Tetraethylammonium-lnduced Synaptic Marc R. PeUetier1 and John J. Hablitz Plasticity in Rat Neocortex Neurobiology Research Center and Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294, USA 'Present address: Playfair Neuroscience Unit, The Toronto Hospital, Western Division, 399 Bathurst Street, Toronto, Ontario, Canada M5T 2S8

Recordings were obtained from neurons in layer 11/111 of slices of rat LTPK, and possesses features distinct from tetanic stimulation- induced LTP observed in the CA1 region of the hippocampus frontal cortex maintained in vitro. We investigated whether brief Downloaded from https://academic.oup.com/cercor/article/6/6/771/363868 by guest on 30 September 2021 application of the potassium channel blocker tetraethylammonium (Aniksztejn and Ben-Ari, 1990, 1991). A concentration of TEA (TEA), which induces a novel form of synaptic plasticity in the CA1 sufficient to block the delayed rectifier potassium current (IDR), region of the hippocampus referred to as LTPK, evokes similar a potassium current that has been demonstrated to participate in responses in neocortex. Consistent with previous findings, TEA the repolarization of action potentials in rat sympathetic neurons produced a persistent enhancement of excitatory transmission, (Belluzzi and Sacchi, 1988) and cat sensorimotor cortex neurons which was independent of NMDA receptor activation but required 2+ (Spain et al, 1991), is required for LTPK. Blockade of IDR the activation of nifedipine-sensitive voltage-dependent Ca produces a prolongation of action potential width and the channels (VDCC), presumably the L-type. We also observed a appearance of Ca2* spikes, which should permit increased entry persistent enhancement of presumptive Cl~-dependent GABAA 2 of Ca * into presynaptic terminals and enhance transmitter receptor-mediated transmission. Enhancement of excitatory and release (Augustine, 1990). LTPK is thought to be mediated via an inhibitory synaptic transmission did not require activation of synapses with electrical stimulation during TEA application. The increase in the release of glutamate, which binds to non-NMDA enhancement of excitatory, but not inhibitory synaptic transmission, receptors and depolarizes the postsynaptic neuron sufficiently to was blocked when the Caz+ chelator 1,2-bis(2-aminophenoxy)- activate high-voltage threshold activation VDCCs (Aniksztejn ethane /v\A/,/V",W-tetraacetic acid (BAPTA) was included in the and Ben-Ari, 1991). Recent reports challenge the exclusivity of recording electrode. Under voltage clamp conditions that minimized these mechanisms. Hanse and Gustafsson (1994) observed TEA the activation of L-type channels robust enhancement of both enhancement of the slope and amplitude of the field excitatory excitatory and inhibitory transmission was still observed. No postsynaptic potential (EPSP) in the CA1 region of the enhancement of excitatory synaptic transmission was observed hippocampus in the presence of the L-type VDCC antagonist in the presence of NiCI2, a putative T-type channel blocker. nifedipine (20 nM) either alone or in combination with another The possible involvement of kinase activation was studied by blocker, flunarizine (30 uM). including the non-specific and competitive kinase inhibitor There appears to be a degree of overlap in the mechanisms (± )-1 -(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride underlying LTP and LTPK, because the magnitude of (H-7) in the patch pipette. H-7 retarded the time course and reduced the magnitude of the enhancement of excitatory transmission. These TEA-induced enhancement of field EPSPs recorded from results suggest that TEA-induced enhancement of excitatory hippocampal CA1 neurons was reduced when application of transmission in the neocortex requires entry of Ca2+ into the TEA was preceded by tetanic stimulation (Huang and Malenka, postsynaptic neuron via VDCCs and possibly the activation of a 1993; Hanse and Gustafsson, 1994). Hanse and Gustafsson kinase. (1994) suggest that the mechanism underlying the increase in the initial slope, which was dependent upon NMDA-receptor activation, is common to both LTP and LTPK, but that distinct Introduction mechanisms underlie the increase in EPSP amplitude. Laerum Persistent modification of synaptic transmission in the nervous and Storm (1994) reported an additional mechanistic difference system has been demonstrated by a variety of experimental between these forms of synaptic plasticity. TEA produced a manipulations. The mechanisms underlying experimentally transient increase in the repolarization time of the presynaptic induced forms of synaptic plasticity are believed to be volley, whereas tetanic stimulation did not. A transient increase representative of those occurring during certain forms of in duration of the presynaptic volley after TEA application has learning (Bliss and Lynch, 1988). Long-term potentiation (LTP) been reported previously (Huang and Malenka, 1993; but see induced by brief episodes of tetanic stimulation in the CA1 Hanse and Gustafsson, 1994). region of the hippocampus has been studied most rigorously Although the mechanisms for the induction of LTP in the (Bliss and Collingridge, 1993). Although the locus for the critical neocortex (Artola and Singer, 1987) were initially suggested to mechanisms is debated (Malinow, 1994) there is consensus that be substantially different from those required in the hippo- an increase in Ca2* concentration in the postsynaptic neuron is campus (Komatsu et al, 1988; Perkins and Teyler, 1988), LTP necessary for the induction of LTP in this region. Ca2* entry is reminiscent of what is typically observed in the hippocampus believed to be via the AT-methyl-D-aspartic acid (NMDA) subtype has been observed in the neocortex (Sutor and Hablitz, 1989; of glutamate receptor (Madison et al, 1991), or under certain Kirkwood etaL, 1993). Previous studies investigating LTPK were circumstances (Grover and Teyler, 1990), voltage-dependent restricted to the CA1 region of the hippocampus. We therefore 2 Ca * channels (VDCC). examined whether application of TEA in a brain slice A novel form of synaptic plasticity produced by brief preparation of rat frontal neocortex produces enhancement of application of the potassium channel blocker tetra- synaptic transmission. A preliminary account of some of these ethylammonium (TEA) has been described, is referred to as results has appeared (Pelletier and Hablitz, 1994).

Cerebral Cortex Nov/Dec 1996;6:771-780; 1047-3211/96/$4.00 Materials and Methods conducted with independent Mests. Differences were considered The method for preparation of brain slices has been described previously significant at P < 0.05. Results are expressed as mean ± SEM. (Sutor and Hablitz, 1989). Briefly, Sprague-Dawley rats of both sexes (14-42 days old) were decapitated under anesthesia (100 mg/kg Ketamine). The brains were removed rapidly then placed in ice-cold Results artificial cerebrospinal fluid (ACSF) for -1 min. Slices with a nominal thickness of 500 um were prepared from frontal neocortex using a Passive Membrane Properties Vibroslice. After storage for a minimum of 1 h at room temperature, The RMP and RN of the neocortical neurons impaled with individual slices were transferred to an interface-type chamber and intracellular electrodes in this study were -82.6 ±1.4 mV and warmed slowly to the recording temperature of 33 ± 1°C. ACSF was 27.5 ± 2.8 MQ (n = 36) respectively. TEA (25 mM), bath applied perfused continuously from below at a rate of 1 ml/min, which consisted for 7 min, produced reversible effects on the passive membrane of (in mM): 125 Nad, 35 KC1, 2.5 CaCl2, 1.3 MgCl2, 26 NaHCO3 and 10 properties and firing characteristics of neocortical neurons. TEA. D-glucose. The ACSF was bubbled continuously with 95% O2/5% CO2 to maintain a steady-state oxygen level and a pH value of 7.4. produced a depolarization of die RMP of 4.8 ± 0.7 mV and an increase in RN of 16.8 ± 4.7%. At early time points after return to Intracellular recordings from rats 18-42 days old were obtained from Downloaded from https://academic.oup.com/cercor/article/6/6/771/363868 by guest on 30 September 2021 layer II/III pyramidal neurons using 4 M potassium acetate-filled control ACSF, if the RMP had not yet returned to control, microelectrodes (resistance 80-120 Mli). Intracellular signals were hyperpolarizing current was injected when I/O relations were recorded and amplified using an Axoclamp-2A amplifier in bridge determined. As shown in Figure L4 (left), injection of supra- mode. Whole-cell patch-clamp recording techniques were used as threshold depolarizing current during the control period evoked described previously (Burgard and Hablitz, 1993). Briefly, patch pipettes an action potential. Injection of depolarizing current in the (resistance 2-4 Mii) were filled with a solution consisting of (in presence of TEA produced a broadening of action potential 2+ mM): 125 KMsethionate, 10 KCI, 0.5 ethylene glycol-bis@-aminoethyl width and Ca spikes, which persisted beyond die termination ether)Jv',JV,JV,JV'-tetraacetic acid (EGTA), 10 W-2-hydroxyethylpiperazine- of the current pulse (Fig. L4, middle). After return to control AT-2-ethanesulfonic acid (HEPES), 2 MgATP and 0.2 NaGTP. The ACSF for 30 min the action potential width returned to control osmolality of this solution was 270-280 mOsm, and the pH adjusted to (shorter latency to spike due to slighdy larger current pulse). We 7.2. Voltage-clamp recordings from rats 14-35 days old were obtained observed no difference in action potential threshold attributable using a discontinuous single-electrode voltage-clamp amplifier (NPI to TEA. In our experiments, return to control ACSF for 30-40 SEC1L, Tamm, Germany) at a switching frequency of 27 kHz and a 25% minutes was typically required for die return to control action duty cycle. Series resistance ranged from 10 to 15 MQ. potential spike width and RN, at which point we defined changes Postsynaptic responses were evoked (0.05 Hz) via a bipolar in synaptic transmission as persistent and not attributable to stimulating electrode located directly below the recording electrode in cortical layer IV/V. Input-output (I/O) relations were determined every changes in passive membrane properties. 15 min by varying the intensity of electrical stimulation. I/O relations comprised four or five progressively greater stimulation intensities (e.g. Enhancement of Synaptic Potentials weak < test < intermediate < strong). The effect of TEA on synaptic EPSPs evoked in response to electrical stimulation are shown in responses is presented typically at three representative stimulation intensities: weak, evoked small amplitude responses with no failures; test, Figure IB Geft). Control responses to weak stimulation in this evoked responses -50% of maximal amplitude; strong, evoked maximal neuron consisted of a small EPSP. Increasing the strength of amplitude responses. Three to five responses were measured at each stimulation produced an increase in EPSP amplitude and intensity. When I/O relations were not being determined postsynaptic prolongation in duration. After bath application of TEA there responses were evoked with the test stimulation intensity. The recorded was an increase in amplitude of die response evoked by weak signals were filtered at 1 kHz, stored on videotape (Neuro-Corder DR stimulation. We also observed an enhancement of the amplitude 484), and digitized using pClamp software (Axon Instruments). For and a prolongation of the duration of a late depolarizing intracellular recording the resting membrane potential (RMP) and input component on die decay phase of the potential evoked by strong resistance (RN; determined from the voltage deflection resulting from a stimulation (Fig. IB, middle). As seen in Figure IB (right) these 0.3-0.5 nA, 50 ms hyperpolarizing current pulse) were monitored changes were persistent after returning to control ACSF for 45 continuously during the experiments. For voltage-clamp recording min. The time course of the enhancement of the EPSP amplitude neurons were clamped at -75 mV and access resistance was assessed by evoked by the test intensity from nine experiments is monitoring the capacitative transients and the current produced by a summarized in Figure \C. The enhancement commenced during 5.0 mV, 50 ms hyperpolarizing voltage pulse. the application of TEA and persisted for up to 75 min in control The following drugs were used: TEA (25 mM; Sigma, St Louis, MO), ACSF, the longest time recorded (n = 3). As shown in Figure ID, D-2-amino-5-phosphonovaleric acid (APV; 20 uM; Tocris Neuramin, the magnitude of the increase in amplitude was greatest for Bristol, UK), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 |iM; Tocris Neuramin), l,2-bis(2-aminophenoxy)ethaneJV^V^V^v*-tetraacetic acid EPSPs evoked by weak stimulation and decreased progressively (BAPTA; 11 or 200 mM; Calbiochem, La Jolla, CA), nifedipine (20 \iM; as the stimulation intensity increased. After return to control Sigma), NiCh (200 uM; Fisher, Norcross, GA) and (±>l{5-isoquinoline- ACSF for 45 min, the amplitudes for EPSPs evoked by the weakest sulfonyl>2-methylpiperazine dihydrochloride (H-7; 100 uM; Calbiochem). to the strongest stimulation respectively ranged from 205.1 ± Drugs were dissolved in ACSF, except for CNQX and nifedipine, which 52.7% (P < 0.05) to 113.6 ± 17.0% of control (n = 9). The were dissolved in dimethyl sulfoxide (final concentration in ACSF 0.1 %). response evoked by strong stimulation comprises a mixed APV, CNQX, BAPTA, nifedipine and H-7 were prepared as stock solutions EPSP/IPSP and die reduction in the magnitude of enhancement and stored frozen. TEA and NiCh were prepared dairy. Drugs not included with increasing stimulation intensity is probably a consequence in the intracellular solution were bath applied. In our experiments, TEA of a shunting effect of the conductance associated widi the IPSP. was added directly to the bathing solution because Huang and Malenka The response evoked by weak stimulation is less contaminated (1993) reported that TEA-induced synaptic enhancement was not altered by EPSPs, and tiierefore exhibits a greater magnitude of when the osmolality of the bathing solution was controlled by reducing enhancement. the concentration of Nad. Differences in synaptic transmission (amplitude) attributable to the A late, depolarizing, component of die response evoked by application of TEA were determined with dependent f-tests (each neuron strong stimulation had an equilibrium potential of -73-6 ± 0.6 served as its own control). Comparisons between experiments were mV. This is consistent with the equilibrium potential expected

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Figure 1. TEA produces reversible effects on firing properties and persistent enhancement of synaptic transmission. (4) (Left) Action potential produced by depolarizing current (+0.8 nA) and voltage deflection produced by hyperpolarizing current (-0.5 nA) in a neocortical pyramidal neuron during the control period. (Center) Bath application of 25 mM TEA for 7 min produces an increase in width of action potential and additional spikes. Note, greater timescale applies to center records only. (Right) Action potential evoked (+0.9 nA) after return to control ACSF for 40 min is similar to spike evoked during the control period. (fl) (Left) Recording under control conditions of responses to weakest, intermediate, and strong stimulation intensities (RMP -86 mV|. (Center) Responses obtained after TEA application. (Right) Responses obtained after return to control ACSF for 45 min. Amplitude of EPSP evoked by weakest stimulation is enhanced. Note, also, enhancement of late component of the response evoked by strong stimulation. EPSPs evoked by weakest stimulation prior to TEA application and after return to control ACSF for 45 min are superimposed in the inset. (C) Summary of the time course of the enhancement of the amplitude of EPSPs evoked by test stimulation (n = 9). TEA application denoted by bar. (D) Plot of the effect of TEA on the amplitude of PSPs evoked by weakest (filled squares), intermediate (filled triangles), strong (filled diamonds) stimulation and late component (measured 50 ms from peak of responses evoked by strong stimulation; open squares). Symbols apply to this and all subsequent figures.

for the CT-dependent GABAA receptor-mediated inhibitory (n = 6). Presumptive GABAA receptor-mediated IPSPs were postsynaptic potential (IPSP) in neocortical neurons (Weiss and 234.0 ± 44.6% of control (P < 0.05). Hablitz, 1984; Howe etaL, 1987; Connors etaL, 1988). It should To test further the activity dependence of LTPK we applied be noted that the RMP we typically record in these neurons is TEA in the absence of electrical stimulation. Under control hyperpolarized compared to the equilibrium potential for Cl"; conditions (Fig. 2/4, left) typical PSPs were evoked. After return therefore, GABAA receptor-mediated IPSPs are depolarizing. to control ACSF for 15 min stimulation was again applied (Fig. After return to control ACSF for 45 min, the magnitude of the 2/4, center). An increase in the amplitude of the EPSP evoked by increase in amplitude of the presumptive IPSPs (measured 50 ms weak stimulation and a prolongation of the depolarizing from the peak of the potential evoked by the strongest presumptive IPSP evoked by intermediate and strong stimulation stimulation) was robust (180.3 ± 10.0 of control; P < 0.05) and was observed. Action potentials were evoked by strong approximated that observed for the EPSP evoked by weak stimulation (3/9), as observed in experiments described above stimulation. using stimulation. EPSP and IPSP enhancement persisted after 45 Activation of the NMDA subtype of glutamate receptor is min of control ACSF (Fig. 2A, right). EPSP enhancement ranged necessary for the induction of synaptic plasticity in various brain from 201.1 ± 50.0% (P < 0.05) to 113.6 ± 5.0% of control for the regions (Gustafcson etaL, 1987; Kirkwood and Bear, 1994). We weakest to the strongest stimulation respectively (n = 6). The tested whether LTPK in the neocortex observed this requirement average increase in the amplitude of the presumptive GABAA by evaluating the effect of TEA on synaptic transmission in the receptor-mediated IPSPs was 262.8 ± 76.1% of control (P< 0.05). presence of the NMDA receptor antagonist, APV (20 uM). The relation between TEA enhancement of amplitude and Consistent with what has been reported previously (Aniksztejn stimulation intensity from these experiments is shown in Figure and Ben-Ari, 1991; Huang and Malenka, 1993), LTPK was 28. The results are similar to those observed in experiments unaffected by APV. That is, LTPK induction in the neocortex does where electrical stimulation occurred for the duration of the not require the activation of NMDA receptors. After return to experiment. The greatest enhancement in every experiment was control ACSF for 45 min, the amplitudes of EPSPs evoked by the observed for EPSPs evoked by weak stimulation and presumptive weakest to the strongest stimulation intensity respectively IPSPs evoked by maximal stimulation. These experiments ranged from 179.3 ± 398 (P < O.O5) to 106.2 ± 5.8% of control indicate that the induction of LTPK requires neither the

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Figure 2. Enhancement of synaptic transmission produced by TEA does not require simultaneous activation of synapses with electrical stimulation. 14) (left) Recording under control conditions of responses evoked by three successively greater stimulation intensities (RMP -87 mV). (Center) Recording of responses after return to control ACSF for 15 min. Note enhancement of amplitude of EPSP evoked by weak stimulation and action potentials evoked by strong stimulation. (Right) Enhancement of synaptic transmission persists after return to control ACSF for 45 min. EPSPs evoked by weak stimulation prior to TEA application and after return to control ACSF for 45 min are superimposed in the inset. (S) Summary of the time course of TEA enhancement from these experiments {n = 6). Thickest solid bars denote orthodromic stimulation, thinner solid bar denotes TEA application, and dotted lines indicate absence of electrical stimulation. activation of NMDA receptors nor the simultaneous activation of from neurons impaled with electrodes filled with 200 mM synapses during TEA application, two features which make this BAPTA, a Ca2* chelator (Tsien, 1980). To confirm that BAPTA form of synaptic plasticity distinct from tetanus-induced LTP had been injected, we monitored the amplitude of the slow observed in the CA1 region of the hippocampus. afterhyperpolarization (sAHP), which is mediated by a The enhancement in the amplitude of GABAA receptor- Ca2*-dependent K* current in hippocampal (Lancaster and mediated IPSPs after TEA application has not been reported Adams, 1986; Lancaster and Nicoll, 1987; Storm, 1987) and previously. We therefore examined directly evoked, presumably neocortical neurons (Schwindt et aL, 1988). Figure 44 (left) monosynaptic, IPSPs elicited in the absence of excitatory illustrates the sAHP that is produced after a train of action transmission. As seen in Figure 3A Qeft), direct stimulation of potentials. This neuron was depolarized to a potential of -70 mV interneurons in the presence of 10 uM CNQX and 20 uM APV, to to enhance the sAHP. The amplitude of the sAHP was decreased block non-NMDA and NMDA receptors respectively, produced a after 20 min of impalement with the BAPTA-containing depolarizing PSP (RMP of this neuron -86 mV). The PSPs electrode (Fig. 4/4, center). This can be seen most clearly in recorded under these conditions had an equilibrium potential of Figure 4-4 (right) where the two records are superimposed. The -72.6 ± O.9 mV (n = 6), consistent with the expected equilibrium decrease in the sAHP typically required 20-30 min, at which potential for Cl", suggesting that they were GABAA receptor- point we applied TEA. PSPs evoked under these conditions (Fig. mediated IPSPs. Application of TEA produced an increase in the 42?, left) appeared similar to those recorded with electrodes not amplitude of the IPSP (Fig. 3A, center), which persisted after containing BAPTA. EPSPs evoked by weak stimulation were not return to control ACSF for 45 min (Fig. 5A, right). The increase enhanced in the presence of TEA (Fig. 4B, center, 105.1 ± 19-1% in the amplitude of the IPSP can be seen clearly in Figure 3-B. of control; P > 0.05) and were depressed slightly after return to where the IPSP during the control period is superimposed upon control ACSF for 20 min (Fig. 4B, right; 86.4 ±11.3% of control; the IPSP obtained after return to control ACSF for 45 min. The P 0.05; n = 4). This is seen most clearly in the inset where the time course of the TEA-induced IPSP enhancement is presented EPSP evoked prior to TEA application is superimposed on the in Figure 3C (n = 6). Note that, similar to what we described EPSP evoked after 20 min of control ACSF. In contrast, the above for EPSPs, IPSP enhancement commenced during the amplitude of the presumptive IPSP evoked by strong stimulation application of TEA. IPSP enhancement was 172.9 ± 30.9% of was 1835 ± 40.6% and 193.4 ± 43.3% of control (both P < 0.05) control (P < 0.05) when measured after return to control ACSF when measured at the same time points. The blockade of for 45 min. TEA-induced enhancement of the amplitude of the EPSPs evoked by the test stimulation intensity is shown clearly in Figure AC, Co2*- dependence of LTPK which summarizes four experiments. In the CA1 region of the hippocampus, the induction of LTPK is Pharmacological antagonism of L-type VDCCs prevents the dependent upon an increase in the postsynaptic concentration induction of LTPK in the hippocampus (Aniksztejn and Ben-Ari, of Ca2* (Aniksztejn and Ben-Ari, 1991; Huang and Malenka, 1991; Huang and Malenka, 1993; but see Hanse and Gustaffson, 1993). To test whether an increase in postsynaptic Ca2* is 1994). Ca2* entry into the postsynaptic neuron via this class of required for the induction of LTPK in the neocortex, we recorded VDCC is believed to be critical for the induction of LTPK- TO test

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Figure 3. TEA-induced enhancement of GABAA receptor-mediated transmission evoked by direct activation of inhibitory interneurons. (4) (Left) IPSP recorded in the presence of 10 |xM CNQX and 20 \>M APV prior to TEA application (RMP -86 mV). CNQX and APV were present for the duration of the experiment. (Center) The amplitude of the IPSP is increased by TEA. (Right) The increase in the amplitude of the IPSP persisted after return to control ACSF for 45 min. (B) The IPSPs prior to TEA application and after return to control ACSF for 45 min are superimposed. (C) Summary of the time course of TEA-induced enhancement of IPSP amplitude (n = 6). TEA application is denoted by the solid bar.

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Figure 4. Enhancement of EPSP amplitude is dependent upon an increase in postsynaptic Ca2+. (4) (Left) Repetitive firing of action potentials produced by injection of depolarizing current (+0.6 nA for 300 ms) through a BAPTA-containing electrode. This record was obtained after the neuron had been impaled for 8 min. The RMP was maintained at -70 mV with depolarizing DC current to enhance the sAHP. (Center) The amplitude of the sAHP is reduced after 20 min of impalement with BAPTA-containing electrode. (Right) Responses after 8 and 20 min are superimposed. (B) (Left) Responses recorded from a neuron 20 min after being impaled with a BAPTA-containing electrode (different neuron than in A; RMP -89 mV), prior to TEA application. (Center) Responses recorded after application of TEA. No enhancement of the amplitude of the EPSP evoked by weak stimulation was observed. Note, robust enhancement of depolarizing presumptive IPSP evoked by strong stimulation. (Right) Responses recorded after return to control ACSF for 20 min. EPSPs evoked by weakest stimulation intensity prior to TEA application and after return to control ACSF are superimposed in the inset. Note that the amplitude of EPSP evoked by the weakest stimulation showed no enhancement however, the enhancement of the presumptive IPSP evoked by strong stimulation was persistent (C) Summary of the time course of the experiments using BAPTA-containing electrodes (n = 4).

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Figure 5. TEA enhancement requires activation of nifedipine-sensitive VDCCs. Insets show records from a representative experiment of responses evoked with the test intensity at times indicated by numbers on graph: 1, control; 2, after 10 min application of nrfedipine (20 uA/l); 3. after return to control ACSF for 30 min. Summary of the time course of the experiments where nifedipine and TEA were applied simultaneously (n = 5). Nifedipine and TEA application denoted by solid bars.

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Figure 6. TEA enhances the amplitude of both EPSCs and IPSCs. (A) (Left) Recording under control conditions of an inward current evoked by weak stimulation (neuron clamped at -75 mV). (Center) EPSC amplitude is increased by TEA. (Right) Increase in EPSC amplitude persists after return to control ACSF for 30 min. (B) Same neuron as in (4), EPSC evoked by strong stimulation. (C) Summary of the time course of TEA enhancement of the amplitude of the EPSCs evoked by weak stimulation (n = 5). TEA application is denoted by bar. (D) (Upper) Both inward and outward currents can be evoked by strong stimulation when the neuron is clamped at -50 mV (different neuron than \nA). (Lower) Note enhancement of outward current after application of TEA. {£) Current-voltage relation from same neuron as in (£7). TEA produces an increase in outward current but has no effect on the apparent C!~ equilibrium potential (control, filled squares; return to control ACSF for 10 min. open squares). the involvement of L-type VDCCs for neocortical LTPK we simultaneous application of nifedipine and TEA (89-1 ± 6.6% of recorded responses evoked first in the presence of the L-type control). A summary of five experiments is presented in Figure 5. VDCC channel blocker nifedipine (20 uM), followed by simul- TEA failed to produce persistent enhancement of responses taneous application of both nifedipine and TEA. Amplitudes of evoked at any stimulation intensity. The amplitudes of responses responses evoked by the test intensity were not different from measured after return to control ACSF for 30 min evoked by control when measured in the presence of nifedipine alone (91.8 weak and strong stimulation were 899 ± 11.8% and 86.0 ± 13.1% ± 3-8% of control) or return to control ACSF for 30 min after of control respectively. The amplitude of the presumptive IPSPs

776 TEA-Induccd Plasticity in Ncocortcx • Pdfctier and Hablitz measured at the same time point was 106.7 ± 157% of control. These results suggest that Ca2* entry via nifedipine-sensitive 300 llmM BAPTA VDCCs, presumably the L-type, is necessary for TEA-induced 200

synaptic enhancement in the neocortex. ( tude( 100 Voltage-Clamp Analysis O/LITPK We next investigated whether TEA would produce enhancement Ampl i of synaptic transmission under voltage-clamp conditions 5 10 15 20 25 30 designed to prevent the activation of L-type VDCCs. A critical Time (min) time period for the induction of LTP when patch pipettes are 300 used has been suggested (Kato et at, 1993). The critical time B 200|iMNiCl2 period (15 min after attaining whole cell) was attributed to the 200 removal from the cytoplasm of essential constituents, but could be extended (to 30 min) if ATP and GTP were included in the patch pipette. We therefore included routinely in the pipette 100 i Downloaded from https://academic.oup.com/cercor/article/6/6/771/363868 by guest on 30 September 2021 solution 2 mM MgATP and 0.2 mM NaGTP and ensured that TEA Amplitud e (° / was applied -15 min after attaining the whole-cell configuration. Figure &AJi (left) illustrates the synaptic currents evoked by 5 10 15 20 25 30 35 40 weak and strong stimulation respectively prior to the application Time (min) of TEA. In the presence of TEA, EPSC amplitudes were increased 300 significantly (Fig. 6AM, center), and the enhancement persisted 100uMH7 after return to control ACSF for 30 min (Fig. 6AJB, right). As described above for EPSPs, the increase in amplitude was 200 greatest for the EPSC evoked by weak stimulation. After return to itud e ( Jt control ACSF for 20 min EPSCs evoked by weak stimulation were 100 ••- 216.9 ± 70.6% of control (w = 5; P < 0.05). The time course of the 1 enhancement of amplitude for EPSCs is presented in Figure 6C. At the beginning of the return to control ACSF EPSCs evoked by 5 10 15 20 25 30 the test intensity were 184.6 ± 29.0% of control (/> < 0.05). The Time (min) EPSC enhancement persisted, and increased to 211.7 ± 51.4% of control (P < 0.05), after return to control ACSF for 20 min. Figure 7. Enhancement of EPSC amplitude is dependent upon an increase in To evaluate further the enhancement of IPSCs, we clamped 2+ the membrane potential depolarized to the expected equili- postsynaptic Ca . (4) Summary of the time course of blockade of enhancement of amplitude of EPSCs recorded with patch pipettes containing 11 mM BAPTA/1 mM brium potential for Cl", which in these experiments was -69 mV. CaClz |n = 8). TEA application is denoted by solid bar. (5) Summary of the time course As shown in Figure 6D (upper, control) at -50 mV, strong of blockade of enhancement of EPSC amplitude when 200 |iM N1CI2 was included in stimulation evokes both an inward EPSC and an outward IPSC. the ACSF to block T-type channels (n = 10). (C) Inclusion of 100 pM H7, a non-specific, After return to control ACSF for 10 min (Fig. 6D, lower) an competitive kinase inhibitor, retards and reduces TEA-induced enhancement of increase in the amplitude of the presumptive IPSC is seen clearly. amplitude of EPSCs (n = 8). IPSCs were 172.3 ± 26.6% of control (P < 0.05) when measured after return to control ACSF for 15 min. The current-voltage voltage-damp conditions that should functionally eliminate the relation of the IPSC from the neuron presented in Figure 6C, contribution of high-voltage threshold activation VDCCs, during control (filled squares) and after return to control ACSF suggests that activation of a different subtype of VDCC might be 10 min (open squares), is presented in Figure 6E. It can be seen sufficient to induce LTPK. A likely candidate might be the that there is an increase in the slope conductance after low-voltage threshold activation T-type VDCC (Tsien et at, application of TEA, with no change in the apparent equilibrium 1988). To test this hypothesis we conducted experiments potential. under voltage-clamp conditions in the presence of 200 jiM NiCh In additional experiments BAPTA was included in the patch to block T-type VDCCs (Mogul and Fox, 1991). A high pipette solution (11 mM BAPTA/1 mM CaCh; intracellular Ca2* concentration of NiCb was used to ensure complete block of buffered to 20 nM; Pethig et at, 1989) to examine further the T-type channels despite a loss of specificity. Because a holding Ca2*-dependence of LTPK- Under these conditions we observed potential of -60 mV was used, significant activation of high a reduction of the sAHP within 5 min after attaining the threshold Ca2* channels was unlikely. As summarized in Figure whole-cell configuration. Figure 1A illustrates a relation similar IB, we observed no EPSC enhancement when measured either to what we observed when PSPs were recorded under at the beginning (104.8 ± 24.6% of control) or after return to current-clamp conditions with BAPTA-containing electrodes. control ACSF for 20 min (93.2 ± 11.8% of control; n = 10; both P EPSCs were not enhanced when measured either at the > 0.05). These results suggest that Ca2* entry in the postsynaptic beginning of the return to control ACSF after TEA application neuron via a subtype of low-voltage threshold activation VDCC, (105.6 ± 197% of control; P > 0.05) or after 15 min in control possibly the T-type, might also participate in the enhancement ACSF (116.3 ± 17.4% of control; n = 8;P> 0.05). However, IPSCs of excitatory transmission we have observed. were increased (142.6% of control; n = 3). These results confirm Protein phosphorylation is important for a variety of 2 the critical dependence on Ca * entry in the postsynaptic neuron neuronal functions, including synaptic transmission. Activation for the induction of TEA-induced enhancement of excitatory of kinases promotes phosphorylation, whereas activation of transmission. phosphatases promotes dephosphorylation. Ca entry into a The observation of robust enhancement of EPSCs under neuron can activate a variety of intracellular signalling pathways

Cerebral Cortex Nov/Dccl996, V6N 6 777 or can itself act as a messenger by inducing its release from An important issue concerning LTPK induction is the point of intracellular stores (for reviews see Miller, 1988; Penner et al, entry for Ca2* into the postsynaptic neuron. Previous studies 1993). In order to assess the contribution of kinase activation in have suggested that Ca2* entry via the L-type VDCC is critical for neocortical LTPK, we included in the patch pipette solution, the the induction of LTPK (Aniksztejn and Ben-Ari, 1991; Huang and competitive, non-selective kinase inhibitor H-7 (100 jiM; Hidaka Malenka, 1993). hi contrast, Hanse and Gustafsson (1994) et al., 1984; Boulis and Davis, 1990; Takahashi et al, 1990). observed an enhancement of both slope and amplitude of field When measured at the beginning of the return to control ACSF EPSPs recorded in the CA1 region in the presence of the organic after TEA application, EPSCs evoked by the test intensity were L-type channel blockers, nifedipine alone, or in combination 126.1 ± 28.0% of control (Fig. 1Q. After return to control ACSF with flunarizine. We observed no TEA-induced synaptic for 15 min, EPSCs were 1398 ± 60.0% of control (n = 8; both P > enhancement in the presence of nifedipine, indicating that 0.05). Blockade of kinase activation with H-7 both retarded the L-type VDCCs are necessary in neocortex. time course and reduced the magnitude of the enhancement by Two pieces of evidence suggest that VDCCs other than the TEA of the EPSC amplitude (cf. Fig. 6C). Taken together, these L-type might be sufficient for LTPK induction in the neocortex. data suggest that Ca -dependent activation of a kinase, whose Under voltage-clamp conditions designed to functionally prevent identity remains to be elucidated, might be necessary for TEA the activation of L-type VDCCs, robust enhancement of synaptic Downloaded from https://academic.oup.com/cercor/article/6/6/771/363868 by guest on 30 September 2021 enhancement of excitatory transmission in the neocortex. transmission was observed and the blockade of synaptic enhancement by NiCh, a putative T-type channel blocker. L-type channels are thought to be located most densely on the soma and Discussion at the base of proximal dendrites in neocortical neurons, and We observed that brief (7 min) application of TEA (25 mM) only sparsely on more distal dendrites (Ahlijanian et al, 1990; produced a persistent enhancement of excitatory synaptic Hell et al, 1993); however, if the spatial localization of L-type transmission in a slice preparation of rat neocortex. Several channels is more widespread than suggested by these studies features of the neocortical TEA-induced synaptic enhancement activation of L-type channels may be contributing under our we observed are similar to those described previously in the voltage-clamp conditions due to poor spatial control of voltage. CA1 region of the hippocampus, including NMDA receptor We used a relatively high concentration of NiCh (200 |iM) in an independence, requirement of an increase in the concentration attempt to block maximally T-type channels, although at the 2 of Ca * in the postsynaptic neuron and dependence upon expense of selectivity. Therefore, it cannot be ruled out that in nifedipine-sensitive VDCCs, presumably the L-type. An addition to T-type channels L-type channels were blocked as important consideration is whether the persistent enhancement well. It appears that die most tenable hypothesis supported by of synaptic transmission is due to factors other than the our observations is that Ca2* entry via the L-type VDCC is continued presence of TEA. Return to control ACSF for -30 min necessary for LTPK in the neocortex. Hypotheses that remain to was required for action potential duration, RMP and RNto return be tested include determining the involvement of N-type to control values—our operational definition of TEA removal. VDCCs, which have been reported to be located on the soma Although our group data is reported after return to control ACSF and throughout the dendritic arbor of hippocampal pyramidal for 45 min, we observed persistent effects after >75 min (n = 3). neurons (Mills et al, 1994), or whether TEA has either a direct Aniksztejn and Ben-Ari (1991) reported that the threshold effect, or an indirect effect mediated by an intracellular signaling concentration of TEA required to produce a persistent pathway, on Ca2* channels, which would promote the entry of enhancement of synaptic transmission was 15 mM. It is unlikely Ca2* into the postsynaptic neuron. that any residual TEA would be at a concentration sufficient to explain the persistent effects reported here. Neocortical LTPK did not require the simultaneous activation We observed also a novel persistent enhancement of of synapses with electrical stimulation. This observation is at presumptive cr-dependent GABAA receptor-mediated inhibit- variance with Petrozzino and Connor (1994) who reported that ory transmission. Failure to observe an enhancement of in the absence of low-frequency stimulation (0.05 Hz) EPSP inhibitory transmission in previous studies can be attributed to enhancement in the CA1 region was decremental and decayed back to baseline after 60 min of control ACSF. Petrozzino and the inclusion of picrotoxin to block GABAA receptors (Hanse and 2 Gustaffson, 1994), recording of field potentials in the absence of Connor (1994) suggest LTPK requires both Ca * entry via L-type pharmacological blockade of excitatory transmission (e.g. channels and co-activation of metabotropic glutamate receptors Aniksztejn and Ben-Ari, 1991), or perhaps stimulating at an (mGluR). A reconciliation of our observations with those of intensity insufficient to evoke IPSPs. Similar to TEA, the Petrozzino and Connor (1994) might be that TEA produces an potassium channel blocker 4-aminopyridine (4-AP) enhances increase in frequency of spontaneous PSPs or action potentials, which would depolarize the postsynaptic neuron sufficiently to both EPSPs and IPSPs (Rutecki et al, 1987; Perreault and Avoli, 2 1991, 1992); however, 4-AP also produces epileptiform dis- activate VDCCs. Spontaneous Ca * spikes might also be sufficient 2 charges, an effect we did not observe with TEA. Features of the to fulfil the Ca * requirement of LTPK. We did not monitor enhancement of the presumptive IPSPs we observed (e.g. action spontaneous PSPs systematically, and TEA-induced spontaneous 2 potentials associated with the depolarizing GABAA potential) action potentials or Ca * spikes were observed only infrequently. are more reminiscent of the enhancement of GABAergic TEA did produce a reversible depolarization (4.8 ± 0.7 mV) and transmission reported for zinc (Zhou and Hablitz, 1993). increase in RN (16.8 ± 4.7%), which would increase the Enhancement of the presumptive inhibitory responses was excitability of neocortical neurons. An increase in frequency of not dependent upon an increase in postsynaptic Ca2* concen- spontaneous events, in combination with the effects on passive tration: the enhancement was unaffected when BAPTA was membrane properties described above, might participate in the included in either the intracellular electrode or patch pipette. activation of VDCCs. Consistent with this hypothesis is the However, whether an increase in Ca2* in neurons providing observation that in dendrites of neocortical neurons single excitatory input onto GABAergic interneurons is necessary subthreshold EPSPs can activate low-voltage threshold Ca2* cannot be ruled out. channels (Markram and Sakmann, 1994).

778 TEA-induced Plasticity in Neocortex • Pdletier and Hablitz Kinase activation has been reported to be necessary for LTPK potentiation in the hippocampus using depolarizing potentials as the in the hippocampus (Petrozzino and Connor, 1994). The conditioning stimulus to single volley synaptic potentials. J Neurosci enhancement of EPSC amplitude was both retarded in time 7:774-780. course and reduced in magnitude when we included H-7 in the Hanse E, Gustaffsson, B (1994) TEA elicits two distinct potentiations of synaptic transmission in the CA1 region of the hippocampal slice. J pipette to inhibit kinase activity, however, due to the extreme Neurosci 14:5028-5034. varability in the responses we recorded with H-7 containing Hell JW, Westenbroek RE, Warner C, Ahlijanian MK, Prystay W, Gilbert pipettes, a statement concerning whether activation of a kinase MM, Snutch TP, Catterall WA (1993) Identification and differential is necessary for neocortical LTPK cannot be made with sibcellular localization of the neuronal class C and class D L-type confidence at this time. calcium channel al subunits. J Cell Biol 123:949-962. Hidaka H, Inagaki M, Kawamoto S, Sasaki Y (1984) Isoquinoline- The characteristics of TEA-induced synaptic enhancement we sulfanomides, novel and potent inhibitors of cyclic nucleotide observed in neocortex are similar in many respects to that dependent protein kinase C. Biochemistry 23:5036-5041. described in the CA1 region of hippocampus, including NMDA Howe JR, Sutor B, Zieglansberger W (1987) Characteristics of long- receptor independence and requirement of Ca2* entry via L-type duration inhibitory postsynaptic potentials in rat neocortical neurons VDCCs, although synaptic activity evoked with electrical in vitro. Cell Mol Neurobiol 7:1-18.

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780 TEA-Induccd Plasticity in Neoconex • Pdletier and Hablitz