HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 98/6/3143    most recent 00337.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhou, J. Articles by Pfaff, D. W. Search for Related Content PubMed PubMed Citation Articles by Zhou, J. Articles by Pfaff, D. W.

Histamine-Induced Excitatory Responses in Mouse Ventromedial Hypothalamic Neurons: Ionic Mechanisms and Estrogenic Regulation

Laboratory of Neurobiology and Behavior, The Rockefeller University, New York, New York

Submitted 26 March 2007; accepted in final form 12 October 2007

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Histamine is capable of modulating CNS arousal states by regulating neuronal excitability. In the current study, histamine action in the ventromedial hypothalamus (VMH), its related ionic mechanisms, and its possible facilitation by estrogen were investigated using whole cell patch-clamp recording in brain slices from ovariectomized female mice. Under current clamp, a bath application of histamine (20 µM) caused membrane depolarization, associated with an increased membrane resistance. In some cells, the depolarization was accompanied by action potentials. Histamine application also significantly reduced the latency of action potential evoked by current steps. Histamine-induced depolarization was not affected by either tetrodotoxin or Cd²⁺. However, after blocking K⁺ channels with tetraethylammonium, 4-aminopyridine, and Cs⁺, depolarization was significantly decreased. Under voltage clamp, histamine-induced depolarization was associated with an inward current. The current–voltage relationship revealed that this inward current reversed near E_K. The histamine effect was mimicked by a histamine receptor 1 (H₁) agonist, but not a histamine receptor 2 (H₂) agonist. An H₁ antagonist, but not H₂ antagonist, abolished histamine responses. When ovariectomized mice were treated with estradiol benzoate (E2), histamine-induced depolarization was significantly enhanced with an increased percentage of cells showing action potential firing. These results suggest that histamine depolarized VMH neurons by attenuating a K⁺ leakage current and this effect was mediated by H₁ receptor. E2 facilitated histamine-induced excitation of VMH neurons. This histamine effect may present a potential mechanism by which estrogens modulate the impact of generalized CNS arousal on a sexual arousal–related neuronal group.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
The ventromedial nucleus of the hypothalamus (VMH) is a cell group involving a wide range of neuroendocrinological brain functions (Pfaff et al. 2002). In particular, VMH is closely involved in lordosis and sexual arousal. Its roles in governing sexual behavior and related hormone regulation have been extensively studied in our laboratory (Kow and Pfaff 1998; Pfaff 1980, 1999; Zhou et al. 2005). In exploring the molecular mechanisms underlying VMH-mediated arousal, several neurotransmitter systems have emerged in the literature. For example, at least three neurotransmitters signaling generalized CNS arousal affect electrical activity in VMH neurons that are essential for normal lordosis behavior and sexual arousal. They are histamine, norepinephrine, and the opioid peptide enkephalin (Lee et al. 2006).

As one of the major arousal-related neurotransmitter systems (Pfaff 2006), histamine is produced by neurons in the tuberomammillary nucleus of the hypothalamus. The efferent fibers of histaminergic neurons project widely throughout the brain (Schwartz et al. 1991; Takada et al. 1987). High densities of histaminergic fibers are found in the hypothalamus, with all nuclei including VMH receiving a strong or moderate innervation (Martinez-Mir et al. 1990; Terao et al. 2004). In CNS, histamine is synthesized from histidine by a specific enzyme, histidine decarboxylase (HDC), and signals through three receptor subtypes: histamine receptor 1, 2, and 3 (H₁, H₂, and H₃). All histamine receptor subtypes present in the CNS were found in the hypothalamus (Brown et al. 2001; Schwartz et al. 1991), with high densities of H₁ receptors in the VMH (Bouthenet et al. 1988; Palacios et al. 1981). In fact, histamine acting in the VMH can increase lordosis behavior (Donoso and Broitman 1979).

Histamine functions detected as neurotransmitter-like or neuromodulator-like have been identified in many brain areas. A typical action of histamine is that it excites neurons by producing a depolarization and a subsequent increase in firing frequency (Brown et al. 2001). The mechanisms underlying histamine effects are diverse, depending on brain area and neuronal type (Haas and Panula 2003). The ionic mechanisms include the inhibition of a background potassium conductance (Li and Hatton 1996; McCormick and Williamson 1991), stimulation of a chloride current (Starodub and Wood 2000), stimulation of a nonspecific cation channel or electrogenic Na⁺/Ca²⁺ exchanger (Smith and Armstrong 1996), and an increase of intracellular calcium levels (Leopoldt et al. 1997; Leurs et al. 1994). Different histamine receptor subtypes are involved in these diverse mechanisms.

Even though histamine actions have been studied in the VMH (Alvarez and Donoso 1981; Jang et al. 2001; Kow et al. 2005) and the literature indicates its potential in modulating VMH functions, such as sexual behavior (Donoso and Broitman 1979) and feeding behavior (Aou et al. 1995; Magrani et al. 2004; Sakata and Yoshimatsu 1995), electrophysiological characterization of histamine action in the VMH is very limited. In our current study, we used whole cell patch-clamp recording to investigate the effect of histamine on VMH neurons. Our particular interest highlighted its ionic and receptor mechanisms. Because estrogens act as a crucial modulator in VMH-related sexual arousal function, we wondered whether the general arousal function of histamine would interact with the modulator effect of estrogens on sexual arousal in the VMH. Thus by comparing histamine effects on VMH neurons from estrogen- versus vehicle-treated animals, the possible action of estrogen treatment on histaminergic function was examined. We found that histamine increased the excitability of VMH neurons as indicated by membrane depolarization and increased firing rate. This effect is produced by the inhibition of potassium leakage currents through the H₁ receptor. Estrogen treatment facilitated histamine-induced excitation, indicating a potential interaction between general arousal and specific sexual arousal functions.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Slice preparation

Four- to 5-wk-old, ovariectomized Swiss-Webster female mice (Taconic Farm, Hudson, NY) were used to prepare VMH brain slices. After mice were deeply anesthetized by intraperitoneal injection of urethane (40%, 1–1.5 g/kg), brains were rapidly removed and placed in an ice-cold oxygenated slicing solution consisting of (in mM): 210 sucrose, 3.5 KCl, 1 CaCl₂, 4 MgCl₂, 1.25 NaH₂PO₄, and 10 D-glucose, pH 7.3. Coronal brain slices (250 µm in thickness) containing VMH were prepared using a vibratome (Leica VT1000, Wetzlar, Germany). Slices were then transferred to a room-temperature oxygenated bath solution and allowed to recover 1 h at room temperature before electrophysiology recording.

During the estrogen effect study, ovariectomized mice received daily injections of either estradiol benzoate [E2, 10 µg/0.1 ml oil, administered subcutaneously (sc)] or sesame oil (0.1 ml) for 2 days before the recording. The dose of estradiol used achieves proestrous levels of estrogen in the blood, 60–90 pg/ml. All animals were cared for in accordance with the Rockefeller University Animal Care and Use Committee protocol.

Electrophysiology

Whole cell patch-clamp recordings were performed at room temperature (22–25°C) from VMH slices using a MultiClamp 700A amplifier (Axon Instruments, Foster City, CA). Patch pipettes were pulled from thin-walled borosilicate glass pipettes (Warner Instrument, Hamden, CT) and had a resistance of between 3 and 5 M. The recording chamber was continuously perfused with an artificial cerebrospinal fluid (ACSF) of the following composition (in mM): 126 NaCl, 5 KCl, 2 MgCl₂, 2 CaCl₂, 10 D-glucose, 1.25 MaH₂PO₄, and 26 NaHCO₃. ACSF was aerated with 95% O₂-5% CO₂ to a final pH of 7.3 (osmolarity 300–310 mOsm). The internal pipette solution contained (in mM): 140 K-gluconate, 10 HEPES, 0.6 NaHCO₃, 2 KCl, 1 CaCl₂, 2 MgATP, 2 Na₂ATP, 0.3 Na₂GTP, 8 sucrose, and 5 EGTA (pH 7.3, osmolarity 285–290 mOsm). The series resistance was typically 10–20 M, which was frequently checked during and at the end of the recording. Data were not included if changes were >30% from the starting series resistance. The holding potential for the current clamp was set at –55 mV. Data were acquired by pCLAMP 9.0 software and analyzed by Clampfit software (Axon Instruments).

Drugs and solutions

When the ionic mechanism was examined, tetrodotoxin (TTX, 0.5 µM) or CdCl₂ (100 µM) were added to the ACSF to block Na⁺ or Ca²⁺ channels. To block the K⁺ channel, tetraethylammonium (TEA, 20 mM) and 4-aminopyridine (4-AP, 5 mM) were added to ACSF and the K⁺ in the pipette solution was replaced by Cs⁺ (120 mM). When required, 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA, 11 mM) was added into the internal pipette solution to block Ca²⁺ release from internal Ca²⁺ storage. When Ca²⁺-free ACSF was required, Ca²⁺ in the ACSF was replaced by the equally molar Mg²⁺.

Histamine, betahistine (H₁ receptor agonist, 100 µM; Tocris), mepyramine (H₁ receptor antagonist, 1 µM; Tocris), dimaprit (H₂ receptor agonist, 50 µM), and cemitidine (H₂ receptor antagonist, 30 µM) were applied by bath perfusion to the slices in respective experiments. All drugs were purchased from Sigma except as indicated. All drugs were diluted in fresh ACSF to final concentration before experiments.

Statistical analysis

Data are represented as means ± SE. Statistical analysis was accessed using different statistical tests in different experiments. Student's paired t-test was used when drug effects were compared on the same neuron; Mann–Whitney U test was used to compare histamine responses between oil- and E2-treated neurons; the chi-square test was used to compare action potential firing between oil- and E2-treated neurons. P < 0.05 was taken to indicate statistical significance.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
In our study, the recordings were acquired from cells located in the ventrolateral part of the VMH, where estrogen receptors are highly expressed (Li et al. 1993; Mitra et al. 2003; Pfaff and Keiner 1973). These neurons can be generally classified as midsize, multidendrite neurons. A total of 171 neurons from 55 different mice were collected in this study. The average resting membrane potential of these cells was –59.2 ± 0.6 mV.

Histamine-induced responses in ventromedial hypothalamic neurons

We first examined the effect of histamine on the membrane potential of VMH neurons under current-clamp conditions. Figure 1A1 shows a typical example of membrane potential recorded from VMH neurons under current-clamp conditions. Membrane potentials were usually held at –55 mV. Bath applications of histamine (20 µM, 2 min) reversibly depolarized every VMH neuron recorded. The depolarization started within 10 s after histamine was delivered into the recording chamber and lasted 3 to 6 min, after which the cells repolarized to holding potential during washout. To estimate the membrane resistance, hyperpolarizing current steps (–100 pA, 150-ms duration, 3-s interstep interval) were applied before, during, and after histamine application. Figure 1A2 shows an example in which a reversible increase in membrane resistance was observed with histamine application (baseline: 135 ± 25 M; histamine: 165 ± 30 M; n = 6, P < 0.05). In all 12 neurons recorded, the membrane potential was significantly depolarized by histamine application (5.2 ± 0.6 mV, n = 12, P < 0.001; Fig. 1A3). The depolarization was accompanied by action potential firing in 3 of 12 cells. Under voltage-clamp conditions (with holding potential at –55 mV), histamine application produced an inward current (10.9 ± 1.5 pA, n = 20, P < 0.001; Fig. 1, B1 and B2) with the same time course as the depolarization shown in the current clamp. Therefore histamine-induced depolarization is associated with an inward current and increased membrane resistance.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Histamine-induced membrane depolarization and inward current in ventromedial hypothalamus (VMH) neurons. A1: sample of a continuous whole cell current-clamp recording from a VMH neuron showing reversible membrane depolarization with 20-µm histamine application. Holding potential was –55 mV. A2: sample of a continuous current-clamp recording from another VMH neuron showing membrane depolarization and concomitant action potential firing. Negative deflections were produced by hyperpolarizing rectangular wave current steps (–100 pA, 150-ms duration, 3-s interstep interval) injected before, during, and after histamine application. A reversible increase in membrane resistance was observed during histamine application indicated by the increase in amplitude of electrotonic potentials (negative deflections of the records). A3: summary graph illustrating membrane potential before (baseline) and during histamine application in VMH neurons. Mean value (n = 12) of baseline membrane potential and the peak membrane potential in response to histamine are plotted. Error bars represent SE. B1: sample of a continuous whole cell voltage-clamp recording showing histamine-induced inward current. B2: summary graph illustrating membrane current before and during histamine application. Mean value (n = 20) of baseline membrane current and the peak inward current in response to histamine are shown (means ± SE).

View larger version (29K): [in this window] [in a new window]   FIG. 2. Histamine decreased the evoked action potential latency in VMH neurons. A: samples of whole cell current-clamp recording from a VMH neuron in response to current steps before (baseline) and in the presence of histamine. Current steps consisted of a series of current injections from –40 to 80 pA. In the presence of 20 µM histamine, action potentials were evoked more rapidly by current steps compared with baseline condition. B: histogram illustrating threshold stimulus to evoke action potential in the absence (baseline) and in the presence of histamine. Threshold current stimulus to evoke action potential was significantly decreased in the presence of histamine (n = 10, P < 0.01). C: plot of current stimulus vs. action potential latency in the absence (baseline) and in the presence of histamine.

We then investigated the ionic basis for histamine-induced depolarization. In this experiment, histamine response was tested when neurons were preperfused with different channel blockers. Shown in Fig. 3A, when VMH neurons were perfused with ACSF containing TTX (0.5 µM), Cd²⁺ (100 µM), or combined TTX and Cd²⁺, respectively, histamine still depolarized the neurons. Therefore it is unlikely that Na⁺ and Ca²⁺ channels were involved in histamine-induced depolarization. However, after blocking K⁺ channels with TEA (20 mM) and 4-AP (5 mM) in the ACSF solution and Cs⁺ (120 mM) in the pipette solution, histamine failed to induce depolarization. Intracellular Ca²⁺ from the endoplasmic reticulum has been shown to play an important role in mediating histamine signaling (Brown et al. 2001). To test Ca²⁺ dependence in histamine responses, calcium chelator BAPTA (11 mM) was added to the pipette solution and extracellular Ca²⁺ in ACSF was replaced by an equal molarity of Mg²⁺. As shown in Fig. 3A, BAPTA and Ca²⁺-free ACSF did not affect histamine-induced depolarization. Statistical analysis (Fig. 3B) suggests that only K⁺ channel blockers significantly reduced histamine-induced membrane responses (0.1 ± 0.1 mV; ACSF control, 4.2 ± 1.1 mV; n = 6; P < 0.01). Neither TTX (3.1 ± 0.6 mV; ACSF control, 3.8 ± 0.5 mV; n = 8; P = 0.342), Cd²⁺ (3.1 ± 0.7 mV; ACSF control, 3.6 ± 0.6 mV; n = 7; P = 0.308), nor the combined TTX and Cd²⁺ (2.9 ± 0.5 mV; ACSF control, 3.7 ± 0.9 mV; n = 9; P = 0.461) inhibited the histamine-induced depolarization. Moreover, neither BAPTA (3.0 ± 0.6 mV; control, 3.8 ± 0.6 mV; n = 8; P = 0.195) nor BAPTA combined with Ca²⁺-free ACSF (4.3 ± 0.6 mV; control, 3.8 ± 0.5 mV; n = 9; P = 0.436) had significant effect on histamine-induced depolarization. These results suggest that the ionic mechanism underlying histamine responses is likely mediated through K⁺ currents.

View larger version (18K): [in this window] [in a new window]   FIG. 3. Histamine-induced membrane depolarization is blocked by potassium channel blocker. A: samples of continuous whole cell current-clamp recording from VMH neurons showing histamine response in the presence of different channel blockers and Ca2+ chelator. VMH neurons were pretreated with tetrodotoxin (TTX, 0.5 µM), Cd2+ (100 µM), tetraethylammonium (TEA, 20 mM) plus 4-aminopyridine (4-AP, 5 mM), 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA, 11 mM), and Ca2+-free ACSF, respectively, and their membrane potential responses to histamine (20 µM) were shown. Histamine-induced membrane depolarization was abolished by TEA and 4-AP (with Cs+ internal solution), but not affected by TTX, Cd2+, BAPTA, and Ca2+-free ACSF. B: summary histogram illustrating the pharmacological profile of histamine-induced depolarization in the presence of different channel blockers and Ca2+ chelator. Only potassium channel blocker significantly reduced histamine effect (n = 6, P < 0.01). Data shown as means ± SE.

View larger version (13K): [in this window] [in a new window]   FIG. 4. Potassium current underlying histamine-induced membrane responses in VMH neurons. A: plot of current–voltage (I–V) relationships before (baseline) and during histamine application. I–V relationships reveal a decrease in the slope of the I–V relationship in the presence of histamine, indicating a decrease in membrane conductance. Note the reversal potential is around –90 mV, close to that for potassium ions under our recording conditions. B: subtracting I–V relationship obtained in the presence of histamine from that obtained under baseline condition reveals that the current shows a relatively linear manner with membrane potential and has a reversal potential near –90 mV.

Both H₁ and H₂ receptors have been indicated to involve histamine effect on neuronal activity (McCormick and Williamson 1991). To test which particular histamine receptor mediates histamine-induced response in VMH neurons, agonists of H₁ and H₂ receptors were applied to the VMH neuron. Figure 5A shows that a bath application of H₁ receptor agonist betahistine (100 µM) depolarized VMH neurons in a similar way as did histamine. However, when H₂ receptor agonist dimaprit (50–100 µM) was applied through a bath perfusion, no membrane depolarization was observed. Moreover, when histamine (20 µM) was applied in the presence of H₁ antagonist meyperamine (1 µM), histamine failed to induce depolarization. On the other hand, application of H₂ antagonist cimetidine (50 µM) did not affect histamine-induced depolarization. A histogram (Fig. 5B) shows that betahistine (4.1 ± 0.5 mV; histamine control, 3.1 ± 0.5 mV; n = 12; P = 0.118), but not dimaprit (1.0 ± 0.4 mV; histamine control, 3.8 ± 0.6 mV, n = 13; P < 0.01), has a membrane depolarization effect similar to that of histamine; mepyramine (0.4 ± 0.2 mV; histamine control, 3.2 ± 0.7 mV; n = 8; P < 0.001), but not cimetidine (5.0 ± 1.0 mV; histamine control 3.8 ± 0.7 mV; n = 7; P > 0.3), significantly blocked histamine response. These results indicate that histamine-induced membrane depolarization in VMH neurons was mediated by the H₁ receptor.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Histamine-induced membrane responses are blocked by histamine receptor 1 (H1) but not histamine receptor 2 (H2) receptor antagonist. A: samples of continuous whole cell current-clamp recording from VMH neurons showing effect of H1, H2 receptor agonists and antagonists on histamine-induced membrane responses. Application of H1 receptor antagonist mepyramine (1 µM) blocked the histamine-induced depolarization. Application of H1 receptor agonist betahistine (100 µM) produced a depolarization effect similar to that of histamine. H2 receptor antagonist cimetidine (50 µM) was without effect on histamine-induced membrane response. H2 receptor agonist dimaprit (50–100 µM) did not have an effect on membrane potential similar to that of histamine. B: summary histogram illustrating the pharmacological profile of histamine receptor-mediated responses in VMH neurons. Histamine-induced membrane responses in the presence of H1 or H2 receptor antagonist and the membrane response to H1 or H2 receptor agonist are shown. Data shown as means ± SE.

Estrogen action in the VMH has been intensively studied for its regulatory function in neuroendocrinologic processes. Our previous study (Zhou et al. 2005) showed that estrogen treatment modulated neuronal network activity by increasing spontaneous activity in cultured VMH neurons from female rats. As a generalized arousal neurotransmitter, histamine was shown earlier to trigger membrane depolarization and increased excitability. Previous study in our lab found bath applications of estrogen potentiated histamine-induced spikes in VMH neurons (Kow et al. 2005). We then examined the effect of estrogen treatment on the response of VMH neurons to histamine. Ovariectomized (OVX) animals received either estradiol benzoate (E2, 10 µg/0.1 ml, sc) or vehicle (sesame oil) 48 h before experiments. Figure 6A1 shows current-clamp recordings of histamine response in VMH neurons from oil-treated and E2-treated animals. In neurons from E2-treated animals, the amplitude of histamine-induced depolarization is higher than that in neurons from oil-treated animals. In addition, during depolarization E2-treated neurons showed more action potential firing compared with oil-treated neurons. Figure 6A2 shows that the histamine-induced depolarization was significantly increased (7.9 ± 0.8 mV, n = 17; P < 0.01) in neurons from E2-treated animals compared with that of oil-treated animals (4.7 ± 0.5 mV). Out of 17 neurons recorded in each group, only 5 neurons from the oil-treated group showed action potential firing during histamine-induced depolarization. In the E2-treated group, 12 of 17 neurons showed action potential firing. As shown in Fig. 6A3, the percentage of cells showing action potential firing during histamine application was significantly higher in the E2-treated group (70.6%) compared with the oil-treated group (29.4%, n = 17; ² = 5.764, P < 0.05). Under voltage-clamp conditions, the amplitude of inward current produced by histamine application was higher in neurons from E2-treated animals than in those from oil-treated animals (Fig. 6B1). Statistical analysis (Fig. 6B2) showed that histamine-induced inward currents were significantly increased in E2-treated neurons (14.8 ± 1.4 pA) compared with oil-treated neurons (9.2 ± 1.0 pA; n = 18; P < 0.01).

View larger version (15K): [in this window] [in a new window]   FIG. 6. Estradiol benzoate (E2) treatment increased histamine-induced change of membrane potential and inward current in VMH neurons. A1: samples of continuous current-clamp recording of VMH neurons from oil- and E2-treated animals. Holding potential was –55 mV. Neuron from E2-treated animal showed larger depolarization compared with neuron from oil-treated animal. A2: summary histogram illustrating histamine-induced membrane depolarization was significantly enhanced in neurons from E2-treated than oil-treated animals (n = 17, P < 0.01). A3: summary histogram illustrating that the proportion of neurons showing action potential firing in response to histamine application was significantly higher in E2-treated than in oil-treated group (n = 17, P < 0.05). B1: samples of continuous voltage-clamp recording of VMH neurons from oil- and E2-treated animals. B2: summary histogram illustrating histamine-induced inward current was significantly enhanced in neurons from E2-treated than oil-treated animals (n = 18, P < 0.01).

View larger version (24K): [in this window] [in a new window]   FIG. 7. E2 treatment facilitated histamine effect on action potential latency in VMH neurons. A and B: samples of current-clamp recording from oil- and E2-treated VMH neurons in response to current steps before (baseline) and in the presence of histamine. Current steps consisted a series of current injections from –40 to 80 pA. A: in neuron from oil-treated animal, histamine application decreased the action potential latency. B: in neuron from E2-treated animal, histamine application further decreased action potential latency. C: quantification analysis illustrating that histamine-induced action potential latency changes were significantly enhanced in neurons from E2- than oil-treated animals. Data shown as means ± SE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Histamine enhanced excitability of VMH neurons by membrane depolarization and increased firing frequency

As one of the neurotransmitter systems related to brain arousal (Pfaff 2006), histaminergic neurons are featured by their abundant efferent fibers to almost all brain areas. In many neuronal networks, histamine showed powerful excitatory action on neuronal activity by depolarizing neurons. Depolarization, a typical response to histamine, has been shown not only in areas of the CNS such as the cortex (Reiner and Kamondi 1994), the thalamus (McCormick and Williamson 1991), and the hypothalamic nucleus (Smith and Armstrong 1996), but also in peripheral nervous systems such as sympathetic preganglionic neurons (Whyment et al. 2006) and intracardiac neurons (Hardwick et al. 2006). Depending on different brain areas and neuronal types, histamine-induced depolarization can mediate important local functions. For example, in the supraoptic nucleus, histamine-induced depolarization might be related to increased vasopressin release (Dogterom et al. 1976; Li and Hatton 1996; Smith and Armstrong 1996).

As one of the neuroendocrine functions managed by VMH neurons, sexual behavior has been studied for decades in our laboratory (Pfaff 1999). Our data here show that histamine application increasing VMH neuron excitability might be the mechanism through which the arousal-related histamine regulates these VMH-related functions, including lordosis.

Receptor mechanisms of histamine-induced depolarization in VMH neurons

Even though histamine-induced depolarization is a common phenomenon in many areas, the mechanism behind it is far from simple and unique. Setting aside the recently cloned ionotropic receptors in insect eyes, histamine has three receptor subtypes (H₁, H₂, and H₃) in CNS that all belong to the G-protein–coupled receptor family. These histamine receptors are coupled to different types of G proteins and various second-messenger pathways. This fact could explain histamine's versatile actions. Among histamine receptors, both H₁ and H₂ receptors have been shown to mediate histamine-induced depolarization (Haas and Panula 2003). Compared with more limited distribution of H₂ receptors (Vizuete et al. 1997), H₁ receptors have a widespread distribution throughout the brain, with especially high densities in areas of the hypothalamus such as the preoptic area, the ventromedial, and most posterior nuclei (Bouthenet et al. 1988). In examining the receptor mechanism of histamine-induced depolarization in VMH neurons, both agonists and antagonists of H₁ and H₂ receptors were tested. The results suggested that histamine response was well mimicked by the H₁ receptor agonist and abolished by the H₁ antagonist. In addition, the H₂ receptor agonist could not induce a histamine-like response and the H₂ receptor antagonist failed to block histamine response. Therefore histamine-induced depolarization in VMH neurons is mediated by the H₁ receptor. This matches that fact that compared with the high density of the H₁ receptor, the H₂ receptor showed only weak density in the hypothalamus.

The primary signal transduction event induced by the H₁ receptor action is the activation of phospholipase C (PLC) by a pertussis toxin-insensitive G_q/11 protein (Leopoldt et al. 1997; Leurs et al. 1994). The activation of the H₁-receptor–coupled G_q/11 protein leads to the stimulation of PLC, which in turn hydrolyzes phosphatidyl-4,5-biphosphate (PIP₂) to DAG and IP₃. IP₃ binds to its own receptors located on the endoplasmic reticulum, allowing the release of stored Ca²⁺ into cytoplasm. Among other signaling pathways activated by H₁ receptor activation, many of them appear secondary to changes in intracellular Ca²⁺ concentration. Ca²⁺ dependence in histamine-induced depolarization in VMH neurons was tested by: 1) applying calcium chelator BAPTA within a pipette solution, 2) using Ca²⁺ channel blocker Cd²⁺ in ACSF, and 3) depriving extracellular calcium by Ca²⁺-free ACSF. None of these attempts affects histamine response, therefore indicating that calcium is not required in histamine-induced depolarization in VMH neurons.

Ionic mechanisms of histamine-induced excitability in VMH neurons

Another notable H₁ receptor signaling is that histamine directly blocks a background/leaking potassium current (I_KL), which leads to depolarization and/or an increase in firing frequency. I_KL is characterized by a lack of voltage and time dependence, and with a linear current–voltage relationship (Patel and Honore 2001). Background/leaking potassium selective channels play an essential role in setting the resting membrane potential, tuning the action potential duration, and modulating the responsiveness to synaptic inputs. Regulation of background potassium channels by neurotransmitters and second messengers is central for synaptic function (Belardetti and Siegelbaum 1988; Hawkins et al. 1993). The role of I_KL in histamine-induced depolarization has been found in many brain areas such as the cortex (Reiner and Kamondi 1994), thalamus (McCormick and Williamson 1991), hypothalamus (Li and Hatton 1996), and striatum (Munakata and Akaike 1994) and might be the mechanism for antihistamine-induced sedation in the human brain (Reiner and Kamondi 1994). In VMH slices, current–voltage relationships revealed that histamine-induced depolarization was associated with an inward current that reversed near E_K. This current displayed a linear relationship with voltage, and therefore was not voltage dependent. These characteristics indicated that the depolarization was due to a decrease in potassium current. The possibility that the observed effect was due to a reduction in chloride conductance was ruled out for two reasons: 1) low chloride ACSF did not affect histamine-induced depolarization and 2) low chloride ACSF did not affect I–V curve changes of histamine response. Therefore decreased background/leaking potassium current by histamine application depolarized cell membranes and increased the excitability of VMH neurons. One feature of leaking potassium channel is that it is insensitive to most classical potassium channel blockers including TEA and 4-AP. Our data showed that TEA and 4-AP, together with Cs, actually blocked depolarization. This finding may suggest the involvement of other types of potassium channels.

Potential caveats

These recordings were performed at room temperature. The difference between this temperature and normal body temperature would not only affect chemical kinetics but also might influence histamine dose–response curves and other physiological parameters. However, by recording from VMH neurons over the years we have produced a large body of data on norepinephrine effects in which electrophysiological results gathered at room temperature are consistent not only with each other but also with behavioral results (Kow et al. 1992). Further, in an ongoing whole cell recording study with VMH neurons we found no difference in their responses between room temperature and 34°C. In fact, in our experience, hypothalamic slices do not remain healthy as long when maintained at 36°C. We know that higher bath temperatures would be associated with reduced oxygen solubility, but do not know whether that is the only cause of problems with tissue slice health when maintained at higher temperatures.

Estrogenic regulation of histamine response in VMH neurons

We propose that neurons in the ventrolateral corner of the VMH are responsible for the integration of nutritional signals with estrogenic signaling because, together, they influence female reproductive behavior. This proposal makes sense in that it would not be biologically adaptive for females to reproduce at times when they do not have an adequate food supply.

Estrogens and histamine are both strongly implicated in feeding and energy metabolism, even though the interactions between estrogen and histamine action are less investigated. Hypothalamically caused obese animals produced by VMH and mammillary nuclei lesions showed very low plasma estrogen and estrogen replacement reduced food intake in those animals (Jaccoby et al. 1995). During food restriction, estrogen-receptor–containing cell numbers in the VMH decreased (Hileman et al. 1999), which may represent one mechanism whereby undernutrition enhances the ability of estrogens to foster reproduction. Histamine also modulated feeding behavior through its receptors in the VMH. As shown by Sakata (Sakata and Yoshimatsu 1995), food intake was suppressed and drinking was accelerated by either activation of H₁ receptors or inhibition of H₃ receptors in the VMH. Pharmacological blockage of both H₁ and H₂ VMH receptors significantly increased overnight food intake and decreased water intake, which may be specifically attributed to the set of histaminergic receptors situated within the VMH (Magrani et al. 2004).

Estrogens also have long been studied in the VMH for their role in facilitating hormone-dependent mating behaviors. Lordosis behavior is a classic model used to examine estrogenic regulation in sexual behavior (Pfaff 1999). The neurons at the ventrolateral quadrant of the VMH express both estrogen receptors, alpha and beta (Ikeda et al. 2003; Li et al. 1993; Pfaff and Keiner 1973), which mediate estrogen-dependent action in governing lordosis behavior. Several neurotransmitter systems have been implicated in estrogenic regulation of lordosis behavior, such as norepinephrine, acetylcholine, and serotonin (Kow and Pfaff 1985). Notably, Donoso and Broitman (1979) have implicated hypothalamic histamine in lordosis behavior performance. At least two ways were found for estrogen engaging its modulation action in VMH neurons: neuronal resting activity and specific neurotransmitter-induced responses. In VMH cultures (Zhou et al. 2005), estrogen treatment increased spontaneous synaptic events in neurons derived from females. In the meantime, the frequency of miniature inhibitory postsynaptic currents was decreased in these neurons with estrogen application, suggesting that estrogen-induced changes in GABAergic inhibition could at least partially explain estrogen effects on neuronal activity. Estrogen treatment also potentiates excitatory responses of VMH neurons to specific neurotransmitters. Extracellular recording showed that estrogen application potentiated N-methyl-D-aspartate (NMDA) or histamine induced an increased spiking rate even though estrogen itself did not have any significant effect on resting activity (Kow et al. 2005). In our study, estrogen treatment did not change membrane potential itself (data not shown); however, it facilitated histamine-induced depolarization. This result indicates that estrogen has multiple cellular mechanisms in controlling VMH neuronal activity.

Supporting specific types of CNS arousal, such as sexual arousal, are the mechanisms of generalized arousal (Pfaff 2006). This fact raises questions regarding exactly how generalized arousal modulators affect VMH neurons managing sexual behavior, a specific form of arousal-dependent motivated behavior. Histamine-induced responses shown in this study indicate a possible mechanism through which the generalized arousal factor (histamine) can affect neurons in a local brain area (VMH), which is crucial for a specific motivated behavior (lordosis). Furthermore, we addressed this question by examining whether a general arousal modulator, histamine, interacts with a hormonal modulator, estrogen, in VMH neurons. Our results are remarkable in the sense that they show a possible pivotal point where generalized arousal influences and supports a specific (sexual) arousal state. As a generalized arousal neurotransmitter, histamine's excitatory impact on VMH neurons was further potentiated by estrogen. Thus the interaction between histamine and estrogen might be a key mechanism linking generalized arousal with specific (sexual) arousal.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. Zhou, Neuroscience Biology, CNS Discovery, AstraZeneca Pharmaceuticals, Wilmington, DE 19809 (E-mail: jin.zhou{at}astrazeneca.com)

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Alvarez EO, Donoso AO. Effects of histamine implants in several brain regions on the release of prolactin in conscious adult male rats. J Endocrinol 88: 351–358, 1981.[Abstract/Free Full Text]

Aou S, Shiramine K, Ma J, Matsui H, Hori T. Hypothalamus regulates calcium metabolism in rats. Neurobiology (Bp) 3: 339–350, 1995.[Medline]

Belardetti F, Siegelbaum SA. Up- and down-modulation of single K⁺ channel function by distinct second messengers. Trends Neurosci 11: 232–238, 1988.[CrossRef][Web of Science][Medline]

Bouthenet ML, Ruat M, Sales N, Garbarg M, Schwartz JC. A detailed mapping of histamine H1-receptors in guinea-pig central nervous system established by autoradiography with [125I]iodobolpyramine. Neuroscience 26: 553–600, 1988.[CrossRef][Web of Science][Medline]

Brown RE, Stevens DR, Haas HL. The physiology of brain histamine. Prog Neurobiol 63: 637–672, 2001.[CrossRef][Web of Science][Medline]

Dogterom J, van Wimersma Greidanus TB, De Wied D. Histamine as an extremely potent releaser of vasopressin in the rat. Experientia 32: 659–660, 1976.[CrossRef][Web of Science][Medline]

Donoso AO, Broitman ST. Effects of a histamine synthesis inhibitor and antihistamines on the sexual behavior of female rats. Psychopharmacology (Berl) 66: 251–255, 1979.[CrossRef][Medline]

Haas H, Panula P. The role of histamine and the tuberomamillary nucleus in the nervous system. Nat Rev Neurosci 4: 121–130, 2003.[Web of Science][Medline]

Hardwick JC, Kotarski AF, Powers MJ. Ionic mechanisms of histamine-induced responses in guinea pig intracardiac neurons. Am J Physiol Regul Integr Comp Physiol 290: R241–R250, 2006.[Abstract/Free Full Text]

Hawkins RD, Kandel ER, Siegelbaum SA. Learning to modulate transmitter release: themes and variations in synaptic plasticity. Annu Rev Neurosci 16: 625–665, 1993.[Web of Science][Medline]

Hileman SM, Lubbers LS, Jansen HT, Lehman MN. Changes in hypothalamic estrogen receptor-containing cell numbers in response to feed restriction in the female lamb. Neuroendocrinology 69: 430–437, 1999.[CrossRef][Web of Science][Medline]

Ikeda Y, Nagai A, Ikeda MA, Hayashi S. Sexually dimorphic and estrogen-dependent expression of estrogen receptor beta in the ventromedial hypothalamus during rat postnatal development. Endocrinology 144: 5098–5104, 2003.[Abstract/Free Full Text]

Jaccoby S, Arnon E, Snapir N, Robinzon B. Effects of estradiol and tamoxifen on feeding, fattiness, and some endocrine criteria in hypothalamic obese hens. Pharmacol Biochem Behav 50: 55–63, 1995.[CrossRef][Web of Science][Medline]

Jafri MS, Moore KA, Taylor GE, Weinreich D. Histamine H1 receptor activation blocks two classes of potassium current, IK(rest) and IAHP, to excite ferret vagal afferents. J Physiol 503: 533–546, 1997.[Abstract/Free Full Text]

Jang IS, Rhee JS, Watanabe T, Akaike N. Histaminergic modulation of GABAergic transmission in rat ventromedial hypothalamic neurones. J Physiol 534: 791–803, 2001.[Abstract/Free Full Text]

Kow LM, Easton A, Pfaff DW. Acute estrogen potentiates excitatory responses of neurons in rat hypothalamic ventromedial nucleus. Brain Res 1043: 124–131, 2005.[CrossRef][Web of Science][Medline]

Kow LM, Pfaff DW. Estrogen effects on neuronal responsiveness to electrical and neurotransmitter stimulation: an in vitro study on the ventromedial nucleus of the hypothalamus. Brain Res 347: 1–10, 1985.[CrossRef][Web of Science][Medline]

Kow LM, Pfaff DW. Mapping of neural and signal transduction pathways for lordosis in the search for estrogen actions on the central nervous system. Behav Brain Res 92: 169–180, 1998.[CrossRef][Web of Science][Medline]

Kow LM, Weesner GD, Pfaff DW. Alpha 1-adrenergic agonists act on the ventromedial hypothalamus to cause neuronal excitation and lordosis facilitation: electrophysiological and behavioral evidence. Brain Res 588: 237–245, 1992.[CrossRef][Web of Science][Medline]

Lee AW, Devidze N, Pfaff DW, Zhou J. Functional genomics of sex hormone-dependent neuroendocrine systems: specific and generalized actions in the CNS. Prog Brain Res 158: 243–272, 2006.[Web of Science][Medline]

Leopoldt D, Harteneck C, Nurnberg B. G proteins endogenously expressed in Sf 9 cells: interactions with mammalian histamine receptors. Naunyn Schmiedebergs Arch Pharmacol 356: 216–224, 1997.[CrossRef][Web of Science][Medline]

Leurs R, Traiffort E, Arrang JM, Tardivel-Lacombe J, Ruat M, Schwartz JC. Guinea pig histamine H1 receptor. II. Stable expression in Chinese hamster ovary cells reveals the interaction with three major signal transduction pathways. J Neurochem 62: 519–527, 1994.[Web of Science][Medline]

Li HY, Blaustein JD, De Vries GJ, Wade GN. Estrogen-receptor immunoreactivity in hamster brain: preoptic area, hypothalamus and amygdala. Brain Res 631: 304–312, 1993.[CrossRef][Web of Science][Medline]

Li Z, Hatton GI. Histamine-induced prolonged depolarization in rat supraoptic neurons: G-protein-mediated, Ca(2+)-independent suppression of K⁺ leakage conductance. Neuroscience 70: 145–158, 1996.[CrossRef][Web of Science][Medline]

Magrani J, de Castro e Silva E, Varjao B, Duarte G, Ramos AC, Athanazio R, Barbetta M, Luz P, Fregoneze JB. Histaminergic H1 and H2 receptors located within the ventromedial hypothalamus regulate food and water intake in rats. Pharmacol Biochem Behav 79: 189–198, 2004.[CrossRef][Web of Science][Medline]

Martinez-Mir MI, Pollard H, Moreau J, Arrang JM, Ruat M, Traiffort E, Schwartz JC, Palacios JM. Three histamine receptors (H1, H2 and H3) visualized in the brain of human and non-human primates. Brain Res 526: 322–327, 1990.[CrossRef][Web of Science][Medline]

McCormick DA, Williamson A. Modulation of neuronal firing mode in cat and guinea pig LGNd by histamine: possible cellular mechanisms of histaminergic control of arousal. J Neurosci 11: 3188–3199, 1991.[Abstract]

Mitra SW, Hoskin E, Yudkovitz J, Pear L, Wilkinson HA, Hayashi S, Pfaff DW, Ogawa S, Rohrer SP, Schaeffer JM, McEwen BS, Alves SE. Immunolocalization of estrogen receptor β in the mouse brain: comparison with estrogen receptor . Endocrinology 144: 2055–2067, 2003.[Abstract/Free Full Text]

Munakata M, Akaike N. Regulation of K⁺ conductance by histamine H1 and H2 receptors in neurones dissociated from rat neostriatum. J Physiol 480: 233–245, 1994.[Abstract/Free Full Text]

Palacios JM, Wamsley JK, Kuhar MJ. The distribution of histamine H1-receptors in the rat brain: an autoradiographic study. Neuroscience 6: 15–37, 1981.[CrossRef][Web of Science][Medline]

Patel AJ, Honore E. Properties and modulation of mammalian 2P domain K⁺ channels. Trends Neurosci 24: 339–346, 2001.[CrossRef][Web of Science][Medline]

Pfaff D, Keiner M. Atlas of estradiol-concentrating cells in the central nervous system of the female rat. J Comp Neurol 151: 121–158, 1973.[CrossRef][Web of Science][Medline]

Pfaff DW. Estrogen and Brain Function: Neural Analysis of a Hormone Controlled Mammalian Reproductive Behavior. New York: Springer, 1980.

Pfaff DW. Drive: Neurobiological and Molecular Mechanisms of Sexual Motivation. Cambridge, MA: MIT Press, 1999.

Pfaff DW. Brain Arousal and Information Theory: Neural and Genetic Mechanisms. Cambridge, MA: Harvard Univ. Press, 2006.

Pfaff DW, Arnold A, Etgen AM, Fahrbach SE, Rubin RT. Hormones, Brain, and Behavior. San Diego, CA: Academic Press, 2002.

Reiner PB, Kamondi A. Mechanisms of antihistamine-induced sedation in the human brain: H1 receptor activation reduces a background leakage potassium current. Neuroscience 59: 579–588, 1994.[CrossRef][Web of Science][Medline]

Sakata T, Yoshimatsu H. Homeostatic maintenance regulated by hypothalamic neuronal histamine. Methods Find Exp Clin Pharmacol 17 Suppl. C: 51–56, 1995.[Web of Science][Medline]

Schwartz JC, Arrang JM, Garbarg M, Pollard H, Ruat M. Histaminergic transmission in the mammalian brain. Physiol Rev 71: 1–51, 1991.[Free Full Text]

Smith BN, Armstrong WE. The ionic dependence of the histamine-induced depolarization of vasopressin neurones in the rat supraoptic nucleus. J Physiol 495: 465–478, 1996.[Abstract/Free Full Text]

Starodub AM, Wood JD. Histamine H(2) receptor activated chloride conductance in myenteric neurons from guinea pig small intestine. J Neurophysiol 83: 1809–1816, 2000.[Abstract/Free Full Text]

Takada M, Li ZK, Hattori T. A direct projection from the tuberomammillary nucleus to the spinal cord in the rat. Neurosci Lett 79: 257–262, 1987.[CrossRef][Web of Science][Medline]

Terao A, Steininger TL, Morairty SR, Kilduff TS. Age-related changes in histamine receptor mRNA levels in the mouse brain. Neurosci Lett 355: 81–84, 2004.[CrossRef][Web of Science][Medline]

Vizuete ML, Traiffort E, Bouthenet ML, Ruat M, Souil E, Tardivel-Lacombe J, Schwartz JC. Detailed mapping of the histamine H2 receptor and its gene transcripts in guinea-pig brain. Neuroscience 80: 321–343, 1997.[CrossRef][Web of Science][Medline]

Whyment AD, Blanks AM, Lee K, Renaud LP, Spanswick D. Histamine excites neonatal rat sympathetic preganglionic neurons in vitro via activation of H1 receptors. J Neurophysiol 95: 2492–2500, 2006.[Abstract/Free Full Text]

Zhou J, Pfaff DW, Chen G. Sex differences in estrogenic regulation of neuronal activity in neonatal cultures of ventromedial nucleus of the hypothalamus. Proc Natl Acad Sci USA 102: 14907–14912, 2005.[Abstract/Free Full Text]

This article has been cited by other articles:

				B. Liu, H. Liang, L. Liu, and H. Zhang Phosphatidylinositol 4,5-bisphosphate hydrolysis mediates histamine-induced KCNQ/M current inhibition Am J Physiol Cell Physiol, July 1, 2008; 295(1): C81 - C91. [Abstract] [Full Text] [PDF]

				A. W. Lee, A. Kyrozis, V. Chevaleyre, L.-M. Kow, N. Devidze, Q. Zhang, A. M. Etgen, and D. W. Pfaff Estradiol modulation of phenylephrine-induced excitatory responses in ventromedial hypothalamic neurons of female rats PNAS, May 20, 2008; 105(20): 7333 - 7338. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 98/6/3143    most recent 00337.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Zhou, J. Articles by Pfaff, D. W. Search for Related Content PubMed PubMed Citation Articles by Zhou, J. Articles by Pfaff, D. W.