The histamine H₄ receptor is functionally expressed on neurons in the mammalian CNS

Abstract

Background and purpose:

The histamine H₄ receptor is the most recently identified of the G protein-coupled histamine receptor family and binds several neuroactive drugs, including amitriptyline and clozapine. So far, H₄ receptors have been found only on haematopoietic cells, highlighting its importance in inflammatory conditions. Here we investigated the possibility that H₄ receptors may be expressed in both the human and mouse CNS.

Methods:

Immunological and pharmacological studies were performed using a novel anti-H₄ receptor antibody in both human and mouse brains, and electrophysiological techniques in the mouse brain respectively. Pharmacological tools, selective for the H₄ receptor and patch clamp electrophysiology, were utilized to confirm functional properties of the H₄ receptor in layer IV of the mouse somatosensory cortex.

Results:

Histamine H₄ receptors were prominently expressed in distinct deep laminae, particularly layer VI, in the human cortex, and mouse thalamus, hippocampal CA4 stratum lucidum and layer IV of the cerebral cortex. In layer IV of the mouse somatosensory cortex, the H₄ receptor agonist 4-methyl histamine (20 µmol·L⁻¹) directly hyperpolarized neurons, an effect that was blocked by the selective H₄ receptor antagonist JNJ 10191584, and promoted outwardly rectifying currents in these cells. Monosynaptic thalamocortical CNQX-sensitive excitatory postsynaptic potentials were not altered by 4-methyl histamine (20 µmol·L⁻¹) suggesting that H₄ receptors did not act as hetero-receptors on thalamocortical glutamatergic terminals.

Conclusions and implications:

This is the first demonstration that histamine H₄ receptors are functionally expressed on neurons, which has major implications for the therapeutic potential of these receptors in neurology and psychiatry.

Introduction

The histamine H₃ and H₄ receptors are two closely related and recently discovered members of the histamine receptor family, both targets for the new-generation of ‘anti-histamine’ drugs (de Esch et al., 2005). The H₃ receptor is a presynaptic auto- and hetero-receptor, reported to be abundantly expressed in the CNS of different mammalian species (Chazot et al., 2001; Pillot et al., 2002; Cannon et al., 2006), including human (Coge et al., 2001a).

The human histamine H₄ receptor (hH₄ receptor) is the most recently discovered member of the G protein-coupled receptor subfamily of histamine receptors (Nakamura et al., 2000; Oda et al., 2000; Liu et al., 2001; Morse et al., 2001; Nguyen et al., 2001; Zhu et al., 2001). The H₄ receptor is predominantly expressed in haematopoietic cells and is suggested to play a role in inflammation (O'Reilly et al., 2002; Hofstra et al., 2003; Dijkstra et al., 2007; 2008; Bäumer et al., 2008) and allergy (Dunford et al., 2006). The H₄ receptor has also been linked with rheumatoid arthritis (Ikawa et al., 2005), colon cancer (Cianchi et al., 2005; Varga et al., 2005) and breast cancer (Maslinska et al., 2006). Our recent data have suggested that the H₄ receptor is not exclusively expressed on haematopoietic cells (Grandi et al., 2008) and, consequently, this receptor has potential as a new drug target in a number of therapeutic areas. Several early studies could not detect H₄ receptors in the brain (e.g. Nakamura et al., 2000), but others have detected mRNA for these receptors in discrete brain regions (Coge et al., 2001b; Liu et al., 2001), but these authors do speculate whether the relatively sparse CNS labelling may reflect immune cell infiltration of the CNS. Here, we use our previously validated anti-hH₄ receptor antibody (van Rijn et al., 2006; Dijkstra et al., 2007) combined with patch clamp electrophysiology and selective pharmacological probes to locate human and rodent H₄ receptor protein in the CNS and address the important hypothesis that the H₄ receptor protein is functionally expressed on neurons in the brain.

This study demonstrates, for the first time, the topological and functional expression of H₄ receptors in mammalian brain, allowing neuroscientists and neurologists to profile its roles (in the healthy and diseased brains) and appraise its potential for future drug development strategies.

Methods

Antibodies

Anti-hH₄ receptor 374-390 antibodies were produced and validated for detecting both human and rodent H₄ receptors in the School of Biological and Biomedical Sciences, Durham University (van Rijn et al., 2006; 2008; Dijkstra et al., 2007; 2008; Bäumer et al., 2008; Grandi et al., 2008; Morini et al., 2008). The human 374-390 peptide sequence shares over 50% homology with rodent sequences.

Immunological studies

Immunoblotting and immunohistochemistry were performed using brains from 6 week C3H/HeJ mice as described in our previous studies (Chazot et al., 2001; Cannon et al., 2006; van Rijn et al., 2006; Dijkstra et al., 2007; Grandi et al., 2008). The normal human post-mortem cortex material for the immunoblots was obtained with full ethical approval under the UK Newcastle and Tyneside LREC 2002/295.

Immunoblotting

Sodium dodecyl sulphate polyacrylamide gel electrophoresis was carried out using 7.5% (w/v) polyacrylamide slab gels under reducing conditions. Samples of membranes (20–50 µg protein) were prepared using a chloroform/methanol method of protein precipitation, and immunoblotting was performed as previously described (Chazot et al., 2001; Bakker et al., 2006; van Rijn et al., 2006). Blots were probed with our validated rabbit anti-hH₄ receptor 374-390 antibodies at 1 µg·mL⁻¹, in the absence and presence of 50-fold excess 374-390 peptide (van Rijn et al., 2006; Dijkstra et al., 2007; 2008). Horseradish peroxidase-conjugated goat anti-rabbit antibodies (1:2000) were used as secondary antibodies (Little Chalfont, Buckinghamshire, England).

Immunohistochemistry

The human brain tissue for the immunohistochemistry study was obtained from the Neurological Foundation of New Zealand Human Brain Bank (Department of Anatomy with Radiology, University of Auckland). The University of Auckland Human Participants Ethics Committee approved the protocols used in these studies and all tissue was obtained with full consent of the families. Brain tissue was obtained from three neurologically normal cases, with an average age of 63 years (range 53–77 years), with no history of neurological disease and no evidence of neuropathology. The cases had a post-mortem interval between 16 and 23 h after death (mean post-mortem interval 18.6 h). For the immunohistochemistry studies, the human brains were processed as previously described (Waldvogel et al., 2006). In brief, the human brains were fixed by perfusion through the basilar and internal carotid arteries, first with phosphate-buffered saline (PBS) with 1% (w/v) sodium nitrite, followed by 15% (v/v) formalin in 0.1 mol·L⁻¹ phosphate buffer, pH 7.4. After perfusion, blocks from the basal ganglia were carefully dissected out and kept in the same fixative for 24 h. The tissue blocks were cryoprotected in 20% (w/v) sucrose in 0.1 mol·L⁻¹ phosphate buffer with 0.1% (w/v) sodium azide for 2–3 days, and then in 30% (w/v) sucrose in 0.1 mol·L⁻¹ phosphate buffer with 0.1% (w/v) sodium azide for a further 2–3 days. The blocks were sectioned on a freezing microtome (50 µm) and the sections were stored in PBS with 0.1% (w/v) sodium azide.

Adjacent series of sections were selected and processed free-floating in tissue culture wells using standard immunohistochemistry procedures. Sections were washed in PBS and 0.2% (v/v) Triton-X (PBS-triton) and pretreated for antigen retrieval using standard protocols (Waldvogel et al., 2004) before being processed. Briefly, sections for antigen retrieval were transferred to six-well tissue culture plates and incubated overnight in 0.1 mol·L⁻¹ sodium citrate buffer, pH 4.5, transferred to 10 mL of fresh sodium citrate buffer solution, microwaved in a 650 W microwave oven for 30 s and allowed to cool before washing (3 × 15 min) in PBS-triton. The sections were then incubated for 20 min in 50% (v/v) methanol and 1% (v/v) H₂O₂, washed (3 × 15 min) in PBS-triton, and incubated in primary antibodies for 2–3 days on a shaker at 4^°C. The primary antibodies were washed off (3 × 15 min, PBS-triton) and the sections incubated overnight in biotinylated sheep anti-rabbit at 1:500 (secondary antibody, Chemicon, USA). The secondary antibodies were washed off (3 × 15 min, PBS-triton), the sections incubated for 4 h at room temperature in ExtrAvidin™, 1:1000 (Sigma) and reacted in 0.05% (w/v) 3,3-diaminobenzidine tetrahydrochloride (Sigma) and 0.01% (v/v) H₂O₂ in 0.1 mol·L⁻¹ phosphate buffer, pH 7.4, for 15–30 min to produce a brown reaction product. The sections were washed in PBS, mounted on gelatine chrom-alum-coated slides, rinsed in distilled water, dehydrated through a graded alcohol series to xylene, and coverslipped with DPX (BDH, Poole, England, UK).

Control sections were routinely processed to determine non-specific staining using the same immunohistochemical procedures detailed above, except that the primary antibody was omitted from the procedure. In addition, some sections were Nissl-stained with cresyl violet according to standard techniques.

For mouse studies, in brief, perfusion fixation was performed with 4% (w/v) paraformaldehyde (PFA)/PBS pH 7.4. Cardiac perfusion fixation in the mice was performed initially with PBS/0.01% (w/v) sodium nitrite, followed by ice-cold 4% (v/v) PFA/PBS pH 7.4, and brains dissected. The brains were then incubated overnight in ice-cold 4% (w/v) PFA/PBS pH 7.4, prior to sequential PBS/20% and 30% (w/v) sucrose/PBS pH 7.4 for 2 days as described above. Following sucrose infiltration, the samples were frozen in iso-pentane at −70^oC for 1 min and sectioned in a cryostat at −26^oC (20–30 µm sections). The immunohistochemistry for the mouse brain slices was performed as described in Chazot et al. (2001). Slices were probed with the rabbit anti-hH₄ receptor 374-390 antibodies at 1 µg·mL⁻¹, in the absence and presence of 50-fold excess 374-390 peptide (van Rijn et al., 2006; Dijkstra et al., 2007; 2008).

Electrophysiological and pharmacological studies

The 4- to 6-week-old male C3H/HeJ mice were given a lethal dose of pentobarbital (120 mg·kg⁻¹; i.p.), according to University of Otago animal welfare protocol ET27/07. Brains were rapidly dissected out into ice-cold sucrose artificial CSF (aCSF) of the following composition (in mmol·L⁻¹): 248 sucrose, 3 KCl, 2 MgCl₂, 1 CaCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃ and 10 glucose (saturated with 95% O₂, 5% CO₂). Thalamocortical slices containing the somatosensory cortex were prepared as described by Agmon and Connors (1991). Briefly, using a vibratome (VT1000S; Leica, Ora, Italy) 200–300 µmol·L⁻¹ thick slices were cut at 50° to the coronal plane, rotated through the caudo-rostral axis and placed in a holding chamber at 35°C in aCSF of the following composition (in mmol·L⁻¹): 124 NaCl, 3 KCl, 1 MgCl₂, 2 CaCl₂, 1.25 NaH₂PO₄, 26 NaHCO₃, 10 glucose, 1 sodium ascorbate, 3 sodium pyruvate (bubbled with 95% O₂, 5% CO₂) for 30 min and then allowed to return to room temperature. Slices were incubated under these conditions for at least 30 min before recording began. Slices were placed in a custom-made recording chamber on the stage of a differential interference contrast microscope (E600FM DIC; Nikon, Tokyo, Japan) and perfused with room temperature (20–24°C) aCSF (Lees et al., 2006; Payne et al., 2006).

Whole-cell patch clamp recordings were made using 2–5 MΩ thin-walled electrodes filled with the following solution (in mmol·L⁻¹): 120 potassium gluconate, 10 EGTA, 10 HEPES, 1 CaCl₂, 2 MgCl₂, 4 phosphocreatine, 2 Na₂ATP, 0.2 NaGTP. During voltage clamp experiments, series resistance was regularly monitored and, if it rose above 30 MΩ or changed by more than 20%, the cell was excluded from further analysis. Series resistance was compensated by 70–80%. Neurons were designated as pyramidal cells, fast-spiking interneurons or low-threshold interneurons using standard morphological and electrophysiological characteristics (Kawaguchi and Kubota, 1993; Povysheva et al., 2006). 4-Methylhistamine (4-MH), mepyramine maleate and cimetidine (Tocris, UK) were dissolved in aCSF, and JNJ 10191584 (Tocris, UK) was dissolved in DMSO. All solutions were made freshly each day. The final constant concentration of 1:10 000 DMSO was also present during control and wash phases.

Data analysis

Results are shown as means ± SEM and were analysed by Student's t-test or one-way anova with Dunnett's post hoc test, as appropriate. P-values less than 0.05 were taken to show significant differences between means. Data analysis assumed normality and used Prism 4 software from Graphpad (CA, USA).

Results

Immunological studies

We used established immunoblotting and immunohistochemistry approaches (van Rijn et al., 2006; Dijkstra et al., 2007; Morini et al., 2008) to seek evidence that the H₄ receptor protein is expressed in both the human and mouse brains. On immunoblots, a specific major protein species (approximately M_r 75 000) was detected in human and mouse brain samples, which is coincident with the N-glycosylated dimeric species detected in human lymphocytes and recombinant hH₄ receptors expressed in HEK 293 cells (Figure 1, lanes 1 and 2; van Rijn et al. (2006; 2008). These immunoreactive species were greatly suppressed by prior incubation with the respective oligopeptide (Figure 1, lanes 3 and 4).

Figure 1.

Immunological evidence for the presence of histamine H₄ (hH₄) receptors in the human and mouse brains. Immunoblotting studies were performed using brains from 6 week C3H/HeJ mice and post-mortem tissue from normal human brain. Human (lane 1) and mouse (lane 2) cortex membranes were applied to 7.5% (w/v) sodium dodecyl sulphate polyacrylamide gel electrophoresis gels under reducing conditions, subjected to immunoblotting and probed with rabbit anti-hH₄ receptor 374-390 antibodies at 1 µg·mL⁻¹, in the absence (lanes 1 and 2) and presence of 50-fold excess 374-390 peptide (lanes 3 and 4). A coincident immunoreactive species (M_r 75 000) was detected, which corresponds well with both the recombinant dimeric hH₄ receptor and native dimeric species detected in human lymphocytes (van Rijn et al., 2006; 2008).

In order to investigate the anatomical topography of the expression of H₄ receptors, immunohistochemical studies were performed using human cortical and mouse whole brain slices. Clear punctate labelling was observed decorating the cell bodies and processes of neurons within sections of healthy human insular cortex in multiple deep laminae (Figure 2), particularly prominent in layer VI, which may indicate a new signalling role for H₄ receptors in grey matter. In the mouse forebrain, a distinct unique expression pattern of anti-H₄ receptor immunoreactivity was revealed, with notable prominent expression in the thalamus (particularly in the posterior nuclei), layer IV of the cerebral cortex, the entorhinal cortex and stratum lucidum of the CA4 (Figures 3 and 4, Table 1). In contrast, very low expression was seen in the striatum and the remaining subfields of the hippocampus (Figure 4C). All positive immunoreactivity was greatly suppressed by pre-absorption with oligopeptide (e.g. Figure 3D for cortical labelling).

Figure 2.

Immunological evidence for the presence of histamine H₄ receptors in the human cortex. Fixed post-mortem normal human brain slices (described in Methods section and Waldvogel et al., 2006) were permeabilized and probed with rabbit anti-hH₄ receptor 374-390 antibodies at 1 µg·mL⁻¹. (A) Insular cortex layers III–V lateral to the basal ganglia (scale bar = 50 µm). (B) Insular cortex layers V–VI lateral to the basal ganglia (scale bar = 50 µm). (C) and (D) Magnified layer VI immunoreactive human cortical cell showing punctate decoration of both cell soma and processes (scale bar = 100 µm).

Figure 3.

Immunological evidence for the presence of histamine H₄ receptors in the mouse cortex. Perfusion-fixed C3H mouse horizontal brain slices were permeabilized and subjected to immunohistochemical analysis as described in Chazot et al. (2001), probed with rabbit anti-hH4 receptor 374-390 antibodies at 1 µg·mL⁻¹. Images focusing on layer IV cerebral cortex, (A) magnification ×100, scale bar = 100 µm; (B) magnification ×200, scale bar = 50 µm; (C) anti-hH₄ receptor 374-390 antibodies (magnification ×400; scale bar = 50 µm); (D) anti-hH₄ receptor 374-390 antibodies at 1 µg·mL⁻¹ pretreated with 50-fold excess peptide hH₄ receptor 374-390 (magnification ×400; scale bar = 50 µm), magnification ×200, scale bar = 50 µm.

Figure 4.

Immunological evidence for the presence of histamine H₄ receptors in the mouse thalamus and hippocampal formation. Perfusion-fixed 4–6 week C3H mouse horizontal brain slices were permeabilized and subjected to immunohistochemical analysis as described in Chazot et al. (2001), probed with rabbit anti-hH4 receptor 374-390 antibodies at 1 µg·mL⁻¹. Images focusing on: (A) layer IV cerebral cortex, striatum and thalamus, scale bar = 150 µm; (B) thalamus (strong labelling of posterior pole) and striatum, scale bar = 200 µm; (C) hippocampal formation CA4 (prominent labelling)/dentate gyrus, scale bar = 200 µm.

Table 1.

Comparison of histamine H₃ and H₄ receptor expression in the mouse brain

Brain structure	Expression levels
	H4	H3
Cerebral cortex
Lamina I	+	(+)
II	++	++
III	+	+
IV	+++	++
V	++	+++
VI	+	+
Hippocampal formation
CA1	+	(+)
CA2	+	(+)
CA3/4	++	+
Dentate gyrus	+	+
Entorhinal cortex	+++	+++
Cerebellum
Granule cell layer	+++	+
Purkinje cell layer	(+)	+++
Molecular cell layer	+	(+)
Thalamus	+++	+
Striatum	(+)	+++

Qualitative summary of the relative levels of histamine H₃ and H₄ receptors in selected C3H mouse brain structures: +++ high, ++ moderate, + low overall intensity of staining, (+) light diffuse or scattered profiles. Data from immunohistochemical experiments using anti-H₃ and anti-H₄ receptor antibodies (Chazot et al., 2001; Cannon et al., 2006; van Rijn et al., 2006).

Functional electrophysiological and pharmacological studies

To evaluate whether the presence of H₄ receptors in the most heavily labelled neurons was of functional significance, we performed an electrophysiological study using recently developed hH₄ receptor selective compounds (Jablonowski et al., 2003; Lim et al., 2005).

Whole cell voltage and current clamp recordings were taken from layer IV somatosensory cortex cells in adult mouse brain slices. In the presence of tetrodotoxin (500 nmol·L⁻¹) to block multiquantal neurotransmitter release, application of the H₄ receptor agonist 4-MH (20 µmol·L⁻¹) produced a significant reversible hyperpolarization in the majority of the neurons tested (17/24; control, −64.8 ± 1.0 mV; 4-MH, −68.9 ± 1.0 mV, P < 0.0001, paired t-test; Figure 5A). A small number of neurons were unresponsive (4/24) or responded with a ∼4 mV depolarization (3/24). There was no apparent correlation between the nature of the response and whether the neuron was a pyramidal cell, a fast-spiking or low-threshold spiking interneuron. The hyperpolarizing response was significantly and reversibly reduced by the highly selective H₄ receptor antagonist, JNJ 10191584 (1 µmol·L⁻¹; control, −3.9 ± 0.8 mV; JNJ, −2.2 ± 0.6 mV; wash, −4.4 ± 0.4 mV; control vs. JNJ P < 0.05, n = 4, Dunnett's test; Figure 5B). The depolarizing response was mediated by histamine H₂ receptors as it was blocked by the selective H₂ receptor antagonist cimetidine (50 µmol·L⁻¹), and was unaffected by the H₁ receptor antagonist mepyramine (10 µmol·L⁻¹) or the H₄ receptor antagonist JNJ 10191584 (1 µmol·L⁻¹). Indeed, cimetidine revealed a small, presumably H₄ receptor-mediated, hyperpolarizing response that was previously masked by the H₂ receptor-mediated depolarization (Figure 6). Under voltage clamp (V_h−70 mV), 4-MH (20 µmol·L⁻¹) induced an apparent outward current and a significant increase in input resistance (control, 131 ± 16.2 MΩ; 4-MH, 168.3 ± 26.8 MΩ, P < 0.05, n = 9, paired t-test; Figure 5C). A slow voltage ramp (7.5 mV·s⁻¹) demonstrated that 4-MH (20 µmol·L⁻¹) reduced a largely voltage-independent current between −90 and −45 mV, but at potentials more depolarized than this the H₄ receptor agonist markedly enhanced an outward voltage-gated current (Figure 5D).

Figure 5.

The histamine H₄ receptor was functionally active in the mouse layer IV somatosensory cortex: activation of H₄ receptors produced a hyperpolarizing response in layer IV somatosensory cortex neurons of 4- to 6-week-old C3H mice by closing an ion channel. (A) A representative trace showing the effect of the H₄ receptor agonist 4-methylhistamine (4-MH, 20 µmol·L⁻¹) on resting membrane potential, and the partial block of this by the selective H₄ receptor antagonist JNJ 10191584 (JNJ, 1 µmol·L⁻¹). (B) JNJ significantly reduced the hyperpolarizing effect of 4-MH (control, −3.9 ± 0.8 mV; JNJ, −2.2 ± 0.6 mV; wash, −4.4 ± 0.4 mV; control vs. JNJ P < 0.05, n = 4, Dunnett's test). (C) Under voltage clamp, 4-MH produced a significant increase in input resistance (control, 131 ± 16.2 MΩ; 4-MH, 168.3 ± 26.8 MΩ, P < 0.05, n = 9, paired t-test). (D) A slow voltage ramp revealed that the hyperpolarizing effect of 4-MH was due to modulation of a largely voltage-independent current, and that at depolarized potentials, 4-MH enhanced an outward current. *P < 0.05.

Figure 6.

4-Methylhistamine (4-MH) induced a depolarization in a small number of cells via the histamine H₂ receptor. In three out of 24 cells, 4-MH (20 µmol·L⁻¹) produced a depolarizing response. (A) A representative trace showing the lack of effect of the H₄ receptor antagonist JNJ 10191584 (1 µmol·L⁻¹). (B) The H₁ receptor antagonist mepyramine (10 µmol·L⁻¹) had no effect on the depolarizing response induced by 4-MH (20 µmol·L⁻¹). (C) Cimetidine (50 µmol·L⁻¹) blocked the depolarizing response induced by 4-MH (20 µmol·L⁻¹) and revealed a small hyperpolarizing response.

Because the layer IV cortical neurons, which are the major site for the termination of thalamocortical fibres (Herkenham, 1980), were selectively labelled for H₄ receptors, it was possible that some of the H₄ receptor immunoreactivity was due to H₄ receptors expressed on thalamocortical terminals. To address this possibility we evoked monosynaptic thalamocortical excitatory postsynaptic potentials by stimulating the ventrobasal thalamus. However, the amplitude of excitatory postsynaptic potentials, sensitive to CNQX, was not significantly altered by 4-MH (20 µmol·L⁻¹) (control, 112 ± 5.2% of baseline; 4-MH, 116 ± 17% of baseline, P > 0.05, n = 4, paired t-test; Figure 7).

Figure 7.

Activation of histamine H₄ receptors did not modulate thalamocortical synaptic traffic. (A) Corticothalamic excitatory postsynaptic potentials (EPSPs) in the 4- to 6-week-old C3H mouse were CNQX-sensitive (averaged trace in insert, control in grey, CNQX in black, scale bars 1 mV, 10 ms). (B) The amplitude of thalamicocortical EPSPs were not modulated by 4-MH (20 µmol·L⁻¹; n = 4). (C) Representative overlays of thalamocortical EPSPs before and after the application of 4-MH.

Discussion

Based on the selectivity of our antibodies (van Rijn et al., 2006; 2008; Dijkstra et al., 2007; 2008; Bäumer et al., 2008; Grandi et al., 2008; Morini et al., 2008)) and pharmacological probes (Terzioglu et al., 2004; Varga et al., 2005; Venable et al., 2005), these findings provide strong evidence that the H₄ receptor was both expressed, and was functionally active, on neurons in the mammalian CNS. The unique expression pattern indicates that the H₄ receptor may have an important role to play in the central histaminergic system in addition to the other well-described members of the histamine receptor family (Table 1, Brown et al., 2001; Haas et al., 2008). The presence of labelling in the cerebral cortex in both the human and mouse brains contrasts with the apparent lack of the corresponding mRNA in the human cerebral cortex (Coge et al., 2001b) and implies that, in the human cortex, expression of H₄ receptors is on fibres emanating from other brain regions (most likely the hippocampus or thalamus). Our electrophysiological data demonstrated that 4-MH (20 µmol·L⁻¹) induced a ∼4 mV hyperpolarization in layer IV neurons, and that this effect was blocked by the highly selective H₄ receptor antagonist JNJ 10191584. At this concentration, 4-MH is likely to act as an agonist at histamine H₂ receptors (Lim et al., 2005; Breunig et al., 2007) and potentially also at H₃ receptors (Lim et al., 2005). Therefore, it is not surprising that in three out of 24 neurons tested, 4-MH (20 µmol·L⁻¹) produced a H₂ receptor-mediated depolarization. It is unlikely that the hyperpolarization can be attributed to 4-MH acting at other histamine receptors, as histamine H₃ receptor ligands have no effect on the resting potential of cortical neurons and H₁ receptor agonists are known to depolarize cortical neurons (W.M. Connelly, unpubl. obs.; Reiner and Kamondi, 1994). The pattern of expression of H₄ receptors shows some similarities to the H₃ receptors, such as overlap in entorhinal cortex and deep laminae of the cortex, but contrasts particularly in the relatively high expression in the thalamus (Chazot et al., 2001; Pillot et al., 2002). Recent published in vivo pharmacological studies would concur with a role for H₄ receptors in the CNS, in control of pain transmission at the level of the spinal cord (Cowart et al., 2008; Strakhova et al., 2009). Indeed, we also have preliminary evidence for H₄ receptor expression in the rodent spinal cord and dorsal root ganglia, which yields an anatomical framework for this proposed nociceptive role, and for the potential of H₄ receptors as a novel analgesic therapeutic target (not shown).

Our results indicate that activation of H₄ receptors directly hyperpolarized cortical neurons but concurrently enhanced the input resistance of the cells. Such actions are opposed to those of activated H₁ receptors, which depolarize cortical neurons by closing leak potassium currents (Reiner & Kamondi, 1994). The mechanism for this inhibitory response and the contributory ion channels and second messengers will be the focus of future studies in both the Otago and Durham laboratories. It is interesting to note that H₄ receptors are much more sensitive sensors for histamine than H₁ receptors (affinites of 1–50 nmol·L⁻¹ vs. 2–10 µmol·L⁻¹; Liu et al., 2001; Nguyen et al., 2001; Zhu et al., 2001; Booth et al., 2002; Seifert Wenzel-Seifert et al., 2003; Haas et al., 2008). This may indicate that H₄ receptors are able to sense the low level of histamine present during sleep, while the higher concentration of histamine during wakefulness may allow the depolarizing influence of H₁ receptors to predominate (Strecker et al., 2002; Chu et al., 2004). It would be interesting to explore the systems and behavioural effects of co-activation of H₁ and H₄ receptors by histamine on neuronal excitability. The growing availability of brain-permeant selective agonists and antagonists for H₄ receptors will help unravel the roles for the new receptor and its potential as a therapeutic target.

Our study suggests that H₃ receptors (already established as a predominately presynaptic species regulating the release of histamine and a range of other transmitters) and H₄ receptors subserve distinct and contrasting roles in the mammalian brain. Whether alternative splicing in H₄ receptors has functional relevance in the brain, as indicated for the H₃ receptor (Bakker et al., 2006; van Rijn et al., 2006), remains to be seen. It is fascinating to reflect that many of the drugs that bind to hH₄ receptors in the low micromolar range, such as amitriptyline and clozapine, are frequently used as neurological or psychiatric disease modifiers (Nguyen et al., 2001). Furthermore, these results demonstrate the urgent need to re-evaluate previously published studies in the CNS, which utilized first-generation ligands, which are now known to bind both H₃ and H₄ receptors, including thioperamide and clobenpropit (Lim et al., 2005). Revealing the functional presence of this receptor in the CNS for the first time will allow a broader neuropharmacological community to characterize roles for this G protein-coupled receptor, appraise its involvement in brain diseases (and drug action) and to evaluate its potential as a target for new drugs particularly in neurological diseases, notably inflammatory pain disorders and dementias where inflammation (the potential of hH₄ receptors in immunology and inflammation is already established) plays a prominent role.

Glossary

Abbreviations:

CNQX

6-cyano-7-nitroquinoxaline-2,3-dione

EPSP

excitatory postsynaptic potential

JNJ 10191584

(1-[(5-chloro-1H-benzimidazol-2-yl)carbonyl]-4-methylpip erazine maleate)