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 micromol x 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 micromol x 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.

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).

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.

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 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 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.

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 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.