Neurons in the brain receive precise, continuously updatedinformation from other neurons via thousands of specializedsynaptic contacts on their somata and dendrites. At anygiven time, only a subset of these synaptic contacts will beactive, i.e. releasing neurotransmitter. Regulation of theprobability of transmitter release is a major mechanism bywhich the brain processes incoming information andinternally generated messages. Release probability variesaccording to the history of activation of the synapse as aresult of the time course of the intracellular cascadesinvolved in the release process (Zucker, 1989). In addition,however, release probability can be varied by neuro-transmitters or modulators which bind to receptors locatedin the presynaptic membrane (Thompson et al. 1993; Wu &Saggau, 1997).

The principal neurons in the dentate gyrus region of thehippocampus are the granule cells, which receive theirprimary excitatory afferent input from layer II stellate cellsof the entorhinal cortex, containing highly processed

multimodal information (Amaral & Witter, 1989). Thestellate cell axons form the so-called perforant path input tothe dentate gyrus, synapsing on the outer two-thirds of thegranule cell dendritic tree. A number of different neuro-transmittersÏmodulators act presynaptically on thesesynapses, thereby altering the subset of synapses whichrelease transmitter. Glutamate, the main transmitter releasedat these synapses, itself acts upon at least two types ofpresynaptic receptors (Brown & Reymann, 1995; Macek et

al. 1996). Upon high-frequency stimulation, the peptide,dynorphin is released from these synapses, depressingglutamate release via ê-opioid receptors (Wagner et al. 1993).The purine adenosine, which is released locally from neuronsin a manner dependent on metabolic activity, depressestransmission via A1 receptors (Dolphin & Archer, 1983).Finally, these synapses are controlled by transmitters whichare released in a global manner in the brain and whichprovide effectively the ‘context’ in which the afferentinformation should be processed, e.g. the state of arousal or

Journal of Physiology (1999), 515.3, pp.777—786 777

On the mechanism of histaminergic inhibition of glutamate

release in the rat dentate gyrus

Ritchie E. Brown and Helmut L. Haas

Institut f�ur Neurophysiologie, Heinrich-Heine-Universit�at, D_40001 D�usseldorf, Germany

(Received 27 July 1998; accepted after revision 15 December 1998)

1. Histaminergic depression of excitatory synaptic transmission in the rat dentate gyrus wasinvestigated using extracellular and whole-cell patch-clamp recording techniques in vitro.

2. Application of histamine (10 ìÒ, 5 min) depressed synaptic transmission in the dentategyrus for 1 h. This depression was blocked by the selective antagonist of histamine H×receptors, thioperamide (10 ìÒ).

3. The magnitude of the depression caused by histamine was inversely related to theextracellular Ca¥ concentration. Application of the N_type calcium channel blockerù_conotoxin (0·5 or 1 ìÒ) or the PÏQ_type calcium channel blocker ù_agatoxin (800 nÒ) didnot prevent depression of synaptic transmission by histamine.

4. The potassium channel blocker 4-aminopyridine (4_AP, 100 ìÒ) enhanced synaptictransmission and reduced the depressant effect of histamine (10 ìÒ). 4_AP reduced theeffect of histamine more in 2 mÒ extracellular calcium than in 4 mÒ extracellular calcium.

5. Histamine (10 ìÒ) did not affect the amplitude of miniature excitatory postsynapticcurrents (mEPSCs) and had only a small effect on their frequency.

6. Histaminergic depression was not blocked by an inhibitor of serineÏthreonine proteinkinases, H7 (100 ìÒ), or by an inhibitor of tyrosine kinases, Lavendustin A (10 ìÒ).

7. Application of adenosine (20 ìÒ) or the adenosine A1 agonist N É-cyclopentyladenosine (CPA,0·3 ìÒ) completely occluded the effect of histamine (10 ìÒ).

8. We conclude that histamine, acting on histamine H× receptors, inhibits glutamate release byinhibiting presynaptic calcium entry, via a direct G-protein-mediated inhibition of multiplecalcium channels. Histamine H× receptors and adenosine A1 receptors act upon a commonfinal effector to cause presynaptic inhibition.

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Keywords:

motivation. One example of these transmitters is the biogenicamine histamine, which is released from varicosities ofaxons originating in the tuberomammillary nucleus of thehypothalamus.

Three receptors for histamine have been identified. Thehistamine H1 and Hµ receptors are postsynaptically locatedand are coupled to phospholipase C and to stimulation ofadenylyl cyclase, respectively (Schwartz et al. 1991). Thehistamine H× receptor, which was first described in 1983(Arrang et al. 1983) as an autoreceptor regulating histaminerelease, has subsequently been shown to inhibit the releaseof other transmittersÏmodulators (Schwartz et al. 1991). Inthe dentate gyrus, histamine H× receptors are located onperforant path terminals and inhibit the release ofglutamate (Brown & Reymann, 1996). The mechanism bywhich H× receptors inhibit transmitter release, however,remains unclear.

Two main mechanisms for inhibition of transmitter releasehave been proposed (Thompson et al. 1993; Wu & Saggau,1997). Firstly, they may inhibit calcium influx during thepresynaptic action potential, either by blocking presynapticcalcium channels, or by enhancing potassium currents.Secondly, they may act downstream of the calcium influx onthe proteins of the transmitter release machinery. Themechanism of presynaptic inhibition can have importantfunctional consequences. For instance, G-protein-mediatedinhibition of voltage-gated calcium channels is relieved bydepolarization (Dolphin, 1998) and will thus be less during atrain of high-frequency action potentials than during low-frequency stimulation. In this study we present evidencethat histamine, acting via histamine H× receptors depressesglutamate release via the first mechanism. Furthermore, weinvestigate the signal transduction pathway involved in thiseffect and compare the actions of adenosine and histamine.Preliminary reports of this work have been presented(Brown & Haas, 1997, 1998).

METHODS

Hippocampal slices were prepared from 3- to 5-week-old maleWistar rats. All experiments were conducted in compliance withGerman law and with the approval of the BezirksregierungD�usseldorf. The animals were quickly decapitated and the brainquickly transferred to a modified artificial cerebrospinal fluid(ACSF), in which all NaCl had been replaced by isomolar sucrose(Aghajanian & Rasmussen, 1989). Horizontal, 400 ìm thick wholebrain slices were cut using a vibroslicer (Campden Instruments,Loughborough, UK). Subsequently, the hippocampi were isolatedunder a dissecting microscope before being transferred to aprechamber with standard ACSF containing (mÒ): 124 NaCl, 2·8KCl, 1·2 KHµPOÚ, 4 MgSOÚ, 4 CaClµ, 25·6 NaHCO×, 10 ª_glucose,0·05 picrotoxin. In some experiments the CaClµ concentration wasreduced to 2 or 1 mÒ. Slices remained in a holding chamber at roomtemperature (18—22°C) until use, when they were transferredindividually to a recording chamber and perfused with ACSF at aflow rate of 2·5 ml min¢. Extracellular recordings were made at28°C, and recordings of miniature excitatory postsynapticcurrents (mEPSCs) were made at room temperature or at 28°C.

Constant voltage bipolar pulses (1—20 V, 0·2 ms duration) wereapplied every 30 s through a monopolar, lacquer-coated, platinumor NiÏCr electrode placed in the middle third of the stratummoleculare of the dentate gyrus to stimulate the medial perforantpath input. For recording of field excitatory postsynaptic potentials(fEPSPs) a glass electrode filled with ACSF was inserted into themolecular layer at the same level as the stimulation electrode. Theinitial slope of the fEPSP was used as the measure of this potential.The medial perforant path was distinguished according to thekinetics of the synaptic potentials and the presence of paired-pulsedepression (Abraham & McNaughton, 1984).

Intracellular recordings were made from dentate granule cells usingthe ‘blind’ whole-cell patch-clamp technique (Staley et al. 1992).Intracellular signals were recorded using an Axoclamp-2B amplifier(Axon Instruments) in the continuous single-electrode voltage-clamp mode. Patch pipettes (3—6 MÙ) were normally filled with anintracellular solution containing (mÒ): 135 caesium methane-sulphonate, 6 MgClµ, 10 Hepes, 10 EGTA, 1 CaClµ, 2 NaµATP(pH 7·25 with CsOH). In some experiments the patch solutioncontained (mÒ): 135 CsCl, 2 MgClµ, 0·2 EGTA, 5 NaCl, 2 NaµATP.Results were similar with the two solutions and have been pooled.mEPSCs were recorded at a holding potential of −75 mV. Accessresistance was calculated from the instantaneous current measuredat the beginning of a 5 mV, 100 ms hyperpolarizing step from theholding potential and input resistance from the steady-statecurrent remaining at the end of the step. Neurons accepted for thisstudy had resting membrane potentials of −71·7 ± 0·5 mV(measured immediately after obtaining access to the whole-cell,uncorrected for liquid junction potentials); access resistances were22·2 ± 1·4 MÙ and input resistances 213 ± 13 MÙ (n = 15).Experiments in which the access or input resistance changed bymore than 15% during the course of the experiment werediscarded. mEPSCs were analysed using the N event-analysissoftware kindly provided by Dr S. Traynelis (Emory University,Atlanta, GA, USA). mEPSCs (negative deflections with a fast risingphase and exponential decay) were detected by eye; a marker waspositioned at the start of the rising phase and their peak amplitude(average of 2 most negative points) measured relative to a baselineperiod (mean of 30 points) immediately preceding them. Eventswere accepted if they exceeded a 3 pA threshold value and weresignificantly more negative than the baseline period. Amplitudedistributions were compared with the Kolmogorov—Smirnoff test.

Drugs used in this study were: 4-aminopyridine (Fluka, Buchs,Switzerland), adenosine (Merck, Darmstadt, Germany), CdClµ(Fluka), H7 (Alexis Biochemicals, Gr�unberg, Germany), histaminedihydrochloride (Sigma), Lavendustin A (Alexis Biochemicals), N É-cyclopentyladenosine (CPA, Research Biochemicals International),nifedipine (Sigma), ù_agatoxin (Alomone Labs, Jerusalem, Israel),ù_conotoxin GVIA (Sigma), picrotoxin (Sigma), tetrodotoxin(Sigma), thioperamide maleate (Tocris Cookson, Bristol, UK).Drugs were dissolved in distilled water (except nifedipine,Lavendustin A — dissolved in DMSO), titrated to pH 7·4 usingNaOH and stored as stock solutions. Drugs were bath applied. Allvalues are given as the mean ± s.e.m. Statistical comparisons weremade using Student’s unpaired or paired t test, as appropriate.

RESULTS

Bath application of histamine (10 ìÒ) for 5 min under controlconditions (4 mÒ extracellular calciumÏmagnesium, 50 ìÒpicrotoxin) led to a rapid depression of fEPSPs recordedfrom the medial perforant pathway of the dentate gyrus; it

R. E. Brown and H. L. Haas J. Physiol. 515.3778

required up to 1 h for complete washout (Fig. 1). A repeatapplication of histamine, 1 h following the first, led to asimilar-sized depression as seen following the first application(n = 3), i.e. there was no homologous desensitization of thisresponse. Application of the selective H× receptor antagonistthioperamide (10 ìÒ, n = 4) completely blocked thedepression caused by histamine (10 ìÒ).

The magnitude of the depression of synaptic transmissionby histamine was dependent on the extracellular calciumconcentration. In the presence of 4 mÒ extracellular calcium,the depression measured 10 min after starting histamineapplication was to 77·9 ± 2·0% of baseline (n = 10) whereasin the presence of 2 and 1 mÒ calcium the depression was to59·6 ± 2·6% (n = 7) and 56·5 ± 3·5% (n = 6) of baseline,respectively.

Multiple presynaptic calcium channels, including theN_type, Q_type and a so far poorly characterized subtype,control the release of glutamate in the hippocampus (Dunlapet al. 1995; Wu & Saggau, 1997). In our experiments, abrief application of the selective N_type calcium channelblocker ù_conotoxin GVIA (0·5 ìÒ, n = 4) depressed thefEPSP to a new stable level around 60% of baseline.Subsequent application of histamine (10 ìÒ) 30 minfollowing conotoxin administration depressed the fEPSPfurther, similar to control conditions. A higher concentrationof conotoxin (1 ìÒ, n = 2) did not depress the fEPSP to agreater extent than the lower concentration and also did notblock the histamine effect, so the results have been pooled(Fig. 2). Taking the period immediately preceding histamineapplication as 100%, histamine depressed the fEPSP to75·1 ± 4·7% in the presence of conotoxin (n = 6) ascompared with 77·9 ± 2·0% in control (n = 10).

Application of a high concentration of the selective PÏQ_typecalcium channel blocker ù_agatoxin IVA (800 nÒ, n = 4)

also depressed the fEPSP to around 50% of baseline(Fig. 3). Application of histamine (10 ìÒ) 30 min followingagatoxin application still depressed synaptic transmissionand in fact the magnitude of the depression under theseconditions was slightly enhanced (P = 0·053). Taking theperiod immediately preceding histamine application as100%, histamine depressed the fEPSP to 69·7 ± 3·6% inthe presence of agatoxin (n = 4) as compared with77·9 ± 2·0% in control (n = 10).

Application of the L-type calcium channel blocker nifedipine(10 ìÒ, n = 2) did not affect synaptic transmission and didnot reduce the histamine-mediated depression of synaptictransmission.

The compound 4-aminopyridine (4_AP) blocks a number ofdifferent types of voltage-dependent potassium channels,including inwardly rectifying channels of the type activatedby pertussis toxin-sensitive G-proteins. Bath application of4_AP (100 ìÒ), led, as expected (Buckle & Haas, 1982), to abroadening of the presynaptic fibre volley and a substantialenhancement in the size of fEPSPs to 300 ± 25% ofbaseline (n = 7). Application of histamine (10 ìÒ) 45 minfollowing the addition of 4_AP, depressed the fEPSP to262 ± 23% of baseline. Taking the points immediatelypreceding histamine application as 100%, histaminedepressed the fEPSP to 87·8 ± 1·6% of baseline (n = 7,Fig. 4B), as compared with 77·9 ± 2·0% (n = 10) in control,i.e. 4_AP reduced the histaminergic depression by 45%.This reduction of the histaminergic depression by 4_APcould indicate that histamine is acting through voltage-dependent potassium channels blocked incompletely by4_AP. Alternatively, the reduction could arise due to theincreased influx of calcium through the different types ofpresynaptic calcium channels in the presence of 4_AP(Wheeler et al. 1996). To try and distinguish between these

Presynaptic inhibition by histamineJ. Physiol. 515.3 779

Figure 1. The extent of the depression of synaptic

transmission by histamine is dependent on the the external

calcium concentration

A,mean data showing the time course of depression of the fEPSPwith 4 mÒ (n = 10), 2 mÒ (n = 7) or 1 mÒ (n = 6) extracellularcalcium. B, analog traces from an experiment in 2 mÒ externalcalcium before and after histamine application.

R. E. Brown and H. L. Haas J. Physiol. 515.3780

Figure 2. The depressant effect of histamine is not

occluded by the N_type calcium channel blocker

ù_conotoxin

A, a short application of ù_conotoxin (0·5 ìÒ, n = 4 or1 ìÒ, n = 2) depresses the fEPSP but does not prevent afurther depression by histamine (10 ìÒ). Inset, analogtraces recorded at the times indicated by the lower caseletters. Scale bar, 0·5 mV, 10 ms. B, comparison of thedepression of the fEPSP under control conditions (n = 10)or in the presence of ù_conotoxin (n = 6).

Figure 3. The depressant effect of histamine is not

occluded by the PÏQ_type calcium channel blocker

ù_agatoxin

A, a short application of ù_agatoxin (800 nÒ, n = 4)depresses the fEPSP but does not prevent a furtherdepression by histamine (10 ìÒ). Inset, analog tracesrecorded at the times indicated by the lower case letters.Scale bar, 0·5 mV, 10 ms. B, comparison of the depression ofthe fEPSP under control conditions (n = 10) or in thepresence of ù_agatoxin (n = 4).

two possibilities we performed the same experiment in thepresence of 2 mÒ extracellular calcium. If histamine isacting by enhancing voltage-dependent potassium channelsthen one would expect a similar degree of inhibition of theeffect by 4_AP under the two conditions. In 2 mÒextracellular calcium 4_AP potentiated the fEPSP to330 ± 33% of control and application of histamine 45 minlater in the continued presence of 4_AP reduced the fEPSPto 298 ± 29% of baseline (n = 6, Fig. 4A). Taking thepoints immediately preceding histamine application as100%, histamine depressed the fEPSP to 88·5 ± 3·0% ofbaseline (n = 6, Fig. 4B), as compared with 59·6 ± 2·6%(n = 7) in control, i.e. 4_AP reduced the histaminergicdepression by 72%, suggesting that enhancement ofpresynaptic potassium channels is not responsible forhistaminergic presynaptic inhibition. (This large differencebetween the effect of 4_AP in 4 and 2 mÒ calcium remains,even if one corrects for the slight increase in baseline presentin the 4_AP experiments before the application of histamine.)

The dependence of the histamine-mediated depression onthe extracellular calcium concentration and the differenteffects obtained with the calcium or potassium channelblockers suggests that histamine may be acting by reducingpresynaptic calcium influx. Alternatively, or in addition,histamine may affect processes downstream of the calcium

influx. To examine this possibility we examined the effect ofhistamine on miniature excitatory postsynaptic currents(mEPSCs). In general, the frequency of these spontaneousevents is not affected by blockade of presynaptic calciumchannels (e.g. by cadmium), but is affected by substanceswhich act downstream of the presynaptic calcium influx.

mEPSCs were recorded from the somata of granule cellsusing the whole-cell configuration of the patch-clamptechnique at a holding potential of −75 mV. The externalmedium contained 2 mÒ calcium and 0·5 ìÒ TTX. In controlperiods mEPSCs occurred at a frequency of 0·82 ± 0·09 Hzat room temperature (n = 15) and were completely blocked bythe AMPA receptor antagonist CNQX (10 ìÒ). mEPSCsranged in size from 3 pA (threshold value) to 41 pA with anamplitude distribution which was skewed towards highervalues (i.e. non-Gaussian). In control experiments, the non-specific calcium channel blocker CdClµ (100 ìÒ, n = 4) didnot affect the frequency of mEPSCs although at thisconcentration it rapidly, completely and irreversibly blockedextracellularly recorded fEPSPs. Histamine (10 ìÒ), waswithout effect on the amplitude of mEPSCs (n = 9, Fig. 5),but slightly reduced mEPSC frequency from 0·72 ± 0·12 Hzin control to 0·66 ± 0·11 Hz (P = 0·014, paired t test). Incontrast, adenosine (20 ìÒ) reduced mEPSC frequency by55%, without affecting mEPSC amplitude (n = 2).

Presynaptic inhibition by histamineJ. Physiol. 515.3 781

Figure 4. 4-Aminopyridine reduces the depression

of synaptic transmission caused by histamine

A, 4-aminopyridine (100 ìÒ) causes a large enhancementof synaptic transmission. Administration of histamine(10 ìÒ) 45 min after beginning 4-aminopyridineapplication depresses synaptic transmission, but by asmaller amount than in control. Inset, analogue tracesfrom a single experiment recorded at the times indicatedby the lower-case letters. B, normalized mean data for thedepression of synaptic transmission in the presence of4_aminopyridine with either 4 mÒ (n = 7) or 2 mÒ(n = 6) extracellular Ca¥.

To exclude the possibility that these results were an artefactof recording at room temperature we performed a furtherseries of experiments at 28°C — under these conditions,mEPSC frequency was slightly higher under baselineconditions (0·90 ± 0·04 Hz, n = 9). Histamine (10 ìÒ)again had only a minor effect on mEPSC frequency,reducing it to 97·7 ± 2·4% of control (n = 5) whereasadenosine (20 ìÒ) strongly reduced it to 47·8 ± 3·1% ofcontrol (n = 4, P < 0·05).

At the present time it is unclear which signal transductionmechanisms are utilized by the histamine H× receptor. Onepossible mechanism of action of histamine could be toinhibit protein kinases since activation of protein kinaseshas been shown to enhance synaptic transmission atglutamatergic synapses (Capogna et al. 1995). To determineif protein kinases mediate the histamine H× receptor-mediated depression we tested two broad-spectrum blockersfor their ability to inhibit histamine’s effect on synaptictransmission. We applied the serineÏthreonine kinaseblocker H7 at a concentration (100 ìÒ) which is high enoughto inhibit the calcium-dependent kinase, PKC, the cAMP-dependent kinase, PKA, and the cGMP-dependent kinase,PKG. We applied H715 min before, during and after theapplication of histamine (10 ìÒ). H7 did not affect themagnitude of depression elicited by histamine (n = 6,Fig. 6A and C).

Recently, it has been shown that one pathway for inhibitionof calcium channels by noradrenaline involves tyrosinekinases (Diverse Pierluissi et al. 1997). To test for aninvolvement of tyrosine kinases we applied the potenttyrosine kinase inhibitor Lavendustin A, which has beenshown to block long-term potentiation in the CA1 regionof the hippocampus when applied extracellularly (O’Dell et al.1991). Lavendustin A (10 ìÒ) did not affect the magnitude ofthe histamine-induced depression but did appear to delaythe onset of the effect (n = 7, Fig. 6B and D). Anothertyrosine kinase inhibitor, genistein, could not be used forthis purpose in the dentate gyrus since it (as well as theinactive analogue, daidzein) depressed synaptic transmissionat low concentrations (50% reduction at 20 ìÒ) by itself.

Finally, we investigated whether the histamine-induceddepression could be occluded by the potent endogeneousinhibitor of excitatory synaptic transmission, adenosine. Aconcentration of adenosine (20 ìÒ) was chosen which wouldgive a depression of fEPSPs of around 50%. Application of20 ìÒ adenosine rapidly depressed synaptic transmission(Fig. 7A). Application of histamine, in the continuedpresence of adenosine was completely without effect,i.e. adenosine occluded the histaminergic depression (n = 7).Bath application of the adenosine A1 receptor agonistN É_cyclopentyladenosine (CPA, 0·3 ìÒ, n = 7) alsodepressed evoked fEPSPs and prevented a furtherdepression by histamine (Fig. 7B).

R. E. Brown and H. L. Haas J. Physiol. 515.3782

Figure 5. Histamine action on miniature excitatory postsynaptic current (mEPSC) amplitude

and frequency (n = 9)

A, traces are 2·5 s long and are separated by 40 pA. B, amplitude distribution for the experimentcorresponding to the data in A. Continuous line, control; dotted line, histamine (10 ìÒ). C, mean data forthe frequency of mEPSCs in control and in the presence of histamine.

DISCUSSION

We have studied the depression of synaptic transmissionat the perforant path input to the dentate gyrus by thebiogenic amine histamine. The selective H× receptorantagonist thioperamide blocked the depression, confirmingprevious findings that this is a response mediated bypresynaptically located H× receptors (Brown & Reymann,1996). The histamine H× receptor has not yet been clonedbut is thought to be coupled to a pertussis toxin-sensitiveG_protein (Gé or Gï), like many other presynaptic inhibitoryreceptors. H× receptor agonists stimulated GTPã

35

S bindingin rat cerebrocortical membranes, and this binding wasprevented by pretreatment with pertussis toxin (Clark &Hill, 1996). Furthermore, pertussis toxin blocks thehistamine-mediated inhibition of noradrenaline releasefrom sympathetic endings onto the heart (Endou et al.

1994) and the H×-receptor-mediated inhibition of excitatoryjunction potentials in the guinea-pig mesenteric artery(Nozaki & Sperelakis, 1989).

Activation of neurotransmitter receptors coupled topertussis toxin-sensitive G-proteins can lead to threepossible effects which could explain their inhibition ofevoked transmitter release (Thompson et al. 1993):

(i) blockade of high-threshold, voltage-dependent calciumchannels; (ii) facilitation of voltage-dependent potassiumchannels; and (iii) a direct modulation of the neurotrans-mitter release machinery.

Here we have investigated the potential contribution ofthese three effects to the histamine H× receptor-mediateddepression of glutamate release.

Effects on calcium influxÏcalcium channels

The amount of depression caused by histamine was foundto be inversely related to the extracellular calciumconcentration, i.e. a larger depression was observed at lowerexternal calcium concentrations. A similar relationship haspreviously been demonstrated for H× receptor-mediateddepression of histamine (Arrang et al. 1985) andnoradrenaline release (Schlicker et al. 1994). Such arelationship is consistent with an effect of histamine oncalcium influx (i.e. mechanisms (i) and (ii) above) but couldalso be explained by a modulation of one of the calcium-sensitive steps in the release process or by the calcium-dependent binding of histamine to its receptor. Extracellularcalcium promotes conversion of the H× receptor to the low-affinity (inactive?) form (Arrang et al. 1990).

Presynaptic inhibition by histamineJ. Physiol. 515.3 783

Figure 6. Protein kinase inhibitors do not block the histamine-mediated depression

A and B, application of the inhibitor of serineÏthreonine protein kinases H7 (100 ìÒ, n = 6) or thetyrosine kinase inhibitor Lavendustin A (5, ìM, n = 7), respectively, do not reduce the magnitude of thehistamine-mediated depression. C and D, comparison of the depression caused by histamine in the presenceor absence of the inhibitors.

Multiple high-threshold calcium channels are responsible forglutamate release in the CA1 region of the hippocampus. Inour experiments in the dentate gyrus, both the N_typecalcium channel blocker ù_conotoxin and the PÏQ_typeblocker ù_agatoxin substantially depressed synaptic trans-mission implicating an involvement of these two channeltypes. In addition, it is likely that a third component (R-type) is also present as in CA1 (Wu & Saggau, 1997),although we have not specifically tested this possibility here.In contrast, L-type channels are not involved in synaptictransmision at this synapse since nifedipine was withouteffect. To determine if the histamine effect was mediated viaa selective inhibition of either N_type or PÏQ-type channels,we applied histamine to slices which had been treated withone of the two toxins described above and compared thedepression with that observed under control conditions. Thedepression observed in the presence of the toxins was notreduced in comparison with control — in fact in the presence

of agatoxin it was slightly enhanced. Therefore, histaminedoes not act by selectively inhibiting one channel type. Theseresults do not exclude the possibility, however, that histamineinhibits all the high-threshold presynaptic calcium channelsby a similar amount (with the PÏQ_type channel beingsomewhat less inhibited).

Blockade of potassium channels

Application of the unspecific potassium channel blocker4_aminopyridine potentiated synaptic transmission (Buckle& Haas, 1982) and reduced the histaminergic depression.The reduction in the histaminergic depression by 4_APcould have two possible explanations. Firstly, the reductioncould be because 4_AP blocks a potassium channel which isenhanced by H× receptor activation. Secondly, the reductioncould be due to the enhanced calcium influx caused by aprolongation of the presynaptic action potential. Recently ithas been shown that changes in action potential durationfollowing administration of 4_AP to slices alters the relianceof excitatory synaptic transmission on multiple types ofcalcium channels (Wheeler et al. 1996), i.e. under theseconditions the calcium influx through any of the channeltypes alone is sufficient to trigger transmitter release. Thus,the presynaptic inhibition will be determined by theamount that the least inhibited channel is blocked. Ifhistamine is acting by enhancing a presynaptic potassiumconductance then one would expect that the amount ofinhibition of the effect by 4_AP would remain fairlyconstant when the extracellular calcium concentrationchanges. However, this was not found to be the case. 4_APinhibited the histamine effect by 45% in 4 mÒ extracellularcalcium but by 72% in 2 mÒ extracellular calcium. Thus, weconclude that it is more likely that histamine inhibits severaltypes of presynaptic calcium channels rather thanactivating presynaptic potassium channels.

Studies on the histamine H×-mediated inhibition of corticalnoradrenaline release also concluded that potassiumchannels were not responsible for the effect (Schlicker et al.

1994). Blockers or activators of ATP-sensitive potassiumchannels did not affect the histaminergic inhibition. Tetra-ethylammonium, another broad-spectrum inhibitor ofpotassium channels reduced the histamine effect (like 4_AP)but when the extracellular calcium concentration washalved, histamine inhibited noradrenaline release by asimilar amount as in control.

Action on mEPSCs

To test whether an inhibition of the transmitter releasemachinery could account for the histamine H× receptor-mediated presynaptic inhibition of evoked release weexamined the effect of histamine on mEPSCs. mEPSCswere recorded in the presence of TTX, to block voltage-gated sodium channels; they were blocked by the AMPAreceptor antagonist CNQX and were unaffected by thevoltage-gated calcium channel blocker cadmium. Thus, theseevents are action potential independent, do not require aninflux of calcium ions through voltage-gated calcium

R. E. Brown and H. L. Haas J. Physiol. 515.3784

Figure 7. Adenosine occludes the depressant effect of

histamine

A, adenosine (20 ìÒ) depressed the fEPSP and occluded thedepressant effect of histamine (10 ìÒ, n = 7). Inset, analogtraces recorded at the times indicated by the lower caseletters. Scale bar, 0·5 mV, 10 ms. B, the adenosine A1

receptor agonist N É-cyclopentyladenosine (CPA, 0·3 ìÒ,n = 7) reproduced the effects of adenosine — it depressed thefEPSP and occluded the histamine-mediated depression.

channels and are mediated by the AMPA subtype ofpostsynaptic glutamate receptors. We found that histaminehad only a minor effect on the frequency of mEPSCs, whichcould not account for the inhibition of evoked release (40%)under these conditions. Therefore, we conclude that a directaction of histamine on the transmitter release machinery isnot a major factor in histaminergic presynaptic inhibition ofglutamate release.

Interaction with adenosine receptors

Another potent inhibitor of synaptic transmission in thehippocampus is the purine adenosine (Thompson et al.

1992), which also acts on a receptor (A1) coupled to apertussis toxin-sensitive G-protein. We found that inaddition to depressing synaptic transmission itself,adenosine completely occluded the histaminergic depression.These actions of adenosine were mimicked by the selectiveagonist of adenosine A1 receptors, CPA. This suggests thatadenosine, acting through A1 receptors, and histamine,acting through H× receptors, converge onto a final commonpathway. The action of adenosine stands in contrast to theclass III metabotropic glutamate receptor (mGluR) agonist¬_AP4, which depressed synaptic transmission but did notprevent a further depression of the fEPSP by histamine(Brown & Reymann, 1996). Class III mGluRs may use adifferent mechanism from histamine or may be located on adifferent subset of presynaptic terminals.

A recent study (Luscher et al. 1997) using mice lacking thegene encoding the inward rectifier potassium channelGIRK2 showed that the postsynaptic hyperpolarizationcaused by activation of adenosine A1 receptors was absent inthese mice, whereas A1 receptor-mediated presynapticinhibition was intact, suggesting that activation ofpotassium channels was not responsible for this presynapticeffect. If histamine and adenosine act via a common finaleffector then this conclusion would also hold for histamineH× receptors. Presynaptic inhibition caused by adenosineacting on A1 receptors is reduced by 4_AP in the CA1 region(Klapstein & Colmers, 1992) and the absolute amount ofpresynaptic inhibition is not enhanced by reducing theextracellular calcium concentration (similar to what we haveobserved here for histamine). Unfortunately, in that studydepression in the low calcium condition alone (without 4_AP)was not measured so a comparison of the amount ofinhibition by 4_AP under the two conditions was notpossible. However, this study highlights the similarity of thehistamine H× receptor- and adenosine A1 receptor-mediatedeffects.

Role of second messengersÏprotein kinases

The possible coupling of the histamine H× receptor to aparticular signal transduction cascade is still a matter fordebate. H× receptor-mediated presynaptic inhibition isblocked by pertussis toxin (Nozaki & Sperelakis, 1989; Endouet al. 1994) or N_ethylmaleimide (Schlicker et al. 1994), agentswhich inactivate Gi and Go G-proteins. Gi G_proteins arecoupled to inhibition of adenylyl cyclase. Since activation of

PKA can facilitate transmitter release (Chavez Noriega &Stevens, 1994) an inhibition of cAMP production mightaccount for the observed inhibition of transmitter release.However, application of a high concentration of H7, whichblocks protein kinases A, C and G, did not affect thedepression of the fEPSP by histamine.

Recent data suggest that there is a pathway for inhibition ofvoltage-dependent calcium channels via GéÏGï coupledreceptors which involves a tyrosine kinase of the src family(Diverse Pierluissi et al. 1997). To investigate a role fortyrosine kinases we applied the tyrosine kinase inhibitorLavendustin A at a concentration which was high enough toblock LTP in the CA1 region. However, this drug waswithout effect on the histaminergic depression.

Although in some cell types receptors coupled to GéÏGïG_proteins appear to inhibit high-threshold calciumchannels via PKC or tyrosine kinases (Diverse Pierluissi etal. 1997), under most conditions the inhibition involves adirect action of the âã subunits of the G-protein on theâ_subunit of the voltage-dependent calcium channels(Dolphin, 1998). Since in our experiments the histamineaction was not blocked by inhibitors of serineÏthreonine ortyrosine kinases a direct action of âã subunits is most likely.

Conclusion

Recent data suggest that inhibition of calcium influx duringthe presynaptic action potential, most likely to be viainhibition of presynaptic calcium channels, is a majormechanism by which presynaptic neurotransmitter receptorsinhibit transmitter release (Wu & Saggau, 1997). Directmeasurements of calcium concentrations in presynapticterminals, coupled with recording of synaptic potentials,demonstrated a close relationship between the magnitude ofdepression of the presynaptic calcium transient and thedepression of synaptic potentials for a variety ofpresynaptic receptors, including the adenosine A1 receptor(Wu & Saggau, 1994). In addition, it was found using patch-clamp recording from presynaptic terminals that thedepression of transmitter release caused by activation ofclass III metabotropic glutamate receptors could beentirely accounted for by inhibition of presynaptic calciumcurrent (Takahashi et al. 1996). The present data suggestthat histamine, acting on H× receptors, also utilizes thismechanism, most likely via a direct action of theâã G_protein subunits.

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Acknowledgements

We wish to thank Dr Stephen Traynelis for kindly providing theN event-analysis software. This work was supported by DeutscheForschungsgemeinschaft (Grant DEG HA1525Ï1-3).

Corresponding author

R. Brown: Institut f�ur Neurophysiologie, Heinrich-Heine-Universit�at, D-40001, D�usseldorf, Germany.

Email: brown@uni-duesseldorf.de

R. E. Brown and H. L. Haas J. Physiol. 515.3786