Effects of histamine on the spontaneous activity of preoptic GABAergic neurons
Preoptic GABAergic neurons were identified by using a transgenic mouse line GAD65-GFP which expresses enhanced green fluorescent protein (eGFP) under the control of the regulatory region of mouse glutamic acid decarboxylase (GAD) 65 gene. Most GABAergic neurons recorded displayed spontaneous “pacemaker” activity (19 out of 22), i.e. they presented regular firing patterns and the action potentials were preceded by depolarizing prepotentials (e.g. ). The average firing rate of the neurons was 7.5 ± 2.6 Hz (n=19). In 12 out of 22 GABAergic neurons studied histamine (3 μM) applied locally via a perfusion pencil (diameter 100 μm) reduced the spontaneous firing rates by 62 ± 11 % (n=12, paired t-test P<0.05) (). All neurons inhibited by histamine displayed pacemaker activity (). Higher concentrations of histamine (20 μM) had similar effect, a reduction in firing rate by 66 ± 13 % (n=4). This value was not statistically different to the one obtained with 3 μM histamine (ANOVA, P>0.1), suggesting that the effect saturates at low concentrations of the neurotransmitter. The reduction in firing rate by histamine was blocked by the H3R antagonist thioperamide () in all neurons tested (n=5). presents the average firing rate of three neurons in response to histamine, before and after thioperamide incubation. The H3 antagonist appeared to remove an H3R tone in 2 out of 6 neurons tested: the firing rate increased by 14 ± 5 % (n=2) as compared to the initial firing rate of the neuron. The H3R specific agonist R-α-methylhistamine (1μM) also decreased the firing rate by 64 ± 17 % in 5 out of 9 GABAergic preoptic neurons tested (). Together with the block by thioperamide, these results indicated that the histamine-induced reduction in firing of GABAergic preoptic neurons is mediated by activation of the H3Rs.
Histamine reduces the firing rate of preoptic GABAergic neurons in slices by acting at H3 subtype receptors
The effect described above was not associated with any obvious change in the frequency of spontaneous (s) IPSPs or sEPSPs. We further studied possible synaptic effects in 5 GABAergic neurons inhibited by the H3R agonist and in 14 GABAergic neurons which did not respond to it, by recording synaptic events in the absence of action potentials at a −50 mV holding potential. The properties (frequency, amplitude and kinetics) of sIPSCs and sEPSCs in these neurons were not affected by either histamine (n=5) or the H3 agonist (n=5) (). The holding current and the input resistance of the neurons were not affected by either histamine or the H3R agonist (). To further address the possibility of synaptic mechanisms we have also studied the effects of R-α-methylhistamine (1μM) in the absence of fast synaptic events which were blocked by adding CNQX (20 μM), AP-5 (100 μM) and bicuculline (20 μM) to the extracellular solution. In the presence of the antagonists, all 5 neurons studied maintained their spontaneous firing and the H3R agonist decreased the spike frequency by 71 ± 22 % in 3 out of the 5 neurons studied, a value which was not statistically different to the one obtained with the H3 agonist alone (ANOVA, P>0.1).
We also tested the response of preoptic GABAergic neurons to the H1R agonist 2-pyridylethylamine (100 μM) and to the H2R specific agonist dimaprit (10 μM). The firing rates, input resistance, synaptic activity of the neurons tested were not affected (n=10 and n=9, respectively). Data are summarized in ().
We then questioned whether the preoptic GABAergic neurons that were inhibited by histamine were thermosensitive. The thermosensitivity of the neurons (see Methods) was measured before and during incubation with R-α-methylhistamine (1μM) (). We found that out of 9 GABAergic neurons studied 4 were warm-sensitive with an average thermal coefficient of 0.9 ± 0.1 impulses s−1 °C−1 (n=4). Three of them were inhibited by the H3R specific agonist, and their thermal coefficients decreased to 0.4 ± 0.2 impulses s−1 °C−1 (p<0.05, paired t-test). Recordings from one of these neurons are presented in . The temperature-insensitive neurons displayed an average thermal coefficient of 0.4 ± 0.3 impulses s−1 °C−1 (n=5). Conversely, the firing rate of only one out of the 5 temperature-insensitive neurons tested was reduced by R-α-methylhistamine (1μM). Its thermosensitivity decreased from 0.4 to 0.3 impulses s−1 °C−1.
Histamine acting at H3 subtype receptors reduces the firing rate and thermosensitivity of preoptic GABAergic neurons
Histamine actions on the spontaneous activity of preoptic non-GABAergic neurons
None of 29 preoptic non-GABAergic (i.e. GFP-negative) neurons studied were inhibited by histamine (3 μM)(n=14) or R-α methylhistamine (1μM)(n=11), however, a population of GFP-negative neurons (5 out of 29) was excited by histamine. The spontaneous firing rates averaged 3.5 ± 2.7 Hz (n=5) and was increased by the neurotransmitter by 94 ± 43 % (n=5). This excitation was blocked by the H1R specific antagonist trans
triprolidine (1μM) in all neurons tested (n=5) (). The maximal excitatory effect was reached at histamine concentrations of 20 μM or higher. At 20 μM histamine increased the firing rate by 361 ± 137 % (n=12, ANOVA P<0.05) in 12 out of 45 neurons tested. The excitatory effect was associated with a depolarization of 2–7 mV (average 3.1 ± 2.0, n=12) in all neurons (). The actions of histamine (20 μM) were blocked by trans
triprolidine (1μM, n=3) or mepyramine (100 nM, n=5). In contrast, the H2R antagonist tiotidine (100 nM) did not affect the histamine effects in all neurons tested (n=5). The H1R agonist 2-pyridylethylamine (100 μM) mimicked these responses: an increase in firing rate by 286 ± 191% (n=8) and a depolarization by 4.0 ± 2.7 (n=8) (). Also the actions of 2-pyridylethylamine (100 μM) were fully blocked by the H1R antagonist mepyramine (100 nM) and were not affected by the H2R antagonist tiotidine (100 nM). At a lower concentration, 2-pyridylethylamine (10 μM) increased the firing rate by 215± 89 % (n=4). Finally, we have also tested betahistine a partial H1R agonist/H3 antagonist that diplays little affinity for the H2R (Arrang et al., 1985
). Betahistine (100 μM) excited 4 of 15 GFP-negative neurons tested. The betahistine effects were fully blocked by the H1R antagonist mepyramine (100 nM) and were not affected by the H2R antagonist tiotidine (100 nM)(data not shown). Non-GABAergic neurons recorded in voltage-clamp mode displayed an inward current in response to the H1R agonist (), that averaged 21 ± 8 pA (n=8) at −50 mV holding potential. In a subset of these neurons (5 out of 8) we also noticed an increase in the frequency and amplitude of sEPSCs (). The frequency of sEPSCs increased by 94 ± 41% (n=5, paired t-test, P<0.05) () while their average amplitude increased by 105 ± 37% (n=5, paired t-test, P<0.05). It is interesting to note that in GFP-negative neurons in which H1R agonist did not induce an inward current we did not find any effects on sEPSCs (n=11). In the presence of TTX, histamine or the H1R agonist induced an inward current (average 16 ± 5 pA, n=6) and decreased the input resistance of the neuron by 24 ± 7% (n=6, paired t-test, P<0.05) but did not affect the properties of miniature (m)EPSCs in all neurons studied (n=6)(). Finally, histamine activated an inward current (average 17 ± 7 pA, n=3) when ionotropic glutamate receptors were blocked with CNQX (20 μM) and AP-5 (50 μM). These results indicate that the histamine depolarizes non-GABAergic preoptic neurons by a postsynaptic mechanism. Our data also suggest that some H1R expressing preoptic neurons are glutamatergic and present reciprocal connections and/or present recurrent collaterals.
Histamine depolarizes and increases the firing rates of a population of GFP-negative neurons by activating H1 subtype receptors.
Effects of H1 subtype receptor activation in preoptic GFP-negative neurons
We also investigated whether the excitatory effects of histamine were present selectively in warm-sensitive or in temperature-insensitive neurons. We found, however that all non-GABAergic neurons tested were temperature-insensitive. In spite of the increase in firing rate induced by 2-pyridylethylamine (100 μM) in 5 out of 26 neurons studied, the thermal coefficients of the neurons were not significantly affected: their thermosensitivities averaged 0.4 ± 0.3 impulses s−1 °C−1 (n=5) and 0.5 ± 0.2 impulses s−1 °C−1 (n=5) (paired t-test, p>0.1) in control and during H1R agonist application, respectively. The characteristics of sIPSCs were not affected by the H1R agonist in the GFP-negative neurons studied (n=9) ().
Finally, the H2R specific agonists dimaprit (10 μM, n=8) and amthamine (1 μM, n=5 and 10 μM, n=5) was without effect on the membrane potential, firing properties or synaptic activity of non-GABAergic preoptic neurons.
Lack of effects of the H3R agonist on the spontaneous release of glutamate and GABA
Since H3Rs are present at presynaptic terminals in some central neurons (Garduno-Torres et al., 2007
) we have studied the effects of the H3 agonist on the properties of mIPSCs in a group of preoptic neurons. These recordings were carried out using the Cs pipette solution at a holding potential of −10 mV as previously described (Tabarean et al., 2006
). In either GFP-positive (n=6) or GFP-negative neurons (n=5) the H3R agonist did not affect the characteristics (frequency, amplitude and kinetics) of the mIPSCs (). Finally, the H3R agonist did not affect the properties of mEPSCs in any neurons studied (n=7 GFP- positive and n=6 GFP-negative) ().
Histamine effects in cultures of PO/AH neurons
We have previously characterized PO/AH neurons in culture and found that they share most properties of PO/AH neurons in slices (Tabarean et al., 2005
). Here we have studied the effects of histamine in this culture system and obtained very similar results to those presented above. Briefly, histamine (10 μM) increased the firing rates of PO/AH neurons by 232 ± 110 % (n=10), effects accompanied by depolarization (2–5 mV) and/or increased frequencies and amplitudes of sEPSCs in 14% of neurons studied (9 out of 62 tested). This action was mimicked by 2-pyridylethylamine (100 μM) and blocked by trans
triprolidine (1μM) in all neurons tested (n=8) suggesting it was caused by activation of H1Rs. Blocking fast synaptic activity (by adding 20 μM CNQX, 100 μM AP-5 and 20 μM bicuculline to the extracellular solution) did not affect the excitatory action of histamine (10 μM): 3 of 14 neurons tested displayed a depolarization of 2–5 mV and increased their firing rates by 195 ± 85 % (n=3).
In a distinct population of cultured PO/AH neurons (11 out of 62, ~18%) the firing rate was reduced by histamine (1–10 μM) acting at H3Rs: the effect was mimicked by R-α-methylhistamine (1 μM) (data not shown) and blocked by thioperamide. Also this effect of histamine was not dependent on synaptic activity since it was not affected when fast synaptic activity was blocked. The reduction in firing rate averaged 55 ± 32 % (n=11) and 60 ± 25 (n=3) in control and during synaptic block, respectively (ANOVA P>0.1). As it was the case in slices, the properties of mIPSCs and mEPSCs were not affected by histamine or by the H1R or H3R specific agonist (data not shown). The H3R agonist reduced the frequency of sIPSCS in ~ 22% (7 out of 31) of neurons studied by 46 ± 23% (n=7) (data not shown) and did not affect sEPSCs in the neurons studied (n=21). This discrepancy with the data recorded in slices may be due to different networking: in slices the H3R- expressing GABAergic neurons may send few local projections, while in dissociated cultures all projections are “local”.
Finally, the H2R specific agonists dimaprit (10 μM, n=12) and amthamine (1 μM, n=6 and 10 μM, n=8) were without effect on the membrane potential, firing properties or synaptic activity of PO/AH neurons. Thus, it appears that the postsynaptic responses to histamine in the two preparations were very similar, and consequently we used the cell culture preparation for [Ca2+]i imaging and immunocytochemistry experiments aimed at understanding the signaling pathways involved in the histamine actions.
Signaling pathways activated by H1Rs and H3Rs in preoptic neurons
In previous studies H3R signaling involved either PKA, the extracellular regulated MAP kinase (ERK) or Ca2+
release from intracellular stores (Drutel et al., 2001
; Chen et al., 2003
; Haas and Panula, 2003
). To determine which pathway(s) are involved in H3R signaling in PO/AH neurons we have carried out Ca2+
imaging experiments, immunocytochemistry for pERK and phosphorylated CRE binding protein (pCREB) (as a measure of the activation of the PKA pathway), as well as pharmacological experiments to block the phosphorylation of ERK.
We have first investigated whether blocking the phosphorylation of ERK with the MEK1/2 inhibitors U0126 or PD98059 prevents the electrophysiological effects induced by H3R activation. After measuring the initial inhibitory effect of the H3R agonist, we applied a MEK1/2 inhibitor for 3–5 min. We found that the treatment gradually decreased the firing rate of the neuron and occluded the effect of the H3R agonist (). Similar results were obtained in 8 other preoptic GABAergic neurons in slices as well as in 7 PO/AH cultured neurons. The results with U0126 (20 μM) and PD98059 (10 μM) were very similar and therefore were pooled together. The MEK1/2 inhibitors decreased the firing rate by 71 ± 17 % (n=9) in slices and 85 ± 21 % (n=7) in cultures. Interestingly non-GABAergic preoptic neurons were not affected by the two inhibitors (n=11). The responses of non-GABAergic preoptic neurons to the H1 agonist 2-pyridylethylamine (100 μM) were not affected by pre-incubation with the MEK1/2 inhibitors (n=3, data not shown).
A decrease in the level of phosphorylated ERK accounts for the effects of histamine at H3 subtype receptors.
We then examined the effect of histamine on the intracellular Ca2+ concentrations [Ca]i in cultured PO/AH neurons loaded with fura-2AM. Fast synaptic activity was blocked as above. We found again two patterns of responses in different populations of neurons: in 15% of neurons (49 out of 327) histamine induced a robust increase in [Ca]i (see below) while in 22% of neurons (72 out of 327) we saw a clear decrease in [Ca]i. The latter responses were mimicked by the H3R agonist () while the former were mimicked by the H1R agonist (see below). Interestingly, TTX (1 μM) decreased [Ca]i to a larger extent, in the same neurons and prevented the effect of the H3R agonist (). Similarly, incubation with the MEK1/2 antagonists resulted in a decrease in [Ca]i and prevented a further decrease in response to the H3R agonist (). The effect of the MEK1/2 antagonists on [Ca]i was abolished in the presence of TTX (data not shown). These results suggested that activation of H3Rs reduces the firing rate of PO/AH neurons by an ERK-dependent mechanism an that the measured decrease in [Ca]i reflects less firing activity (and consequently less Ca2+ entry through voltage-gated Ca2+ channels) rather than an effect on Ca2+ stores. Changes in level of pERK and of pCREB in response to H3R agonist incubation (1 μM, 3 min, fast synaptic activity was blocked as above) were measured using immunocytochemistry. We found no evidence for changes in the level of pCREB, however that of pERK was clearly affected (). In control conditions 48 ± 8% of neurons (n= 372) were pERK positive (see Methods) while the percentage dropped to 27 ± 6% (n=405) after treatment with H3R agonist () (ANOVA, P<0.05). Almost no pERK-positive cells could be found in the presence of TTX (3 ± 2%), suggesting that in most PO/AH neurons a high level of pERK reflects firing activity. No effect of the H3R agonist on the level of pERK could be detected in the presence of TTX ().
As mentioned above histamine induced a robust increase in [Ca2+]i in a distinct population of PO/AH neurons. This effect was mimicked by 2-pyridylethylamine (100 μM) (), was blocked by trans triprolidine (1 μM) in 25 out of 25 tested neurons (not shown) and was observed in normal extracellular medium as well as in the presence of TTX (), suggesting that it reflects Ca2+ release from intracellular stores, rather than an increase in firing rate. The responses to 2-pyridylethylamine (100 μM) were blocked by mepyramine (100 nM, n=15) and were not affected by the H2R antagonists tiotidine (100 nM, n=15) (data not shown). Betahistine (100 μM) also activated an increase in [Ca2+]i that was blocked by mepyramine (100 nM, n=11) and was insensitive to tiotidine (100 nM, n=11) (data not shown).
Activation of H1 subtype receptors results in activation of PLC and Ca2+ release from intracellular stores.
Since H1R signaling is usually associated with the PLC pathway (reviewed in (Haas and Panula, 2003
)) we have tested the effect of PLC antagonists on the H1R agonist-induced increase in [Ca2+
. The [Ca2+
responses were abolished by preincubation with the PLC antagonists U73122 (5 μM) () or 1-O-Octadecyl-2-O-methyl-sn
-glycero-3-phosphorylcholine (10 μM) in all cells studied (n=157). Similarly, the H1R agonist- induced inward current was blocked by either of the PLC antagonists () in all neurons studied in slices (n=6) and in culture (n=9). These results suggest that both the inward currents and the [Ca2+
responses to H1R activation are mediated by the activation of the PLC pathway.
H1 and H3 histamine receptors expression in preoptic neurons
To reveal the cellular distribution of these receptors we have attempted immunohistochemistry experiments, however the commercially available antibodies tried by us did not yield specific binding in our preparations. We then carried out single cell reverse transcription- PCR (sc RT/PCR) analysis of the H1, H2 and H3 histamine receptor as well as of the GAD67 mRNA transcripts in 19 preoptic neurons in slices from 4 different wild-type C57/bl6 mice. Negative (−) control was amplified from a harvested cell without reverse-transcription, and positive control (+) was amplified using 15 ng of hypothalamic mRNA. Other controls, including samples of the pipette and bath solutions were negative after RT-PCR (data not shown). As shown in H1 and H3 receptors were expressed in different subpopulations of preoptic neurons from slices. The H2R could not be detected in any of the 19 neurons (). Out of the 19 preoptic, 9 neurons were GAD67 positive (47%), 4 were H3-positive (21 %) and 2 neurons were H1-positive (10%).
H1R, H2R, H3R and GAD67 transcripts are present in preoptic neurons in slices
We then carried out similar experiments in 21 preoptic neurons in slices from GAD65 GFP mice. In this set of neurons we have characterized the electrophysiological responses to histamine (20 μM) before harvesting the cytoplasm in the patch pipette. In these cells we have also analysed the presence of GAD65 transcripts. Cells 1–9 and 16–21 were GFP-negative and among them cells 3, 4, and 5 were depolarized by histamine (20 μM) while the rest were not affected. Cells 9–16 and were GFP-positive. Histamine (20 μM) decreased the spontaneous firing rate in cells 9–12 and was without effect in the rest. The sc RT/PCR analysis revealed again that H1 and H3 receptors were expressed in distinct sets of neurons and that the excitatory effects of histamine were present in H1 positive neurons (cells 3 and 4, i.e. in 2 out of 3 cells) while the inhibitory effects of the neurotransmitter were associated with the presence of the H3R (cells 9–12, i.e. 4 out of 4 cells) (). The H3R was expressed only in 4 GFP positive cells while the H1R was present in two GFP negative cells (). GFP positive neurons expressed GAD 65 (8 out of 8) as well as GAD 67 (7 out of 8)(). GAD65 was absent in all 13 GFP-negative neurons while GAD 67 was detected in one GFP-negative neuron (not shown). All H1-positive neurons were GAD65 and GAD 67 negative (2 out of 2) while all the H3-positive neurons expressed both GAD65 and GAD67 (4 out of 4). H2R was not detected in any of the 21 recorded preoptic neurons (). Since previous reports suggested the presence of H2R binding sites and transcripts in the PO/AH (Ruat et al., 1990
; Vizuete et al., 1997
) we have been surprised by their absence in our studies and carried out some additional positive controls. Using the same experimental conditions and primers we have been able to detect H2R transcripts in granule cells of the dentate gyrus (2 out of 5 cells) and dorsomedial hypothalamus (1 out of 3 cells)( ). Nevertheless we cannot rule out the expression of low levels of the H2R transcripts in preoptic neurons that are not detectable by sc RT/PCR, or their presence in only a very small proportion of neurons.
H1R, H2R, H3R, GAD65 and GAD67 transcripts in preoptic neurons recorded in slices from GAD65-GFP transgenic mice.
Figure 9 H1R, H2R, H3R transcripts in hippocampal, DMH, and preoptic neurons in slices from GAD65-GFP mice. Representative gels illustrating the expression of H2Rs (A), H1Rs (B), H2R (C) in 2 granule neurons from the dentate gyrus (1–2), three DMH neurons (more ...)
We have also studied the presence of transcripts of the five genes in a set of 10 cultured PO/AH neurons after recording their responses to histamine (20 μM). In two (out of two) neurons excited by histamine we have detected H1Rs and none of the other 4 genes. In two (out of two) neurons inhibited by histamine (20 μM) we have detected H3Rs, GAD65 and GAD67 but not H1R or H2R. Out of 6 neurons not affected by histamine none expressed H1R, H2R or H3R, two expressed both GAD65 and GAD67, one neuron expressed GAD65 only, while the remaining three did not present transcripts of either isoform.
Effects of intra-MnPO injection of histamine, H1R agonist and H3R agonist on core body temperature and motor activity
In order to assess its effect on core body temperature (CBT) and motor activity (MA), histamine (10 or 30 μM) or aCSF (control) were injected in the MnPO via a cannula (see Methods). Experiments were carried out in parallel in 3 groups of 6 mice. The neurotransmitter induced a dose-dependent hyperthermia when injected in the MnPO (, upper panel), an effect which was not accompanied by increased motor activity (, lower panel), suggesting that it was due to changes in thermoregulation (e.g. BAT thermogenesis). Within 1 h of the injection (0.2 μL) of 10 or 30 μM histamine the CBT started to increase and reached a maximum after ~2.5 h. In contrast, aCSF injections (0.2 μL, control) did not result in hyperthermia. The responses to histamine were statistically different to the control (ANOVA, P<0.05 and P<0.01 for the two doses of histamine, respectively).
Intra-MnPO injection of histamine receptor agonists induces hyperthermia
To determine the receptor subtypes involved we have then injected histamine receptor subtype specific agonists. The H3R agonist (10 μM, 0.2 μL) induced a rapid increase in body temperature which was almost identical with that induced by histamine (30 μM) with no effect in MA (). The response to H3R agonist was not statistically different to that induced by histamine (30 μM) (ANOVA, P>0.1). Since inhibition of ERK phosphorylation mimicked the effects of H3R agonist in our in vitro experiments we also injected the MEK-1 inhibitors PD98069 (20 μM, 0.2 μL) or U0126 (30 μM, 0.2 μL) intra MnPO. PD98069 induced a hyperthermia of similar timecourse to that of the H3 agonist but of smaller amplitude, with no changes in MA (). Nevertheless, the response was different to the control starting at 1 h after injection and up to 6 h later (P<0.05). Similar results were obtained also with the other MEK-1 inhibitor U0126 (not shown).
Intra-MnPO injection of H1R agonist (100 μM, 0.2 μL) or aCSF (control) was carried out in parallel in other two groups of cannulated mice (n=6 each). The agonist induced a persistent hyperthermia (ANOVA, P<0.01 when compared to the aCSF control) which reached a maximum at ~2h after injection and lasted for at least 8 h (, upper panel). No changes in the MA were induced by the H1R agonist (, lower panel).