Tetraethylammonium-lnduced Synaptic Plasticity in ...

Tetraethylammonium-lnduced Synaptic Plasticity in Rat Neocortex

Introduction Persistent modification of synaptic transmission in the nervous system has been demonstrated by a variety of experimental manipulations. The mechanisms underlying experimentally induced forms of synaptic plasticity are believed to be representative of those occurring during certain forms of learning (Bliss and Lynch, 1988). Long-term potentiation (LTP) induced by brief episodes of tetanic stimulation in the CA1 region of the hippocampus has been studied most rigorously (Bliss and Collingridge, 1993). Although the locus for the critical mechanisms is debated (Malinow, 1994) there is consensus that an increase in Ca2* concentration in the postsynaptic neuron is necessary for the induction of LTP in this region. Ca2* entry is believed to be via the AT-methyl-D-aspartic acid (NMDA) subtype of glutamate receptor (Madison et al, 1991), or under certain circumstances (Grover and Teyler, 1990), voltage-dependent Ca2* channels (VDCC). A novel form of synaptic plasticity produced by brief application of the potassium channel blocker tetraethylammonium (TEA) has been described, is referred to as

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

LTPK, and possesses features distinct from tetanic stimulationinduced LTP observed in the CA1 region of the hippocampus (Aniksztejn and Ben-Ari, 1990, 1991). A concentration of TEA sufficient to block the delayed rectifier potassium current (IDR), a potassium current that has been demonstrated to participate in the repolarization of action potentials in rat sympathetic neurons (Belluzzi and Sacchi, 1988) and cat sensorimotor cortex neurons (Spain et al, 1991), is required for LTPK. Blockade of IDR produces a prolongation of action potential width and the appearance of Ca2* spikes, which should permit increased entry of Ca2* into presynaptic terminals and enhance transmitter release (Augustine, 1990). LTPK is thought to be mediated via an increase in the release of glutamate, which binds to non-NMDA receptors and depolarizes the postsynaptic neuron sufficiently to activate high-voltage threshold activation VDCCs (Aniksztejn and Ben-Ari, 1991). Recent reports challenge the exclusivity of these mechanisms. Hanse and Gustafsson (1994) observed TEA enhancement of the slope and amplitude of the field excitatory postsynaptic potential (EPSP) in the CA1 region of the hippocampus in the presence of the L-type VDCC antagonist nifedipine (20 nM) either alone or in combination with another blocker, flunarizine (30 uM). There appears to be a degree of overlap in the mechanisms underlying LTP and LTPK, because the magnitude of TEA-induced enhancement of field EPSPs recorded from hippocampal CA1 neurons was reduced when application of TEA was preceded by tetanic stimulation (Huang and Malenka, 1993; Hanse and Gustafsson, 1994). Hanse and Gustafsson (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 mechanisms underlie the increase in EPSP amplitude. Laerum and Storm (1994) reported an additional mechanistic difference between these forms of synaptic plasticity. TEA produced a transient increase in the repolarization time of the presynaptic volley, whereas tetanic stimulation did not. A transient increase in duration of the presynaptic volley after TEA application has been reported previously (Huang and Malenka, 1993; but see Hanse and Gustafsson, 1994). Although the mechanisms for the induction of LTP in the neocortex (Artola and Singer, 1987) were initially suggested to be substantially different from those required in the hippocampus (Komatsu et al, 1988; Perkins and Teyler, 1988), LTP reminiscent of what is typically observed in the hippocampus has been observed in the neocortex (Sutor and Hablitz, 1989; Kirkwood etaL, 1993). Previous studies investigating LTPK were restricted to the CA1 region of the hippocampus. We therefore examined whether application of TEA in a brain slice preparation of rat frontal neocortex produces enhancement of synaptic transmission. A preliminary account of some of these results has appeared (Pelletier and Hablitz, 1994). Cerebral Cortex Nov/Dec 1996;6:771-780; 1047-3211/96/$4.00

Downloaded from cercor.oxfordjournals.org by guest on July 15, 2011

Recordings were obtained from neurons in layer 11/111 of slices of rat frontal cortex maintained in vitro. We investigated whether brief application of the potassium channel blocker tetraethylammonium (TEA), which induces a novel form of synaptic plasticity in the CA1 region of the hippocampus referred to as LTPK, evokes similar responses in neocortex. Consistent with previous findings, TEA produced a persistent enhancement of excitatory transmission, which was independent of NMDA receptor activation but required the activation of nifedipine-sensitive voltage-dependent Ca2+ channels (VDCC), presumably the L-type. We also observed a persistent enhancement of presumptive Cl~-dependent GABAA receptor-mediated transmission. Enhancement of excitatory and inhibitory synaptic transmission did not require activation of synapses with electrical stimulation during TEA application. The enhancement of excitatory, but not inhibitory synaptic transmission, was blocked when the Caz+ chelator 1,2-bis(2-aminophenoxy)ethane /v\A/,/V",W-tetraacetic acid (BAPTA) was included in the recording electrode. Under voltage clamp conditions that minimized the activation of L-type channels robust enhancement of both excitatory and inhibitory transmission was still observed. No enhancement of excitatory synaptic transmission was observed in the presence of NiCI2, a putative T-type channel blocker. The possible involvement of kinase activation was studied by including the non-specific and competitive kinase inhibitor (± )-1 -(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) in the patch pipette. H-7 retarded the time course and reduced the magnitude of the enhancement of excitatory transmission. These results suggest that TEA-induced enhancement of excitatory transmission in the neocortex requires entry of Ca2+ into the postsynaptic neuron via VDCCs and possibly the activation of a kinase.

Marc R. PeUetier1 and John J. Hablitz

772 TEA-induced Plasticity in Neocortcx • Pdlrtier and Hablitz

conducted with independent Mests. Differences were considered significant at P < 0.05. Results are expressed as mean ± SEM.

Results

Passive Membrane Properties The RMP and RN of the neocortical neurons impaled with intracellular electrodes in this study were -82.6 ±1.4 mV and 27.5 ± 2.8 MQ (n = 36) respectively. TEA (25 mM), bath applied for 7 min, produced reversible effects on the passive membrane properties and firing characteristics of neocortical neurons. TEA. 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 control ACSF, if the RMP had not yet returned to control, hyperpolarizing current was injected when I/O relations were determined. As shown in Figure L4 (left), injection of suprathreshold depolarizing current during the control period evoked an action potential. Injection of depolarizing current in the presence of TEA produced a broadening of action potential width and Ca2+ spikes, which persisted beyond die termination of the current pulse (Fig. L4, middle). After return to control ACSF for 30 min the action potential width returned to control (shorter latency to spike due to slighdy larger current pulse). We observed no difference in action potential threshold attributable to TEA. In our experiments, return to control ACSF for 30-40 minutes was typically required for die return to control action potential spike width and RN, at which point we defined changes in synaptic transmission as persistent and not attributable to changes in passive membrane properties. Enhancement of Synaptic Potentials EPSPs evoked in response to electrical stimulation are shown in Figure IB Geft). Control responses to weak stimulation in this neuron consisted of a small EPSP. Increasing the strength of stimulation produced an increase in EPSP amplitude and prolongation in duration. After bath application of TEA there was an increase in amplitude of die response evoked by weak stimulation. We also observed an enhancement of the amplitude and a prolongation of the duration of a late depolarizing component on die decay phase of the potential evoked by strong stimulation (Fig. IB, middle). As seen in Figure IB (right) these changes were persistent after returning to control ACSF for 45 min. The time course of the enhancement of the EPSP amplitude evoked by the test intensity from nine experiments is summarized in Figure \C. The enhancement commenced during the application of TEA and persisted for up to 75 min in control ACSF, the longest time recorded (n = 3). As shown in Figure ID, the magnitude of the increase in amplitude was greatest for EPSPs evoked by weak stimulation and decreased progressively as the stimulation intensity increased. After return to control ACSF for 45 min, the amplitudes for EPSPs evoked by the weakest to the strongest stimulation respectively ranged from 205.1 ± 52.7% (P < 0.05) to 113.6 ± 17.0% of control (n = 9). The response evoked by strong stimulation comprises a mixed EPSP/IPSP and die reduction in the magnitude of enhancement with increasing stimulation intensity is probably a consequence of a shunting effect of the conductance associated widi the IPSP. The response evoked by weak stimulation is less contaminated by EPSPs, and tiierefore exhibits a greater magnitude of enhancement. A late, depolarizing, component of die response evoked by strong stimulation had an equilibrium potential of -73-6 ± 0.6 mV. This is consistent with the equilibrium potential expected

Downloaded from cercor.oxfordjournals.org by guest on July 15, 2011

Materials and Methods The method for preparation of brain slices has been described previously (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 artificial cerebrospinal fluid (ACSF) for -1 min. Slices with a nominal thickness of 500 um were prepared from frontal neocortex using a Vibroslice. After storage for a minimum of 1 h at room temperature, individual slices were transferred to an interface-type chamber and warmed slowly to the recording temperature of 33 ± 1°C. ACSF was perfused continuously from below at a rate of 1 ml/min, which consisted of (in mM): 125 N a d , 3 5 KC1, 2.5 CaCl2, 1.3 MgCl2, 26 NaHCO3 and 10 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. Intracellular recordings from rats 18-42 days old were obtained from layer II/III pyramidal neurons using 4 M potassium acetate-filled microelectrodes (resistance 80-120 Mli). Intracellular signals were recorded and amplified using an Axoclamp-2A amplifier in bridge mode. Whole-cell patch-clamp recording techniques were used as described previously (Burgard and Hablitz, 1993). Briefly, patch pipettes (resistance 2-4 Mii) were filled with a solution consisting of (in mM): 125 KMsethionate, 10 KCI, 0.5 ethylene glycol-bis@-aminoethyl ether)Jv',JV,JV,JV'-tetraacetic acid (EGTA), 10 W-2-hydroxyethylpiperazineAT-2-ethanesulfonic acid (HEPES), 2 MgATP and 0.2 NaGTP. The osmolality of this solution was 270-280 mOsm, and the pH adjusted to 7.2. Voltage-clamp recordings from rats 14-35 days old were obtained using a discontinuous single-electrode voltage-clamp amplifier (NPI SEC1L, Tamm, Germany) at a switching frequency of 27 kHz and a 25% duty cycle. Series resistance ranged from 10 to 15 MQ. Postsynaptic responses were evoked (0.05 Hz) via a bipolar stimulating electrode located directly below the recording electrode in cortical layer IV/V. Input-output (I/O) relations were determined every 15 min by varying the intensity of electrical stimulation. I/O relations comprised four or five progressively greater stimulation intensities (e.g. weak < test < intermediate < strong). The effect of TEA on synaptic responses is presented typically at three representative stimulation intensities: weak, evoked small amplitude responses with no failures; test, evoked responses -50% of maximal amplitude; strong, evoked maximal amplitude responses. Three to five responses were measured at each intensity. When I/O relations were not being determined postsynaptic responses were evoked with the test stimulation intensity. The recorded signals were filtered at 1 kHz, stored on videotape (Neuro-Corder DR 484), and digitized using pClamp software (Axon Instruments). For intracellular recording the resting membrane potential (RMP) and input resistance (RN; determined from the voltage deflection resulting from a 0.3-0.5 nA, 50 ms hyperpolarizing current pulse) were monitored continuously during the experiments. For voltage-clamp recording neurons were clamped at -75 mV and access resistance was assessed by monitoring the capacitative transients and the current produced by a 5.0 mV, 50 ms hyperpolarizing voltage pulse. The following drugs were used: TEA (25 mM; Sigma, St Louis, MO), D-2-amino-5-phosphonovaleric acid (APV; 20 uM; Tocris Neuramin, 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 (BAPTA; 11 or 200 mM; Calbiochem, La Jolla, CA), nifedipine (20 \iM; Sigma), NiCh (200 uM; Fisher, Norcross, GA) and (±>l{5-isoquinolinesulfonyl>2-methylpiperazine dihydrochloride (H-7; 100 uM; Calbiochem). Drugs were dissolved in ACSF, except for CNQX and nifedipine, which were dissolved in dimethyl sulfoxide (final concentration in ACSF 0.1 %). APV, CNQX, BAPTA, nifedipine and H-7 were prepared as stock solutions and stored frozen. TEA and NiCh were prepared dairy. Drugs not included in the intracellular solution were bath applied. In our experiments, TEA was added directly to the bathing solution because Huang and Malenka (1993) reported that TEA-induced synaptic enhancement was not altered when the osmolality of the bathing solution was controlled by reducing the concentration of Nad. Differences in synaptic transmission (amplitude) attributable to the application of TEA were determined with dependent f-tests (each neuron served as its own control). Comparisons between experiments were

Control

20

30 40 Time (min)

50

60

Control TEA

15 30 Time

45

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 postsynaptic potential (IPSP) in neocortical neurons (Weiss and Hablitz, 1984; Howe etaL, 1987; Connors etaL, 1988). It should be noted that the RMP we typically record in these neurons is hyperpolarized compared to the equilibrium potential for Cl"; therefore, GABAA receptor-mediated IPSPs are depolarizing. After return to control ACSF for 45 min, the magnitude of the increase in amplitude of the presumptive IPSPs (measured 50 ms from the peak of the potential evoked by the strongest stimulation) was robust (180.3 ± 10.0 of control; P < 0.05) and approximated that observed for the EPSP evoked by weak stimulation. Activation of the NMDA subtype of glutamate receptor is necessary for the induction of synaptic plasticity in various brain regions (Gustafcson etaL, 1987; Kirkwood and Bear, 1994). We tested whether LTPK in the neocortex observed this requirement by evaluating the effect of TEA on synaptic transmission in the presence of the NMDA receptor antagonist, APV (20 uM). Consistent with what has been reported previously (Aniksztejn and Ben-Ari, 1991; Huang and Malenka, 1993), LTPK was unaffected by APV. That is, LTPK induction in the neocortex does not require the activation of NMDA receptors. After return to control ACSF for 45 min, the amplitudes of EPSPs evoked by the weakest to the strongest stimulation intensity respectively ranged from 179.3 ± 3 9 8 (P < O.O5) to 106.2 ± 5.8% of control

(n = 6). Presumptive GABAA receptor-mediated IPSPs were 234.0 ± 44.6% of control (P < 0.05). To test further the activity dependence of LTPK we applied TEA in the absence of electrical stimulation. Under control conditions (Fig. 2/4, left) typical PSPs were evoked. After return to control ACSF for 15 min stimulation was again applied (Fig. 2/4, center). An increase in the amplitude of the EPSP evoked by weak stimulation and a prolongation of the depolarizing presumptive IPSP evoked by intermediate and strong stimulation was observed. Action potentials were evoked by strong stimulation (3/9), as observed in experiments described above using stimulation. EPSP and IPSP enhancement persisted after 45 min of control ACSF (Fig. 2A, right). EPSP enhancement ranged from 201.1 ± 50.0% (P < 0.05) to 113.6 ± 5.0% of control for the weakest to the strongest stimulation respectively (n = 6). The average increase in the amplitude of the presumptive GABAA receptor-mediated IPSPs was 262.8 ± 76.1% of control (P< 0.05). The relation between TEA enhancement of amplitude and stimulation intensity from these experiments is shown in Figure 28. The results are similar to those observed in experiments where electrical stimulation occurred for the duration of the experiment. The greatest enhancement in every experiment was observed for EPSPs evoked by weak stimulation and presumptive IPSPs evoked by maximal stimulation. These experiments indicate that the induction of LTPK requires neither the

Cerebral Cortex Nov/Dec 1996. V6N 6 773

Downloaded from cercor.oxfordjournals.org by guest on July 15, 2011

10

Wash

TEA

Control

B g litud

u

1

45 min Wash

15 min Wash

6mV 250-

20 ms

200150 100Control

15 Time

30

45

activation of NMDA receptors nor the simultaneous activation of synapses during TEA application, two features which make this form of synaptic plasticity distinct from tetanus-induced LTP observed in the CA1 region of the hippocampus. The enhancement in the amplitude of GABAA receptormediated IPSPs after TEA application has not been reported previously. We therefore examined directly evoked, presumably monosynaptic, IPSPs elicited in the absence of excitatory transmission. As seen in Figure 3A Qeft), direct stimulation of interneurons in the presence of 10 uM CNQX and 20 uM APV, to block non-NMDA and NMDA receptors respectively, produced a depolarizing PSP (RMP of this neuron -86 mV). The PSPs recorded under these conditions had an equilibrium potential of -72.6 ± O.9 mV (n = 6), consistent with the expected equilibrium potential for Cl", suggesting that they were GABAA receptormediated IPSPs. Application of TEA produced an increase in the amplitude of the IPSP (Fig. 3A, center), which persisted after return to control ACSF for 45 min (Fig. 5A, right). The increase in the amplitude of the IPSP can be seen clearly in Figure 3-B. where the IPSP during the control period is superimposed upon the IPSP obtained after return to control ACSF for 45 min. The time course of the TEA-induced IPSP enhancement is presented in Figure 3C (n = 6). Note that, similar to what we described above for EPSPs, IPSP enhancement commenced during the application of TEA. IPSP enhancement was 172.9 ± 30.9% of control (P < 0.05) when measured after return to control ACSF for 45 min. Co2*- dependence of LTPK In the CA1 region of the hippocampus, the induction of LTPK is dependent upon an increase in the postsynaptic concentration of Ca2* (Aniksztejn and Ben-Ari, 1991; Huang and Malenka, 1993). To test whether an increase in postsynaptic Ca2* is required for the induction of LTPK in the neocortex, we recorded 774 TEA-induced Plasticity in Neocortex • Pdleticr and Hablitz

from neurons impaled with electrodes filled with 200 mM BAPTA, a Ca2* chelator (Tsien, 1980). To confirm that BAPTA had been injected, we monitored the amplitude of the slow afterhyperpolarization (sAHP), which is mediated by a Ca2*-dependent K* current in hippocampal (Lancaster and Adams, 1986; Lancaster and Nicoll, 1987; Storm, 1987) and neocortical neurons (Schwindt et aL, 1988). Figure 4 4 (left) illustrates the sAHP that is produced after a train of action potentials. This neuron was depolarized to a potential of -70 mV to enhance the sAHP. The amplitude of the sAHP was decreased after 20 min of impalement with the BAPTA-containing electrode (Fig. 4/4, center). This can be seen most clearly in Figure 4-4 (right) where the two records are superimposed. The decrease in the sAHP typically required 20-30 min, at which point we applied TEA. PSPs evoked under these conditions (Fig. 42?, left) appeared similar to those recorded with electrodes not containing BAPTA. EPSPs evoked by weak stimulation were not enhanced in the presence of TEA (Fig. 4B, center, 105.1 ± 19-1% of control; P > 0.05) and were depressed slightly after return to control ACSF for 20 min (Fig. 4B, right; 86.4 ±11.3% of control; P 0.05; n = 4). This is seen most clearly in the inset where the EPSP evoked prior to TEA application is superimposed on the EPSP evoked after 20 min of control ACSF. In contrast, the amplitude of the presumptive IPSP evoked by strong stimulation was 1835 ± 40.6% and 193.4 ± 43.3% of control (both P < 0.05) when measured at the same time points. The blockade of TEA-induced enhancement of the amplitude of the EPSPs evoked by the test stimulation intensity is shown clearly in Figure AC, which summarizes four experiments. Pharmacological antagonism of L-type VDCCs prevents the induction of LTPK in the hippocampus (Aniksztejn and Ben-Ari, 1991; Huang and Malenka, 1993; but see Hanse and Gustaffson, 1994). Ca2* entry into the postsynaptic neuron via this class of VDCC is believed to be critical for the induction of LTPK- TO test

Downloaded from cercor.oxfordjournals.org by guest on July 15, 2011

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.

A

45 min Wash

TEA

Control

10 HM CNQX + 20 JIM APV 4mV

B 2mV

g.

10 ms .Wash

••a

itrol

150

10

20

30

40

50

Time (min)

A

200 mM BAPTA 8 min

20 min BAPTA - - f—

30

35

40 45 Time (min)

50

— — — -urn, ^ ^ y » - f

55

Figure 4. Enhancement of EPSP amplitude is dependent upon an increase in postsynaptic Ca 2+ . (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).

Cerebral Cortex Nov/Dec 1996, V 6 N 6 775

Downloaded from cercor.oxfordjournals.org by guest on July 15, 2011

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.

T

10

15

20

25

30

35

40

45

50

55

60

65

Time (min) 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.

Control

30 min Wash

TEA

-75 raV-4 - - -

B 80 pA 30 ms

D Control

300

A

-50 mV

5

PA too

mV

10 15 20 25 30 35 40 i

Time (min) 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 recorded responses evoked first in the presence of the L-type VDCC channel blocker nifedipine (20 uM), followed by simultaneous application of both nifedipine and TEA. Amplitudes of responses evoked by the test intensity were not different from control when measured in the presence of nifedipine alone (91.8 ± 3-8% of control) or return to control ACSF for 30 min after 776 TEA-Induccd Plasticity in Ncocortcx • Pdfctier and Hablitz

simultaneous application of nifedipine and TEA (89-1 ± 6.6% of control). A summary of five experiments is presented in Figure 5. TEA failed to produce persistent enhancement of responses evoked at any stimulation intensity. The amplitudes of responses measured after return to control ACSF for 30 min evoked by weak and strong stimulation were 8 9 9 ± 11.8% and 86.0 ± 13.1% of control respectively. The amplitude of the presumptive IPSPs

Downloaded from cercor.oxfordjournals.org by guest on July 15, 2011

A

measured at the same time point was 106.7 ± 157% of control. These results suggest that Ca2* entry via nifedipine-sensitive VDCCs, presumably the L-type, is necessary for TEA-induced synaptic enhancement in the neocortex.

Ampli tude((

300

Voltage-Clamp Analysis O/LITPK

200 100 5 300

Amplitude (°/

B

10

30

15 20 25 Time (min)

200|iMNiCl 2

200

i

100

5 10 15 20 25 30 35 40 Time (min) itude (

300

1

100uMH7

200

Jt ••-

100 5

10

15 20 25 Time (min)

30

Figure 7. Enhancement of EPSC amplitude is dependent upon an increase in postsynaptic Ca 2 + . (4) Summary of the time course of blockade of enhancement of amplitude of EPSCs recorded with patch pipettes containing 11 mM BAPTA/1 m M CaClz |n = 8). TEA application is denoted by solid bar. (5) Summary of the time course of blockade of enhancement of EPSC amplitude when 200 | i M N1CI2 was included in the ACSF to block T-type channels (n = 10). (C) Inclusion of 100 p M H7, a non-specific, competitive kinase inhibitor, retards and reduces TEA-induced enhancement of amplitude of EPSCs (n = 8).

voltage-damp conditions that should functionally eliminate the contribution of high-voltage threshold activation VDCCs, suggests that activation of a different subtype of VDCC might be sufficient to induce LTPK. A likely candidate might be the low-voltage threshold activation T-type VDCC (Tsien et at, 1988). To test this hypothesis we conducted experiments under voltage-clamp conditions in the presence of 200 jiM NiCh to block T-type VDCCs (Mogul and Fox, 1991). A high concentration of NiCb was used to ensure complete block of T-type channels despite a loss of specificity. Because a holding potential of -60 mV was used, significant activation of high threshold Ca2* channels was unlikely. As summarized in Figure IB, we observed no EPSC enhancement when measured either at the beginning (104.8 ± 24.6% of control) or after return to control ACSF for 20 min (93.2 ± 11.8% of control; n = 10; both P > 0.05). These results suggest that Ca2* entry in the postsynaptic neuron via a subtype of low-voltage threshold activation VDCC, possibly the T-type, might also participate in the enhancement of excitatory transmission we have observed. Protein phosphorylation is important for a variety of neuronal functions, including synaptic transmission. Activation of kinases promotes phosphorylation, whereas activation of phosphatases promotes dephosphorylation. Ca entry into a neuron can activate a variety of intracellular signalling pathways Cerebral Cortex Nov/Dccl996, V6N 6 777

Downloaded from cercor.oxfordjournals.org by guest on July 15, 2011

We next investigated whether TEA would produce enhancement of synaptic transmission under voltage-clamp conditions designed to prevent the activation of L-type VDCCs. A critical time period for the induction of LTP when patch pipettes are used has been suggested (Kato et at, 1993). The critical time period (15 min after attaining whole cell) was attributed to the 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 solution 2 mM MgATP and 0.2 mM NaGTP and ensured that TEA was applied -15 min after attaining the whole-cell configuration. Figure &AJi (left) illustrates the synaptic currents evoked by weak and strong stimulation respectively prior to the application of TEA. In the presence of TEA, EPSC amplitudes were increased significantly (Fig. 6AM, center), and the enhancement persisted after return to control ACSF for 30 min (Fig. 6AJB, right). As described above for EPSPs, the increase in amplitude was greatest for the EPSC evoked by weak stimulation. After return to control ACSF for 20 min EPSCs evoked by weak stimulation were 216.9 ± 70.6% of control (w = 5; P < 0.05). The time course of the enhancement of amplitude for EPSCs is presented in Figure 6C. At the beginning of the return to control ACSF EPSCs evoked by the test intensity were 184.6 ± 29.0% of control (/> < 0.05). The EPSC enhancement persisted, and increased to 211.7 ± 51.4% of control (P < 0.05), after return to control ACSF for 20 min. To evaluate further the enhancement of IPSCs, we clamped the membrane potential depolarized to the expected equilibrium potential for Cl", which in these experiments was -69 mV. As shown in Figure 6D (upper, control) at -50 mV, strong stimulation evokes both an inward EPSC and an outward IPSC. After return to control ACSF for 10 min (Fig. 6D, lower) an increase in the amplitude of the presumptive IPSC is seen clearly. IPSCs were 172.3 ± 26.6% of control (P < 0.05) when measured after return to control ACSF for 15 min. The current-voltage relation of the IPSC from the neuron presented in Figure 6C, during control (filled squares) and after return to control ACSF 10 min (open squares), is presented in Figure 6E. It can be seen that there is an increase in the slope conductance after application of TEA, with no change in the apparent equilibrium potential. In additional experiments BAPTA was included in the patch pipette solution (11 mM BAPTA/1 mM CaCh; intracellular Ca2* buffered to 20 nM; Pethig et at, 1989) to examine further the Ca2*-dependence of LTPK- Under these conditions we observed a reduction of the sAHP within 5 min after attaining the whole-cell configuration. Figure 1A illustrates a relation similar to what we observed when PSPs were recorded under current-clamp conditions with BAPTA-containing electrodes. EPSCs were not enhanced when measured either at the beginning of the return to control ACSF after TEA application (105.6 ± 197% of control; P > 0.05) or after 15 min in control ACSF (116.3 ± 17.4% of control; n = 8;P> 0.05). However, IPSCs were increased (142.6% of control; n = 3). These results confirm the critical dependence on Ca2* entry in the postsynaptic neuron for the induction of TEA-induced enhancement of excitatory transmission. The observation of robust enhancement of EPSCs under

l l m M BAPTA

or can itself act as a messenger by inducing its release from intracellular stores (for reviews see Miller, 1988; Penner et al, 1993). In order to assess the contribution of kinase activation in neocortical LTPK, we included in the patch pipette solution, the competitive, non-selective kinase inhibitor H-7 (100 jiM; Hidaka et al., 1984; Boulis and Davis, 1990; Takahashi et al, 1990). When measured at the beginning of the return to control ACSF after TEA application, EPSCs evoked by the test intensity were 126.1 ± 28.0% of control (Fig. 1Q. After return to control ACSF for 15 min, EPSCs were 1398 ± 60.0% of control (n = 8; both P > 0.05). Blockade of kinase activation with H-7 both retarded the time course and reduced the magnitude of the enhancement by TEA of the EPSC amplitude (cf. Fig. 6C). Taken together, these data suggest that Ca -dependent activation of a kinase, whose identity remains to be elucidated, might be necessary for TEA enhancement of excitatory transmission in the neocortex.

Discussion

778 TEA-induced Plasticity in Neocortex • Pdletier and Hablitz

Downloaded from cercor.oxfordjournals.org by guest on July 15, 2011

We observed that brief (7 min) application of TEA (25 mM) produced a persistent enhancement of excitatory synaptic transmission in a slice preparation of rat neocortex. Several features of the neocortical TEA-induced synaptic enhancement we observed are similar to those described previously in the CA1 region of the hippocampus, including NMDA receptor independence, requirement of an increase in the concentration of Ca2* in the postsynaptic neuron and dependence upon nifedipine-sensitive VDCCs, presumably the L-type. An important consideration is whether the persistent enhancement of synaptic transmission is due to factors other than the continued presence of TEA. Return to control ACSF for -30 min was required for action potential duration, RMP and RNto return to control values—our operational definition of TEA removal. Although our group data is reported after return to control ACSF for 45 min, we observed persistent effects after >75 min (n = 3). Aniksztejn and Ben-Ari (1991) reported that the threshold concentration of TEA required to produce a persistent enhancement of synaptic transmission was 15 mM. It is unlikely that any residual TEA would be at a concentration sufficient to explain the persistent effects reported here. We observed also a novel persistent enhancement of presumptive cr-dependent GABAA receptor-mediated inhibitory transmission. Failure to observe an enhancement of inhibitory transmission in previous studies can be attributed to the inclusion of picrotoxin to block GABAA receptors (Hanse and Gustaffson, 1994), recording of field potentials in the absence of pharmacological blockade of excitatory transmission (e.g. Aniksztejn and Ben-Ari, 1991), or perhaps stimulating at an intensity insufficient to evoke IPSPs. Similar to TEA, the potassium channel blocker 4-aminopyridine (4-AP) enhances both EPSPs and IPSPs (Rutecki et al, 1987; Perreault and Avoli, 1991, 1992); however, 4-AP also produces epileptiform discharges, an effect we did not observe with TEA. Features of the enhancement of the presumptive IPSPs we observed (e.g. action potentials associated with the depolarizing GABAA potential) are more reminiscent of the enhancement of GABAergic transmission reported for zinc (Zhou and Hablitz, 1993). Enhancement of the presumptive inhibitory responses was not dependent upon an increase in postsynaptic Ca2* concentration: the enhancement was unaffected when BAPTA was included in either the intracellular electrode or patch pipette. However, whether an increase in Ca2* in neurons providing excitatory input onto GABAergic interneurons is necessary cannot be ruled out.

An important issue concerning LTPK induction is the point of entry for Ca2* into the postsynaptic neuron. Previous studies have suggested that Ca2* entry via the L-type VDCC is critical for the induction of LTPK (Aniksztejn and Ben-Ari, 1991; Huang and Malenka, 1993). hi contrast, Hanse and Gustafsson (1994) observed an enhancement of both slope and amplitude of field EPSPs recorded in the CA1 region in the presence of the organic L-type channel blockers, nifedipine alone, or in combination with flunarizine. We observed no TEA-induced synaptic enhancement in the presence of nifedipine, indicating that L-type VDCCs are necessary in neocortex. Two pieces of evidence suggest that VDCCs other than the L-type might be sufficient for LTPK induction in the neocortex. Under voltage-clamp conditions designed to functionally prevent the activation of L-type VDCCs, robust enhancement of synaptic 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 at the base of proximal dendrites in neocortical neurons, and only sparsely on more distal dendrites (Ahlijanian et al, 1990; Hell et al, 1993); however, if the spatial localization of L-type channels is more widespread than suggested by these studies activation of L-type channels may be contributing under our voltage-clamp conditions due to poor spatial control of voltage. We used a relatively high concentration of NiCh (200 |iM) in an attempt to block maximally T-type channels, although at the expense of selectivity. Therefore, it cannot be ruled out that in addition to T-type channels L-type channels were blocked as well. It appears that die most tenable hypothesis supported by our observations is that Ca2* entry via the L-type VDCC is necessary for LTPK in the neocortex. Hypotheses that remain to be tested include determining the involvement of N-type VDCCs, which have been reported to be located on the soma and throughout the dendritic arbor of hippocampal pyramidal neurons (Mills et al, 1994), or whether TEA has either a direct effect, or an indirect effect mediated by an intracellular signaling pathway, on Ca2* channels, which would promote the entry of Ca2* into the postsynaptic neuron. Neocortical LTPK did not require the simultaneous activation of synapses with electrical stimulation. This observation is at variance with Petrozzino and Connor (1994) who reported that in the absence of low-frequency stimulation (0.05 Hz) EPSP enhancement in the CA1 region was decremental and decayed back to baseline after 60 min of control ACSF. Petrozzino and Connor (1994) suggest LTPK requires both Ca2* entry via L-type channels and co-activation of metabotropic glutamate receptors (mGluR). A reconciliation of our observations with those of Petrozzino and Connor (1994) might be that TEA produces an increase in frequency of spontaneous PSPs or action potentials, which would depolarize the postsynaptic neuron sufficiently to activate VDCCs. Spontaneous Ca2* spikes might also be sufficient to fulfil the Ca2* requirement of LTPK. We did not monitor spontaneous PSPs systematically, and TEA-induced spontaneous action potentials or Ca2* spikes were observed only infrequently. TEA did produce a reversible depolarization (4.8 ± 0.7 mV) and increase in RN (16.8 ± 4.7%), which would increase the excitability of neocortical neurons. An increase in frequency of spontaneous events, in combination with the effects on passive membrane properties described above, might participate in the activation of VDCCs. Consistent with this hypothesis is the observation that in dendrites of neocortical neurons single subthreshold EPSPs can activate low-voltage threshold Ca2* channels (Markram and Sakmann, 1994).

Kinase activation has been reported to be necessary for LTPK in the hippocampus (Petrozzino and Connor, 1994). The enhancement of EPSC amplitude was both retarded in time course and reduced in magnitude when we included H-7 in the pipette to inhibit kinase activity, however, due to the extreme varability in the responses we recorded with H-7 containing pipettes, a statement concerning whether activation of a kinase is necessary for neocortical LTPK cannot be made with confidence at this time. The characteristics of TEA-induced synaptic enhancement we observed in neocortex are similar in many respects to that described in the CA1 region of hippocampus, including NMDA receptor independence and requirement of Ca2* entry via L-type VDCCs, although synaptic activity evoked with electrical stimulation in the presence of TEA. is not necessary for LTPK in neocortex. An interesting feature not reported in hippocampus was the enhancement of presumptive Cl"-dependent GABAA receptor-mediated responses. Tetanic stimulation-induced LTP of inhibitory synaptic transmission, with properties similar to those known for LTP of excitatory responses in this region, has been described in rat visual cortex (Komatsu, 1994). Further characterization of TEA-induced enhancement of inhibitory transmission in neocortex and hippocampus, and its Ca2* dependence, is warranted.

References Ahlijanian MK, Westenbroek RE, Catterall WA (1990) Subunit structure and localization of dihydropyridine-sensitive calcium channels in mammalian brain, spinal cord and retina. Neuron 4:819-832. Aniksztejin L, Ben-Ari Y (1990) NMDA-independent form of long term potentiation produced by tetraethylammonium in the hippocampal CA1 region. Eur J Pharmacol 181:157-158. Aniksztejn L, Ben-Ari Y (1991) Novel form of long-term potentiation produced by a K channel blocker in the hippocampus. Nature 349:67-69. Artola A, Singer W (1987) Long-term potentiation and NMDA receptors in rat visual cortex. Nature 330:649-652. Augustine GJ (1990) Regulation of transmitter release at the squid giant synapse by presynaptic delayed rectifier potassium current. J Physiol 431:343-364. Belluzzi O, Sacchi O (1988) The interactions between potassium and sodium currents in generating action potentials in the rat sympathetic neurone. J Physiol 397:127-147. Bliss TVP, Collingridge GL (1993) A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361:31-39. Bliss TVP, Lynch MA (1988) Long-term potentiation of synaptic transmission in the hippocampus: properties and mechanisms. In: Long-term potentiation: from biophysics to behavior (Landfield PW, Deadwyler SA, eds), pp 3-72. New York: Alan R liss. Boulis NM, Davis M (1990) Blockade of the spinal excitatory effect of cAMP on the startle reflex by intrathecal administration of the isoquinoline sulfonamide H-8: comparison to the protein kinase C inhibitor H-7. Brain Res 525:198-204. Burgard EC, Hablitz JJ (1993) Developmental changes in NMDA and non-NMDA receptor-mediated synaptic potentials in rat neocortex. J Neurophysiol 69:230- 240. Connors BW, Malenka RC, Silva LR (1988) Two inhibitory postsynaptic potentials and GABAA and GABAs receptor-mediated responses in neocortex of rat and cat. J Physiol 406:443-468. Grover LM, Teyler TJ (1990) Two components of long-term potentiation induced by different patterns of afferent activation. Nature 347:477-479. Gustafsson B, Wigstrom H, Abraham WC, Huang Y-Y (1987) Long-term

53:39-47. Kirkwood A, Bear MF (1994) Hebbian synapses in visual cortex. J Neurosci 14:1634-1645. Kirkwood A Dudek SM, Gold JT, Aizenman CD, Bear MF (1993) Common forms of synaptic plasticity in the hippocampus and neocortex in vitro. Science 260:1518-1521. Komatsu Y (1994) Age-dependent long-term potentiation of inhibitory synaptic transmission in rat visual cortex. J Neurosci 14:6488-6499. Komatsu Y, Fuji K, Maeda J, Sakaguchi H, Toyama K (1988) Long-term potentiation of synaptic transmission in kitten visual cortex. J Neurophysiol 59:124-141. Laerum H, Storm JF (1994) Hippocampal long-term potentiation is not accompanied by presynaptic spike broadening, unlike synaptic potentiation by K* channel blockers. Brain Res 637:349-355. Lancaster B, Adams PR (1986) Calcium-dependent current generating the afterhyperpolarization of hippocampal neurons. J Neurophysiol 55:1268-1282. Lancaster B, Nicoll RA (1987) Properties of two calcium-activated hyperpolarizations in rat hippocampal neurones. J Physiol 389:187-203. Madison DV, Malenka RC, Nicoll RA (1991) Mechanisms underlying long-term potentiation of synaptic transmission. Annu Rev Neurosci 14:379-397. Malinow R (1994) LTP: desperately seeking resolution. Science 266:1195-1196. Markrara H, Sakmann B (1994) Calcium transients in dendrites of neocortical neurons evoked by single subthreshold excitatory postsynaptic potentials via low-voltage-activated calcium channels. Proc Natl Acad Sci USA 91:5207-5211. Miller RJ (1988) Calcium signalling in neurons. Trends Neurosci 11:415-419. Mills LR, Niesen CE, So AP, Carlen PL, Spigelman I, Jones OT (1994) N-type Ca2* channels are located on somata, dendrites, and a subpopulation of dendritic spines on live hippocampal pyramidal neurons.J Neurosci 14:6815-6824. Mogul DJ, Fox AP (1991) Evidence for multiple types of Ca2* channels in acutely isolated hippocampal CA3 neurones of the guinea-pig. J Physiol 433:259-281. Pelletier MR, Hablitz JJ (1994) TEA produces a persistent enhancement of synaptic transmission in rat neocortex. Soc Neurosci Abstr 20:1512. Penner R, Fasolato C, Hoth M (1993) Calcium influx and its control by calcium release. Curr Opin Neurobiol 3:368-378. Perkins AT, Teyler TJ (1988) A critical period for long-term potentiation in the developing rat visual cortex. Brain Res 439:222-229. Perreault P, Avoli M (1991) Physiology and pharmacology of epileptiform activity induced by 4-aminopyridine in rat hippocampal slices. J Neurophysiol 65:771-785. Perreault P, Avoli M (1992) 4-aminopyridine-induced epileptiform activity and a GABA-mediated long-lasting depolarization in the rat hippocampus. J Neurosci 12:104-115. Pethig R, Kuhn M, Payne R, Adler E, Chen T-H, Jaffe LF (1989) On the dissociation constant of BAPTA-type calcium buffers. Cell Calcium 10:491-498.

Cerebral Cortex Nov/Dec 1996,V6N6 779

Downloaded from cercor.oxfordjournals.org by guest on July 15, 2011

Acknowledgements This work was supported in part by NIH grant NS18145. Address correspondence to John J. Hablitz, Neurobiology Research Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA

potentiation in the hippocampus using depolarizing potentials as the conditioning stimulus to single volley synaptic potentials. J Neurosci 7:774-780. Hanse E, Gustaffsson, B (1994) TEA elicits two distinct potentiations of synaptic transmission in the CA1 region of the hippocampal slice. J Neurosci 14:5028-5034. Hell JW, Westenbroek RE, Warner C, Ahlijanian MK, Prystay W, Gilbert MM, Snutch TP, Catterall WA (1993) Identification and differential sibcellular localization of the neuronal class C and class D L-type calcium channel a l subunits. J Cell Biol 123:949-962. Hidaka H, Inagaki M, Kawamoto S, Sasaki Y (1984) Isoquinolinesulfanomides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase C. Biochemistry 23:5036-5041. Howe JR, Sutor B, Zieglansberger W (1987) Characteristics of longduration inhibitory postsynaptic potentials in rat neocortical neurons in vitro. Cell Mol Neurobiol 7:1-18. Huang Y-Y, Malenka RC (1993) Examination of TEA-induced synaptic enhancement in area CA1 of the hippocampus: the role of voltage-dependent Ca2* channels in the induction of LTP. J Neurosci 13:568-576. Kato K, Clifford DB, Zorumski CF (1993) Long-term potentiation during whole cell recording in rat hippocampal slices. Neuroscience

Pettrozzino JJ, Connnor JA (1994) Dendritic Ca2* accumulations and metabotropic glutamate receptor activation associated with an JV-methyl-D-aspartate receptor-independent long-term potentiation in hippocampal CA1 neurons. Hippocampus 4:546-558. Rutecki PA, Lebeda FJ, Johnston D (1987) 4-Aminopyridine produces epileptiform activity in hippocampus and enhances synaptic excitation and inhibition. J Neurophysiol 57:1911-1924. Schwindt PC, Spain WJ, Foehring RC, Stafetrom CE, Chibb MC, Crill WE (1988) Multiple potassium conductances and their functions in neurons from cat sensorimotor cortex in vitro. J Neurophysiol 59:424-449. Spain WJ, Schwindt PC, Crill WE (1991) Two transient potassium currents in layer V pyramidal neurones from cat sensorimotor cortex. JPhysiol 434:591-607. Storm JF (1987) Action potential repolarization and a fast afterhyperpolarization in rat hippocampal pyramidal cells. J Physiol 434:591-607.

Sutor B, Hablitz JJ (1989) EPSPs in rat neocortical neurons in vitro. I. Hectrophysiological evidence for two distinct EPSPs. J Neurophysiol 61:607-620. Takahashi I, Yashi EK, Nakano H, Murakata C, Saitoh H, Suzuki K, Tamaoki T (1990) Potent selective inhibition of 7-O-methyl UCN-01 against protein kinase C.J Pharmacol Exp Ther 255:1218-1221. Tsien RY (1980) New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis and properties of prototype structures. Biochemistry, 19:2396-2404. Tsien RW, Upscombe D, Madison DV, Bley KR, Fox AP (1988) Multiple types of neuronal calcium channels and their selective modulation. Trends Neurosci 11:431-438. Weiss DS, Hablitz JJ (1984) Interaction of penicillin and pentobarbital with inhibitory synaptic mechanisms in neocortex. Cell Mol Neurobiol 4:301-317. Zhou F-M, Hablitz JJ (1993) Zinc enhances GABAergic transmission in rat neocortical neurons. J Neurophysiol 70:1264-1269.

Downloaded from cercor.oxfordjournals.org by guest on July 15, 2011

780 TEA-Induccd Plasticity in Neoconex • Pdletier and Hablitz