|Home | About | Journals | Submit | Contact Us | Français|
The preoptic area/anterior hypothalamus (PO/AH), a region that contains neurons that control thermoregulation, is the main locus at which histamine affects body temperature. Here we report that histamine reduced the spontaneous firing rate of GABAergic preoptic neurons by activating H3 subtype histamine receptors. This effect involved a decrease in the level of phosphorylation of the extracellular signal-regulated kinase (ERK) and was not dependent on synaptic activity. Furthermore, a population of nonGABAergic neurons was depolarized and their firing rate was enhanced by histamine acting at H1 subtype receptors. In our experiments, activation of the H1R receptors was linked to the PLC pathway and Ca2+ release from intracellular stores. This depolarization persisted in TTX or when fast synaptic potentials were blocked indicating that it represents a postsynaptic effect. Single-cell reverse transcription –PCR analysis revealed expression of H3 receptors in a population of GABAergic neurons while H1 receptors were expressed in nonGABAergic cells. Histamine applied in the median preoptic nucleus induced a robust, long lasting hyperthermia effect that was mimicked by either H1 or H3 histamine receptor subtype specific agonists. Our data indicate that histamine modulates the core body temperature by acting at two distinct populations of preoptic neurons which express H1 and H3 receptor subtypes, respectively.
A role of CNS histamine signaling in thermoregulation has been found in various organisms from invertebrates (Hong et al., 2006) to lower vertebrates (Leger and Mathieson, 1997) and mammals (Green et al., 1976). The neurons of the tuberomammillary nucleus represent the main source of histamine in the brain. They project their axons throughout the brain and control arousal, attention, energy expenditure, feeding, and thermoregulation. Histaminergic fibers are especially dense in the cortex, hypothalamus, amygdala and striatum (reviewed in (Haas and Panula, 2003). In the hypothalamus they are particularly dense in the anterior part (Wada, 1992). The preoptic area/anterior hypothalamus (PO/AH), region which contains temperature-sensitive neurons and regulates the thermoregulatory “set point”, appears to be the main locus in which histamine affects body temperature (Colboc et al., 1982). Histamine injected in the PO/AH induces hyperthermia. Similarly, intra-PO/AH injection of a histamine-N-methyltransferase inhibitor, which results in a local increase of histamine concentration, also produces hyperthermia (Gatti and Gertner, 1984). Warm-sensitive neurons in the PO/AH are involved in thermoregulation by sensing local temperature, by integrating peripheral thermal information and by responding to endogenous pyrogens and cryogens (reviewed in Simon, 2000; Boulant, 2006). However, the neurochemical properties and the local networks formed by preoptic warm-sensitive neurons are not known. Recent studies have revealed that preoptic GABAergic neurons project to, and control the activity of thermoregulatory neurons in the rostral raphe pallidus (rRPa) and dorsomedial hypothalamus (DMH) (Morrison et al., 2008).
Histamine actions in the brain are mainly excitatory and involve H1 or H2 subtype receptors. Activation of H1 receptors (H1R) results in depolarization and increased firing rate by either the activation of a cationic current (Gorelova and Reiner, 1996; Smith and Armstrong, 1996; Zhou et al., 2006) or by a decrease in a K leak conductance (McCormick and Williamson, 1991; Munakata and Akaike, 1994; Reiner and Kamondi, 1994; Whyment et al., 2006). Similarly, activation of H2 receptors increases excitability by inhibiting K currents (Greene and Haas, 1990; Munakata and Akaike, 1994; Atzori et al., 2000). H3Rs are present at presynaptic terminals and have inhibitory actions on the release of various neurotransmitters in the brain (reviewed in (Brown et al., 2001)). However, postsynaptic inhibitory actions have also been described (Zhou et al., 2006).
An early extracellular study has shown that histamine has mainly excitatory effects on preoptic neurons (Tsai et al., 1989). The cellular and molecular mechanisms underlying these actions are not known. While histamine influences thermoregulation by acting in several hypothalamic regions, we focus in this study on histamine signaling in the median preoptic nucleus (MnPO) because this region appears to be critical in basal thermoregulation as well as in the fever response (Lazarus et al., 2007; Nakamura and Morrison, 2007). Here we study the effects of histamine on identified preoptic GABAergic and non-GABAergic neurons as well as the histamine receptors and signaling pathways involved in these actions to test the hypothesis that the neurotransmitter modulates core body temperature (CBT) by modifying the activity of warm-sensitive preoptic neurons. We also investigate the histamine receptor subtypes involved in the hyperthermic response to histamine.
Coronal tissue slices containing the median preoptic nucleus (MnPO) were prepared from GAD65-GFP mice (28–42 days old) housed in standard conditions. This transgenic mouse line expresses enhanced green fluorescent protein (eGFP) under the control of the regulatory region of mouse glutamic acid decarboxylase (GAD) 65 gene. The mice were a kind gift from Dr Gabor Szabo (Hungarian Academy of Sciences, Budapest). An animal was anesthetized using isoflurane and sacrificed by decapitation, according to procedures approved by the TSRI Animal Welfare Committee. The brain was quickly removed and placed in ice cold artificial cerebrospinal fluid (aCSF) for 2 min. A tissue block containing the hypothalamus was prepared, glued to the stage of a Vibratome 3000 tissue slicer and placed into the bath of the slicer containing ice-cold oxygenated ACSF. 300 μm thick slices were cut and quickly transferred to the recording bath where they were continuously perfused with oxygenated ACSF and allowed to equilibrate for 2 h at 36–37 C before recording. Patch clamping under visual control was performed with an upright microscope (BX-50, Olympus) equipped with an ×40 water immersion objective and with fluorescent illumination. We must note that we have selected only brightly fluorescent neurons throughout this study as “GFP positive” and neurons lacking any fluorescence as “GFP-negative”. Neurons displaying slight fluorescence were not studied. The slice used in our recordings corresponded to the sections located from 0.5 mm to 0.25 mm from Bregma in the mouse brain atlas (Paxinos, 2001) in order to coincide with the canulla location used in our in vivo experiments (see below). Recordings were made from neurons in the median preoptic nucleus (MnPO) and nearby medial preoptic area (MPA) since these regions are not clearly separated in these sections. Although the in vivo injections (a volume of 0.2 μL) were directed to the MnPO (see below) some spread to nearby MPA neurons is expected. No differences were noticed between recordings from MnPO and MPA neurons and thus results were pooled together and the neurons were referred to as “preoptic neurons”.
PO/AH cultures were prepared as previously described (Tabarean et al., 2005). The cultures were used at 4–5 weeks after plating. Since at embryonic stage the preoptic area cannot be distinguished from the anterior hypothalamus we refer to these cells as “cultured PO/AH neurons”.
The artificial cerebrospinal fluid (aCSF) contained: 130 NaCI, 3.5 KCI, 1.25 NaH2PO4, 24 NaHCO3, 2 CaCI2, 1 MgSO4, 10 glucose osmolarity of 300–305 mOsm, equilibrated with 95% O2 and 5% CO2 (pH 7.4). Other salts and agents were added to this medium. A K+ pipette solution containing 130 K-gluconate, 5 KCI, 10 HEPES, 2 MgCI2, 0.5 EGTA, 2 ATP, 1 GTP (pH7.3) was used in most experiments. For recordings of miniature IPSCs, at a holding potential of −10 mV a Cs pipette solution containing 135 Cs-methanesulphonate, 5 CsCI, 10 HEPES, 2 MgCI2, 0.5 EGTA, 2 ATP, 1 GTP (pH 7.4) was used. For cell-attached recordings the pipette was filled with aCSF. Glass micropipettes were pulled with a horizontal puller (Sutter Instruments) using Sutter borosilicate glass capillaries with filament. The electrode resistance after back-filling was 2–4 MΩ. No holding current was applied to neurons when recording spontaneous activity in response to temperature changes. All histamine receptor ligands were purchased from Tocris while the other substances were purchased from Sigma. The recording chamber was constantly perfused with aCSF (2–3 mL/min). In electrophysiological experiments all histamine receptor agonists were applied locally using a perfusion pencil system (tip diameter 100 μm, Automate Scientific) driven by gravity. All the other treatments were bath-applied.
The temperature of the external solution was controlled with an HCC-100A heating/cooling bath temperature controller (Dagan Corporation) equipped with a Peltier element. To prevent changes induced in the electrode reference potential, the ground electrode was thermally isolated in a separate bath connected to the recording bath by a filter paper bridge.
Data was acquired with a MultiClamp 700B amplifier (Axon Instruments) digitized using a Digidata 1320A (Axon Instruments) interface and the Pclamp9.2 software package (Axon Instruments). The sampling rate for the continuous recordings of spontaneous activity was 50 kHz. After establishing whole-cell configuration (or cell-attached) the spontaneous activity of the neuron was recorded for 2–4 min to determine its control behavior at (36–37 °C). The spontaneous firing activity of the neurons was recorded in either whole-cell mode (current clamp, I=0) or in cell-attached mode (voltage-clamp, V=0). The thermal coefficient of a neuron is defined as the slope of the linear regression fitted to the firing rate vs temperature plot. As in previous reports (Tabarean et al, 2004, 2005) the temperature was varied in the temperature range 34–40 °C and the thermal coefficient was determined over a temperature range of at least 3 °C. A neuron having a thermal coefficient higher than 0.8 impulses s−1 °C was defined as warm-sensitive. The values of the membrane potential indicated in the figures are those of the potential at the beginning of the illustrated sweeps or, where indicated, of the horizontal reference dotted lines. The input resistance of the neurons was determined as the slope of the fit to the linear region of the I–V curve, obtained by injections of hyperpolarizing square current pulses.
Synaptic potentials were detected automatically using the Mini Analysis program (Synaptosoft). The frequency, amplitudes, and kinetics of synaptic events were analyzed using MiniAnalysis software. The statistical significance of differences between cumulative distributions was estimated with the Kolmogorov-Smirnov (K-S) parametric test.
The values reported are presented as mean ± standard deviation (S.D.). Statistical significance of the results pooled from several cells was assessed with paired t-test using Sigmaplot software (Systat, Richmond, CA, USA). Non-parametric one way analysis of variance (ANOVA, Kruskal-Wallis) with Dunn’s post hoc test (P<0.05) was used for comparison of multiple groups (Prism4, GraphPad Software).
Fura-2 fluorescence signals were acquired with a CCD camera (Hamamatsu ORCA-ER) connected to its frame grabber operating in 8-bit mode and driven by Slidebook software (Intelligent Imaging Innovations, Denver CO USA). An ultra high speed wavelength switcher Lambda DG-4 (Sutter Instruments) equipped with model 340HT15 and 380HT15 filters provided alternating excitation for ratiometric fura-2 measurements. The illumination source was a standard Xenon lamp. The sampling frequency of 0.5 Hz was sufficiently fast given the relatively slow responses to histamine or histamine receptor agonists. At this excitation frequency photobleaching and phototoxicity were minimal. Fura-2AM (5mM) was loaded by incubation for 30–60 min at room temperature, in the dark, in 1.5 mM of pluronic acid (Molecular Probes, Eugene, OR) in a HEPES-buffered external solution (155 NaCl, 3.5 KCl, 2CaCl2,1 MgSO4, 10 glucose, 10 HEPES, pH=7.4). Coverslips containing cell cultures were placed in a laminar flow perfusion chamber (Warner Instrument Corp.) and constantly perfused with HEPES-buffered saline (HBS). During Ca2+ imaging experiments solutions were exchanged by switching the bath solution. Following subtraction of background fluorescence, the ratio of fluorescence intensity at the two excitation wavelengths was calculated using Slidebook software.
Coverslips were washed by immersion in HBS. The H3 agonist R-α-methylhistamine (1 μM) or aCSF (control) were applied for 1 min, after which the coverslips were washed in HBS, fixed in ice-cold 4% paraformaldehyde for 30 min, and then incubated for 10 min at room temperature in PBS containing 0.25% Triton X-100. Nonspecific sites were blocked by incubation in PBS containing 10% normal goat/horse serum. For double immunostaining, the coverslips were incubated in 2% normal goat serum containing an antibody against phosphorylated CRE binding protein (pCREB ) (1:1000 dilution, Cell Signaling Technology, Beverly, MA), and antibodies against phospho-extracellular signal-regulated kinase (pERK) (1:400 dilution, Cell Signaling Technology, Beverly, MA) for 2 h at 37°C. The specificity of the antibodies was checked by the supplier and by previous studies by others. Specific binding was detected by using secondary antibodies conjugated to AlexaFluor dyes(594, red; 488, green; Molecular Probes). Images were collected on a Delta Vision Optical Sectioning Microscope consisting of an Olympus (Melville, NY) IX-70 microscope. A Photometrics CH 350 cooled CCD camera and a high-precision motorized XYZ stage were used to acquire multiple consecutive optical sections at a 0.24 μm interval for each of the fluorescent probes by using a 60× oil objective. The settings of the confocal were kept constant so that the images could be analyzed by densitometry and the different conditions could be compared. The experiments were repeated on two different cultures of PO/AH neurons. For cell imaging, 10 different fields in each coverslip were captured, and all neurons in the pictures were analyzed for their green and red fluorescence content. Backgrounds were defined as the fluorescence intensity in non-neuronal fields and subtracted from the values obtained for each neuron. After background subtraction some neurons displayed little pERK immunofluorescence while others displayed a very high level of immunofluorescence. In contrast, in the presence of TTX (1 μM), all neurons displayed only a small level of immunofluorescence. Using Image J software (NIH) we have quantified the intensity of the fluorescent signal as integrated density for all the neurons studied. The integrated density values for the pERK immunofluorescence in the presence of TTX were similar to the lower ones obtained in normal external solution. The average of the integrated density values in TTX was defined as the “basal” level and neurons displaying values of at least 50% higher than this level were considered “pERK positive”. In contrast, using a similar analysis, we found that PO/AH neurons displayed a wide range of values of pCREB immunofluorescence, that were not affected by TTX.
Cultured PO/AH neurons were patch-clamped and then harvested into the patch pipette by applying negative pressure. The content of the pipette was expelled in a PCR tube. First strand buffer (Invitrogen), 50 ng random primers (Invitrogen) and H2O were added to each cell to a volume of 16 μl. The samples were incubated at 65°C for 5 min and then put on ice for 3 min. dNTPs (0.5 mM, Invitrogen), DTT (5 mM, Invitrogen), RNaseOUT (40 U, Invitrogen) and SuperScriptIII (200 U, Invitrogen) were added to each sample to a volume of 20 μl followed by incubation at room temperature for 5 min, at 50°C for 30 min and then at 70°C for 15 min. After reverse transcription samples were immediately put on ice.
Gene specific primers for GAD65, GAD67, H1R, H2R, and H3R were designed using Bisearch (http://bisearch.enzim.hu). All primers are displayed in Table 1. cDNAs were amplified in a volume of 25 μl using a high-fidelity Platinum Taq polymerase kit (Invitrogen) and 0.4 mM dNTPs (Invitrogen). For each gene 1/10 of the cDNA from each cell was subjected to a first round of PCR using the outer primer pair and a thermal cycling program with an initial denaturation at 94°C for 2 min, 35 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 30 s and extension at 68°C for 45 s followed by a final extension at 68°C for 10 min. 1 μl of the PCR product was subjected to a second round of PCR using the inner primer pair and 40 cycles of the same thermal cycling program as above with the extension time reduced to 30 s. PCR products were visualized by ethidium bromide stained 2% agarose gel electrophoresis. For each gene the identity of the PCR product was confirmed by sequencing (The Scripps Research Institute Center for Nucleic Acid Research) after extraction of the product cDNA from the electrophoresis gel using a Quiaquick Gel Extraction kit (Quiagen).
Using sterile surgical techniques an incision was made on the abdomen to separate the skin. The anterior abdominal wall was opened and the radio transmitter was implanted into the peritoneal cavity. Following appropriate wound closure, the animals were allowed two weeks for full recovery prior to the study. Animal maintenance and experimental procedures are in accordance with the “National Institutes of Health Guide for the Care and Use of Laboratory Animals” (Publication No. 85-23, revised 1985). The telemetry system for monitoring vital signs consists of a surgically implanted radio transmitter (TA10TA-F20™, Data Sciences, Inc.) and a receiver (RPC-1™, Data Sciences, Inc.). Core body temperature (CBT) and motor activity (MA) sensors were located in the transmitter implant. The cages were positioned onto the receiver plates. Radio signals from the animals’ CBT and MA were continuously monitored by a fully automated data acquisition system (Dataquest™ A.R.T.™, Data Sciences, Inc). C57/Bl6 adult males (12–14 weeks old) were used for the in vivo experiments.
Mice were anaesthetized (induction 3–5%, maintenance 1–2% isoflorane) via a nose cone and placed into a stereotaxic apparatus. Body temperature was monitored and maintained at 37 ± 0.1 °C by a feedback regulated heating pad. Using sterile surgical techniques an incision was made to separate the skin. The skull was exposed and three holes were drilled. Two of them were used for screws (diameter 2 mm and length 5 mm) and one of them for the cannula implant (27 ga. 16 mm length). Coordinates for cannula implants in the median preoptic nucleus (MnPO): from Bregma: 0.38 mm and ventral 4.6 mm (Paxinos, 2001). After placement the cannula was secured using dental cement filling out the spaces including the screws. Following surgery each mouse was housed in individual Plexiglas recording cages placed in environmentally controlled chambers. The ambient temperature was maintained at ~30 ± 0.5 °C in a 12:12-h lightdark cycle controlled room (lights on 6:00 am). Food and water was available ad libitum. Following surgical implantation and appropriate wound closure, the animals were allowed to recover for two weeks prior to the study. Histamine receptor agonists or the MEK1 inhibitors (U0126 and PD98059) were injected into the MnPO. All substances injected were dissolved in sterile aCSF. Mice were handled for at least three days before the injection during 5 minutes every day for habituation. On the day of injections, mice were held and the injector (cannulae 33 Ga, 17 mm length) was placed inside the cannula. The injector was connected to a microsyringe (1.0 μL). The injected volume was 0.2 μL (rate 0.1 μL/min). After this procedure the animal was returned to the home cage. In some experiments we first injected aCSF and after 30 min we carried out a second injection with aCSF or histamine agonist. This sequence allows for a smaller handling–induced hyperthermic spike in response to the second injection and thus allows a better observation of the early phase of the response to histamine agonists. Animals were used for a maximum of two experiments, with an interval of 10 days. Injections were always performed at 10 am local time, during the “subjective light period”.
The cannula placement was always checked histologically at the end of the experiments. To this end, blue pontamine dye was injected through the cannulae. The animals were then sacrificed and the brains were removed and processed for cryosectioning. The placement of the cannula in the MnPO was confirmed for all mice used in this study by visualizing the sections with an inverted microscope.
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. Fig 1B). 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) (Fig 1A). All neurons inhibited by histamine displayed pacemaker activity (Fig 1B). 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 (Fig 1A) in all neurons tested (n=5). Fig 1D 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 (Fig 1C). 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.
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) (Table 2). The holding current and the input resistance of the neurons were not affected by either histamine or the H3R agonist (Table 2). 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 (Table 2).
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) (Fig 2A). 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 Fig 2. 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.
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) (Figure 3A). 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 (Fig 3A). 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) (Fig 3B). 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 (Figure 4A), 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 (Fig 4A–C). The frequency of sEPSCs increased by 94 ± 41% (n=5, paired t-test, P<0.05) (Table 2) 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)(Table 1). 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.
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) (Table 2).
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.
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 (Table 2). Finally, the H3R agonist did not affect the properties of mEPSCs in any neurons studied (n=7 GFP- positive and n=6 GFP-negative) (Table 2).
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.
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 (Fig 5A). 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).
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 (Fig 5B) 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 (Fig 5B). 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 (Fig 5C). 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 (Fig 5D). 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 (Fig 5D) (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 (Fig 5D).
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) (Fig 6A), 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 (Fig 6A), 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).
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+]i. The [Ca2+]i responses were abolished by preincubation with the PLC antagonists U73122 (5 μM) (Fig 6A) 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 (Fig 6B) in all neurons studied in slices (n=6) and in culture (n=9). These results suggest that both the inward currents and the [Ca2+]i responses to H1R activation are mediated by the activation of the PLC pathway.
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 Fig 7 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 (Fig 7C). Out of the 19 preoptic, 9 neurons were GAD67 positive (47%), 4 were H3-positive (21 %) and 2 neurons were H1-positive (10%).
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) (Figure 8A, B). The H3R was expressed only in 4 GFP positive cells while the H1R was present in two GFP negative cells (Fig 8A, B). GFP positive neurons expressed GAD 65 (8 out of 8) as well as GAD 67 (7 out of 8)(Fig 8D, E). 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 (Fig 8C). 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)( Fig 9). 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.
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.
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 (Fig 10A, upper panel), an effect which was not accompanied by increased motor activity (Fig 10A, 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).
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 (Fig 10B). 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 (Fig 10B). 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 (Fig 10C, upper panel). No changes in the MA were induced by the H1R agonist (Fig 10C, lower panel).
Previous studies have shown that the preoptic area sends tonic GABAergic inhibition to thermoregulatory centers. Here we report that the majority of identified GABAergic neurons displayed pacemaker activity (86 %), in agreement with such a tonic inhibitory output. We also report for the first time that preoptic warm-sensitive neurons or at least a subpopulation are GABAergic. The rate of spontaneous firing of most (55%) preoptic GABAergic neurons decreased in the presence of histamine or of an H3R selective agonist. These agonists also decreased the thermosensitivity of the neurons further confirming its plasticity (Tabarean et al., 2004). Since H3Rs are usually present at presynaptic terminals we were surprised to find that the mechanism of inhibition was postsynaptic, however, postsynaptic H3Rs have been recently found also in substantia nigra GABAergic neurons (Zhou et al., 2006). Activation of H1Rs did not affect the firing rate of identified GABAergic preoptic neurons but depolarized and increased the firing rate of a population (17%) of GFP-negative neurons. The frequencies and amplitudes of sEPSCs increased in some of these neurons. These results suggest that H3Rs are present only on preoptic GABAergic neurons while H1Rs are present on putative glutamatergic neurons. It is interesting to note that only subpopulations of the GABAergic and non-GABAergic neurons were affected by the H3 and H1 agonists, respectively, suggesting heterogeneity among these neuronal subclasses. [Ca]i imaging also revealed two types of histamine responses when fast synaptic activity was blocked: in some neurons [Ca]i increased, response attributed to activation of H1Rs, while in others [Ca]i decreased, response attributed to activation of H3Rs. Together these data suggest that two distinct classes of PO/AH neurons express H3Rs and H1Rs, respectively. Indeed, using single cell RT/PCR we found that both in slices and in cultures, H1 and H3 receptors were present in different neurons, the latter being present in GABAergic cells. A similar histamine receptor distribution may be present also in ventromedial hypothalamic nucleus neurons since the neurotransmitter has opposite effects on the firing rates of populations of neurons (Renaud, 1976).
In PO/AH neurons activation of H3Rs resulted, surprisingly, in reduced levels of pERK immunoreactivity. We found no evidence of involvement of the PKA pathway, or of Ca2+ release from intracellular stores. The MEK1/2 antagonists U0126 and PD (which decrease the level of pERK) mimicked the effects of the H3R agonist and occluded the effect of the agonist (or of histamine). In contrast with the result in substantia nigra neurons we did not record outward currents or hyperpolarization in response to the H3R agonist (Zhou et al., 2006). An alternative mechanism for the decrease in firing rate could be that reduced levels of pERK may result in changes in the phosphorylation state of ion channels involved in “pacemaker” firing activity (e.g. a decrease in the phosphorylation state of Kv4.2 results in increased A-type K currents and consequently in a reduced firing frequency (Yuan et al., 2005)). Another surprising finding was that in cultured PO/AH neurons the level of pERK was dependent on neuronal activity and that the H3R agonist was without effect on the pERK level when neuronal activity was blocked. This result raises the possibility that the reduced pERK level reflects a decrease in the firing rate of the neuron and not a direct effect of H3R activation. However, the fact that both MEK1/2 antagonists mimic the effect of the H3R agonist (or histamine) on firing rate and prevent its effect strongly suggests a causal relationship between a decrease in the pERK level and the reduction in firing rate. Taken together our results indicate that the action of the H3R agonist is activity dependent: potently inhibitory when the neuron is active, and with little effect when the neuron is silent. In agreement with this interpretation was also the finding that no electrophysiological responses to the H3R agonist or to the MEK1/2 antagonists were recorded when neuronal activity was blocked with TTX. [Ca]i responses mirrored the effects on firing activity: [Ca]i decreased in response to H3R activation (or to MEK1/2 antagonists) only in the presence of neuronal activity but was not affected in the presence of TTX. These results point to the possibility that the [Ca]i is a physiological modulator which induces changes in the pERK level as a function of neuronal firing. A similar connection was recently suggested for activity-dependent changes in pERK in DRG neurons (Fukui et al., 2007).
In a distinct set of neurons, H1R activation resulted Ca2+ release from intracellular stores and the activation of an inward current via the PLC pathway. The inward current may be due to activation of Ca-sensitive nonselective cationic channels (Zhou et al., 2006) or by a decrease in a “leak” K conductance (Li and Hatton, 1996; Whyment et al., 2006). The decrease in input resistance in the presence of the H1 agonist suggests that the former possibility is more likely.
We have not detected any H2R activity or H2R transcripts in the preoptic neurons studied here. This finding is in agreement with early studies that suggested the presence of H2Rs in medial or periventricular hypothalamic regions that control heat loss mechanisms, while H1Rs were placed in the rostral hypothalamus (Green et al., 1975; Bugajski and Zacny, 1981).
Little is known about the local neuronal networks comprising preoptic neurons involved in thermoregulation. We found that a population of preoptic GABAergic neurons are inhibited by activation of postsynaptic H3Rs. The H3R agonist did not change the frequency of sIPSCs recorded in preoptic neurons (GFP-positive or GFP-negative) in slices which suggests sparse local projections of the H3R positive GABAergic neurons. In contrast, the fact that the H1R agonist–induced inward current was often accompanied by an increase in the frequency of sEPSCs suggests that the putative H1R-positive glutamatergic neurons form reciprocal connections or present recurring collaterals. We must note that although the agonists were applied locally, it is possible that the cell bodies of nearby neurons were also exposed to them. Thus the changes in sEPSC frequency may be due to activation of H1Rs on either the cell bodies or axons of the presynaptic neuron. On the other hand, the lack of effects of the H1R agonist on the frequency of sEPSCs recorded in GABAergic neurons suggests that putative H1R- positive glutamatergic neurons do not project to nearby GABAergic neurons.
The hypothalamic concentration of histamine displays a circadian rhythm (Prast et al., 1992) and is dependent on the animal’s arousal state, therefore may be involved in the circadian changes in CBT and/or in those induced by arousal (Valdes et al., 2005; Valdes et al., 2006). We report here that intra MnPO histamine induced a dose-dependent hyperthermia. Activation of either H1Rs or H3Rs in the MnPO resulted in hyperthermia of similar amplitude. An interpretation of our results is that warm-sensitive preoptic GABAergic neurons that express H3Rs contribute to the inhibitory tone sent to neurons in the DMH and/or rRPA that control thermogenesis. Thus, in the presence of histamine a decrease in the frequency of their tonic firing results in diminished inhibition of downstream thermoregulatory neurons and increased thermogenesis. Our findings may explain the fact that transgenic mice lacking H3Rs display a lower CBT (Takahashi et al., 2002): lack of inhibition by histamine at H3Rs would result in increased activity of preoptic GABAergic neuron which project to rRPA neurons and consequently in decreased thermogenesis.
Interestingly, activation of H1Rs in the MnPO also induced a hyperthermia of similar amplitude to that induced by histamine. Our data in vitro showed that activation of H1Rs in preoptic nonGABAergic neurons resulted in increased glutamate release. However, local GABAergic neurons were not affected by it. Indeed if preoptic GABAergic neurons were excited by H1R activation (directly or via activation of presynaptic neurons) one would expect a decrease in CBT. Taken together our data strongly suggest that preoptic glutamatergic neurons control at least one thermoregulatory mechanism without having the preoptic GABAergic neurons as intermediate. The wiring of preoptic glutamatergic neurons within thermoregulatory networks is not known. One possibility is the network comprising vasodilator neurons that receive excitatory input from the PO/AH (Nagashima et al., 2000). However, it would be expected that activation of H1Rs in preoptic glutamatergic neurons would result in vasodilation and a decreased CBT, i.e. an effect opposite to that observed. Another possibility is that preoptic glutamatergic neurons project to rRPA neurons controlling thermogenesis directly or via the DMH, in a similar fashion to preoptic GABAergic neurons. Such a network may also account for the observation that the maximal hyperthermia reached in response to activation of either H1Rs or H3Rs in the MnPO is similar to that induced by histamine. Either treatment may result in maximal activation of the rRPA neurons, thus their effects are not additive. The lower affinity of histamine to H1Rs suggests that their activation occurs when the neurotransmitter’s concentration is elevated (e.g. during arousal).
We would like to thank Professor Henri Korn (Institut Pasteur, Paris, France) for suggesting a study of histamine effects on preoptic neurons and for comments on an early version of the manuscript. We would like to thank also Professor Tamas Bartfai (TSRI, La Jolla, USA) for comments and support during the initial period of this study. We would like to thank Dr Gabor Szabo (The Hungarian Academy of Sciences, Budapest, Hungary) for providing the GAD65-GFP transgenic line. We also would like to acknowledge Brendon Ross for excellent technical support. This work was supported by NIH grant NS060799 (I.V.T.).