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Opposite Functions of Histamine H₁ and H₂ Receptors and H₃ Receptor in Substantia Nigra Pars Reticulata

¹Department of Pharmacology and ²Department of Neurology, University of Tennessee College of Medicine, Memphis, Tennessee

Submitted 12 February 2006; accepted in final form 25 May 2006

	ABSTRACT

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The substantia nigra pars reticulata (SNr) is a key basal ganglia output nucleus. Inhibitory outputs from SNr are encoded in spike frequency and pattern of the inhibitory SNr projection neurons. SNr output intensity and pattern are often abnormal in movement disorders of basal ganglia origin. In Parkinson’s disease, histamine innervation and histamine H₃ receptor expression in SNr may be increased. However, the functional consequences of these alterations are not known. In this study, whole cell patch-clamp recordings were used to elucidate the function of different histamine receptors in SNr. Histamine increased SNr inhibitory projection neuron firing frequency and thus inhibitory output. This effect was mediated by activation of histamine H₁ and H₂ receptors that induced inward currents and depolarization. In contrast, histamine H₃ receptor activation hyperpolarized and inhibited SNr inhibitory projection neurons, thus decreasing the intensity of basal ganglia output. By the hyperpolarization, H₃ receptor activation also increased the irregularity of the interspike intervals or changed the pattern of SNr inhibitory neuron firing. H₃ receptor–mediated effects were normally dominated by those mediated by H₁ and H₂ receptors. Furthermore, endogenously released histamine provided a tonic, H₁ and H₂ receptor–mediated excitation that helped keep SNr inhibitory projection neurons sufficiently depolarized and spiking regularly. These results suggest that H₁ and H₂ receptors and H₃ receptor exert opposite effects on SNr inhibitory projection neurons. Functional balance of these different histamine receptors may contribute to the proper intensity and pattern of basal ganglia output and, as a consequence, exert important effects on motor control.

	INTRODUCTION

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The substantia nigra pars reticulata (SNr) is a key output nucleus of basal ganglia motor circuitry (DeLong 1990; Hikosaka et al. 2000; Parent et al. 2000; Wilson 2004). -Aminobutyric acid (GABA)–containing SNr projection neurons are tonically active and spike spontaneously at high frequencies (Atherton and Bevan 2005; Gulley et al. 2002; Maurice et al. 2003; Nakanishi et al. 1987; Schultz 1986; Wichmann et al. 1999; Wilson et al. 1977). Their axons innervate and inhibit the thalamus along with oculormotor and other brain stem motor structures (Cebrian et al. 2005; Chevalier and Deniau 1990; Hikosaka et al. 2000). In Parkinson’s disease and movement disorders of basal ganglia origin, SNr output is often altered in its intensity and/or pattern (Bergman et al. 1998; Hutchison et al. 2004; Nevet et al. 2004; Obeso et al. 2000; Walters et al. 2000). Therefore regulation of SNr neurons is likely to influence overall basal ganglia output and motor control.

Previous anatomical studies showed that histamine fibers originating in the tuberomamillary histamine neurons innervate SNr (Airaksinen and Panula 1988; Panula et al. 1989; Schwartz and Arrang 2002). Histochemical studies indicated that histamine H₁, H₂, and H₃ receptors are expressed in rat and guinea pig substantia nigra including SNr (Bouthenet et al. 1988; Pillot et al. 2002; Ryu et al. 1995; Traiffort et al. 1994; Vizuete et al. 1997). Some of the receptors, H₃ receptor in particular, may also be expressed on afferent terminals (Pillot et al. 2002; Threlfell et al. 2004; Vizuete et al. 1997). Postmortem studies indicate that histamine innervation of SNr is increased in Parkinson’s disease, although the precise relationship between histamine and Parkinson’s disease is unknown (Anichtchik et al. 2000). Injection of an H₃ receptor agonist into SNr affected turning behavior in rats (Garcia-Ramirez et al. 2004). In a monkey Parkinson’s disease model, systemic administration of an H₃ receptor agonist increased parkinsonian symptoms (Gomez-Ramirez et al. 2006). These results indicate that H₃ receptor may regulate SNr neuron activity critical to motion control.

In other brain areas, activation of H₁ receptor may increase neuronal excitability by blocking a leak K⁺ conductance (Bell et al. 2000; Gorelova and Reiner 1996; McCormick and Williamson 1991). H₂ receptor activation may also increase neuronal excitability by inhibiting Ca²⁺-activated K⁺ channels that mediate afterhyperpolarizations (AHPs) (Haas and Konnerth 1983; McCormick and Williamson 1989) and increasing a cation conductance (McCormick and Williamson 1991). In hippocampal interneurons, H₂ receptor activation may inhibit voltage-gated K⁺ channels and alter the output from these interneurons (Atzori et al. 2000). H₃ receptor activation may inhibit neuronal excitability, Ca²⁺ influx, and neurotransmitter release (Brown et al. 2001).

We hypothesize that histamine may directly increase SNr neuron firing by activation of H₁ and H₂ receptors. Histamine may also directly inhibit these neurons by H₃ receptor activation. This inhibition may reduce spike frequency and render the spike firing less regular. Herein, we tested these hypotheses with patch-clamp techniques in well-identified SNr GABA projection neurons.

	METHODS

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Patch clamp

Wild-type, 16- to 25-day-old male and female C57BL/6J mice were used. Animal handling and use followed National Institutes of Health guidelines. These mice were kept at the animal facility of the University of Tennessee Health Science Center in Memphis. They had free access to food and water. The room light was on 7:00 AM to 7:00 PM and off for the night. Under deep halothane anesthesia, mice were decapitated and their brains were quickly dissected out. Coronal midbrain slices (300 µm thickness) containing the midrostral part of substantia nigra were prepared according to well-established procedures (Bonci and Malenka 1999; Richards et al. 1997). Coronal sections were chosen to maximally sever afferent fibers such that SNr neurons can be studied in relative isolation. The cutting solution contains (in mM): 220 sucrose, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 0.5 CaCl₂, 7 MgCl₂, and 20 D-glucose. The slices were then transferred to a holding chamber containing the normal extracellular solution (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 2.5 CaCl₂, 1.3 MgCl₂, and 20 D-glucose. The solution was continuously bubbled with 95% O₂-5%CO₂ to supply oxygen and keep pH at 7.4. Recordings were made at 30°C under visual guidance of a video microscope (Olympus BX51W1) equipped with Nomarski optics and x60 water immersion lens. Relatively large (the longest dimension of the soma was about 25 µm) oval or spindle-shaped SNr neurons were chosen for recording. These characteristics are typical of rodent SNr GABA projection neurons (Grofova et al. 1982; Juraska et al. 1977). This selection was biased against smaller neurons that are potential interneurons.

Conventional whole cell patch-clamp techniques were used (Zhou and Hablitz 1999). Patch electrodes had resistances of 2–3 M when filled with an internal solution containing (in mM): 130 KCl, 0.5 EGTA, 10 HEPES, 2 Mg-ATP, 0.2 Na-GTP, and 4 Na-phosphocreatine. pH was adjusted to 7.3 with NaOH. Axopatch 200B and Multiclamp 700B amplifiers, pClamp 9.2 software, and Digidata 1322A interface (Axon Instruments) were used to acquire and analyze data. Signals were digitized at 5–20 kHz and analyzed off-line. The Mini Analysis Program (Synaptosoft, Fort Lee, NJ) was also used to analyze spontaneous events. Recordings with access resistance increase of >15% were rejected. Whole cell conductance was measured by 100-ms voltage pulses, from –70 to –80 mV.

Histology

Neurobiotin (0.2%) was dissolved in the pipette solution before each experiment and allowed to passively diffuse into neurons from the recording electrode (Zhou and Hablitz 1996). After electrophysiological recordings, brain slices were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at 4°C overnight. Without resectioning, slices were then processed for visualization of neurobiotin-filled neurons. Endogenous peroxidases were quenched with 10% methanol and 3% H₂O₂ in phosphate-buffered saline (PBS) for 5 min at room temperature (RT). Brain slices were rinsed well, permeabilized with 0.5% Triton X-100 (Sigma) for 2 h at RT, incubated in streptavidin conjugated with horseradish peroxidase (Vector Laboratories, Burlingame, CA) at 4°C overnight, and visualized with nickel-intensified diaminobenzidine (Vector) for 10 min. Between each step, slices were thoroughly rinsed three times in PBS over 15 min. Slices were mounted onto glass slides, coverslipped with a 1:1 mix of glycerol and PBS (pH 7.4), and the edges sealed with nail polish.

For Nissl staining, mice were overdosed with pentobarbital (100 mg/kg, administered intraperitoneally), intracardially perfused with 0.9% NaCl saline and then 4% paraformaldehyde in 0.1 M PB. Brains were postfixed in 4% paraformaldehyde in 0.1 M PB for 2 h at 4°C, blocked, and incubated in a cryoprotectant solution (30% sucrose/0.1% sodium azide/0.1 M PB, pH 7.4) for 48 h. Tissue cryosections (20 µm) were dehydrated in 95% ethanol for 3 min, followed by xylene for 10 min. After rehydration, sections were stained with cresyl violet solution (Sigma) for 5 to 10 min, dehydrated, cleared in xylene, and coverslipped with Permount (Sigma).

All chemicals including D-2-amino-5-phosphonopentanoic acid (D-AP5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and bicuculline (BIC) were purchased from Sigma–Aldrich (St. Louis, MO) or Tocris Cookson (Ballwin, MO). D-AP5, CNQX, and BIC were present when examining action potential firing, depolarization, and inward current to prevent the complications from synaptic activity.

All values were expressed as means ± SE. Statistical comparisons were performed using paired t-test or Kolmogorov–Smirnov (K-S) test (to compare the distributions of two sets of events such as spontaneous synaptic currents). P < 0.05 is significant.

	RESULTS

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Identification of substantia nigra pars reticulata GABA projection neurons

Substantia nigra pars reticulata (SNr) can be easily identified. As shown in Fig. 1 A, SNr is a fairly large structure ventral to substantia nigra pars compacta (SNc) and dorsal to the cerebral peduncle (Paxinos and Franklin 2001). SNr also has a low cell density compared with the densely packed cells in SNc. SNr is populated largely by two types of neurons: the majority GABA projection neurons and the minority dopamine (DA) projection neurons (Fallon and Loughlin 1995; Tepper et al. 1995). Both cell types are relatively large and often oval-shaped neurons and cannot be distinguished based on their appearances (Deniau et al. 1982; Grofova et al. 1982; Juraska et al. 1977; Nelson et al. 1996). However, they have very different electrophysiological characteristics (Diana and Tepper 2002; Ibanez-Sandoval et al. 2006; Lacey et al. 1989; Richards et al. 1997; Shen and Johnson 1997; Yung et al. 1991).

View larger version (60K): [in this window] [in a new window]   FIG. 1. Identification of substantia nigra pars reticulata (SNr) -aminobutyric acid (GABA) projection neurons. A1: Nissl-stained coronal mouse brain section showing that SNr, as marked by the red circle, is a distinct, relatively large, and uniquely located brain structure. Arrowhead points to the dense cell band of SNc. d, dorsal; m, medial. A2: example of frequently seen SNr neurons that were chosen for recording. This neuron fired fast spikes and therefore was a presumed GABA projection neuron. Roughly 85% of the SNr neurons chosen this way were fast-spiking neurons or presumed GABA projection neurons and the rest were typical of dopamine (DA) neurons. A3: photograph showing the neuron in A2 stained with neurobiotin. Scale bar in A2 applies to A3. Gross morphology is typical of SNr GABA projection neurons, although it is not distinctively different from SNr DA neurons. B: scatterplot showing that SNr DA and GABA neurons can be unequivocally distinguished based on their spontaneous action potential duration and frequency. GABA neurons formed a cluster with high frequency and short duration, whereas DA neurons formed another cluster with low frequency and long duration. Note that these 2 clusters do not overlap. Inset: example action potentials from a DA neuron and a GABA neuron, respectively. Scale bar: 2 ms and 20 mV. C: at their natural membrane potential, SNr GABA projection neurons lack the slow afterhyperpolarization (sAHP). D: injection of –100-pA hyperpolarizing current brought the membrane potential to –70 mV and the SNr neuron ceased to fire spontaneous action potentials. In the absence of the interference of spontaneous spikes, there was still no significant sAHP after a train of spikes evoked by a pulse of depolarizing current.

Also, SNr GABA projection neurons did not show any significant slow afterhyperpolarization (sAHP) after a train of high-frequency spikes evoked from their natural membrane potentials by injecting depolarizing current pulses (Fig. 1C). To remove any potential interference from the spontaneous spikes, hyperpolarizing holding currents were applied to bring the membrane potential to –65 to –70 mV, such that the SNr neurons completely ceased to fire spontaneous action potentials. Under this condition, there was still no significant sAHP after a train of spikes evoked by depolarizing current pulses (Fig. 1D).

Histamine excites SNr GABA projection neurons by inducing depolarization and inward current

High-frequency spike firing encodes the output from SNr GABA projection neurons (Hikosaka et al. 2000). Therefore our first goal was to investigate whether histamine affected action potential firing in well-identified and synaptically isolated SNr neurons. In the presence of 20 µM D-AP5, 10 µM CNQX, and 10 µM BIC to remove complications of synaptic activity, bath application of histamine (10 µM) reliably increased the firing rate of SNr neurons by 37.2 ± 3.5%, from 10.4 ± 0.8 to 14.1 ± 1.0 Hz (n = 11, P < 0.001, Fig. 2, A and C). This effect was fully reversed after prolonged wash. Histamine did not affect spike amplitude (67.8 ± 3.4 mV under control vs. 67.4 ± 3.6 mV during histamine, n = 11) and base duration (0.97 ± 0.07 vs. 0.98 ± 0.08 ms). The fast AHP (fAHP, 20.1 ± 2.1 vs. 19.9 ± 2.4 mV) and medium AHP (mAHP, 10.6 ± 1.5 vs. 10.6 ± 1.7 mV) were also not affected (Fig. 2B). These results indicate that histamine was not affecting voltage-gated Na⁺ and K⁺ channels or Ca²⁺-activated K⁺ channels that are responsible for spike generation and repolarization.

View larger version (31K): [in this window] [in a new window]   FIG. 2. Histamine excites SNr GABA projection neurons. A: sample recordings of spontaneous action potentials in an SNr GABA projection neuron under control and during 10 µM bath-applied histamine. In this and all following figures, spikes were truncated. B: 2 individual spikes under control and during bath application of 10 µM histamine displayed alone or superimposed at slow and fast timescales, showing that histamine did not affect spike duration and amplitude, the fast AHP (fAHP), or the medium AHP (mAHP). C: group data showing that the SNr GABA neuron firing frequency was clearly increased during histamine application. Frequency was binned every 12 s and normalized to better illustrate the change induced by histamine. D: example of 10 µM histamine-induced depolarization in an SNr GABA neuron under current-clamp recording condition. Synaptic transmission and action potentials were blocked with 20 µM D-2-amino-5-phosphonopentanoic acid (D-AP5), 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 10 µM bicuculline (BIC), and 0.5 µM tetrodotoxin (TTX). E: an example of 10-µM histamine-induced inward current in an SNr GABA neuron voltage clamped at –70 mV. Holding current was relatively large in SNr GABA neurons and much of it was attributed to a physiologically important tonic inward current. Note that the histamine-induced firing increase, depolarization, and inward current have similar time courses.

The depolarization was associated with an increase in whole cell conductance, indicating that histamine was opening ion channels in SNr neurons. To test this idea, SNr neurons were voltage clamped at –70 mV. At this holding potential, bath application of histamine (10 µM) induced an inward current (39.6 ± 4.0 pA, n = 10), increasing the holding current from –164.9 ± 20.9 to –204.5 ± 23.3 pA (Fig. 2E). The majority of the relatively large baseline holding current arose from a tonic inward current (Atherton and Bevan 2005). Whole cell conductance, monitored with 10-mV voltage pulses, was also significantly increased from 5.32 ± 0.46 nS under control to 7.21 ± 0.75 nS (n = 19, P < 0.01) during histamine application, suggesting an opening of ion channels. Voltage ramp experiments revealed that histamine increased the whole cell current. The current was linear between –90 and –20 mV with no signs of voltage-dependent activation or inactivation and reversed its polarity at –42.8 ± 2.1 mV (n = 8, Fig. 3A). These results indicate that histamine was enhancing a tonic, voltage-independent current.

View larger version (34K): [in this window] [in a new window]   FIG. 3. Voltage ramp experiments reveal the current–voltage (I–V) relationships of the currents induced by activation of different histamine receptors. Net current was obtained by digital subtraction of the whole cell current under a histamine ligand by the whole cell current under control condition. A: 10 µM histamine increased the whole cell current and the net current was linear with no signs of voltage-dependent activation or inactivation. B and C: H1 receptor activation and H2 receptor agonist amthamine also increased whole cell current that was linear and voltage independent. D: H3 receptor agonist imetit reduced the whole cell current and the net current was linear. Note the direction of H3 receptor–induced current is opposite to H1 and H2 receptor–mediated currents. Because imetit reduced the whole cell current, H3 receptor was inhibiting an existing current that is the mirror image of apparent H3 receptor–induced current as indicated by the gray trace. Also, all these currents reversed their polarity near –40 mV.

View larger version (30K): [in this window] [in a new window]   FIG. 4. Histamine increases spontaneous inhibitory postsynaptic currents (sIPSCs) in SNr GABA projection neurons. A and B: specimen recordings of sIPSCs in an SNr GABA projection neuron voltage clamped at –70 mV under control conditions (A) and during bath application of 10 µM histamine (B). C and D: group data showing that histamine increased the amplitude (C) and frequency (D) of sIPSCs in SNr GABA neurons. Also shown here are the effects of H1, H2, and H3 receptor activation. H1 receptor activation was achieved by histamine after blocking H2 and H3 receptors because there is no commercially available specific H1 agonist. Amthamine and imetit are agonists for H2 and H3 receptors, respectively. H1 and H2 receptor activation increased sIPSC amplitude and frequency, whereas H3 receptor activation decreased them. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

Histamine H₁ receptor activation excites SNr GABA projection neurons

No selective H₁ receptor agonists are commercially available. Therefore we studied the potential effects of H₁ receptor activation by first blocking H₂ receptor with 5 µM ranitidine or tiotidine and H₃ receptor with 100 nM clobenpropit (van der Goot and Timmerman 2000). As will be discussed in later sections, these H₂ and H₃ receptor antagonists at the concentrations used here completely blocked H₂ and H₃ receptor agonist–induced effects, indicating that these H₂ and H₃ receptor antagonists were able to fully inhibit H₂ and H₃ receptors. Consequently, after incubation with these H₂ and H₃ receptor antagonists, only H₁ receptor can still respond to histamine. Under these conditions, bath application of 10 µM histamine increased the firing rate of SNr neurons by 19.6 ± 2.6%, from 11.1 ± 1.5 to 13.4 ± 2.0 Hz (n = 10, P < 0.01, Fig. 5, A and B). This enhancement was blocked by 2 µM trans-triprolidine, a specific H₁ antagonist (van der Goot and Timmerman 2000), further confirming that H₁ receptor activation was responsible for this histamine-induced excitation of SNr neurons. Also, under these conditions, histamine did not significantly affect action potential shape or the fAHP or mAHP, suggesting that the main effect of H₁ receptor activation was not that of affecting voltage-gated Na⁺ and K⁺ channels and Ca²⁺-activated K⁺ channels in SNr neurons.

View larger version (29K): [in this window] [in a new window]   FIG. 5. Histamine H1 receptor activation excites SNr GABA projection neurons. A: sample recordings of spontaneous action potentials recorded in an SNr GABA projection neuron under control and during 10 µM histamine after blocking H2 and H3 receptors. This arrangement was necessary because there is no commercially available specific H1 agonist. B: group data showing that SNr GABA projection neuron firing frequency was clearly increased during histamine application after blocking H2 receptor with 5 µM ranitidine and H3 receptor with 100 nM clobenpropit. These 2 blockers were present during the entire recording. Increase in firing was blocked when a specific H1 receptor antagonist trans-triprolidine (2 µM) was applied. Frequency was binned every 12 s and normalized to better illustrate the histamine-induced change. C and D: after blocking H2 and H3 receptors with ranitidine and clobenpropit and action potentials with 0.5 µM TTX, 10 µM histamine, by activation of H1 receptor, induced a depolarization under current clamp (C) or an inward current under voltage clamp (at –70 mV) (D).

Consistent with the excitatory effect of H₁ receptor activation, histamine (10 µM), in the presence of ranitidine (5 µM) and clobenpropit (100 nM) to block both H₂ and H₃ receptors, increased sIPSCs frequency by 15.8 ± 4.6% (n = 5, P < 0.05) and amplitude by 16.6 ± 5.1% (P < 0.01) (Fig. 4, C and D). However, when action potentials were blocked with 0.5 µM TTX, mIPSCs were not significantly altered by H₁ receptor activation, indicating a lack of functional H₁ receptors on GABA axon terminals innervating SNr neurons.

Histamine H₂ receptor activation enhances SNr GABA projection neurons

To examine the potential involvement of H₂ receptor in histamine-induced excitation of SNr neurons, we used the highly selective H₂ receptor agonist amthamine (van der Goot and Timmerman 2000). After establishing a stable baseline recording, bath application of 10 µM amthamine had a clearly significant excitatory effect on SNr neurons (Fig. 6, A–D). The spontaneous action potential firing rate was increased by 33.0 ± 8.8%, from 11.5 ± 1.9 Hz in control to 15.3 ± 2.5 Hz during the treatment of amthamine (n = 7, P < 0.01, Fig. 6, A and B). This effect was blocked by a selective H₂ receptor antagonist ranitidine at 5 µM (Fig. 6B). Clearly, H₂ receptor activation has excitatory effects on SNr neurons.

View larger version (27K): [in this window] [in a new window]   FIG. 6. Histamine H2 receptor activation excites SNr GABA projection neurons. A: sample recordings of spontaneous action potentials recorded in an SNr GABA neuron under control and during 10 µM amthamine, a specific H2 receptor agonist. B: group data showing that the SNr GABA neuron firing frequency was clearly increased during application of amthamine (10 µM). This increase in firing was blocked when a specific H2 receptor antagonist, ranitidine (5 µM), was applied. Frequency was binned every 12 s and normalized to better illustrate the histamine-induced change. C and D: after blocking action potentials with 0.5 µM TTX, 10 µM amthamine induced a depolarization under current clamp (C) or an inward current under voltage clamp (at –70 mV) (D).

Consistent with the excitatory effect of H₂ receptor activation, bath application of H₂ specific agonist amthamine (10 µM) increased sIPSC frequency and amplitude by 28.1 ± 8.7 and 24.2 ± 5.1%, respectively (P < 0.01, Fig. 4, C and D). However, mIPSCs recorded in the presence of 0.5 µM TTX were not significantly increased by amthamine treatment, indicating a lack of functional H₂ receptors on GABA axon terminals innervating SNr neurons.

Histamine H₃ receptor activation inhibits SNr GABA projection neurons

H₃ receptor is known to be an inhibitory autoreceptor on histamine neurons (Brown et al. 2001). Modest levels of H₃ receptor are expressed in SNr (Pillot et al. 2002; Ryu et al. 1995; Vizuete et al. 1997). We hypothesize that H₃ receptor activation may induce a mild, direct inhibition of SNr neurons. To test this hypothesis, we did the following experiments using strategies similar to those for H₂ receptor.

First, we examined the effects of an H₃ receptor agonist, imetit (van der Goot and Timmerman 2000). If H₃ receptor activation produces inhibitory effects, then imetit should inhibit SNr neurons. Indeed, bath application of 100 nM imetit significantly decreased SNr neuron firing rate by 15.6 ± 3.7% (n = 11, P < 0.05, Fig. 7, A and B). This inhibitory effect was recovered after prolonged wash (Fig. 7B). Furthermore, imetit-induced inhibition of SNr GABA neuron firing was completely blocked by a selective H₃ receptor antagonist clobenpropit (100 nM). Histamine (10 µM) induced similar effects in the presence of 2 µM H₁ blocker trans-triprolidine and 5 µM H₂ receptor blocker ranitidine (n = 5). These findings suggested that H₃ receptor activation mildly inhibited SNr neurons. H₃ receptor activation by imetit also did not significantly affect the action potential shape of fAHP or mAHP in SNr GABA neurons.

View larger version (37K): [in this window] [in a new window]   FIG. 7. H3 receptor activation changes SNr GABA projection neuron firing pattern. A: example recordings of spontaneous action potentials under control conditions (left) and during bath application of 100 nM imetit, a specific H3 receptor agonist (right). Note that the firing of spontaneous action potentials was more irregular under imetit than under control condition. B: group data of imetit inhibition of SNr GABA projection neuron firing. C and D: regularity of spontaneous action potential firing was quantified by calculating the interspike interval (ISI). Under control condition, the ISI distribution was narrow (C). Mean ISI was 0.1032 s with CV of 0.1782. Under imetit, ISI distribution became wider (D). Mean ISI was 0.1603 s with CV of 0.2605. E and F: after blocking action potentials with 0.5 µM TTX, 100 nM imetit induced a hyperpolarization under current clamp (E) or an outward current under voltage clamp (at –70 mV) (F).

Histamine H₃ receptor activation increases the irregularity of SNr GABA projection neuron spiking

During imetit treatment, the decrease in firing frequency or increase in interspike interval (ISI) was also accompanied by an increase in irregularity in ISI. To quantify this irregularity, values of the coefficient of variation (CV) of ISI under control and during imetit treatment were compared. By definition, CV was computed by dividing the SD of ISI by the mean ISI (Bennett and Wilson 1999; Motulsky 1995). Under normal conditions, SNr neurons in coronal brain slices fired action potentials in a regular pattern such that ISI distribution is narrow [Fig. 7, A (left) and C]. Bath application of 100 nM imetit increased the CV of ISI from 0.176 ± 0.023 to 0.394 ± 0.046 [n = 12, P < 0.001, Fig. 7, B (right) and D]. Furthermore, the ISI distribution also became much wider under imetit than under control conditions (compare Fig. 7, C and D). These results indicate that H₃ receptor activation increased irregularity of SNr neuron spiking.

To explore how H₃ receptor activation altered the SNr neuron firing pattern, hyperpolarizing currents were directly injected into these neurons. Membrane hyperpolarization decreased the firing frequency (Fig. 8, A and B). More important, the direct hyperpolarizing current injection also made the spike firing significantly more irregular, as indicated by the broadening of ISI distribution and the increased CV of ISI (Fig. 8, C and D). Thus direct hyperpolarizing current injection appeared to mimic the effects of H₃ receptor activation, suggesting that H₃ receptor was altering the firing pattern primarily by hyperpolarizing SNr neurons such that these neurons reach spike threshold less reliably and consequently spike less regularly.

View larger version (25K): [in this window] [in a new window]   FIG. 8. Direct hyperpolarizing current injection increases SNr GABA neuron firing irregularity. A: spontaneous spikes recorded in an SNr GABA neuron under control condition. B: when 45-pA hyperpolarizing current was applied and the neuron was hyperpolarized by about 3 mV, spontaneous spikes became slower in frequency. C: histogram of ISIs under control condition. ISI distribution was narrow. Mean ISI was 0.1062 s with CV of 0.1331. D: histogram of ISI after the neuron was injected with 45-pA hyperpolarizing current. ISI distribution became wider. Mean ISI was now 0.2050 s with CV of 0.2901. Similar changes were seen in the other 3 SNr GABA neurons.

As expected from H₃ receptor’s hyperpolarizing effect on SNr neurons, bath application of H₃ receptor agonist imetit (100 nM) slightly but significantly decreased sIPSCs. The sIPSC frequency was reduced by 17.9 ± 2.5% (n = 5, P < 0.05) and the amplitude by 9.8 ± 3.6% (P < 0.05) (Fig. 4, C and D). These results indicate that a fraction of action potential–dependent sIPSCs disappeared during imetit activation of H₃ receptor.

We also hypothesized that H₃ receptor may act as an inhibitory presynaptic receptor on GABA terminals. To test this idea, mIPSCs were recorded in SNr neurons in the presence of 0.5 µM TTX to block action potentials. Bath application of 100 nM imetit decreased the frequency of mIPSCs from 7.0 ± 1.7 Hz under control to 6.1 ± 1.5 Hz during imetit application (P < 0.001; n = 5, Fig. 9B). This effect was almost fully recovered after washing out imetit (Fig. 9B). mIPSC amplitude was not significantly affected (49.1 ± 10.4 pA in control and 48.3 ± 11.1 pA in imetit treatment) (P > 0.05, Fig. 9C). Imetit inhibition of mIPSCs was prevented by a selective H₃ receptor antagonist clobenpropit (100 nM, n = 3). These results indicate that H₃ receptor may inhibit GABA vesicle release from axon terminals synapsing onto SNr neurons.

View larger version (43K): [in this window] [in a new window]   FIG. 9. H3 receptor activation inhibits miniature IPSCs (mIPSCs) in SNr GABA projection neurons. A: example of mIPSCs under control conditions and during 100 nM imetit application recorded at –70 mV. TTX (0.5 µM) was present during the entire recording. Although the outward current is clear, as indicated by the dotted line, the effect on mIPSCs is small and difficult to discern visually. B and C: quantification of the effects of H3 receptor activation on mIPSCs. H3 receptor agonist imetit increased the interval of mIPSCs [P < 0.001, Kolmogorov–Smirnov (K-S) test, control vs. histamine] but did not alter mIPSC amplitude (P > 0.05, K-S test), indicating a decrease in the frequency of spontaneous vesicular GABA release. Recovery after washing out histamine was obtained.

Like other neurotransmitters, histamine may be released spontaneously from histamine terminals and induce a low level, tonic activation of histamine receptors and exert a tonic influence on SNr neurons. Consequently, blocking histamine receptors may have detectable effects in SNr neurons. We did the following experiments to test this idea.

After establishing stable baseline recording of spontaneous action potential firing, H₁, H₂, and H₃ antagonists (2 µM trans-triprolidine, 5 µM ranitidine, and 100 nM clobenpropit) were individually tested; however, none of the antagonists induced statistically significant change in SNr neuron firing. This is not surprising because even the large doses of exogenous histamine agonists induced only mild effects as described earlier. Because both H₁ and H₂ receptors are excitatory, we reasoned that combined application of H₁ and H₂ receptor antagonists might induce detectable effects. Indeed, a combined application of 2 µM trans-triprolidine (H₁ receptor antagonist) and 5 µM ranitidine (H₂ receptor antagonist) induced a small hyperpolarization of 1.3 ± 0.3 mV (n = 6) and significantly decreased SNr neuron firing frequency by 9.2 ± 3.2% (n = 6, P < 0.05, Fig. 10A). At the same time, the CV for ISI was increased by 11.0% (P < 0.05). These results suggest that spontaneously released endogenous histamine has a modest tonic excitatory effect on SNr neurons by activating H₁ and H₂ receptors that tend to keep these neurons spike more regularly (Fig. 10B).

View larger version (33K): [in this window] [in a new window]   FIG. 10. Tonic histamine receptor activation enhances SNr GABA projection neuron firing. A: simultaneous bath application of H1 and H2 receptor antagonists trans-triprolidine (2 µM) and ranitidine (5 µM) induced a small but significant decrease in the frequency of spontaneous action potential firing in SNr GABA neurons. Insets: examples of spikes under control and during trans-triprolidine and ranitidine. Spikes were truncated for display. Scale bar: 100 ms and 20 mV. B: schematic diagram showing that SNr projections may be tonically influenced by spontaneously released histamine.

	DISCUSSION

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H₁ and H₂ receptor activation increases SNr GABA projection neuron output

We found that activation of H₁ receptors induced an inward current and increased spike firing in SNr neurons (Figs. 3 and 5). These effects were accompanied by increased whole cell conductance, indicating an opening of unknown type(s) of ion channels. In the cortex, hippocampus, septum, striatum, and thalamus, activation of H₁ receptors increases neuronal excitability by blocking a leak K⁺ conductance or decreasing whole cell conductance (Bell et al. 2000; Gorelova and Reiner 1996; McCormick and Williamson 1991).

Activation of H₂ receptors also induced an inward current and enhanced spike firing in SNr neurons (Figs. 3 and 6). These effects were also accompanied by increased whole cell conductance, indicating opening of ion channels. In thalamocortical neurons, H₂ receptor activation enhanced the hyperpolarization-activated I_h current (McCormick and Williamson 1991). However, I_h is very small in SNr neurons. Furthermore, this I_h usually starts to activate when the membrane potential is more negative than –60 mV. In contrast, the histamine-induced current was linear between –90 and –20 mV with no signs of voltage-dependent activation or inactivation (Fig. 3), indicating that I_h is not likely histamine’s target conductance in SNr neurons. Also, neither H₁ nor H₂ receptor activation affected the Ca²⁺-activated K⁺ channel–mediated fAHP and mAHP in SNr neurons. H₂ receptor activation inhibits sAHP in hippocampal and cortical neurons (Haas and Konnerth 1983; McCormick and Williamson 1989; Yanovsky and Haas 1998), but SNr GABA neurons lack sAHP (Fig. 1, C and D). Clearly, H₁ receptor and H₂ receptor in SNr neurons are likely coupled to different effectors or ion channels compared with those in other brain areas.

An extracellular recording study reported that exogenous histamine slightly increased the firing rate of SNr neurons by H₁ receptor activation and that H₂ receptor was not involved, although H₃ receptor’s effects were not studied (Korotkova et al. 2002). Apparently, these authors missed some important histamine effects in SNr.

H₃ receptor activation increases SNr projection neuron spiking irregularity

Our data clearly demonstrate that activation of H₃ receptor induced a small hyperpolarization or a small outward current and consequently an inhibition of SNr neuron firing (Fig. 7). Whole cell conductance was decreased, indicating a closing of an unknown type ion channel. These results suggest that H₃ receptor is on the somatodendritic area of SNr neurons. In addition, H₃ receptor activation also slightly reduced the frequency of mIPSCs in SNr neurons, indicating a low level of H₃ receptor expression on GABA axon terminals synapsing onto SNr neurons. This is consistent with histochemical studies (Pillot et al. 2002; Vizuete et al. 1997) and also with previous findings that H₃ receptor serves as an inhibitory presynaptic receptor (Arrang et al. 1983; Brown and Haas 1999; Jang et al. 2001; Threlfell et al. 2004; for review see Haas and Panula 2003).

Importantly, the small hyperpolarization induced by H₃ receptor activation was able to alter the pattern of SNr neuron firing, making the firing of these neurons more irregular (Fig. 7, A–D). Apparently, under normal conditions SNr neurons are depolarized sufficiently and can spike reliably and regularly. When hyperpolarized by H₃ receptor activation or direct negative current injection (Fig. 8), these neurons are no longer sufficiently depolarized, such that they reach action potential threshold less reliably and consequently spike more irregularly.

The molecular identities of the ion channel(s) affected by H₃ receptor and also those affected by H₁ and H₂ receptors are not known. Our results show that histamine did not affect the usual suspects such as the leak K⁺ conductance and AHPs in SNr GABA neurons. Instead, our observations suggest that histamine may modulate a tonically active, Na⁺-dependent inward current that was critical to the spontaneous firing of SNr GABA neurons (Atherton and Bevan 2005). Specifically, H₁ and H₂ receptors appeared to upregulate this tonic inward current, whereas H₃ receptor seemed to downregulate it. However, a definitive answer requires cloning of the channel conducting the tonic Na⁺-dependent inward current (for an example of histamine regulation of molecularly identified K⁺ channel see Atzori et al. 2000).

Tonic activation of histamine receptors by endogenous histamine

We found that blockade of H₁ and H₂ receptors induced a small hyperpolarization, decreased firing frequency, and increased the firing irregularity in SNr neurons (Fig. 10). These results indicate that spontaneously released endogenous histamine may induce a low-level, tonic activation of histamine receptors and influence SNr neuron activity. This is not surprising because other neurotransmitters such as acetylcholine, glutamate, GABA, dopamine, and serotonin are known to be released spontaneously from axon terminals (Colquhoun and Sakmann 1998; Katz 1969; Zhou et al. 2005). Even though in vivo confirmation will be required, the tonic activation of H₁ and H₂ receptors arising from endogenous histamine release may help to keep SNr neurons sufficiently depolarized and spiking reliably. Furthermore, the results on endogenous histamine are consistent with those obtained with exogenous histamine ligands, suggesting that the observations and conclusions made in this study are physiologically relevant. It should also be pointed out that the potential constitutive activity of H₁ and H₂ receptors may also contribute to the tonic H₁ and H₂ receptor activity detected here (Bakker et al. 2000; Smit et al. 1996).

Functional implications

Our present study indicates that the direct effects of histamine on SNr GABA projection neurons are a mild, H₁ and H₂ receptor-mediated excitation and a weak, H₃ receptor-mediated inhibition. Functional balance of these different histamine receptors may alter the intensity and pattern of SNr GABA neuron activity. Consequently, basal ganglia output and movement control may also be affected. Although the situation is likely to be more complex in vivo because histamine may also affect afferents to SNr neurons (Brown et al. 2001; Hass and Panula 2003; Threlfell et al. 2004), the direct histamine effects on SNr neurons described herein are likely to be important. Indeed, selective activation of H₃ receptors by injection of an H₃ receptor agonist into SNr has been shown to influence motor behavior in rats (Garcia-Ramirez et al. 2004). In addition, a recent study found that systemic administration of an H₃ receptor agonist worsened parkinsonian symptoms in a primate model of Parkinson’s disease (Gomez-Ramirez et al. 2006). In aggregate, these findings indicate that H₃ receptors may regulate the activity of SNr GABA projection neurons and basal ganglia output. In Parkinson’s disease, histamine levels in the substantia nigra (both SNc and SNr) were substantially increased (Anichtchik et al. 2000; Rinne et al. 2002). Nigral H₃ receptor expression was also increased in patients with Parkinson’s disease (Anichtchik et al. 2001) and a rodent model of the disease (Ryu et al. 1994). Because H₃ activation causes hyperpolarization, decreases firing rates, and increases the irregularity of spike firing, abnormally high levels of histamine innervation and H₃ receptor expression in SNr in parkinsonian brain may adversely alter the intensity and pattern of the basal ganglia output and consequently contribute to multiple aspects of movement disorders of basal ganglia origin.

	GRANTS

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This work was supported by grants from the National Alliance for Research on Schizophrenia and Depression and National Institutes of Health Grants MH-067119 to F.-M. Zhou and NS-04858 to M. S. LeDoux.

	DISCLOSURE

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The authors declare no conflict of interest.

	ACKNOWLEDGMENTS

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We thank L. Lu and R. Williams for help with histology and S. Tavalin and S. Matta for comments.

	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: F.-M. Zhou, Department of Pharmacology, University of Tennessee College of Medicine, Memphis, TN 38163 (E-mail: fzhou3{at}utmem.edu)

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