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. 1999 Mar 15;515(Pt 3):777–786. doi: 10.1111/j.1469-7793.1999.777ab.x

On the mechanism of histaminergic inhibition of glutamate release in the rat dentate gyrus

Ritchie E Brown 1, Helmut L Haas 1
PMCID: PMC2269185  PMID: 10066904

Abstract

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

  2. Application of histamine (10 μm, 5 min) depressed synaptic transmission in the dentate gyrus for 1 h. This depression was blocked by the selective antagonist of histamine H3 receptors, thioperamide (10 μm).

  3. The magnitude of the depression caused by histamine was inversely related to the extracellular Ca2+ concentration. Application of the N-type calcium channel blocker ω-conotoxin (0.5 or 1 μm) or the P/Q-type calcium channel blocker ω-agatoxin (800 nm) did not prevent depression of synaptic transmission by histamine.

  4. The potassium channel blocker 4-aminopyridine (4-AP, 100 μm) enhanced synaptic transmission and reduced the depressant effect of histamine (10 μm). 4-AP reduced the effect of histamine more in 2 mm extracellular calcium than in 4 mm extracellular calcium.

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

  6. Histaminergic depression was not blocked by an inhibitor of serine/threonine protein kinases, H7 (100 μm), or by an inhibitor of tyrosine kinases, Lavendustin A (10 μm).

  7. Application of adenosine (20 μm) or the adenosine A1 agonist N6-cyclopentyladenosine (CPA, 0.3 μm) completely occluded the effect of histamine (10 μm).

  8. We conclude that histamine, acting on histamine H3 receptors, inhibits glutamate release by inhibiting presynaptic calcium entry, via a direct G-protein-mediated inhibition of multiple calcium channels. Histamine H3 receptors and adenosine A1 receptors act upon a common final effector to cause presynaptic inhibition.

Neurons in the brain receive precise, continuously updated information from other neurons via thousands of specialized synaptic contacts on their somata and dendrites. At any given time, only a subset of these synaptic contacts will be active, i.e. releasing neurotransmitter. Regulation of the probability of transmitter release is a major mechanism by which the brain processes incoming information and internally generated messages. Release probability varies according to the history of activation of the synapse as a result of the time course of the intracellular cascades involved in the release process (Zucker, 1989). In addition, however, release probability can be varied by neurotransmitters or modulators which bind to receptors located in the presynaptic membrane (Thompson et al. 1993; Wu & Saggau, 1997).

The principal neurons in the dentate gyrus region of the hippocampus are the granule cells, which receive their primary excitatory afferent input from layer II stellate cells of the entorhinal cortex, containing highly processed multimodal information (Amaral & Witter, 1989). The stellate cell axons form the so-called perforant path input to the dentate gyrus, synapsing on the outer two-thirds of the granule cell dendritic tree. A number of different neurotransmitters/modulators act presynaptically on these synapses, thereby altering the subset of synapses which release transmitter. Glutamate, the main transmitter released at these synapses, itself acts upon at least two types of presynaptic receptors (Brown & Reymann, 1995; Macek et al. 1996). Upon high-frequency stimulation, the peptide, dynorphin is released from these synapses, depressing glutamate release via κ-opioid receptors (Wagner et al. 1993). The purine adenosine, which is released locally from neurons in a manner dependent on metabolic activity, depresses transmission via A1 receptors (Dolphin & Archer, 1983). Finally, these synapses are controlled by transmitters which are released in a global manner in the brain and which provide effectively the ‘context’ in which the afferent information should be processed, e.g. the state of arousal or motivation. One example of these transmitters is the biogenic amine histamine, which is released from varicosities of axons originating in the tuberomammillary nucleus of the hypothalamus.

Three receptors for histamine have been identified. The histamine H1 and H2 receptors are postsynaptically located and are coupled to phospholipase C and to stimulation of adenylyl cyclase, respectively (Schwartz et al. 1991). The histamine H3 receptor, which was first described in 1983 (Arrang et al. 1983) as an autoreceptor regulating histamine release, has subsequently been shown to inhibit the release of other transmitters/modulators (Schwartz et al. 1991). In the dentate gyrus, histamine H3 receptors are located on perforant path terminals and inhibit the release of glutamate (Brown & Reymann, 1996). The mechanism by which H3 receptors inhibit transmitter release, however, remains unclear.

Two main mechanisms for inhibition of transmitter release have been proposed (Thompson et al. 1993; Wu & Saggau, 1997). Firstly, they may inhibit calcium influx during the presynaptic action potential, either by blocking presynaptic calcium channels, or by enhancing potassium currents. Secondly, they may act downstream of the calcium influx on the proteins of the transmitter release machinery. The mechanism of presynaptic inhibition can have important functional consequences. For instance, G-protein-mediated inhibition of voltage-gated calcium channels is relieved by depolarization (Dolphin, 1998) and will thus be less during a train of high-frequency action potentials than during low-frequency stimulation. In this study we present evidence that histamine, acting via histamine H3 receptors depresses glutamate release via the first mechanism. Furthermore, we investigate the signal transduction pathway involved in this effect 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 male Wistar rats. All experiments were conducted in compliance with German law and with the approval of the Bezirksregierung Düsseldorf. The animals were quickly decapitated and the brain quickly transferred to a modified artificial cerebrospinal fluid (ACSF), in which all NaCl had been replaced by isomolar sucrose (Aghajanian & Rasmussen, 1989). Horizontal, 400 μm thick whole brain slices were cut using a vibroslicer (Campden Instruments, Loughborough, UK). Subsequently, the hippocampi were isolated under a dissecting microscope before being transferred to a prechamber with standard ACSF containing (mM): 124 NaCl, 2.8 KCl, 1.2 KH2PO4, 4 MgSO4, 4 CaCl2, 25.6 NaHCO3, 10 D-glucose, 0.05 picrotoxin. In some experiments the CaCl2 concentration was reduced to 2 or 1 mM. Slices remained in a holding chamber at room temperature (18–22°C) until use, when they were transferred individually to a recording chamber and perfused with ACSF at a flow rate of 2.5 ml min−1. Extracellular recordings were made at 28°C, and recordings of miniature excitatory postsynaptic currents (mEPSCs) were made at room temperature or at 28°C.

Constant voltage bipolar pulses (1–20 V, 0.2 ms duration) were applied every 30 s through a monopolar, lacquer-coated, platinum or Ni/Cr electrode placed in the middle third of the stratum moleculare of the dentate gyrus to stimulate the medial perforant path input. For recording of field excitatory postsynaptic potentials (fEPSPs) a glass electrode filled with ACSF was inserted into the molecular layer at the same level as the stimulation electrode. The initial slope of the fEPSP was used as the measure of this potential. The medial perforant path was distinguished according to the kinetics of the synaptic potentials and the presence of paired-pulse depression (Abraham & McNaughton, 1984).

Intracellular recordings were made from dentate granule cells using the ‘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 MΩ) were normally filled with an intracellular solution containing (mM): 135 caesium methanesulphonate, 6 MgCl2, 10 Hepes, 10 EGTA, 1 CaCl2, 2 Na2ATP (pH 7.25 with CsOH). In some experiments the patch solution contained (mM): 135 CsCl, 2 MgCl2, 0.2 EGTA, 5 NaCl, 2 Na2ATP. Results were similar with the two solutions and have been pooled. mEPSCs were recorded at a holding potential of −75 mV. Access resistance was calculated from the instantaneous current measured at the beginning of a 5 mV, 100 ms hyperpolarizing step from the holding potential and input resistance from the steady-state current remaining at the end of the step. Neurons accepted for this study 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 were 22.2 ± 1.4 MΩ and input resistances 213 ± 13 MΩ (n = 15). Experiments in which the access or input resistance changed by more than 15 % during the course of the experiment were discarded. mEPSCs were analysed using the N event-analysis software kindly provided by Dr S. Traynelis (Emory University, Atlanta, GA, USA). mEPSCs (negative deflections with a fast rising phase and exponential decay) were detected by eye; a marker was positioned at the start of the rising phase and their peak amplitude (average of 2 most negative points) measured relative to a baseline period (mean of 30 points) immediately preceding them. Events were accepted if they exceeded a 3 pA threshold value and were significantly more negative than the baseline period. Amplitude distributions were compared with the Kolmogorov-Smirnoff test.

Drugs used in this study were: 4-aminopyridine (Fluka, Buchs, Switzerland), adenosine (Merck, Darmstadt, Germany), CdCl2 (Fluka), H7 (Alexis Biochemicals, Grünberg, Germany), histamine dihydrochloride (Sigma), Lavendustin A (Alexis Biochemicals), N6-cyclopentyladenosine (CPA, Research Biochemicals International), nifedipine (Sigma), ω-agatoxin (Alomone Labs, Jerusalem, Israel), ω-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 using NaOH and stored as stock solutions. Drugs were bath applied. All values are given as the mean ±s.e.m. Statistical comparisons were made using Student's unpaired or paired t test, as appropriate.

RESULTS

Bath application of histamine (10 μM) for 5 min under control conditions (4 mM extracellular calcium/magnesium, 50 μM picrotoxin) led to a rapid depression of fEPSPs recorded from the medial perforant pathway of the dentate gyrus; it required up to 1 h for complete washout (Fig. 1). A repeat application of histamine, 1 h following the first, led to a similar-sized depression as seen following the first application (n = 3), i.e. there was no homologous desensitization of this response. Application of the selective H3 receptor antagonist thioperamide (10 μM, n = 4) completely blocked the depression caused by histamine (10 μM).

Figure 1. The extent of the depression of synaptic transmission by histamine is dependent on the the external calcium concentration.

Figure 1

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A, mean data showing the time course of depression of the fEPSP with 4 mM (n = 10), 2 mM (n = 7) or 1 mM (n = 6) extracellular calcium. B, analog traces from an experiment in 2 mM external calcium before and after histamine application.

The magnitude of the depression of synaptic transmission by histamine was dependent on the extracellular calcium concentration. In the presence of 4 mM extracellular calcium, the depression measured 10 min after starting histamine application was to 77.9 ± 2.0 % of baseline (n = 10) whereas in the presence of 2 and 1 mM calcium the depression was to 59.6 ± 2.6 % (n = 7) and 56.5 ± 3.5 % (n = 6) of baseline, respectively.

Multiple presynaptic calcium channels, including the N-type, Q-type and a so far poorly characterized subtype, control the release of glutamate in the hippocampus (Dunlap et al. 1995; Wu & Saggau, 1997). In our experiments, a brief application of the selective N-type calcium channel blocker ω-conotoxin GVIA (0.5 μM, n = 4) depressed the fEPSP to a new stable level around 60 % of baseline. Subsequent application of histamine (10 μM) 30 min following conotoxin administration depressed the fEPSP further, similar to control conditions. A higher concentration of conotoxin (1 μM, n = 2) did not depress the fEPSP to a greater extent than the lower concentration and also did not block the histamine effect, so the results have been pooled (Fig. 2). Taking the period immediately preceding histamine application as 100 %, histamine depressed the fEPSP to 75.1 ± 4.7 % in the presence of conotoxin (n = 6) as compared with 77.9 ± 2.0 % in control (n = 10).

Figure 2. The depressant effect of histamine is not occluded by the N-type calcium channel blocker ω-conotoxin.

Figure 2

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A, a short application of ω-conotoxin (0.5 μM, n = 4 or 1 μM, n = 2) depresses the fEPSP but does not prevent a further depression by histamine (10 μM). Inset, analog traces recorded at the times indicated by the lower case letters. Scale bar, 0.5 mV, 10 ms. B, comparison of the depression of the fEPSP under control conditions (n = 10) or in the presence of ω-conotoxin (n = 6).

Application of a high concentration of the selective P/Q-type calcium channel blocker ω-agatoxin IVA (800 nM, n = 4) also depressed the fEPSP to around 50 % of baseline (Fig. 3). Application of histamine (10 μM) 30 min following agatoxin application still depressed synaptic transmission and in fact the magnitude of the depression under these conditions was slightly enhanced (P = 0.053). Taking the period immediately preceding histamine application as 100 %, histamine depressed the fEPSP to 69.7 ± 3.6 % in the presence of agatoxin (n = 4) as compared with 77.9 ± 2.0 % in control (n = 10).

Figure 3. The depressant effect of histamine is not occluded by the P/Q-type calcium channel blocker ω-agatoxin.

Figure 3

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A, a short application of ω-agatoxin (800 nM, n = 4) depresses the fEPSP but does not prevent a further depression by histamine (10 μM). Inset, analog traces recorded at the times indicated by the lower case letters. Scale bar, 0.5 mV, 10 ms. B, comparison of the depression of the fEPSP under control conditions (n = 10) or in the presence of ω-agatoxin (n = 4).

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

The compound 4-aminopyridine (4-AP) blocks a number of different types of voltage-dependent potassium channels, including inwardly rectifying channels of the type activated by pertussis toxin-sensitive G-proteins. Bath application of 4-AP (100 μM), led, as expected (Buckle & Haas, 1982), to a broadening of the presynaptic fibre volley and a substantial enhancement in the size of fEPSPs to 300 ± 25 % of baseline (n = 7). Application of histamine (10 μM) 45 min following the addition of 4-AP, depressed the fEPSP to 262 ± 23 % of baseline. Taking the points immediately preceding histamine application as 100 %, histamine depressed 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-AP could indicate that histamine is acting through voltage-dependent potassium channels blocked incompletely by 4-AP. Alternatively, the reduction could arise due to the increased influx of calcium through the different types of presynaptic calcium channels in the presence of 4-AP (Wheeler et al. 1996). To try and distinguish between these two possibilities we performed the same experiment in the presence of 2 mM extracellular calcium. If histamine is acting by enhancing voltage-dependent potassium channels then one would expect a similar degree of inhibition of the effect by 4-AP under the two conditions. In 2 mM extracellular calcium 4-AP potentiated the fEPSP to 330 ± 33 % of control and application of histamine 45 min later in the continued presence of 4-AP reduced the fEPSP to 298 ± 29 % of baseline (n = 6, Fig. 4A). Taking the points immediately preceding histamine application as 100 %, histamine depressed the fEPSP to 88.5 ± 3.0 % of baseline (n = 6, Fig. 4B), as compared with 59.6 ± 2.6 % (n = 7) in control, i.e. 4-AP reduced the histaminergic depression by 72 %, suggesting that enhancement of presynaptic potassium channels is not responsible for histaminergic presynaptic inhibition. (This large difference between the effect of 4-AP in 4 and 2 mM calcium remains, even if one corrects for the slight increase in baseline present in the 4-AP experiments before the application of histamine.)

Figure 4. 4-Aminopyridine reduces the depression of synaptic transmission caused by histamine.

Figure 4

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A, 4-aminopyridine (100 μM) causes a large enhancement of synaptic transmission. Administration of histamine (10 μM) 45 min after beginning 4-aminopyridine application depresses synaptic transmission, but by a smaller amount than in control. Inset, analogue traces from a single experiment recorded at the times indicated by the lower-case letters. B, normalized mean data for the depression of synaptic transmission in the presence of 4-aminopyridine with either 4 mM (n = 7) or 2 mM (n = 6) extracellular Ca2+.

The dependence of the histamine-mediated depression on the extracellular calcium concentration and the different effects obtained with the calcium or potassium channel blockers suggests that histamine may be acting by reducing presynaptic calcium influx. Alternatively, or in addition, histamine may affect processes downstream of the calcium influx. To examine this possibility we examined the effect of histamine on miniature excitatory postsynaptic currents (mEPSCs). In general, the frequency of these spontaneous events is not affected by blockade of presynaptic calcium channels (e.g. by cadmium), but is affected by substances which act downstream of the presynaptic calcium influx.

mEPSCs were recorded from the somata of granule cells using the whole-cell configuration of the patch-clamp technique at a holding potential of −75 mV. The external medium contained 2 mM calcium and 0.5 μM TTX. In control periods mEPSCs occurred at a frequency of 0.82 ± 0.09 Hz at room temperature (n = 15) and were completely blocked by the AMPA receptor antagonist CNQX (10 μM). mEPSCs ranged in size from 3 pA (threshold value) to 41 pA with an amplitude distribution which was skewed towards higher values (i.e. non-Gaussian). In control experiments, the non-specific calcium channel blocker CdCl2 (100 μM, n = 4) did not affect the frequency of mEPSCs although at this concentration it rapidly, completely and irreversibly blocked extracellularly recorded fEPSPs. Histamine (10 μM), was without effect on the amplitude of mEPSCs (n = 9, Fig. 5), but slightly reduced mEPSC frequency from 0.72 ± 0.12 Hz in control to 0.66 ± 0.11 Hz (P = 0.014, paired t test). In contrast, adenosine (20 μM) reduced mEPSC frequency by 55 %, without affecting mEPSC amplitude (n = 2).

Figure 5. Histamine action on miniature excitatory postsynaptic current (mEPSC) amplitude and frequency (n = 9).

Figure 5

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A, traces are 2.5 s long and are separated by 40 pA. B, amplitude distribution for the experiment corresponding to the data in A. Continuous line, control; dotted line, histamine (10 μM). C, mean data for the frequency of mEPSCs in control and in the presence of histamine.

To exclude the possibility that these results were an artefact of recording at room temperature we performed a further series of experiments at 28°C - under these conditions, mEPSC frequency was slightly higher under baseline conditions (0.90 ± 0.04 Hz, n = 9). Histamine (10 μM) again had only a minor effect on mEPSC frequency, reducing it to 97.7 ± 2.4 % of control (n = 5) whereas adenosine (20 μM) strongly reduced it to 47.8 ± 3.1 % of control (n = 4, P < 0.05).

At the present time it is unclear which signal transduction mechanisms are utilized by the histamine H3 receptor. One possible mechanism of action of histamine could be to inhibit protein kinases since activation of protein kinases has been shown to enhance synaptic transmission at glutamatergic synapses (Capogna et al. 1995). To determine if protein kinases mediate the histamine H3 receptor-mediated depression we tested two broad-spectrum blockers for their ability to inhibit histamine's effect on synaptic transmission. We applied the serine/threonine kinase blocker H7 at a concentration (100 μM) which is high enough to inhibit the calcium-dependent kinase, PKC, the cAMP-dependent kinase, PKA, and the cGMP-dependent kinase, PKG. We applied H7 15 min before, during and after the application of histamine (10 μM). H7 did not affect the magnitude of depression elicited by histamine (n = 6, Fig. 6A and C).

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

Figure 6

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A and B, application of the inhibitor of serine/threonine protein kinases H7 (100 μM, n = 6) or the tyrosine kinase inhibitor Lavendustin A (5, μM, n = 7), respectively, do not reduce the magnitude of the histamine-mediated depression. C and D, comparison of the depression caused by histamine in the presence or absence of the inhibitors.

Recently, it has been shown that one pathway for inhibition of calcium channels by noradrenaline involves tyrosine kinases (Diverse Pierluissi et al. 1997). To test for an involvement of tyrosine kinases we applied the potent tyrosine kinase inhibitor Lavendustin A, which has been shown to block long-term potentiation in the CA1 region of the hippocampus when applied extracellularly (O'Dell et al. 1991). Lavendustin A (10 μM) did not affect the magnitude of the histamine-induced depression but did appear to delay the onset of the effect (n = 7, Fig. 6B and D). Another tyrosine kinase inhibitor, genistein, could not be used for this purpose in the dentate gyrus since it (as well as the inactive analogue, daidzein) depressed synaptic transmission at low concentrations (50 % reduction at 20 μM) by itself.

Finally, we investigated whether the histamine-induced depression could be occluded by the potent endogeneous inhibitor of excitatory synaptic transmission, adenosine. A concentration of adenosine (20 μM) was chosen which would give a depression of fEPSPs of around 50 %. Application of 20 μM adenosine rapidly depressed synaptic transmission (Fig. 7A). Application of histamine, in the continued presence of adenosine was completely without effect, i.e. adenosine occluded the histaminergic depression (n = 7). Bath application of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA, 0.3 μM, n = 7) also depressed evoked fEPSPs and prevented a further depression by histamine (Fig. 7B).

Figure 7. Adenosine occludes the depressant effect of histamine.

Figure 7

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A, adenosine (20 μM) depressed the fEPSP and occluded the depressant effect of histamine (10 μM, n = 7). Inset, analog traces recorded at the times indicated by the lower case letters. Scale bar, 0.5 mV, 10 ms. B, the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA, 0.3 μM, n = 7) reproduced the effects of adenosine - it depressed the fEPSP and occluded the histamine-mediated depression.

DISCUSSION

We have studied the depression of synaptic transmission at the perforant path input to the dentate gyrus by the biogenic amine histamine. The selective H3 receptor antagonist thioperamide blocked the depression, confirming previous findings that this is a response mediated by presynaptically located H3 receptors (Brown & Reymann, 1996). The histamine H3 receptor has not yet been cloned but is thought to be coupled to a pertussis toxin-sensitive G-protein (Gi or Go), like many other presynaptic inhibitory receptors. H3 receptor agonists stimulated GTPγ35S binding in rat cerebrocortical membranes, and this binding was prevented by pretreatment with pertussis toxin (Clark & Hill, 1996). Furthermore, pertussis toxin blocks the histamine-mediated inhibition of noradrenaline release from sympathetic endings onto the heart (Endou et al. 1994) and the H3-receptor-mediated inhibition of excitatory junction potentials in the guinea-pig mesenteric artery (Nozaki & Sperelakis, 1989).

Activation of neurotransmitter receptors coupled to pertussis toxin-sensitive G-proteins can lead to three possible effects which could explain their inhibition of evoked transmitter release (Thompson et al. 1993): (i) blockade of high-threshold, voltage-dependent calcium channels; (ii) facilitation of voltage-dependent potassium channels; and (iii) a direct modulation of the neurotransmitter release machinery.

Here we have investigated the potential contribution of these three effects to the histamine H3 receptor-mediated depression of glutamate release.

Effects on calcium influx/calcium channels

The amount of depression caused by histamine was found to be inversely related to the extracellular calcium concentration, i.e. a larger depression was observed at lower external calcium concentrations. A similar relationship has previously been demonstrated for H3 receptor-mediated depression of histamine (Arrang et al. 1985) and noradrenaline release (Schlicker et al. 1994). Such a relationship is consistent with an effect of histamine on calcium influx (i.e. mechanisms (i) and (ii) above) but could also 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. Extracellular calcium promotes conversion of the H3 receptor to the low-affinity (inactive?) form (Arrang et al. 1990).

Multiple high-threshold calcium channels are responsible for glutamate release in the CA1 region of the hippocampus. In our experiments in the dentate gyrus, both the N-type calcium channel blocker ω-conotoxin and the P/Q-type blocker ω-agatoxin substantially depressed synaptic transmission implicating an involvement of these two channel types. 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 synaptic transmision at this synapse since nifedipine was without effect. To determine if the histamine effect was mediated via a selective inhibition of either N-type or P/Q-type channels, we applied histamine to slices which had been treated with one of the two toxins described above and compared the depression with that observed under control conditions. The depression observed in the presence of the toxins was not reduced in comparison with control - in fact in the presence of agatoxin it was slightly enhanced. Therefore, histamine does not act by selectively inhibiting one channel type. These results do not exclude the possibility, however, that histamine inhibits all the high-threshold presynaptic calcium channels by a similar amount (with the P/Q-type channel being somewhat less inhibited).

Blockade of potassium channels

Application of the unspecific potassium channel blocker 4-aminopyridine potentiated synaptic transmission (Buckle & Haas, 1982) and reduced the histaminergic depression. The reduction in the histaminergic depression by 4-AP could have two possible explanations. Firstly, the reduction could be because 4-AP blocks a potassium channel which is enhanced by H3 receptor activation. Secondly, the reduction could be due to the enhanced calcium influx caused by a prolongation of the presynaptic action potential. Recently it has been shown that changes in action potential duration following administration of 4-AP to slices alters the reliance of excitatory synaptic transmission on multiple types of calcium channels (Wheeler et al. 1996), i.e. under these conditions the calcium influx through any of the channel types alone is sufficient to trigger transmitter release. Thus, the presynaptic inhibition will be determined by the amount that the least inhibited channel is blocked. If histamine is acting by enhancing a presynaptic potassium conductance then one would expect that the amount of inhibition of the effect by 4-AP would remain fairly constant when the extracellular calcium concentration changes. However, this was not found to be the case. 4-AP inhibited the histamine effect by 45 % in 4 mM extracellular calcium but by 72 % in 2 mM extracellular calcium. Thus, we conclude that it is more likely that histamine inhibits several types of presynaptic calcium channels rather than activating presynaptic potassium channels.

Studies on the histamine H3-mediated inhibition of cortical noradrenaline release also concluded that potassium channels were not responsible for the effect (Schlicker et al. 1994). Blockers or activators of ATP-sensitive potassium channels did not affect the histaminergic inhibition. Tetraethylammonium, another broad-spectrum inhibitor of potassium channels reduced the histamine effect (like 4-AP) but when the extracellular calcium concentration was halved, histamine inhibited noradrenaline release by a similar amount as in control.

Action on mEPSCs

To test whether an inhibition of the transmitter release machinery could account for the histamine H3 receptor-mediated presynaptic inhibition of evoked release we examined the effect of histamine on mEPSCs. mEPSCs were recorded in the presence of TTX, to block voltage-gated sodium channels; they were blocked by the AMPA receptor antagonist CNQX and were unaffected by the voltage-gated calcium channel blocker cadmium. Thus, these events are action potential independent, do not require an influx of calcium ions through voltage-gated calcium channels and are mediated by the AMPA subtype of postsynaptic glutamate receptors. We found that histamine had only a minor effect on the frequency of mEPSCs, which could not account for the inhibition of evoked release (40 %) under these conditions. Therefore, we conclude that a direct action of histamine on the transmitter release machinery is not a major factor in histaminergic presynaptic inhibition of glutamate release.

Interaction with adenosine receptors

Another potent inhibitor of synaptic transmission in the hippocampus is the purine adenosine (Thompson et al. 1992), which also acts on a receptor (A1) coupled to a pertussis toxin-sensitive G-protein. We found that in addition to depressing synaptic transmission itself, adenosine completely occluded the histaminergic depression. These actions of adenosine were mimicked by the selective agonist of adenosine A1 receptors, CPA. This suggests that adenosine, acting through A1 receptors, and histamine, acting through H3 receptors, converge onto a final common pathway. The action of adenosine stands in contrast to the class III metabotropic glutamate receptor (mGluR) agonist L-AP4, which depressed synaptic transmission but did not prevent a further depression of the fEPSP by histamine (Brown & Reymann, 1996). Class III mGluRs may use a different mechanism from histamine or may be located on a different subset of presynaptic terminals.

A recent study (Luscher et al. 1997) using mice lacking the gene encoding the inward rectifier potassium channel GIRK2 showed that the postsynaptic hyperpolarization caused by activation of adenosine A1 receptors was absent in these mice, whereas A1 receptor-mediated presynaptic inhibition was intact, suggesting that activation of potassium channels was not responsible for this presynaptic effect. If histamine and adenosine act via a common final effector then this conclusion would also hold for histamine H3 receptors. Presynaptic inhibition caused by adenosine acting on A1 receptors is reduced by 4-AP in the CA1 region (Klapstein & Colmers, 1992) and the absolute amount of presynaptic inhibition is not enhanced by reducing the extracellular calcium concentration (similar to what we have observed here for histamine). Unfortunately, in that study depression in the low calcium condition alone (without 4-AP) was not measured so a comparison of the amount of inhibition by 4-AP under the two conditions was not possible. However, this study highlights the similarity of the histamine H3 receptor- and adenosine A1 receptor-mediated effects.

Role of second messengers/protein kinases

The possible coupling of the histamine H3 receptor to a particular signal transduction cascade is still a matter for debate. H3 receptor-mediated presynaptic inhibition is blocked by pertussis toxin (Nozaki & Sperelakis, 1989; Endou et al. 1994) or N-ethylmaleimide (Schlicker et al. 1994), agents which inactivate Gi and Go G-proteins. Gi G-proteins are coupled to inhibition of adenylyl cyclase. Since activation of PKA can facilitate transmitter release (Chavez Noriega & Stevens, 1994) an inhibition of cAMP production might account for the observed inhibition of transmitter release. However, application of a high concentration of H7, which blocks protein kinases A, C and G, did not affect the depression of the fEPSP by histamine.

Recent data suggest that there is a pathway for inhibition of voltage-dependent calcium channels via Gi/Go coupled receptors which involves a tyrosine kinase of the src family (Diverse Pierluissi et al. 1997). To investigate a role for tyrosine kinases we applied the tyrosine kinase inhibitor Lavendustin A at a concentration which was high enough to block LTP in the CA1 region. However, this drug was without effect on the histaminergic depression.

Although in some cell types receptors coupled to Gi/Go G-proteins appear to inhibit high-threshold calcium channels via PKC or tyrosine kinases (Diverse Pierluissi et al. 1997), under most conditions the inhibition involves a direct action of the βγ subunits of the G-protein on the β-subunit of the voltage-dependent calcium channels (Dolphin, 1998). Since in our experiments the histamine action was not blocked by inhibitors of serine/threonine or tyrosine kinases a direct action of βγ subunits is most likely.

Conclusion

Recent data suggest that inhibition of calcium influx during the presynaptic action potential, most likely to be via inhibition of presynaptic calcium channels, is a major mechanism by which presynaptic neurotransmitter receptors inhibit transmitter release (Wu & Saggau, 1997). Direct measurements of calcium concentrations in presynaptic terminals, coupled with recording of synaptic potentials, demonstrated a close relationship between the magnitude of depression of the presynaptic calcium transient and the depression of synaptic potentials for a variety of presynaptic receptors, including the adenosine A1 receptor (Wu & Saggau, 1994). In addition, it was found using patch-clamp recording from presynaptic terminals that the depression of transmitter release caused by activation of class III metabotropic glutamate receptors could be entirely accounted for by inhibition of presynaptic calcium current (Takahashi et al. 1996). The present data suggest that histamine, acting on H3 receptors, also utilizes this mechanism, most likely via a direct action of the βγ G-protein subunits.

Acknowledgments

We wish to thank Dr Stephen Traynelis for kindly providing the N event-analysis software. This work was supported by Deutsche Forschungsgemeinschaft (Grant DEG HA1525/1–3).

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