Differential effects of isoquinolinesulfonamide protein kinase inhibitors on CA1 responses in hippocampal slices

I%?ros&?#ce Vol. 44, No. 2, pp, 351-370, Printed in Great Britain

0306-4522191 $3.00 + 0.00 Pergamon Press ple 0 1991 IBRCI

1991

DIFFERENTIAL EFFECTS OF ISO~UINOLINESULFONAMID~ PROTEIN KINASE INHIBITORS ON CA1 RESPONSES IN HIPPOCAMPAL SLICES J. C. LEAHY* and M. L. VA~LANQ

Department

ofPharmacology,State University ofNew York/Health

Science Center, 750 E. Adams Street,

Syracuse, NY 13210, U.S.A. Abstract-The effects of the isoquinolinesulfonamide protein kinase inhibitors I-(%soquinolinylsulfonyl)Z-me~ylp~~r~ne (H-7) and ~~2-~din~thyl)-5-is~~no~nesulfon~de (HAioo$) on CAl responses in hippo~amp~ slices of the rat were examined to clarify their mode of action, and also to further define the role of Ca2+-dependent kinases in long-term ~tentiation. Initially, the inhibitor potencies of H-7 and HAlOW against both protein kinase C and type II Cat+ ~calmodulin~e~ndent kinase were examined in standard in vitro phospba~lation assays. The apparent Kf values of H-7 and HA1004 For protein kinase C were 9 and 57 uM. resnectivelv. In contrast. the K, values of H-7 and HA1004 for type II calcium/ calmodulin-depenhen; prdtein kiiase were 1.56“and 1j p M, respectively. These results in&ate that H-7 is a more effective inhibitor of protein kinase C, whereas HA1004 is a more effective inhibitor of typ I1 ~l~urn~~~~in~~dent protein kinase. Following the induction of long-term potentiation, addition of 50 FM H-7 or HA1004 su~~nti~ly increased the amplitude of the population spike in a control pathway, while producing no change or a slight increase in the spike amplitude in a previously potentiated long-term potentiation pathway. Moreover, H-7 (50 pM), but not HAlW, produced multiple population spikes in both pathways. Addition of a higher concentration of H-7 (300 PM) reduced the amplitude of the initial population spike but still produced multiple spikes. HA1004 (300 FM) typically produced effects similar to those observed with 50 PM H-7, increasing the amplitude of the control population spike and producing multiple spike activity in bath pathways. In contrast to the differential ~n~Rtmtion-de~ndent effects of H-7 on the population spike responses, qualitatively similar efl%cts were observed at both low (50 fi Mf and high (3~~M) ~n~ntrations with regard to synaptic field responses. The initial slope of the population excitatory postsynaptic potential was significantly reduced by H-7, to a similar degree in both pathways. HA1004 produced a modest, but insignificant reduction in both pathways. These results, in conjunction with other reports, suggest that H-7 and HA1004 exert complex concentration-dependent effects which synchranously affect both excitatory and inhibitory synaptic transmission. We hypothesize that reduction of the population excitatory postsynaptic potential and spike (300pM H-7) is d&i to reduction of excitatory inputs, whereas enhancement of the population spike amplitude (50 FM H-7) and the pr~u~tion of multiple spikes are due to the reduction of GABA-m~iated inhibitory inputs. Moreover, the actions of these drugs on synaptic tr~smission were not affected by prior induction of: long-term potentiation, except for the eff&ts of SOpM H-7 on the population spike amplitude. However, the lack of further potentiation of the population spike amplitude with 50 FM H-7 is consistent with recent reports suggesting that reduction of inhibitory mechanisms contributes to spike long-term potentiation. Tee resultibf thk phosphorylation assays suggest that the electrophysiolo&al alterations observed following addition of H-7 and HA1004 can be attributed to inhibition of protein kinase C. The biochemical studies also indicate the potentiai usefulness of HA1004 as a potent idhibitor of type II calcium/~lmodulin-de~ndent protein kinase.

Long-term potentiation (LTP) is a persistent form of synaptic plasticity9 that may be involved in learning and memory processes. The mechanisms underlying the induction of some forms of LTP have been well

characterized and involve activation of postsynaptic N-methyl-r+aspartate (NMDA) receptors’5*20 and subsequent calcium influx through receptor-gated channels.29~33” The bi~hemi~ changes following postsynaptic calcium influx which lead to synaptic enhancement are not well understood. Both presynapti& and postsynaptic alterations have been proposed to mediate the expression and/or maintenance of LTP. Recent efforts have Focused on the importance of ~lcium-de~ndent protein kinases, particularly protein kinase C (PKC) and the type II cal~um/~lmodulin-de~ndeut protein kinase (CaM KII). Both PKC and CaM ICI1 have been implicated in the regulation of neurotransmitter release,4,d4 and activation of presynaptic PKC by phorbol esters results in synaptic enhancement that resembles L-l-P3’

*To whom correspondence should be addressed. KII, type II calcium/calmodulindependent protein kinase; DEAE. diethvlaminoethvl(&llulose); kGTA, ethylene~y~lbis(ami~~thyleth~)tetra-acetate; EPSP, excitatory postsynaptic potential; H-7, 1-(S-isoquinolinyls~fonyl)-Z-methyl-ui~r~ne; HAlOW, ~-(~-~an~in~th~lj-5-is~u~no~i~~ulfon~ amide; IPSP, inhibitory nostsvnantic notentiai: LTP. long-term potentiation;-M-AP-i m&rot;bule-ass&iatd protein 2; NMDA, N-methyl-D-aspartate; PKC, protein kinase C; SDS, sodium dodecyl sulphate; SDS-PAGE, sodium dodecyl suIpha~~lyac~lamide gel electrophoresis.

Abbreuiu?ioans: CaM

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J. C. LEAHYand M. L. VALI.ANO

(but see Refs 19,40). Additionally, inhibition of PKC and CaM KII by bath application of pharmacological agents %39.45or intracellular injection of kinase-specific synthetic peptides”4,36prevents the induction of LTP and abolishes previously established LTP.14.30,35 Due to the availability of potent and specific activators of PKC, and extensive evidence that PKC-specific phosphorylation of protein Fl (also designated growth associated protein-43, P-57, B-SO, pp46) is increased with LTP, most of these reports have focused on the role of PKC in LTP, The isoquinolinesuifonamides~ which are potent inhibitors of protein kinases, are com~titive with respect to ATP and non-competitive with respect to phosphate acceptor. Hidaka et aL2’ have previously reported that the K, vaIue of I-(5isoquinolinylsulfonyl)-2-methylpiperazine (H-7) for PKC was 6 PM, whereas the K, value of N-(2-guanidinoethyl)-5-isoquinoline sulfonamide (HA1004) for PKC was 40 PM. As a consequence, investigators examining the role of PKC in LTP have used these compounds extensively. However, reports describing the effects of H-7 and HA1004 have been at odds, suggesting that these agents have complex effects. While an initial report indicated that high concentrations (300 PM) of H-7, but not HAl~4, selectively inhibited expression of synaptic LTP,35 a more recent study suggests that H-7 (400 PM) reduces synaptic transmission in genera14’ Furthermore, lower concentrations (lo-100 p M) of H-7 have been shown to “disinhibit” CA1 neurons leading to the appearance of multiple population spikes by reduction of GABA-mediated inhibition.16 Similar concentrations of H-7 also increased the initial slope of the population excitatory postsynaptic potential (EPSP),16 although a small reduction in EPSP slope has also been observed.& In order to clarify the mode of action of these compounds, in the present study, we have examined the effects of different concentrations of H-7 and HA1004 on synaptic and population spike responses in previously potentiated and control pathways in area CA1 of rat hippocampal slices. Moreover, since the effects of these compounds on CaM KII activity had not been examined previously, we determined the apparent K, values of H-7 and HA1004 for CaM KII. A preliminary account of these results has been described.‘7 EXPERIMENTAL

PROCEDURES

~aze~i~is A colony of Sprague-Dawley rats was established with breeders from Taco& Farms (Stanton, NY). offspring obtained from this colony (both male and female) were killed at post-natal days 15-60 and used for electrophysiological recording studies. Adult animals (40-60 days) were used for protein kinase purification studies. H-7 and HA1004 were purchased from Toronto Research Chemicals, Inc. (Downsview, Ontario). A&Gel phenothiazine, Affi-Gel calmodulin and most standard chemicals for gel electrophoresis were obtained from Bio-Rad Laboratories (Richmond, CA). Molecular weight standards for gel filtration, S-Sepharose resin and Sephacryl S-300 HR superfine resin were obtained from Pharmacia LKB Biotechnology, Inc. (Piscataway, NJ).

Histone (Type IIIS from calf thymus) and most standard buffers and biochemicals were- purchased from Sigma Chemical Co. (St Louis, MO). ACS liquid scintillation cocktail was purchased from Amersham Corp. (Arlington Heights, IL). [y-32P]ATP(S-10 Ci/mmol) was obtained from ICN Radiochemicals (Irvine, CA). Methods Preparation ofslices. Sprague-Dawley

rats were killed by decapitation and a single hippocampus was rapidly dissected in chilled (4°C) medium containing (in mM): N&l, 124; KCl, 5; KH,PO,, I .25; MgSO, x 2; CaCI,, 2; NaHCO,, 25; and glucose, IO. Transverse slices (SOOpm thick) were cut with a tissue chopper (Stoelting, Chicago, IL) and maintained in medium oxygenated with 95% 0,/S% CO, at 23°C for at least 1h. Individual slices were placed on a nylon net in a submersion chamber and perfused at 4 ml/min with standard medium (33-34”C, pH 7.4). Drug application was performed by replacement of the standard medium with medium containing either H-7 or HA1004 (50 or 3OOyM in 50 ml). Medium containing drug was oxygenated and reperfused using a peristaltic pump. Extracellular recording procedures. Extracellular recordings were obtained from the CA1 region using 2 M NaCl-filled microelectrodes (2-10 MR) and conventional recording techniques. Microelectrodes were placed in both the stratum radiatum and stratum pyramidale for recording the population EPSP and population spike, respectively. Concentric bipolar stainless steel electrodes (SNE- 100, Rhodes Medical Instruments, Woodland Hills, CA) were positioned in stratum radiatum on either side of the recording electrodes for evoking orthodromic responses. The stimulus intensity at both sites was adjusted to evoke a I-mV population spike (measured from the peak of the spike to the peak of the ensuing positive potential). Responses were amplified, digitized (Neuro-corder DR-484, Neuro Data Instruments, New York. NY) and stored on VCR for later analysis (Axotape 1.2, Axon Instruments, Foster City, CA). Test stimuli (O.l-ms pulses) were delivered to both pathways at a rate of 1120s (10 s internulse interval) for 10 min. Foilowing the test period, a high frkquency stimulus (100 Hz, 1 s train) which reliablv elicited LTP was delivered to one pathway (Sl) while the other pathway (S2) received no stimulation. The intensity of stimulation was increased during the train by doubling the stimulus pulse duration (from 0.1 to 0.2 ms). The effect of the high frequency st~ulation on the initial slope of the EPSP and the population spike amplitude was then monitored for 2Omin employing the initial test stimulus pattern. At this point, the effects of medium containing H-7 or HA1004 were examined for 20min. Washout of drug was obtained by perfusion with standard medium until baseline responses were reestablished in the control (S2) pathway.Kinuse acrivitv assays. Inhibition of CaM KII and PKC activities by HI7 and HA1004 (3-3OOpM) was tested in standard phosphorylation assays. The K, values were estimated from Dixon plots. CaM KII from adult rat forebrain was purified approximately 200-300-fold as previously described.5’ CaM KII activity was assayed in a reaction mixture containing 50mM Tri-HCI (nH 7.4). 1OmM MgCl,, 5 mM CaCi,, 10 fig ~modulin~ I6 pg bovine serum albumin, 5yM [32PJATP (4 x lOsc.p.m.), 3Opg high molecular weight microtub~e-ass~at~ protein-2 (MAP-2) substrate, 20-30 ng enzyme in the presence or absence of H-7: HA1004 in a final volume of 100 ~1. In a separate series of experiments designed to test the competitive or non-competitive nature of kinase inhibition by drug, the concentration of ATP or MAP-2 was varied. The calmodulin-dependence of the reaction was assessed in the presence or absence of 0.1 mM trifluoperazine as nreviouslv described.” The phosphorylation reaction was initiated by addition of CaCi, and placement of sample tubes in a 30°C shaking water bath. The reaction was terminated after 30 s by addition of 50 pl

Hippocampal LTP and protein kinase inhibitors PKC

0

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Inhibitory

200 300 Concentration

400 (1.4)

363

of the pre-tetanic, post-tetanic and drug (H-7/HAl004) treatment periods. Values depicted in the text are means f standard error of the mean (S.E.M.) and have been analyzed for their statistical significance using a paired difference t-test,‘9 when applicable. Otherwise, comparisons were obtained using an unpaired r-test. Otherprocedures.A heat-stable fraction enriched in MAP-2 was prepared from thrice-cycled microtubules obtained from cow brain as described by Vallee.53Calmodulin was purified from calf brain by chromatography on DEAE cellulose and At&Gel phenothiazine as described by Marshak et a/.‘* Protein was determined by the method of Bradford” using bovine serum albumin as a standard. RESULTS

HA1004

Biochemistry of kinase inhibitors

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The inhibitory

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Fig. 1. Effects of H-7 and HA1004 on PKC activity as a function of drug concentration. PKC from rat forebrain was partially purified and assayed as described under Experimental Procedures in the presence of the following micromolar concentrations of H-7 (A) or HA1004 (B): 0,3,6, 12, 2550, 100,300. The data were transformed to a Dixon plot in order to estimate the K, values (inset). All gels were run in duplicate and the means f S.E.M. of at least three separate experiments are shown.

NSC&W--E

CaM KII

A =

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of a “stop” solution containing 62.5 mM Tri-HCl, 10% glycerol (v/v), 2% sodium dodecyl sulphate (SDS), 2.5% )?I-mercaptoethanol (v/v) and a trace amount of pyronine Y. Samples were placed in a boiling water bath for 2 min. PKC was partially purified from adult rat forebrain through the diethylaminoethyl(cellulose) (DEAE) chromatography step as previously described.2s PKC activity was assayed in a reaction mixture containing 50 mM Tri-HCl (PH 7.0), 10 mM MgCl,, 0.5 mM CaCl, or 1 mM EGTA, 10 pg phosphatidylserine, 5 pM [‘*P]ATP (4 x lO’c.p.m.), 1OOpg histone substrate, and 3-5 pg enzyme in the presence or absence of H-7/HA1004 in a final volume of 100 ~1. The reaction was initiated by addition of ATP and placement of the sample tube in a 30°C shaking water bath. The reaction was terminated after 5 min by addition of 50 ~1 of stop solution, and samples were boiled for 2 min. Gel electrophoresis. Proteins were resolved on onedimensional sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using 7% (CaM KII) or 12% (PKC) resolving gels and 4 or 5% stacking gels, respectively.X After electrophoresis for l-3 h at constant current (50 mA/ gel), gels were stained with Coomassie Blue reagent, destained and dried on filter paper, followed by autoradiography on Kodak XAR film. The specific activities were assessed by excision of MAP-2 or histone substrate from the gel, solubilization of the slice in 300~1 hydrogen peroxide for IO-12 h at WC, and scintillation spectroscopy in 5 ml of ACS scintillant. All gels were run in duplicate and average values were used to calculate kinaae-specific activities. Data anufysb. Electrophysiological response measurements (EPSP slope/population spike amplitude) were obtained at a tixed interval (one measurement/min) and mean values were calculated from measurements during the last 10 min

potencies

of H-7 versus HA1004 for

PKC and CaM KII activities were tested in standard phosphorylation assays. H-7 was more effective than HA1004 in inhibiting PKC activity (Fig. lA, B). Dixon plots were used to estimate the Ki values. H-7 had a Ki value of 9 p M and HA 1004 had a Ki value of 57 PM. Using an unpaired t-test, these values were significantly different (P < 0.05). Figure 2A and B show that the converse relationship was observed with respect to inhibition of CaM KII (Fig. 2A, B). H-7 had a K, value of 156 GM, whereas HA1004 had a K,

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Fig. 2. Effects of H-7 and HA1004 on CaM KII activity as a function of drug concentration. CaM KII from rat forebrain was purified and assayed as described under Experimental Procedures in the presence of the following micromolar concentrations of H-7 (A) or HA1004 (B): 0,3, 6, 12, 25, 50, 100, 300. The data were transformed to a Dixon plot in order to estimate the Kj values (inset). All gels were run in duplicate and the means f S.E.M. of at least three separate experiments are shown.

364

J. C. LEAHYand M. L. VALLANO

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Fig. 3. Effects of SO/300PM HA1004 or 50 PM H-7 on population spike amplitude. (A) Time-course of population spike amplitude measurements (left) and representative responses during the pre-tetanic (a), post-tetanic (b) and drug (c) periods. The responses depicted in the upper traces of Sl and S2 (right) are from a slice exposed to 50 PM H-7, whereas those depicted in the lower Sl and S2 traces are from a slice exposed to 300 PM HA1004 Each point on the graph (left) represents the average amplitude measurement from five experiments. S.E.Ms are indicated at 5-min intervals. (B) Same as above, except slices were

exposed to 50 PM HA1004 (c) and responses recorded during the washout (d) period are indicated. The asterisk (c, right) indicates a small second response evoked in the presence of HA1004 (50 pM). Each point on the graph represents the average amplitude measurement from four experiments.

value of 13 PM. These values were also significantly different (P < 0.001). Increasing the concentration of ATP used in the phosphorylation assay by IO-fold partially overcame the inhibition of kinase activity (not shown). Increasing the concentration of MAP-2 substrate in the CaM KII phosphorylation reaction was without effect. Studies examining the effect of 300 PM H-7 on purified Na + /I(+ ATPase from dog kidney showed no inhibition of activity (not shown).

Effects of kinase inhibitors: population spike

The effects of H-7/HA1004 were examined in 21 slices exhibiting potentiation of the population spike (> 20%). High frequency stimulation of one of two separate inputs resulted in a selective potentiation of the population spike amplitude in that pathway (219 +_8% vs 105 + 3%; n = 21, P
of 50 PM H-7 20 min after the induction of LTP produced a significant increase in the amplitude of the population spike in the control (S2) pathway [+52 f 19% (163% vs 111%); II = 6, P < 0.051, and only a slight increase of the spike amplitude in the previously potentiated (Sl, LTP) pathway [ + 8 + 24% (222% vs 214%); n = 6, P > 0.051. Additionally, 50 FM H-7 produced multiple population spikes (six of six slices; see Fig. 3A, right). Comparison of the effects of H-7 on the initial population spike amplitude, between the control (S2) and previously potentiated (Sl) pathways, confirmed that 50 PM H-7 affected the former to a greater degree [H-7 means were expressed as a percentage of the post-tetanic (pre-drug) means; +44 + 7%, P < 0.011. The effects of H-7 developed within 10 min, persisted throughout the drug period (Fig. 3A), and were typically washed out after 45-75 min (not shown). To determine whether the effects of H-7 were mediated by inhibition of PKC, we tested the effects

Hippocampai LTP and protein kinase inhibitors

365

stope Slope

0

Tfme

(Minuted

H-7 on the population spike amplitude and EPSP slope. (A) Same as in Fig. 3 except slices were exposed to 300 FM H-7. Each point represents the average of four experiments. (B) Same as in A except the time-course and responses depicted are population EF’SPs. The responses shown in A and B are from the same slice. Fig. 4. Effects of 300 pM

of the less potent PKC inhibitor, HA1004. Like H-7, SOpM HA1004 selectively increased the amplitude

of the population spike in the control pathway f+sg* 13% (173% vs 115%); n -4, P O.OS].However, uniike H-7, 50 PM HA1004 did not produce multiple population spikes (eliciting one additional spike in only one of four slices). Examples of these effects are shown in Fig. 3B. The effects of 50 PM HA1004 developed within 10 min and persisted throughout the drug period, similar to H-7, but were typically washed out within 20-4Omin (Fig, 3B). Although 50pM H-7 enhanced the population spike amplitude, higher concentrations (300 p M) were previously reported to selectively inhibit synaptic LTF?’ In the present series of experiments, 300 PM H-7 substantialiy reduced the amplitude of the population spike in the previously potentiated pathway in eight of 10 slices [-78 + 12% (139% vs 217%); P c O.OOl],but a significant reduction in spike amplitude was also observed in the control pathway [-38 rt 10% (60% vs 98%); n s 8, P < 0,011. Although &e amplitude of the initial population spike was reduced by 300 ~1M H-7, multiple spike activity was still ob-

served (as noted for 50 PM H-7) in both potent&ted and control pathways. Examples of these effects are shown in Fig. 4A. In two of the 10 slices, 3OOgM H-7 increased the control spike amplitude (+ 95%, + 127%) and produced either no change or a reduction (- 81%) in the previously potentiated pathway (not shown), The effects of H-7 and HA1004 on the population spike amplitudes are summarized in Fig. SD. The effects of 300 PM HA1004 on the population spike amplitude were examined in three slices. In Che previously potentiated pathway, 300 PM HA1004 produced an increase in the population spike amplitude in two slices (+41%, +32%) and a decrease in one slice (-60%). HA1004 (300 FM) affected the control pathways in a similar manner and aiso produced multiple ~pu~a~on spikes in both pathways in the two slices exhibiting HAIL-mediated potenCation. An example of this effect is shown in Fig. 3A (lower Sl and S2 traces, right).

Eflects of kinme inhibitors: population excitatory post sJ’mptic potential The effects of H-7/HA1004 were examined in 14 slices exbibiting >20% potentiation of the initial

366

J.C. LEAHYand M. L. VALLANO

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Fig. 5. Effects of 50/~M H-7 and HA1004 on the population EPSP slope and spike amplitude. (A) Representative EPSP responses during the (a) pre-tetanic, (b) post-tetanic and (c) 50 #M H-7 periods. (B) Same as in A except responses in c were evoked in the presence of 50/~M HAl004. (C) Effects of H-7 and HA1004 on EPSP slope. Error bars are +S.E.M.; numbers in parentheses indicate the number of slices from which slope measurements were obtained. (D) Same as in C, except effects of H-7 and HA1004 on population spike amplitude are depicted.

slope of the population EPSP. (Population spike amplitude measurements were also obtained in 10/14 of these slices.) High frequency stimulation delivered to one pathway (S1) resulted in a selective potentiation of the EPSP slope, relative to the control ($2) pathway (175 + 8% vs 96 _+ 3%; n = 14, P < 0.001). Contrary to its effects on the population spike amplitude, 50 ~ M H-7 significantly reduced the EPSP slope in both the previously potentiated pathway [ - 3 1 + 4 % (131% vs 162%); P < 0 . 0 1 ] and the control pathway [-16-1-3% (80% vs 96%); P <0.01] in four slices (Fig. 5A). 5 0 # M HA1004 also reduced the EPSP slope in both pathways; however, this decrease was not significant [S1, - 16 _+ 5% (173% vs 189%) and $2, - 6 _+ 7% (104% vs 110%); n = 3, P > 0.05] (Fig. 5B). A summary of the effects of H-7 and HA1004 on EPSP slope is shown in Fig. 5C. Addition of higher concentrations (300 #M) of H-7 produced a reduction of the initial slope of the EPSP in five of five slices in the LTP pathway [ - 4 4 _+ 8% (134% vs 178%); P < 0.01] and in four of five slices in the LTP pathway [ - 4 2 + 8 % (54% vs 96%); n = 4, P < 0.02]. Examples of these effects are shown in Fig. 4B. HA1004 (300/IM) produced only a slight reduction in the initial slope of the EPSP ( - 5 . 5 % , n = 2) in both the LTP and control pathways.

DISCUSSION

The mechanism of induction of some forms of LTP has been well characterized and involves the activation of NMDA receptors m2° and subsequent influx of Ca 2÷ into the postsynaptic neuron. 29'33'44 The cascade of events following Ca 2÷ influx which underlie synaptic enhancement are not well understood. Highly suggestive evidence obtained from several laboratories indicates that activation of two calcium-dependent protein kinases, PKC and CaM KII, that are concentrated at the synapse mediate the effects of Ca 2÷ on LTP (for review see Refs 24, 42). However, the precise mode of action of PKC and CaM KII in LTP remains to be elucidated and data from different laboratories are somewhat controversial. The majority of these studies investigated the role of PKC in LTP by employing PKC activators, primarily of the phorbol ester type, and PKC inhibitors, primarily the isoquinolinesulfonamides. For example, Routtenberg and associates have provided extensive evidence that translocation of PKC and phosphorylation of protein F1 are correlated with LTP. 3:3'28Consistent with this hypothesis, Malenka e t al. 31 and Colley e t al. 13 observed a long-lasting synaptic enhancement in response to phorbol esters. Malenka e t al. further

Nippocampal LTP and protein kinase inhibitors

demonstrate that phorbol esters enhanced presynaptic glutamate release32 and occluded subsequent induction of LTP.3’ In addition, high concentrations of H-7 (but not HA1004) selectively inhibited established LTP.‘4*35However, Muller et u/.40*41 failed to observe significant inhibition with 100 FM H-7, while observing a non-selective inhibition of both LTP and control pathways with 400 PM H-7, suggesting that PKC is not involved in the persistence of LTF’ (but see Ref. 7). Several factors contribute to the disparate results reported by different laboratories that have employed bath appli~tion of pha~acolo~~l agents to assess the roles of these kinases in LTP: (i) there are no highly specific activators or inhibitors of CaM KII; (ii) the phorbol esters are specific activators of PKC but not truly representative of the physiological mode of activation in that their effects are persistent and not transient; (iii) PKC and CaM KII are localized both presynaptically and postsynaptically thereby making it difficult to selectively affect only one of these compartments; (iv) at least three, and possibly seven, distinct isoforms of PKC with distinct subcellular localizations are present in hippocampus and are differentially affected by PKC activators and inhibitors.2Z43 As a consequence, it is not surprising that induction and expression of LTP or abolition of established LTP has not been satisfactorily reproduced in experiments utilizing these compounds. Undoubtedly, their effects are more widespread than those produced by a brief high frequency stimulation of a specific afferent pathway. Nevertheless, these compounds can be extremely useful in investigating the role of protein kinases in synaptic neurotransmission if attempts to unravel their complex effects on different neuronal populations are made.

367

responses that are sibilantly more sensitive to the inhibitory effects of H-7 relative to HA1004 can be ascribed to inhibition of PKC, whereas the converse relationship would support inhibition of CaM KII. Eflects

of

kinase inhibitors: population spike

At relatively low concentrations (50 PM), both H-7 and HA1004 significantly enhanced the amplitude of the population spike in the control pathway and produced either no change or a small increase in the LTP pathway. Moreover, H-7, but not HA1004, also elicited multiple spike activity. The observed timecourse and magnitude of the effects of H-7 on control responses are similar to those reported by Corradetti et al.,16with 30 PM (10-100 FM) H-7 {see also Ref. 2). In agreement with the observations in the present study, Corradetti et al.16also failed to observe multiple population spikes in the presence of 50 PM HA1004. However, they did not observe potentiation of the population spike amplitude by HA1004 This may have been due to their use of higher stimulus intensities (not specified) for HA1004treated slices (5 mV control spike amplitude, Fig. 2)16relative to the H-7-treated slices (~2 mV, Fig. l).j6 The latter were potentiated to 5 mV in the presence of 30 PM H-7. There are several lines of evidence indicating that the augmentation of spike responses observed with 50 PM H-7 is primarily due to a reduction of GABAmediated inhibition. Firstly, the enhancement of the population spike amplitude and production of multiple spikes by 50pM H-7 mimics the effects of GABA antagonists.4,47 Secondly, H-7 and the GABA antagonist bicuculline depress miniature inhibitory postsynaptic currents,‘6 an effect opposite of phorbol esters which increase the frequency of spontaneous inhibitory postsynaptic potentials (IPSPS).~’ Thirdly, Corradetti ef aLi demonstrated that H-7 abolished B~oche~jstry of kinuse ~hib~tors antidromi~lly evoked, GABA-mediated inhibitory In the present report, the combination of H-7 and postsynaptic currents in CA1 pyramidal neurons, but HA1004, which differ by seven-fold in their k; values had no effect on the inhibitory currents evoked by bath application of GABA. The latter observations for PKC, but are roughly equipotent against cGMPsuggest that H-7 acts presynaptically on inhibitory dependent protein kinase, CAMP-dependent protein kinase, and myosin light chain kinase,” were used to interneurons to reduce GABA release, leading to assess their relative effects on evoked responses in disinhibition of CA1 neurons. Additionally, it should area CA1 in rat hippocampus. In addition, since the be noted that the lack of any further enhancement of inhibitory potencies of these compounds for CaM KII the population spike amplitude in the previously were not previously examined, they were determined potentiated pathway with H-7 and HA1004 treatment from Dixon plots. Interestingly, the K; values were (50 PM) suggests that disinhibition may contribute to 13 p M for HA 1004 and 156 p M for H-7, indicating potentiation (LTP) of spike responses. Such a conthat HA1004 is a more potent CaM KII inhibitor. clusion is consistent with previous reports demonstratNext, the relative inhibitory potencies of these com- ing that the GABA antagonist, picrotoxin, prevents pounds for PKC were determined and Ki values of 9 expression of a component of spike potentiation that and 57 PM were obtained for H-7 and HA1004, can not be accounted for by potentiation of the EPSP respectively. These values are in good agreement with (i.e. the E-S component).‘,” previously reported values of 6 and 40 PM for H-7 Two observations in particular suggest that H-7 and HA1004, respectively.2’ The observation that H-7 and HA1004 mediate their effects by inhibiting PKC is more effective in inhibiting PKC than CaM KII is rather than CaM KII. Firstly, H-7 disinhibited CA1 also consistent with previous reports’7,“s5a (but see neurons at the lower concentration (50 p M), whereas Ref. 36). Thus, a specific prediction in interpretation HA1004, which was less effective than H-7 in inhibitof the experimental data is that electrophysiological ing PKC, only produced multiple responses at

J. C. LEAHYand M. L. VALLANO

36X

300 PM. Secondly, HA1004 (SOPM), which was more effective than H-7 in inhibiting CaM KII, produced potentiation of the control spike amplitude, whereas high concentrations (300 p M) of H-7 did not produce potentiation but rather resulted in a reduction of the population spike amplitude (while still exhibiting multiple spikes). Thus, H-7 preferentially affected the CA1 responses compared to HA1004, consistent with their effects being mediated by inhibition of PKC. Effects ofkinase

inhibitors:poputation

excitatorypost-

synaptic potential

Contrary to the effects of H-7 and HA1004 on the population spike amplitude, these compounds reduced the initial slope of the population EPSP in both pathways, at 50 and 300,~M concentrations. However, only the effects observed in the presence of H-7 were signifi~nt. Previous reports examining the effects of H-7 on EPSP slope have been inconsistent. For example, Corradetti et aLI observed an enhancement of the EPSP slope with 30pM H-7. In contrast, Muller et aL4” observed both a modest, but nonsignificant decrease in the EPSP slope and subsequent positive potentials reflecting multiple spike discharges, with 100 ,uM H-7. The reason for the discrepancies between the effects of 30 p M H-716 on the EPSP slope and those with 10040and 50 FM (present study) H-7 are unclear. The results of the present study suggest that H-7mediated PKC inhibition reduces both excitatory and inhibitory synaptic transmission. Such a conclusion is supported by a recent report demonstrating that phorbol esters (which activate PKC) increase the frequency of both spontaneously occurring EPSPs and IPSPs.” It is likely that the reduction of the EPSP slope observed in the present study with low (50 pM) and high (300 PM) concentrations of H-7 is due to a reduction of excitatory synaptic transmission. The demonstration that the initial slope of the population EPSP is unaffected by application of picrotoxin,54 supports this idea. A reduction of inhibitory inputs

would not be expected to substantially affect early components of synaptic potentials in CA1 neurons since both feed-forward’ and recurrent6 inhibitory inputs are minimally disynaptic. However, Alger and Nicoll’ have demonstrated that feed-forward inhibitory circuits exert a powerful control over celi firing in CA1 neurons, a finding which further supports a role for H-7-mediated disinhibition in the production of multiple spike discharges (above). The differential effects on the amplitude of initial population spikes observed with 50 ktM H-7 (increase) and 300 FM H-7 (decrease) suggest that effects of this compound on excitatory and inhibitory pathways may be concentration dependent. Since there was a tendency for a greater reduction of the EPSP slope at 300 PM versus 50 PM (Fig. 5) addition of low concentrations of H-7 may preferentially reduce inhibitory synaptic transmission, whereas further reduction of excitatory transmission would require higher concentrations. The observed reduction of synaptic and initial spike responses in the presence of 300hM H-7 in both control and previously potentiated pathways, in the present study, suggests that H-7 inhibits synaptic transmission in general?’ rather than specifically inhibiting established LTP.35 These results are consistent with the hypothesis that PKC may not be necessary for the expression of LTP.4’ However, recent reports demonstrating that production of LTP is blocked by postsynaptic injection of H-7 or synthetic peptide inhibitors of PKC/CaM KI134*36 suggest that PKC and/or CaM KII are involved in the induction of LTP.

Acknowledgements-We would like to thank Mr James Gorecki for excellent technical assistance and helpful suggestions in the biochemical studies on CaM KII and PKC. We also thank the laboratory of Dr Joseph D. Robinson for testing the effect of H-7 on the Na+/K’ ATPase. This research was supported by Public Health Service Grant NS 24705.

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