpolarizing pre-pulse of 20 mV amplitude (500 ms duration) from a resting membrane potential of -58 to -65 mV caused a 72 % inhibition of adapted firing rate from 156 + 20 to 44 + 6 Hz (n = 5) which persisted for 425 + 80 ms (ranging between 250 and 625 ms). As the amplitude of the pre-pulse potential was made more negative, a further reduction in the firing rate was observed. For instance, a 40 mV hyperpolarizing prepulse caused 86% inhibition of the firing rate from 156 + 20 to 23+9 Hz. The inhibition of firing rate was also a function of the duration of the conditioning hyperpolarizing pre-pulse. In four neurones, the amplitude of the conditioning prepulse was kept constant (-30 mV) while the duration of the pulse was varied from 20 to 500 ms. The reduction in firing rate started to develop after a pre-pulse of 83 + 5 ms duration (ranging from 50 to 120 ms). The inhibition of firing rate by the conditioning pre-pulse was associated with an increase in the cell input conductance, as shown by the decrease in amplitude of the electrotonic potential in response to a
DELA YED K+ CLTRRENTT IN HIPPOCAMPUS Control
-59 mV
Calcium free
-58 mV
TEA (20 mM)
-58 mV
120 mV
-i -
_
11 nA
200 ms
Fig. 2. Influence of a hyperpolarizing pre-pulse on spike activity. All responses are oscilloscope tracings with membrane potential (top; actual value indicated before each record) and current (bottom). In Ca2+-free medium (containing 2 mM Co2+) the inhibition of firing by a conditioning hyperpolarization was preserved while it was lost in the presence of 20 mm TEA. Spikes are truncated. Caesium (3 mM)
-59 mV
TRH (1
gtM)
-57 mV
4-AP (2 mM) -57 mV
120 mV =
912 nA 200 ms
Fig. 3. Persistence of post-hyperpolarization inhibition of firing activity in the presence of Cs' (3 mM), TRH (1 aM) or 4-AP (2 mM). For further details see legend to Fig. 2.
579
580
A. NISTRI ANAD E. CHER UTBINI
fixed depolarizing current pulse (see also Fig. 1A). This suggests that one (or more) voltage-dependent inhibitory current (probably due to K+), either activated or deactivated by a conditioning hyperpolarization, was contributing to the reduction in cell excitability. In order to gain an insight into the identity of such conductance(s), A
b
a wf7
d
c
l'
Fj d e -12
-52
-22
J-] 2 nA
..
-92
B
3 **
-2
Journal of Physiology (1992), 457, pp. 575-590 With 8 figures Printed in Great Britain
INACTIVATION CHARACTERISTICS OF A SUSTAINED, Ca2+INDEPENDENT K+ CURRENT OF RAT HIPPOCAMPAL NEURONES IN VITRO BY ANDREA NISTRI AND ENRICO CHERUBINI From INSERM U. 29, 123 Boulevard de Port Royal, 75014 Paris, France and the Laboratory of Biophysics, International School for Advanced Studies (SISSA), 34014 Trieste, Italy
(Received 24 July 1991) SUMMARY
1. Current or voltage clamp recordings from CA3 neurones of the adult rat hippocampal slice were performed to study the inactivation properties of a slow outward K+ current identified as the delayed rectifier (IK). 2. In current clamp experiments, burst firing evoked from resting membrane potential by intracellular current injection was reduced or blocked by conditioning hyperpolarizing pre-pulses of 20-40 mV amplitude. This effect was inhibited by tetraethylammonium (TEA; 20 mM) but was unaffected by Cs' (3 mM), 4aminopyridine (4-AP; 2 mm), carbachol (30-50 ,rm), mast cell degranulating peptide (MCDP; 300 nM), thyrotrophin releasing hormone (TRH; 1 /LM) or by a Ca2"-free solution containing Mn2+ or Co2+ (2 mM). 3. Single-electrode voltage clamp experiments were carried out on neurones superfused with Ca2+-free solution, containing tetrodotoxin (TTX; 1 gM), Mn2+ or Co2+ (2 mM), 4-AP (2 mM), Cs' (3 mM) and carbachol (30 ,M). Step depolarizations from a holding potential of -55 mV activated an outward current which reached a plateau after 200 ms, followed by an outward tail current. Such an outward current had the characteristics of IK. 4. The outward currents were significantly potentiated by conditioning hyperpolarizing pre-pulses suggesting the IK was reduced by a voltage-dependent inactivation process. Removal of inactivation was a function of the amplitude of the conditioning hyperpolarizing pre-pulse. At a holding potential of -55 mV removal of inactivation was time dependent with a time constant of 211 ms. High K+ (12'5 or 21-5 mM) solutions did not affect the inactivation characteristics of IK. 5. Tetraethylammonium (20 mM) or low concentrations of Ba21 (0'1 mM) readily depressed the outward current without significantly affecting the inactivation process. Dendrotoxin (200 nM) also depressed such a slow current but, in addition, increased the inactivation process of IK. 6. It is suggested that removal of inactivation of IK by hyperpolarization can modulate cell excitability by fully restoring the ability of IK to inhibit burst firing of CA3 hippocampal neurones. MS 9583
576
A. NISTRI AND E. CHER UBINI INTRODUCTION
Potassium channels play an important role in regulating neuronal excitability. On the basis of their kinetics and pharmacological properties several voltage-dependent K+ currents, activated by membrane depolarization, have been identified in central neurones (for reviews see Rudy, 1988; Storm. 1990). One of them, the delayed rectifier (IK), was originally described by Hodgkin & Huxley (1952) to contribute to repolarization of the action potential of the squid giant axon. Other functional properties of the delayed rectifier in peripheral nerve fibres have been investigated in a number of studies (Rudy, 1988). Recently, in bullfrog sympathetic ganglion cells, pharmacological inhibition of the delayed rectifier had been found to prolong action potential duration, to reduce the fast component of the spike afterhyperpolarization and to enhance spike frequency adaptation (Goh, Kelly & Pennefather, 1989). Furthermore, in bullfrog sensory or autonomic ganglion cells a slowly inactivating delayed K+ current (which is partly active at resting membrane potential) can contribute to the resting membrane conductance (Tokimasa, Tsurusaki & Akasu, 1991). In the case of mammalian brain cells, two voltage-activated outward K+ currents (a fast and a slow one) are observed as 'window' currents already active below threshold for action potential generation by cat cortical neurones (Spain, Schwindt & Crill, 1991 a). The slow current regulates the repetitive firing pattern of these neurones but it also displays voltage-dependent inactivation as suggested by the fact that the accommodation of spike frequency can be enhanced by removing inactivation of the slow K+ current with a hyperpolarizing pre-pulse or can be reduced by pharmacological block of the same current (Spain, Schwindt & Crill, 1991 b). In the hippocampus, the interplay between various ionic conductances of CA3 pyramidal cells is thought to generate 'endogenous' bursts in this region (Hablitz & Johnston, 1981; Wong & Prince, 1981). The bursting behaviour of these neurones is in turn involved in the initiation of synchronized population discharges (Traub & Wong, 1982; Miles & Wong, 1983, 1986). These phenomena are usually prevented by the activation of several K+ currents whose pharmacological block induces intense and sustained bursting activity (Gho, King, Ben Ari & Cherubini, 1986; Cherubini, Neuman, Rovira & Ben Ari, 1988). In the present study on CA3 neurones of the rat hippocampal slice preparation we report a new property of the slowly inactivating, voltage-dependent K+ current, which resembles 'K described in hippocampal neurones either grown in culture (Segal & Barker, 1984) or acutely dissociated (Numann, Wadman & Wong, 1987; Sah, Gibb & Gage, 1988). We have found that, in analogy to the experiments on feline sensorimotor neurones (Spain et al. 1991 b), removal of inactivation of this current by a conditioning hyperpolarizing pre-pulse, largely increased the steady-state amplitude of 'K and reduced burst activity electrically evoked in CA3 neurones. Part of this work has been presented as an abstract (Nistri & Cherubini, 1992). METHODS
Experiments were performed on hippocampal slices obtained from adult Wistar rats (both sexes). The brain was quickly removed from rats under ether anaesthesia and both hippocampi were dissected free. The methods for preparing and maintaining the slices have been extensively
DELA YED K+ CURRENT IN HIPPOCAP1UPLS
577
reported (Nistri & Cherubini, 1991). Briefly, transverse 450 Iam-thick slices were cut and immediately incubated at room temperature (20-22 'C) in artificial cerebrospinal fluid (ACSF) containing (mM): NaCl, 126; KCl, 35; Ca"l2, 2; NaH2PO4, 1-2; MgCl26H2O, 1-3; NaHCO3, 25; glucose, 1 1. Equilibrating the ACSF with 95 %02 and 5 % CO2 gave a pH of 7 3-7 4. The slices were allowed to recover for 1 h before being transferred to a recording chamber in which they were .continuously superfused at 34 'C with oxygenated ACSF at a rate of 25-3 ml min-. Intracellular recordings were obtained from hippocampal CA3 neurones using microelectrodes filled, in most cases, with 3 M KCl (resistance 40-50 MQ). In a few cases CA3 neurones were impaled with microelectrodes containing 2 M potassium methylsulphate (resistance 60-80 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 were reduced to a minimum by negative capacity compensation. In voltage clamp experiments, membrane currents originated mainly from the neuronal soma were recorded via a single-electrode voltage clamp amplifier (Axoclamp 2A), switching between voltage recording and current injection at 3-5 kHz (300% duty cycle). The voltage signal at the head-stage amplifier was continuously monitored on a separate oscilloscope to ensure correct operation of the voltage clamp system. Responses were digitized and displayed on a Nicolet digital oscilloscope and on a chart recorder. The slowly inactivating voltage-dependent K+ current, which we refer to as IK' was studied in isolation in a nominally Ca2+-free solution, containing tetrodotoxin (TTX, 1 ,UM), Co2+ or Mn2+ (2 mM), Cs' (3 mM), 4-aminopyridine (4-AP, 2 mm) and carbachol (30-50 AtM). TTX was used to eliminate fast sodium currents. Co21 or Mn2' replaced Ca2+ in order to eliminate inward Ca21 currents and Ca2+activated K+ currents (Storm, 1990). Cs' was used to suppress the hyperpolarization-activated inward rectification (Halliwell & Adams, 1982) while 4-AP was employed to block fast K+ currents such as 'A (Rogawski, 1985) and ID (Storm, 1988). Carbachol was used to reduce the M-current as well as other K+ currents, including the 'leak' current and the slow Ca2+-dependent K+ current (VAHP; Storm 1990). Such concentrations of carbachol are approximately 10 and 100 times larger than those needed to block by 50 % the M-current and IAHP of hippocampal neurones (Storm, 1990). Under voltage clamp, current-voltage (I-V) plots were constructed by stepping the voltage to various potentials and measuring the current when it had reached a steady-state value (usually at 200 ms). The I-V relation appeared to be linear between -50 and -70 mV (this linearity was maintained when potassium methylsulphate electrodes were used). A least-squares routine was fitted to the linear part of the I-V curve, the slope of which was taken to calculate passive 'leak' conductance. Assuming that the leak conductance was time- and voltage-independent, the I-V relation of IK was plotted after subtracting the observed currents from the extrapolated 'leak' currents at the same level of test potential (original current tracings shown in the figures are not leak subtracted). Drugs were dissolved in ACSF to give the final concentration stated in the text and applied via a three-way tap system. TTX, carbachol, 4-AP, and tetraethylammonium chloride (TEA) were purchased from Sigma; thyrotrophin-releasing hormone (TRH) was kindly supplied by CyanamidTakeda (100% pure compound) while mast cell degranulating pepetide (MCDP) was a gift by Alomone Labs, Jerusalem. Pure dendrotoxin (toxin I) was kindly donated by Dr D. G. Owen (Wyeth Labs). Data are expressed as means+S.E.M. Statistical analysis was performed using Student's paired t test. RESULTS
Stable intracellular recordings were obtained from thirty-eight CA3 pyramidal cells. In keeping with previous studies (Sim & Cherubini, 1990; Neuman, Ben Ari & Cherubini, 1991), all neurones had initial resting membrane potentials more negative than -55 mV, spike overshoots of 15-20 mV and resting input resistances ranging from 25 to 68 MQ. Current clamp experiments CA3 pyramidal neurones, when depolarized from rest by a 50-80 ms depolarizing current pulse, exhibited a burst of action potentials, followed by a slow afterhyperpolarization. Such a burst comprised a slowly depolarizing potential with
578
A. NISTRI AND K CHER UBINI
superimposed spikes. Spike frequency adaptation was also observed as a decline in firing rate from a fast to a slower rate at the end of the pulse. When a conditioning pre-pulse of variable amplitude (ranging from -10 to -60 mV) and/or duration (ranging from 20 to 500 ms) preceded the depolarizing test pulse, a clear reduction A
B
120 mV
-56 mV
!_ _ _ _
_
-
12 nA 100 ms
Fig. 1. Inhibition of cell firing by preceding hyperpolarizing potential. A, two superimposed oscilloscope tracings showing membrane potential (top) and injected current pulses (bottom). Cell membrane potential was -56 mV. Note suppression of firing and reduction in the amplitude of depolarizing electrotonic potential after a hyperpolarizing current pulse. B, superimposed oscilloscope tracings of spike activity evoked at increasing intervals from hyperpolarizing current pulse. The last response on the right was elicited without preceding the hyperpolarization and comprised four action potentials. The response immediately after the hyperpolarizing pre-pulse generated only one spike, while the second and third ones produced two spikes with progressively smaller interspike intervals. A different cell from A held at the same level of membrane potential.
or complete block in firing rate was seen. An example is shown in Fig. IA in which a depolarizing pulse from a membrane potential of -56 mV elicited a burst of action potentials blocked by a conditioning hyperpolarizing pre-pulse to -116 mV. Similar results were obtained in six cells. The inhibiting action of a hyperpolarization on subsequent firing was persistent as shown by the example of Fig. 1 b: as the interval between the end of the conditioning pre-pulse and the depolarizing pulse was decreased from 300 ms to zero, a burst of four closely spaced action potentials (unaffected by the pre-pulse) was replaced first by two spikes and then by just one spike. The inhibition of the burst was also a function of the amplitude and duration of the conditioning hyperpolarizing pre-pulse. Thus, a conditioning hyperpolarizing pre-pulse of 20 mV amplitude (500 ms duration) from a resting membrane potential of -58 to -65 mV caused a 72 % inhibition of adapted firing rate from 156 + 20 to 44 + 6 Hz (n = 5) which persisted for 425 + 80 ms (ranging between 250 and 625 ms). As the amplitude of the pre-pulse potential was made more negative, a further reduction in the firing rate was observed. For instance, a 40 mV hyperpolarizing prepulse caused 86% inhibition of the firing rate from 156 + 20 to 23+9 Hz. The inhibition of firing rate was also a function of the duration of the conditioning hyperpolarizing pre-pulse. In four neurones, the amplitude of the conditioning prepulse was kept constant (-30 mV) while the duration of the pulse was varied from 20 to 500 ms. The reduction in firing rate started to develop after a pre-pulse of 83 + 5 ms duration (ranging from 50 to 120 ms). The inhibition of firing rate by the conditioning pre-pulse was associated with an increase in the cell input conductance, as shown by the decrease in amplitude of the electrotonic potential in response to a
DELA YED K+ CLTRRENTT IN HIPPOCAMPUS Control
-59 mV
Calcium free
-58 mV
TEA (20 mM)
-58 mV
120 mV
-i -
_
11 nA
200 ms
Fig. 2. Influence of a hyperpolarizing pre-pulse on spike activity. All responses are oscilloscope tracings with membrane potential (top; actual value indicated before each record) and current (bottom). In Ca2+-free medium (containing 2 mM Co2+) the inhibition of firing by a conditioning hyperpolarization was preserved while it was lost in the presence of 20 mm TEA. Spikes are truncated. Caesium (3 mM)
-59 mV
TRH (1
gtM)
-57 mV
4-AP (2 mM) -57 mV
120 mV =
912 nA 200 ms
Fig. 3. Persistence of post-hyperpolarization inhibition of firing activity in the presence of Cs' (3 mM), TRH (1 aM) or 4-AP (2 mM). For further details see legend to Fig. 2.
579
580
A. NISTRI ANAD E. CHER UTBINI
fixed depolarizing current pulse (see also Fig. 1A). This suggests that one (or more) voltage-dependent inhibitory current (probably due to K+), either activated or deactivated by a conditioning hyperpolarization, was contributing to the reduction in cell excitability. In order to gain an insight into the identity of such conductance(s), A
b
a wf7
d
c
l'
Fj d e -12
-52
-22
J-] 2 nA
..
-92
B
3 **
-2
