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FASEB J. 2010 Jan; 24(1): 49–57.
doi: 10.1096/fj.09-137034
PMCID: PMC2797035
PMID: 19720621

Dopamine D2-receptor activation elicits akinesia, rigidity, catalepsy, and tremor in mice expressing hypersensitive α4 nicotinic receptors via a cholinergic-dependent mechanism

Rubing Zhao-Shea,* Bruce N. Cohen,† Herwig Just,† Tristan McClure-Begley,‡ Paul Whiteaker,§ Sharon R. Grady,‡ Outi Salminen,‖ Paul D. Gardner,* Henry A. Lester,† and Andrew R. Tapper*,1
Author information Article notes Copyright and License information Disclaimer

Abstract

Recent studies suggest that high-affinity neuronal nicotinic acetylcholine receptors (nAChRs) containing α4 and β2 subunits (α4β2*) functionally interact with G-protein-coupled dopamine (DA) D2 receptors in basal ganglia. We hypothesized that if a functional interaction between these receptors exists, then mice expressing an M2 point mutation (Leu9′Ala) rendering α4 nAChRs hypersensitive to ACh may exhibit altered sensitivity to a D2-receptor agonist. When challenged with the D2R agonist, quinpirole (0.5–10 mg/kg), Leu9′Ala mice, but not wild-type (WT) littermates, developed severe, reversible motor impairment characterized by rigidity, catalepsy, akinesia, and tremor. While striatal DA tissue content, baseline release, and quinpirole-induced DA depletion did not differ between Leu9′Ala and WT mice, quinpirole dramatically increased activity of cholinergic striatal interneurons only in mutant animals, as measured by increased c-Fos expression in choline acetyltransferase (ChAT)-positive interneurons. Highlighting the importance of the cholinergic system in this mouse model, inhibiting the effects of ACh by blocking muscarinic receptors, or by selectively activating hypersensitive nAChRs with nicotine, rescued motor symptoms. This novel mouse model mimics the imbalance between striatal DA/ACh function associated with severe motor impairment in disorders such as Parkinson’s disease, and the data suggest that a D2R–α4*-nAChR functional interaction regulates cholinergic interneuron activity.—Zhao-Shea, R., Cohen, B. N., Just, H., McClure-Begley, T., Whiteaker, P., Grady, S. R., Salminen, O., Gardner, P. D., Lester, H. A., Tapper, A. R. Dopamine D2-receptor activation elicits akinesia, rigidity, catalepsy, and tremor in mice expressing hypersensitive α4 nicotinic receptors via a cholinergic-dependent mechanism.

Keywords: acetylcholine, striatum, nicotine, parkinsonian, interneuron

Motor symptoms associated with Parkinson’s disease, including bradykinesia, rigidity, and resting tremor, are a consequence of the degeneration of dopaminergic neurons in basal ganglia (1). Although many studies show that loss of striatal dopamine (DA) release partially underlies loss of motor control, the striatum also receives a strong acetylcholine (ACh) stimulus (2, 3) from tonically active large aspiny cholinergic interneurons (4, 5). Not surprisingly, ACh neurotransmission is thought to play a critical role in controlling voluntary movement. For example, in patients with Parkinson’s disease, as DA levels in striatum decrease, ACh levels increase, suggesting that a DA/ACh imbalance may underlie motor symptoms (6, 7). Early treatments focused on blocking ACh signaling with muscarinic receptor antagonists. These drugs can relieve symptoms; however, severe side effects vitiate treatment (8). The mechanism of the striatal ACh increase remains unknown, although an early hypothesis stated that loss of DA near D2 receptors (D2Rs) on cholinergic interneurons led to disinhibition of these neurons and thus to the increased ACh release (9, 10). Contrary to this idea, DA depletion in primates fails to increase cholinergic interneuron pacemaker activity, indicating that another mechanism must account for the increase in striatal ACh (11, 12).

Neuronal nicotinic ACh receptors (nAChRs) are ligand-gated cation channels that are activated by the endogenous neurotransmitter, ACh, as well as by the psychoactive component of tobacco, nicotine (13). To date, 12 mammalian nAChR subunits are known (α2–10 and β2–4). The majority of high-agonist-affinity neuronal nAChR subtypes are heteromeric pentamers consisting of α and β subunits, while a subset of lower agonist affinity nAChRs are α7 homopentamers (14, 15). Both nicotine and ACh modulate striatal cholinergic interneuron excitability. Activation of nAChRs by nicotine in striatal slices inhibits pacemaker activity of cholinergic interneurons. Conversely, nAChR antagonists increase burst firing of these neurons, although the mechanism and nicotinic receptor subtypes involved in interneuron modulation is unknown (16). Interestingly, nAChR expression decreases in striatum of patients with Parkinson’s disease, as well as in animal models of the disease (17, 18), suggesting that a loss of nAChRs may contribute to dysregulation of cholinergic interneuron excitability.

Previously, it was shown that the D2R-D3R antagonist raclopride increased striatal DA release in rat, and this effect was blocked by dihydro-β-erythroidine (19), a competitive antagonist selective for β2* nAChRs (* represents subunits other than those indicated that may be present in the heteropentameric receptors). In addition, α4β2* nAChRs could be immunoprecipitated with D2Rs in transiently transfected HEK cell membranes, as well as striatal membranes, suggesting a direct physical interaction between the receptors (19). This is consistent with emerging evidence suggesting that DA receptors may exist as heteromeric complexes with ligand-gated ion channels (20). To explore a functional interaction between D2-like receptors and α4* nAChRs, we challenged mice expressing hypersensitive α4* nAChRs (21, 22) with the D2R agonist quinpirole. We hypothesized that if a functional interaction between α4* nAChRs and D2Rs exists, then activation of D2Rs in these animals would unmask this interaction. Remarkably, D2R activation in mutant, but not wild-type (WT), littermates elicited severe motor impairment characterized by akinesia, rigidity, catalepsy, and tremor.

MATERIALS AND METHODS

Mice

Male and female Leu9′Ala homozygous and WT littermates used in experiments were 8–20 wk of age and were housed 3–4 animals/cage until the start of each experiment. Leu9′Ala mice express α4 nAChR subunits that contain a single-point mutation, a leucine residue mutated to an alanine residue, within the putative pore forming transmembrane domain, M2, that renders nicotinic receptors containing the mutated subunit 50-fold more sensitive to nicotine and ACh (22, 23). The genetic engineering and initial characterization of these mice has been described previously (22). Animals were derived from heterozygous × heterozygous breeding pairs. Data presented are from Leu9′Ala mice that were backcrossed to C57Bl/6J mice >8 generations. Because no significant gender differences were observed between male and female Leu9′Ala mice or WT littermates in preliminary experiments, both genders were used. Animals were kept on a standard 12-h light-dark cycle with lights on at 7:00 AM and off at 7:00 PM. The animals were given food and water ad libitum. All experiments were conducted in accordance with the guidelines for care and use of animals provided by the National Institutes of Health, as well as with an animal protocol approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School, the University of Colorado, and the California Institute of Technology.

Drugs

Quinpirole hydrochloride (0.5–10 mg/kg), nicotine hydrogen bitartrate (0.03 mg/kg), and scopolamine hydrochloride (1 mg/kg) were purchased from Sigma (St. Louis, MO, USA) and dissolved in saline. Mice were injected i.p. at a volume of 5 μl/g body weight.

Behavior analysis

Ambulation

Ambulation (locomotion) was recorded in a novel cage using a cage rack photobeam system (San Diego Instruments, San Diego, CA, USA). A single ambulatory event was measured as disruption of 2 distinct light beams 10 cm apart. For locomotion measurements, mice were injected with quinpirole (5 mg/kg) in their home cage and then placed in the activity cage 60 min later. Locomotor activity was recorded over a 90-min period.

Rigidity

To measure rigidity, mice were suspended by their hind legs from the side of a plexiglass cage. The latency to fall was measured (24, 25).

Akinesia

To measure akinesia, mice were held by the tail with forepaws resting on a flat surface. The number of forward steps taken with both forelimbs was recorded during 30-s trials (24). Each animal received 2 trials every 30 min for 3 h. The 2 trials were averaged for each data point.

Tremor

Tremor was scored visually by using the following scale: 0 , no tremor; 1, isolated twitches; 2, tremor with periods of calm; 3, constant tremor (26). Tremor was scored during tail suspension.

Eye closure

Eye closure was visually scored using the following scale: 1, eyes ¼ closed; 2, eyes ½ closed; 3, eyes ¾ closed, 4, completely closed (27).

Catalepsy

Catalepsy was measured using the horizontal bar method: Mice had their forepaws placed on a horizontal bar 5 cm above a flat surface. Latency to move forepaws off the bar was measured. The cutoff for latency to move paws from the bar was 3 min.

Tissue collection

Mice were heavily anesthetized and decapitated, and brains were removed and placed on a glass plate over ice. The optic nerves, olfactory bulbs, and cerebellum were removed and discarded. The cortical hemispheres were spread apart and separated by cutting the corpus callosum, followed by removal of the hippocampi. The striata (caudate-putamen+nucleus accumbens) were removed without the adjacent cortex.

[3H]-spiperone binding

Plastic tubes (1 ml) set in a 96-well format were used. Buffer consisted of 50 mM HEPES, pH 7.5; 120 mM NaCl; 2 mM CaCl2; 1 mM MgCl2; and 5 mM KCl. Final reaction volume was 600 μl, consisting of mouse striatal membranes prepared as described previously (28) in buffer containing 2.0 nM 3H-spiperone, 20 μM pargyline, 1 mM ascorbic acid, and 50 nM ketanserin (to prevent spiperone binding to 5-HT2 receptors). For nonspecific binding assessment, 18 mM DA was added to the reaction. For D2/D3- vs. D4-binding populations, binding buffer was supplemented with 300 nM raclopride. Reaction mixtures were incubated at room temperature for ≥1 h to reach equilibrium, then binding was terminated by filtration at 4°C onto glass fiber filters previously soaked in 0.5% polyethylenimine, and washed with ice-cold buffer (6×) using an Inotech Cell Harvester (Inotech, Rockville, MD, USA). Filters were dried overnight, and radioactivity was measured in a 1450 MicroBeta scintillation counter after addition of Optiphase Supermix scintillation cocktail (both from Perkin Elmer Life and Analytical Sciences/Wallac Oy, Turku, Finland). Protein levels were determined by the Lowry method using BSA as standard.

Cannulation surgery

Mice were given a buprenorphine injection (0.05–0.1 mg/kg. s.c.) for pain relief before the operation. Mice were anesthetized under isoflurane anesthesia (induction 4.5%; maintenance 1.5–2.5%) and implanted with guide cannulae (CMA/7; CMA/Microdialysis, Stockholm, Sweden). Guide cannulae were positioned at the dorsal striatum based on coordinates from the Franklin and Paxinos (29) mouse brain atlas. Relative to bregma, the coordinates were A/P = +0.6, L/M = +1.8, D/V = −3.0. Cannulae were affixed to the skull with dental cement and 2 stainless steel screws. Postoperatively, 8–12 h after the initial dose, mice received another injection of buprenorphine (0.05–0.1 mg/kg, s.c.) to relieve pain. Mice were allowed to recover for ≥5 d before the start of the experiment.

In vivo microdialysis

At ∼4 PM on the day before the experiment, a microdialysis probe (CMA/7; 1.0-mm membrane, o.d. 0.24 mm; CMA, Stockholm, Sweden) was inserted into the guide cannula, and the probe was infused with a modified Ringer solution (147 mM NaCl, 1.2 mM CaCl2, 2.7 mM KCl, 1.0 mM MgCl2, and 0.04 mM ascorbic acid) at a flow rate of 0.5 μl/min. In the morning of the experiment day (∼8 AM), the flow rate was increased to 2.2 μl/min, and after 1 h, the collection of actual microdialysis samples (every 20 min, 44 μl/sample) was started. After completion of the experiments, the positions of the probes were verified histologically by fixing coronal brain sections on gelatin/chrome-alume coated slides.

High-performance liquid chromatography (HPLC)

The concentrations of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were determined by HPLC with an electrochemical detector (Coulochem II; ESA, Chelmsford, MA, USA) equipped with a model 5014B microdialysis cell, a Pharmacia LKB model 2248 HPLC pump (Pharmacia LKB, Uppsala, Sweden), and a pulse damper (SSI LP-21; Scientific Systems, State College, PA, USA). The column (Spherisorb ODS2; 3 μm, 4.6×100 mm, Waters, Milford, MA, USA) was kept at 40°C with a column heater (Croco-Cil, Bordeaux, France). The mobile phase used consisted of 0.1 M NaH2PO4 buffer, pH 4.0 (adjusted with 1.0 mM citric acid), 0.6–0.8 mM octanesulphonic acid, 12–15% methanol, and 1.2 mM EDTA. The flow rate of the mobile phase was 1.0 ml/min. The dialysate sample (35 μl) was injected onto the column with a CMA/200 autoinjector (CMA). DA was reduced with an amperometric detector (potential −100 mV) after being oxidized with a coulometric detector (+300 mV); DOPAC and HVA were also oxidized with the coulometric detector. The chromatograms were recorded, and the peak heights were analyzed using Azur 4.0 software (Datalys, Theix, France).

Striatal DA content

The brain was removed from the skull and placed on a glass plate over ice. Striata were isolated as described above. Striata were weighed and stored at −80°C until assayed. The striatal concentrations of DA, DOPAC, and HVA were measured by HPLC with electrochemical detection (described above) after Sephadex G-10 gel chromatographic cleanup of samples as described by Haikala (30). As a control, frontal cortex norepinephrine content was measured. Monoamine and metabolite values were calculated as micrograms per gram wet weight of tissue.

Preparation of striatal tissue sections

A separate group of mice was used for immunohistochemistry experiments. Mice were anesthetized with sodium pentobarbital (200 mg/kg, i.p.) 90 min after saline or quinpirole injection (5 mg/kg), and perfused transcardially with 10 ml of 0.1 M phosphate-buffered saline (PBS) followed by 10 ml of 4% paraformaldehyde (pH 7.4). Brains were removed and postfixed for 2 h, and cryoprotected in 30% sucrose until brains sank. Striatal serial sections (20 μm, from bregma 1.1 to 0.60 mm) were cut by using a freezing microtome and collected into 24-well tissue culture plates containing PBS.

Double immunofluorescence

Free-floating sections were washed with PBS 2 × 5 min on an orbital shaker, and incubated in 0.4% Triton X-100 PBS (PBST) for 2 × 2 min followed by 2% BSA/PBS for 30 min. Sections were washed with PBS once and then incubated in a cocktail of primary antibodies for choline acetyltransferase (ChAT; monoclonal, 1:100, sc-55557; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and c-Fos (polyclonal, 1:400, sc-52; Santa Cruz) in 2% BSA/PBS overnight at 4°C. The sections were then washed with PBS 3 × 5 min followed by incubation in secondary fluorescence labeled antibodies (goat anti-rabbit Alexa Fluor® 488 and goat anti-mouse Alexa Fluor 594, 1:250; Molecular Probes, Eugene, OR, USA) at room temperature in the dark for 2 h. After washing with PBS 5 × 5 min, sections were mounted on slides using Vectashield® mounting medium (Vector Laboratories, Burlingame, CA, USA).

Optical density measurements

The number of c-Fos positive neurons was counted under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany) with an ×20 objective using an unbiased approach. The area of each imaged section was 0.42 × 0.32 mm = 0.13 mm2. The immunopositive counts from dorsal striatum were averaged and scaled to provide a mean number of c-Fos positive cells/mm2. ChAT-positive neurons were imaged with an ×63 objective. Striatal ChAT-positive neurons were sparse and displayed a soma size >20 μm in diameter, consistent with striatal cholinergic interneurons that make up ∼1% of neurons in striatum (31). The intensity of fluorescence was quantified using a computer-associated image analyzer (Axiovision 4.6; Carl Zeiss). Neurons were counted as signal positive if intensities were ≥2-fold higher than that of the average value of background (sections stained without primary antibodies). The percentage of c-Fos positive neurons that were also ChAT positive was calculated.

Statistics

Behavioral data were analyzed via 1- or 2-way ANOVAs, as indicated, with repeated measures as needed. For immunohistochemistry, data were analyzed with Student’s t test. Data were analyzed using Origin 7.5 software (OriginLab Corp., Springfield, MA, USA). All values are expressed as means ± se. Values of P < 0.05 were considered significant.

RESULTS

In initial behavioral tests of D2R activation in Leu9′Ala mice, we injected Leu9′Ala homozygous animals with the D2-like receptor agonist, quinpirole. Figure 1 illustrates the responses to D2-like receptor activation 1 h after injection of 5 mg/kg quinpirole in WT and Leu9′Ala mice (Fig. 1). Leu9′Ala mice developed rigidity of the extremities, postural instability, and arched back (Fig. 1D) compared to WT (Fig. 1C). In addition, resting tremor was observed during the onset of motor symptoms, but ceased once rigidity and akinesia became severe. However, when mice were held by the tail, the tremor was readily apparent for the duration of the phenotype. In addition, Leu9′Ala displayed clasped fore- and hindlimbs (Fig. 1B) compared to WT (Fig. 1A). After quinpirole injection, Leu9′Ala mice were significantly hypoactive in a novel environment compared to WT animals (Fig. 2A, average summed activity over 30 min 55.9±10.2 in WT mice vs. 5.29±1.66 in Leu9′Ala homozygous mice; F1,13=20.8, P<0.001). In the catalepsy test (see Materials and Methods), Leu9′Ala mice had a significantly greater latency to remove their forepaws from an elevated horizontal bar compared to WT mice after quinpirole exposure, indicating catalepsy (Fig. 2B, 139.3±23.1 vs. 1.67±0.33 s, respectively; F1,4=35.5, P<0.01). To measure rigidity, mice were subjected to a “grasping” test (Fig. 2C; see Materials and Methods). After quinpirole injection, Leu9′Ala mice grasped the side of a cage significantly longer than WT mice (42.8±6.1 vs. 1.25±0.25 s, respectively; F1,6=46.2, P<0.001). Together, these behavioral data indicate that Leu9′Ala mice develop severe akinesia, rigidity, and tremor after quinpirole injection. Leu9′Ala mice displayed this phenotype with quinpirole doses as low as 0.5 mg/kg, whereas doses as high as 10 mg/kg did not elicit symptoms in WT mice (data not shown).

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The D2-like receptor agonist quinpirole elicits a hypomotor phenotype in Leu9′Ala but not WT mice. Photographs of WT (A, C) and Leu9′Ala mice (B, D) 1 h after quinpirole injection (5 mg/kg, i.p.). Note the clasped fore- and hindlimbs in Leu9′Ala mice compared to WT animals (A, B). D2R activation elicits akinesia, posture instability, and rigidity in Leu9′Ala mice but not WT animals (compare C and D). Also note the arched back in Leu9′Ala mice. (WT mice were held by the tail to prevent locomotion during photo; C.)

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Behavioral analysis of motor symptoms in Leu9′Ala mice. A) Total ambulation in a novel environment 1 h after i.p. injection of quinpirole (5 mg/kg) in WT (left) or Leu9′Ala homozygous mice (right). B) Catalepsy assay: latency to remove paws from an elevated horizontal bar 1 h after i.p. injection of quinpirole in WT (left) or Leu9′Ala homozygous mice (right). C) Rigidity assay: time spent grasping (hanging) from the side of a cage 1 h after quinpirole injection in WT (left) or Leu9′Ala (right) mice. D) Akinesia assay: Number of forward steps taken with both forelimbs during 30-s trials after injection of quinpirole in WT (solid squares) or Leu9′Ala homozygous mice (open squares). Mice received saline at time 0. Each data point represents the average of 2 trials/animal every 30 min postinjection. E) Tremor score (see Materials and Methods) at 30-min intervals after quinpirole injection in WT and Leu9′Ala mice. F) Closed-eye score in WT and Leu9′Ala after saline (t=0) or at 30-min intervals after quinpirole injection. n = 4–8 mice/test. **P<0.01, ***P < 0.001.

To determine the onset, severity, and duration of akinesia, we quantified and recorded the number of forward steps taken with both forelimbs in Leu9′Ala and WT littermates before and after quinpirole injection (Fig. 2D). Steps were measured during 30-s trials (24) every 0.5 h over the course of 3 h following quinpirole injection. Overall, quinpirole induced a significant effect on the akinesia assay postinjection in mutant (F6,21=3.98, P<0.01) but not WT animals (F6,21=0.66, NS). Post hoc analysis revealed a significant effect of quinpirole in Leu9′Ala mice 0.5, 1.0, and 1.5 h after injection, but not after 2.0, 2.5, or 3.0 h, indicating recovery from the drug. Similarly, quinpirole significantly elicited a tremor over time in Leu9′Ala but not WT mice. The tremor became constant ∼ 1 h post-quinpirole injection (F6,21=25.1, P<0.001). Again, post hoc analysis revealed a significant effect of tremor 1.0, 1.5, 2.0, and 2.5 h after injection but no significant effect after 3.0 h (Fig. 2E). A similar timeline occurred for quinpirole-induced eyelid closure (Fig. 2F). Thus, symptoms were reversible; ∼3 h postinjection, Leu9′Ala mice acted indistinguishably from WT.

To determine whether the difference in quinpirole sensitivity between genotypes was due to compensatory changes of D2-like receptor expression in Leu9′Ala mice, we measured D2R levels using radioligand binding. In striatum there was no significant change in raclopride-sensitive (D2R) or -resistant (D4R) [3H]spiperone binding between Leu9′Ala homozygous and WT mice. Raclopride-sensitive binding in WT striatum was 225 ± 16 fmol/mg protein compared to 217 ± 51 fmol/mg protein in Leu9′Ala striatum. Raclopride-resistant binding in WT striatum was 389 ± 24 fmol/mg protein compared to 412 ± 34 fmol/mg protein in Leu9′Ala striatum (n=5/genotype).

Because of the known role of D2R in inhibiting DAergic neuronal activity (32), we measured total striatal tissue DA content in both genotypes. No significant difference was observed in DA tissue content (data not shown). To assay striatal DA release, we utilized in vivo microdialysis and compared DA concentrations in WT and Leu9′Ala mice at baseline and after injections of quinpirole. Baseline DA release was not significantly different between genotypes (26.7±4.3 and 25.3±5.1 fmol/sample in WT vs. Leu9′Ala homozygous mice; n=11/genotype). Using in vivo microdialysis, we compared striatal DA release in WT and mutant mice challenged with 0.5 mg/kg quinpirole. Similar to previous reports, quinpirole significantly reduced DA concentrations in striatum (33). A maximal reduction of DA release was observed 60 min after quinpirole injection in both WT and Leu9′Ala mice to 51.7 ± 8.8 and 40.0 ± 4.4% of baseline, respectively (Fig. 3A). Repeated-measure 2-way ANOVA indicated no significant difference of genotype on this effect of quinpirole. In addition, no significant differences in the effect of quinpirole were observed on DA metabolites HVA and DOPAC between genotypes (Fig. 3B, C). Notably, the parkinsonian phenotype was observed in Leu9′Ala but not WT mice in response to 0.5 mg/kg quinpirole in these experiments (data not shown).

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Striatal release of DA and its metabolites does not differ between WT and Leu9′Ala mice in response to quinpirole. A) Striatal DA release of awake behaving WT and Leu9′Ala mice, as measured by in vivo microdialysis, at baseline and after injection of 0.5 mg/kg quinpirole (arrow). Microdialysis samples were acquired every 20 min. B, C) DOPAC (B)and homovanillic acid release (C) for WT and Leu9′Ala homozygous mice at baseline and after quinpirole as in A. n = 8–11 mice/genotype.

Recently, striatal cholinergic interneurons have reemerged as potential modulators of parkinsonian symptoms (34). To determine whether these neurons were involved in the quinpirole-induced motor phenotype in Leu9′Ala mice, we challenged a separate group of WT and Leu9′Ala mice with quinpirole and examined c-Fos immunoreactivity in ChAT-positive striatal neurons as a gross measure of neuronal activation (35,36,37). In WT mice, the percentage of ChAT, c-Fos-immunopositive neurons was not altered by quinpirole exposure compared to saline (Fig. 4A, B; 24.3±1.5 vs. 23.9±3.8%). However, quinpirole did significantly decrease the number of non-ChAT, c-Fos-immunopositive neurons by 32.6 ± 4.7% compared to saline (P<0.05; n=24–30 slices, 3 mice/treatment, data not shown). In contrast to the effects in WT mice, quinpirole induced a robust, significant increase in the percentage of c-Fos, ChAT-immunopositive striatal neurons in Leu9′Ala mice compared to saline (72.8±0.4 vs. 20.1±1.5%, respectively; P<0.001; Fig. 4A, B). In addition, quinpirole increased the number of non-ChAT, c-Fos-immunopositive neurons by 38.2 ± 14.1% compared to saline, although this was not statistically significant (P>0.05; n=25–30 slices, 3 mice/treatment, data not shown). Together, these data suggest that, although quinpirole decreases DA release in striatum in both genotypes, it increases cholinergic interneuron activity more strongly in Leu9′Ala than in WT mice.

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Quinpirole activates striatal cholinergic interneurons in Leu9′Ala homozygous mice. A) Representative images of cholinergic interneurons within dorsal striatal slices immunostained to detect c-Fos (left, green) or ChAT (middle, red). Right column shows merged images. WT (top 2 rows) and Leu9′Ala mice (bottom 2 rows) were perfused and brains were harvested 90 min after injection of saline (top) or 5 mg/kg quinpirole (bottom). B) Bar graph presents percentage of ChAT-positive neurons that also express c-Fos after a saline (open bars) or quinpirole (filled bars) injection. **P < 0.01.

To determine whether muscarinic ACh actions play a role in the striking akinesia induced by quinpirole in Leu9′Ala mice, we preinjected mice with either saline or the muscarinic antagonist scopolamine 15 min before quinpirole injection. Scopolamine preinjection effectively rescued the phenotype. Leu9′Ala mice that received a scopolamine injection before quinpirole exhibited significantly greater ambulation compared to saline-preinjected mice (Fig. 5A). At 30 min, summed average ambulation was 128.7 ± 25.8 with scopolamine pretreatment compared to only 12 ± 8.6 with saline pretreatment (Fig. 5B; F1,6=41.7, P<0.001).

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Muscarinic antagonism and nicotinic agonism reverses quinpirole-induced motor phenotype. A) Leu9′Ala homozygous animals were injected with the muscarinic antagonist scopolamine (1 mg/kg, i.p., open squares) or saline (solid squares) 15 min before challenge with quinpirole (5 mg/kg). Each data point represents 5 min total ambulation in a novel activity chamber, 1 h after quinpirole injection. B) Summed average ambulatory activity for 30 min from the experiment in panel A. C) Leu9′Ala homozygous mice were injected with 5 mg/kg quinpirole. At 1 h postinjection, when the motor phenotype was readily apparent, mice were injected with saline (solid squares) or 0.030 mg/kg nicotine (open squares) and immediately placed in activity chambers. D) Summed average ambulatory activity for 30 min from the experiment in panel C. **P < 0.01, ***P < 0.001.

Based on previous studies indicating that nicotine can inhibit cholinergic interneuron activity (16), we also selectively activated Leu9′Ala α4* nAChRs with a small dose of nicotine (0.030 mg/kg) in animals already expressing the quinpirole-induced akinetic phenotype. Nicotine produced a robust, significant burst in locomotor activity compared to a saline injection (Fig. 5C). Summed average ambulation during the 30 min immediately after nicotine injection was significantly greater than summed ambulation after a saline injection (60.1±9.7 after nicotine vs. 7.6±4 after saline injection; F1,14=24.3, P<0.001; Fig. 5D).

DISCUSSION

Improved mouse models of voluntary motor disorders such as Parkinson’s disease are critical in order to obtain a better understanding of potential mechanisms underlying motor symptoms. Here we describe a novel pharmacologically reversible hypomotor phenotype in mice expressing α4* nAChRs that are hypersensitive to ACh. This phenotype is revealed by activating D2-like receptors via challenging mutant animals with the D2-like receptor agonist quinpirole. In Leu9′Ala mice, but not WT littermates, quinpirole elicited several motor symptoms that are often associated with a DA-depleted state, including akinesia, rigidity, catalepsy, and tremor.

Although quinpirole induced a dramatic decrease in striatal DA release, presumably via activation of inhibitory D2 autoreceptors in DAergic neurons of the substantia nigra pars compacta (38, 39), the onset and magnitude of this effect did not differ between Leu9′Ala mice and WT littermates. In addition, baseline DA release and striatal tissue content also did not differ between genotypes. This finding is surprising, both because the motor symptoms often indicate a DA-depleted state and because previous studies suggest that there may be crosstalk between D2Rs and α4β2 nAChRs on presynaptic DAergic terminals (19).

If a decrease in striatal DA release alone cannot account for the remarkable motor impairments characterized in quinpirole-challenged Leu9′Ala mice, then what is the explanation? Our data suggest that the mechanism involves striatal cholinergic interneurons. After quinpirole injection, Leu9′Ala, but not WT mice, exhibited robust increases in c-Fos immunoreactivity in ChAT-positive striatal neurons, indicating increased activation of cholinergic interneurons. While quinpirole decreased the number of c-Fos-immunoreactive noncholinergic neurons in dorsal striatum of WT mice, the drug increased the number of c-Fos-immunoreactive neurons in Leu9′Ala mice, supporting the hypothesis that ACh concentrations were increased in these animals. Administration of the acetylcholinesterase inhibitor tacrine, or activation of muscarinic receptors by a nonspecific muscarinic agonist, such as pilocarpine, can increase the number of c-Fos-immunoreactive neurons (40, 41) in dorsal striatum. Furthermore, blocking muscarinic receptors rescued motor impairment. This finding is significant because motor symptoms, like those associated with Parkinson’s disease, are associated with a DA/ACh imbalance (7, 42). As DA decreases and motor symptoms progressively become more severe, striatal ACh levels rise. Cholinergic interneurons express inhibitory D2Rs that, when DA levels decrease, could disinhibit these interneurons (42,43,44). However, recent data suggest that cholinergic disinhibition via DA depletion alone does not account for increased ACh release, but rather is explained by reduction of muscarinic receptor (M4) autoreceptors (45). Our data indicate that neuronal nAChRs and D2Rs may functionally interact to modulate ACh levels during DA depletion.

While most studies in striatum have focused on the role of neuronal nAChRs expressed on DAergic presynaptic terminals and their facilitation of striatal DA release (46,47,48,49), less is known about the expression and function of nAChRs in striatal neurons. Our data suggest that α4* nAChRs contribute, directly or indirectly, to regulating cholinergic interneuron excitability. Application of nicotine in striatal slices inhibits cholinergic interneurons and, conversely, blockade of nAChRs with the nonspecific antagonist mecamylamine increases burst firing (16). Consistent with this finding, selective activation of α4* nAChRs with nicotine rescued motor impairment in Leu9′Ala mice, although we cannot rule out that this rescue was solely due to silencing of cholinergic interneurons, since nicotine would also be predicted to increase striatal DA release (50). In addition, some reports indicate that nAChRs are expressed in fast-spiking GABAergic interneurons, which presumably provide local inhibitory input onto cholinergic interneurons (51, 52), as well as the more predominant medium spiny neurons of striatum. Thus, activation of nAChRs on these interneurons would be expected to reduce cholinergic interneuron excitability. However, we find no evidence that striatal GABAergic or cholinergic interneurons express α4* nAChRs (unpublished results).

How could mice expressing a single-point mutation that renders α4* nAChRs hypersensitive to agonist exhibit such a dramatic behavioral phenotype when challenged with a D2-like receptor agonist? One explanation could be that mice expressing the Leu9′Ala α4 mutation have compensatory increases in D2 autoreceptor expression in DAergic neurons. However, radioligand binding uncovered no such compensation. In addition, in vivo microdialysis revealed little difference in D2-like receptor agonist-mediated DA release in striatum. These data also suggest that a functional interaction between α4* nAChRs and D2Rs is unlikely to exist in DAergic neurons themselves. We cannot rule out the possibility that D2R expression is altered in cholinergic interneurons in Leu9′Ala mice, but increased expression would not be expected to yield neuronal activation in response to a D2 agonist. Possibly the mechanism of this interaction involves known effects of nAChR activation on the frequency dependence of DA release from DAergic terminals; this would escape detection in the dialysis data and must be studied with time-resolved measurements (53, 54). Therefore, future experiments utilizing slice electrophysiology and electrochemistry will focus on uncovering a potential functional interaction between α4* nAChR and D2-like receptors that results, perhaps indirectly, in modulation of striatal cholinergic interneurons.

In summary, our data provide a novel, reversible, genetic, and pharmacological mouse model that mimics the striatal DA/ACh concentration imbalance associated with severe motor impairment. Because this phenotype is elicited by a D2R agonist in a mouse expressing hypersensitive α4* nAChRs, these data also suggest that α4* nAChRs and D2Rs may functionally interact to regulate cholinergic interneuron activity.

Acknowledgments

The authors thank Ms. Marjo Vaha for excellent technical assistance and Dr. T. Petteri Piepponen for advice with the microdialysis experiments. This work was supported in part by the National Institute of Neurological Disorders and Stroke (NS059586), the National Institute on Drug Abuse (DA017279 and DA12242), and the National Institute on Aging (AG033954). Production of mice in Boulder was supported by NIH animal resources grant DA015663.

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