Behavioral Cross-Tolerance between Repeated Intracerebellar Nicotine and Acute Δ⁹-Tetrahydrocannabinol-Induced Cerebellar Ataxia: Role of Cerebellar Nitric Oxide

Several investigators have demonstrated a strong association between the abuse of nicotine and Δ⁹-THC together (Simmons and Tashkin, 1995; Watson et al., 2000). It has also been shown that tobacco abuse may precede the abuse of marijuana (Simmons and Tashkin, 1995). Furthermore, studies have reported that the rewarding properties of nicotine may be dependent on a functioning cannabinoid system (Castañé et al., 2002) and that nicotine possibly modulates numerous subjective and physiological effects of Δ⁹-THC (Penetar et al., 2005). These studies, taken collectively, provide strong evidence for potential functional interactions between nicotine and Δ⁹-THC.

Nicotine and Δ⁹-THC are abused and harmful and have been shown to interact although few studies have addressed the specific behavioral and biochemical consequences of co-administration of nicotine and Δ⁹-THC in animal models, despite the current association of the two substances in humans (Balerio et al., 2004). Le Foll et al. (2006) reported that nicotine pre-exposure may produce some tolerance to the sedative effects of Δ⁹-THC administration. We have shown that acute nicotine-induced attenuation of cerebellar Δ⁹-THC-induced ataxia occurs through the cerebellar α₄β₂ nAChR subtype in a dose-dependent manner (Smith and Dar, 2006). Recently, we have also reported (Smith and Dar, 2007) that the attenuation of Δ⁹-THC-induced ataxia via activation of α₄β₂ nAChR subtype appeared to involve participation of nitric oxide-guanylyl cyclase signaling. Intracerebellar microinfusion of nitric oxide donor and inhibitor of inducible nitric-oxide synthase markedly increased and decreased, respectively, the α₄β₂ nAChR subtype-induced attenuation of Δ⁹-THC ataxia. The attenuation was similarly enhanced and reduced by intracerebellar administration of an activator and inhibitor of guanylyl cyclase, respectively. These behavioral data excellently correlated with changes in the cerebellar nitric oxide concentrations where Δ⁹-THC decreased and the α₄β₂ nAChR subtype agonist increased, respectively, the cerebellar concentrations (Smith and Dar, 2007).

In this study we also evaluated possible involvement of cerebellar nitric oxide production in the cross-tolerance between nicotine or RJR-2403, a α₄β_2-selective nAChR agonist, and Δ⁹-THC. Both nitric oxide and nitric-oxide synthase are expressed in high levels in the cerebellar cortex (Vincent et al., 1998; Hartell et al., 2001). Specifically, the cerebellar Purkinje cells express nitric oxide-stimulated soluble guanylyl cyclase, thereby generating cGMP (Hartell et al., 2001). Nicotine administration resulting in increased locomotor activity has been shown to be blocked by inhibitors of nitricoxide synthase, suggesting the role of nitric oxide in the control of locomotor activity (Shim et al., 2002). Conversely, cannabinoids, such as Δ⁹-THC, have been shown to decrease production of nitric oxide (Lévénés et al., 1998), and the nitric oxide system has been shown to play a significant role in the effect of Δ⁹-THC on locomotor activity (Azad et al., 2001). Furthermore, recent findings in our laboratory have shown the importance of cerebellar nitric oxide in both the development of cerebellar Δ⁹-THC-induced ataxia, as well as its attenuation by nicotine or RJR-2403 in the acute paradigm (Smith and Dar, 2007).

The primary purpose of this investigation was to evaluate the consequence of repeated intracerebellar microinfusion of nicotine or RJR-2403 on acute intracerebellar Δ⁹-THC-induced ataxia. We intended to address: 1) whether cross-tolerance between nicotine or RJR-2403 and Δ⁹-THC exists; 2) the minimum time required for the development of cross-tolerance; 3) the duration over which cross-tolerance to Δ⁹-THC-induced ataxia persists; and 4) whether initial activation of the cerebellar α₄β₂ nAChRs is involved in the development of cross-tolerance between nicotine or RJR-2403 and Δ⁹-THC. Additionally, the possible involvement of cerebellar nitric oxide in the behavioral cross-tolerance between nicotine or RJR-2403 and Δ⁹-THC was investigated. Our hypothesis was that repeated nicotine or RJR-2403 administration attenuates acute cerebellar Δ⁹-THC-induced ataxia through participation of the cerebellar α₄β₂ nAChR subtype, and this attenuation is functionally correlated with changes in the concentration of cerebellar nitric oxide.

Materials and Methods

Animals

Male CD-1 mice were purchased from Charles River Laboratories, Inc. (Raleigh, NC). The mice were 5 to 6 weeks old and ranged in weight from 23 to 28 g at the time of behavioral experiments. The mice were maintained in a housing facility under controlled humidity and temperature (22–24°C) and kept on a 12-h light/dark cycle. Mice were allowed to acclimate to their housing conditions for 2 days before stereotaxic surgery. After the implantation of a stainless steel guide cannula for direct microinfusion into the cerebellum, each animal was housed in its own individual plastic cage. Mice had free access to water and commercial mouse chow. Each animal was used only once in the Rotorod experiment. All animals used in the current study were maintained according to the authors' animal use protocol approved by the East Carolina University Animal Care and Use Committee in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).

Stereotaxic Surgery

The surgical procedure was performed under aseptic conditions, and all surgical tools were sterilized via autoclaving and/or glass beads. Two days after arrival, mice were brought to the laboratory from the housing facility and anesthetized with chloral hydrate (450 mg/kg i.p.) before placement in a small stereotaxic frame (model 900; David Kopf Instruments, Tujunga, CA). The head of each mouse was trimmed of hair, scrubbed with povidone-iodine (The Clinipad Co., Rocky Hill, CT) via swab stick and then wiped clean with an isopropyl alcohol swab. With the skull flat, a 2-cm-long midsagittal incision was made by sterile scalpel to expose the skull. Cannulation of the cerebellum was performed aseptically according to the following coordinates of Slotnick and Leonard (1975): AP –6.4 mm (from bregma); ML ± 0.8 mm; and DV –1.0 mm from the skull surface. The stainless steel guide cannula (22 gauge, 10 mm length) was lowered through a drilled craniotomy hole via a Masterlight Hand Piece (Henry Schein, Port Washington, NY) into the superficial layers of the anterior lobe region of the cerebellum. Durelon cement (Premier Dental Products Co., Norristown, PA) was used to anchor the cannula to the skull surface. A removable stainless steel wire plug was placed inside the guide cannula to prevent occlusion. After surgery, each animal received 3000 units/20 g s.c. of procaine and benzathine penicillin G (Durapen; VEDCO Inc., St. Joseph, MO) to prevent infection during postsurgical recovery. Each animal also received an injection of ketorolac tromethamine (2 mg/kg s.c.; Abbott Laboratories, North Chicago, IL) for analgesia shortly after surgery and again 4 h later. Animals were allowed to recover in their own individual cages in the animal housing facility maintained by the Department of Comparative Medicine for 2 days before the start of repeated drug microinfusions.

Drugs

In general, drug solutions were either prepared on the day of behavioral experiments or prepared 1 day earlier and kept in the deep freezer at –70°C. Chloral hydrate was prepared in distilled water and injected at a volume of 0.1 ml/10 g b.wt. The following drugs were used in the current investigation: CB₁ receptor agonist Δ⁹-THC was provided by the United States Department of Health and Human Services from the National Institute on Drug Abuse Research Triangle Institute (Research Triangle Park, NC); (–)-nicotine-di-l-tartrate, nonselective nicotinic acetylcholine receptor antagonist hexamethonium, and α₄β₂-selective nAChR subtype antagonist dihydro-β-erythroidine hydrobromide (DHβE), were purchased from Sigma-Aldrich (St. Louis, MO); and α₄β₂-selective nAChR subtype agonist N-methyl-4-(3-pyridinyl)-3-buten-1-amine(RJR-2403 fumarate or metanicotine) was bought from Tocris (Ellisville, MO). Δ⁹-THC was dissolved in 100% dimethyl sulfoxide (DMSO), and all nicotinic drugs were dissolved in artificial cerebrospinal fluid (aCSF). The composition of aCSF was the following: 127.65 mM NaCl; 2.55 nM KCl; 0.05 mM CaCl₂; 0.94 mM MgCl₂; and 0.05 nM Na₂S₂O₅ (at pH 7.4). The reagents used in the nitrite assay were as follows: 2,3-diaminonaphthalene (DAN), d-glucose 6-phosphate disodium salt dihydrate (G-6-P), glucose-6-phosphate dehydrogenase (G-6-PDH) (type IX), β-NADPH reduced tetra sodium salt, nitrate reductase [(NAD[P]H) from Aspergillus niger] and sodium nitrite. All reagents were purchased from Sigma-Aldrich. Enzymes and cofactors were stored individually at –70°C. The key reagents, DAN, NADPH, and the standard sodium nitrite solutions were prepared fresh on the day of the total sum of nitrite + nitrate (NO_x) assay and were kept protected from light.

Intracerebellar Microinfusions

All drugs used in the present investigation were administered via intracerebellar microinfusion. A Harvard model 22 (Harvard Apparatus, Holliston, MA) microinfusion syringe pump was used for drug infusions. Drugs were microinfused through PE-10 (Clay Adams; Parsippany, NJ) polyethylene tubing fitted with a 25-μl Hamilton syringe. The sterile stainless steel injection cannula (30 gauge; 0.31 mm diameter) was fitted to the PE-10 tubing so that the total length of exposed injection cannula was 11 mm. This allowed for protrusion of the injection cannula 1 mm beyond the lower tip of the guide cannula. All drugs were infused at a rate of 0.1 μl/min for 1 min, in a total volume of 100 nl, with the exception of Δ⁹-THC, which was given at 1 μl/min, resulting in a total volume of 1 μl. Injection cannulas were left in guide cannulas for 1 additional min to allow for adequate diffusion of the solution. An air bubble separating drug solution and water in the polyethylene tubing was monitored for continuous movement to indicate that blockage was not occurring and that the desired drug dose was administered. The area of the cerebellum targeted for direct microinfusion was the spinocerebellar molecular layer with the following coordinates: AP –6.4 mm (from bregma); ML ± 0.8 mm; DV –1.0 mm from the skull surface. These coordinates were selected because of the ability of Δ⁹-THC to produce significant cerebellar ataxia. Before the beginning of stereotaxic cerebellar microinfusion work, the authors conducted functional mapping of the cerebellar cortex. The results of the mapping study (data not published) indicated marked functional (motor) activity when anteroposterior stereotaxic coordinates were between –6.2 to –6.4 (i.e., a location within the culmen of the anterior lobe of the cerebellum). Microinfusion of Δ⁹-THC, which is known to produce cerebellar ataxia, failed to alter normal motor coordination when microinfused into other cerebellar cortical sites (AP coordinates –5.8, –6.6, –6.8, or –7.0), further supporting the functional significance of the culmen of the anterior lobe.

Protocol for Repeated Intracerebellar Microinfusions

Figure 1 provides a schematic diagram for five different protocols used for studying the development of cross-tolerance between nicotine or RJR-2403 and Δ⁹-THC. Details are as follows.

Dose-Response Relationship (Fig. 1A). This study was conducted to determine whether tolerance after repeated intracerebellar nicotine or RJR-2403 microinfusion to acute intracerebellar Δ⁹-THC-induced ataxia was dose-dependent. Nicotine (1.25, 2.5, or 5 ng), RJR-2403 (250, 500, or 750 ng), or aCSF was microinfused directly into the cerebellum, once daily, for 5 days. Sixteen hours after the last nicotine, RJR-2403, or aCSF microinfusion, Δ⁹-THC (20 μg) was administered and mice were subjected to Rotorod evaluation.

Onset of Tolerance (Fig. 1B). To establish how quickly tolerance to Δ⁹-THC-induced ataxia begins after repeated RJR-2403 microinfusion, mice were repeatedly treated with intracerebellar RJR-2403 (750 ng; once daily for 1, 2, 3, 5, or 7 days) or aCSF followed by Δ⁹-THC (20 μg) 16 h after the last microinfusion of RJR-2403 or aCSF. Animals in all groups were then subjected to Rotorod evaluation.

Duration of Tolerance (Fig. 1C). To determine the duration for which tolerance to Δ⁹-THC-induced ataxia persisted, animals that were treated repeatedly with intracerebellar RJR-2403 (750 ng; once daily for 5 days) or aCSF received Δ⁹-THC (20 μg) at 16, 24, 36, and 48 h after the last intracerebellar microinfusion of RJR-2403 or aCSF. Animals in all groups were then subjected to Rotorod evaluation.

Participation of the nAChR in Tolerance Development (Fig. 1, D and E). To evaluate whether the cerebellar nAChR and, more specifically, the α₄β₂ nAChR subtype, participate in the development of tolerance to acute intracerebellar Δ⁹-THC-induced ataxia after repeated intracerebellar nicotine or RJR-2403 treatment, respectively, nicotine (5 ng) or RJR-2403 (750 ng) was given via intracerebellar microinfusion once daily for 5 days. Chronic hexamethonium (1 μg), a nonselective nicotinic receptor antagonist, or DHβE (500 ng), a α₄β₂-selective nAChR subtype antagonist, was also microinfused (once daily for 5 days) into the cerebellum, either 1) 10 min before daily nicotine or RJR-2403 microinfusion, 2) 10 min after daily nicotine or RJR-2403 microinfusion, or 3) alone. Sixteen hours after the last intracerebellar microinfusion for each treatment group, Δ⁹-THC (20 μg) was administered, and animals were then subjected to Rotorod evaluation.

Rotorod Evaluation

Mice were evaluated for motor coordination using a standard Rotorod treadmill (Ugo Basil, Verese, Italy) set at a fixed speed of 24 rpm. As described previously, normal motor coordination was arbitrarily defined as the ability of a mouse to walk continuously on the Rotorod, without falling off, for 180 s (Dar et al., 1983). All mice were screened before intracerebellar microinjection to ensure that animals exhibited normal motor coordination. Therefore, the mice served as their own controls. Screening was performed the morning of the experiment, typically 20 min before microinjection and subsequent Rotorod evaluations. Any mouse unable to walk 180 s in three attempts during screening was considered to have abnormal coordination and was excluded from the experiment. In the present investigation, all animals passed the Rotorod screening test. Motor coordination experiments were performed between 8 and 11 AM. The Rotorod experiments were conducted 5 days after surgery to allow the animals to recover from the effects of the anesthetic and surgical trauma. The Rotorod evaluation times used were 10, 20, 30, and 40 min starting from the moment of intracerebellar Δ⁹-THC microinjection. After evaluation on the Rotorod at each time point, the mouse was placed back into its original cage until the next evaluation time. Each treatment group consisted of 10 mice. Results are expressed in seconds, and 180 s represents normal motor coordination based on our established criterion. The longer the animals walked on the Rotorod, the lesser the motor incoordination and vice versa. Accentuation or attenuation of intracerebellar Δ⁹-THC-induced ataxia by other drug microinfusion is thus indicated by either a decrease or an increase, respectively, in the time period the animals walked on the Rotorod.

Histology

To confirm the accuracy of drug microinjections, mice were microinfused with 100 nl of Fast Green dye at the end of each Rotorod experiment. The mice were sacrificed by cervical dislocation and decapitation under light isoflurane (IsoFlo; Abbott Laboratories) anesthesia. The guide cannula and brain were carefully removed, and the site of microinjection was then verified by examining the location of the dye in the cerebellar anterior lobe region. Only mice verified to have correct cannula placement were included in data analysis. The cannulation success rate has been in excess of 95% during the past 15 years of work in our laboratory, and the present study resulted in a 100% successful cannulation rate. Representative histological photomicrographs were examined to assess any tissue damage due to permanent implantation of guide cannula. There was minimal variation between and within groups and treatments in the drug dispersion sites of microinfusions and extent of tissue damage due to cannula implantation. It was also important to confirm that the drug dispersion after intracerebellar drug microinfusion remained confined to the cerebellar tissues. Based on previous tissue dispersion studies (Meng and Dar, 1996), the dispersion of drugs microinfused directly into specific brain regions was confined to tissues immediately around the microinfusion site.

Measurement of Cerebellar Nitric Oxide

Changes in cerebellar nitric oxide (NO) concentration was monitored by measurement of NO metabolic breakdown products, nitrite and nitrate (NO_x). The assay is based on the measurement of NO_x, which permits indirect determination of the amount of actual NO present in tissue samples. NO_x was measured in the present investigation by a modification of the fluorometric assay in which the reaction is based on the DAN reagent (Rao et al., 1998). The DAN reagent displays a 50-fold greater sensitivity (nanomolar range) compared with the Griess and most other methods (Misko et al., 1993).

Sample Preparation

All assays were performed on the same day that tissue samples were prepared after animal euthanasia. Mice were euthanized by cervical dislocation and decapitation 10 min after Δ⁹-THC or DMSO microinfusion. This time point for animal euthanasia was chosen because peak Δ⁹-THC ataxia is observed at this post-Δ⁹-THC microinfusion time interval. The assay was conducted via tissue homogenization, for which the tissue was cut out around the drug microinfusion site in the cerebellum. Brains were carefully removed from the skull and placed on a cold plastic dish. The guide cannula mark was used as a landmark for dissecting out cerebellar tissue. Using the tip of a RIB-BACK carbon-steel surgical blade no. 11 (Bard-Parker Becton Dickinson Acute Care, Franklin Lakes, NJ), tissue was dissected as close and as deep to the guide cannula mark as possible. Upon successive experiments and practice, the dissection of the cerebellar tissue was standardized by maintaining tissue weight between approximately 15 and 20 mg. The dissected tissue was immediately frozen in liquid nitrogen. The tissues from two animals were pooled to increase the sample volume and NO_x concentration. The frozen pooled tissue sample was added to a tared plastic homogenizing tube and weighed to obtain net tissue weight. Seven volumes of 20 mM Tris, 10 mM EDTA (pH 7.4) buffer was added, and the tissue sample was homogenized twice at setting 6 for 10 s using a tissue homogenizer (Polytron model PT 10/35; Brinkmann Instruments, Westbury, NY). All samples were subsequently centrifuged at 4000g for 20 min. The supernatant was then filtered through 10,000 molecular weight cutoff microcentrifuge filters (Microcon YM-10; Amicon Bioseparations, Millipore Co., Bedford, MA) by centrifugation at 16,000g for 60 min at 4°C to remove proteins such as hemoglobin that cause interference in the assay (Fernandez-Cancio et al., 2001). A colorless filtrate was obtained after filtration.

Nitrite Assay

Measurement of NO_x concentration in the cerebellar tissue samples involved a two-step reaction procedure. First, the tissue nitrate was converted to nitrite by an enzymatic reaction with NADPH-dependent nitrate reductase. Second, the reaction with the DAN reagent was followed by the measurement of total nitrite present in the sample. The reaction between tissue nitrite and DAN yielded the fluorescent 2,3-naphthotrazole product, which indirectly was the basis for the quantification of total tissue nitrite. NADPH higher than a concentration of 3 μM reduces the sensitivity of the fluorometric assay (Rao et al., 1998). Therefore, the optimal NADPH concentration of 3 μM was maintained by using an NADPH regenerating system comprising G-6-P and G-6-PDH. The actual assay, conducted in triplicate, involved pipetting 100 μl of sample mixture into a separate well in a white 96-well flat-bottom plate (Costar; Corning Glassworks, Corning, NY). Each 100-μl sample mixture consisted of 20 μl of 750 μM G-6-P, 20 μl of G-6-PDH (48 mU), 20 μl of 3 μM NADPH, 20 μl of nitrate reductase (30 mU), and 20 μl of ultrafiltrate sample in a 20 mM Tris-10 mM EDTA buffer, pH 7.4. Samples were incubated for 90 min at 25°C. One hundred microliters of freshly prepared sodium nitrite standard solutions (50–1000 pmol) were transferred into separate wells in triplicate. At the end of the incubation, 30 μl of DAN solution (0.05 mg/ml in 0.62 N HCl) was added to each well and incubated in the dark for an additional 10 min. The reaction was stopped by adding 30 μl of 1.4 N NaOH. The intensity of the fluorescent signal was immediately measured by a fluorometer (FL600 Microplate Reader; Bio-Tek Instruments, Winooski, VT) at λ_ex = 360 and λ_em = 460. The concentration of NO_x in each sample was calculated from a standard curve generated by plotting the fluorescent units with a sodium nitrite concentration in picomoles using KC4 software (Bio-Tek Instruments). Results are expressed in picomoles per milligrams of fresh tissue consistent with others (Seo and Revier, 2003). Total NO_x present in each sample was calculated as follows: total NO_x = [NO_x in sample (picomoles) × total volume added (microliters)/20 μl] per milligram of tissue.

Statistical Analysis

Motor impairment data were analyzed by a two-way repeated measure analysis of variance (ANOVA) to evaluate the effect of various drug doses and time on Rotorod motor coordination using the multivariate criterion of Wilks' Λ. Significance in drug versus time interaction was evaluated with a one-way ANOVA. A Dunnett's C post hoc test was performed whenever significance was found on treatment and/or time. Statistical analyses were performed using SPSS for Windows (version 13.0; SPSS, Inc., Chicago, IL). Area under the curve (AUC) analysis was performed using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA). Data comparing NO_x levels were analyzed by one-way ANOVA and Bonferroni post hoc analysis. P < 0.05 was taken as the level of significance in all statistical tests.

Results

Effect of Repeated Intracerebellar Nicotine Treatment on Acute Cerebellar Δ⁹-THC-Induced Ataxia.Figure 2A illustrates the relationship between repeated (once daily for 5 days) intracerebellar microinfusion of various doses of nicotine (1.25, 2.5, and 5 ng) on acute cerebellar Δ⁹-THC-induced ataxia. Furthermore, repeated microinfusion of aCSF instead of nicotine was followed by acute intracerebellar Δ⁹-THC to serve as the control. Acute Δ⁹-THC was administered 16 h after the last nicotine or aCSF microinfusion. Chronic intracerebellar nicotine administration resulted in a dose-dependent decrease of acute cerebellar Δ⁹-THC-induced ataxia. Nicotine at its highest dose (5 ng) resulted in complete abolishment of Δ⁹-THC-induced ataxia, whereas mice receiving 2.5 and 1.25 ng of nicotine regained normal motor coordination by 20 and 30 min after Δ⁹-THC microinfusion, respectively. Chronic nicotine at its highest dose, 5 ng, followed by DMSO instead of Δ⁹-THC, had no effect on normal motor coordination. The AUC analysis (Fig. 2B) reveals that as the dose of nicotine increases from 1.25 up to 5 ng, the AUC decreases in a dose-dependent manner consistent with a decrease in cerebellar Δ⁹-THC-induced ataxia. Drug treatments from Fig. 2A are shown over the appropriate bar with which the treatment corresponds.

Effect of Repeated Intracerebellar RJR-2403 Treatment on Acute Cerebellar Δ⁹-THC-Induced Ataxia.Figure 3A illustrates the relationship between repeated intracerebellar microinfusion of various doses of RJR-2403 (250, 500, and 750 ng) on acute cerebellar Δ⁹-THC-induced ataxia. Once again, repeated microinfusion of aCSF to a separate group of animals served as the control. Δ⁹-THC was micro-infused 16 h after the last aCSF or RJR-2403 microinfusion. Chronic intracerebellar RJR-2403 administration resulted in a dose-dependent decrease in acute cerebellar Δ⁹-THC-induced ataxia. Animals receiving 750 or 500 ng of RJR-2403 regained normal motor coordination by 20 and 30 min after Δ⁹-THC microinfusion, respectively. When animals were pretreated with the highest dose of RJR-2403 (750 ng) followed by acute intracerebellar DMSO microinfusion, no change in normal motor coordination was observed, indicating a lack of effect of repeated RJR-2403 on normal motor coordination. The AUC analysis (Fig. 3B) reveals that as the dose of RJR-2403 increases from 250 up to 750 ng, the total AUC decreases in a dose-dependent manner consistent with a decrease in cerebellar ataxia. Drug treatments from Fig. 3A are shown over the appropriate bar with which the treatment corresponds.

Onset of Cross-Tolerance between Repeated Intracerebellar RJR-2403 and Acute Cerebellar Δ⁹-THC-Induced Ataxia. To ascertain the time required for the development of tolerance to acute Δ⁹-THC-induced ataxia after repeated RJR-2403 treatment, we evaluated the effect of intracerebellar microinfusion of RJR-2403 for 1, 2, 3, 5, and 7 days on acute intracerebellar Δ⁹-THC-induced ataxia (Fig. 4A). For these experiments, only the 750-ng dose of RJR-2403 was used. RJR-2403 at 2, 3, 5, and 7 days markedly attenuated acute intracerebellar Δ⁹-THC-induced ataxia in a treatment time-related manner. Animals receiving 2 days of RJR-2403 treatment regained normal motor coordination by 30 min after Δ⁹-THC microinfusion, whereas the 3-, 5-, and 7-day treatments resulted in a return to normal motor coordination by 20 min after Δ⁹-THC microinfusion. The AUC analysis (Fig. 4B) reveals that as the duration of RJR-2403 treatment increased from 1 to 7 days, the AUC, and thus the level of ataxia, correspondingly decreased, with the largest decrease seen at 5 days. Drug treatments from Fig. 4A are shown over the appropriate bar with which the treatment corresponds.

Duration of Cross-Tolerance between Repeated Intracerebellar RJR-2403 and Acute Cerebellar Δ⁹-THC-Induced Ataxia. We also wanted to investigate the duration of tolerance to acute ICB Δ⁹-THC-induced ataxia due to repeated ICB RJR-2403 (750 ng) microinfusion. Figure 5A shows the evaluation of acute Δ⁹-THC-induced ataxia at 16, 24, 36, and 48 h after the last RJR-2403 microinfusion. The duration of RJR-2403-induced attenuation of cerebellar Δ⁹-THC-induced ataxia persisted up to 36 h after the last RJR-2403 microinfusion. The animals who received acute Δ⁹-THC 16 and 24 h after the last RJR-2403 microinfusion exhibited a return to normal motor coordination by 20 min after Δ⁹-THC microinfusion. However, animals in the 36-h duration treatment did not regain normal motor coordination until 30 min after Δ⁹-THC administration. AUC analysis (Fig. 5B) illustrates that as the time period between acute Δ⁹-THC administration and the last RJR-2403 microinfusion increased, there is a similar increase in AUC and therefore cerebellar ataxia. Acute Δ⁹-THC treatment 48 h after the last RJR-2403 microinfusion resulted in an AUC virtually identical to that of the “aCSF +Δ⁹-THC” control. Drug treatments from Fig. 5A are shown over the appropriate bar with which the treatment corresponds.

Effect of Repeated Intracerebellar Hexamethonium on Repeated Nicotine- and RJR-2403-Induced Attenuation of Cerebellar Δ⁹-THC Ataxia. We investigated whether the cerebellar nAChRs mediated the observed attenuation of acute Δ⁹-THC-induced ataxia through nAChR activation by repeated ICB microinfusion of nicotine or RJR-2403. Figure 6A demonstrates the effect of repeated intracerebellar hexamethonium (1 μg), a nonselective nAChR antagonist, microinfused 10 min before administration of either nicotine (5 ng) or RJR-2403 (750 ng), on acute cerebellar Δ⁹-THC-induced ataxia. This dose of hexamethonium was selected on the basis of our previous studies (Dar et al., 1994). Sixteen hours after the last nicotine or RJR-2403 microinfusion, acute intracerebellar Δ⁹-THC was administered followed by Rotorod evaluation. Hexamethonium pretreatment virtually abolished both repeated nicotine- or RJR-2403-induced attenuation of acute Δ⁹-THC-induced ataxia. Conversely, we evaluated the effect of repeated hexamethonium administration given 10 min after daily nicotine/RJR-2403 microinfusion (i.e., after nicotine or RJR-2403 microinfusion instead of before nicotine or RJR-2403) on acute intracerebellar Δ⁹-THC-induced ataxia. Hexamethonium, when administered after nicotine or RJR-2403 microinfusion, failed to block either nicotine- or RJR-2403-induced attenuation of acute cerebellar Δ⁹-THC-induced ataxia. Thus, nicotine or RJR-2403 markedly attenuates cerebellar Δ⁹-THC-induced ataxia only when microinfused before hexamethonium. Chronic intracerebellar microinfusion of hexamethonium alone did not alter acute intracerebellar Δ⁹-THC-induced ataxia. The AUC analysis (Fig. 6B) demonstrates that when nicotine or RJR-2403 microinfusion precedes that of hexamethonium, AUC levels are dramatically reduced. However, when hexamethonium is administered before nicotine or RJR-2403, AUC values are significantly increased, nearly approaching that of the aCSF +Δ⁹-THC control, indicating a significant increase in cerebellar ataxia. Drug treatments from Fig. 6A are shown over the appropriate bar with which the treatment corresponds.

Effect of Repeated Intracerebellar DHβE on Repeated Nicotine- or RJR-2403-Induced Attenuation of Cerebellar Δ⁹-THC Ataxia. Upon establishing the involvement of the cerebellar nAChR in the observed behavioral interactions between repeated nicotine or RJR-2403 and acute Δ⁹-THC, we investigated the role of the cerebellar α₄β₂ nAChR in the attenuation of Δ⁹-THC-induced ataxia by nicotine or RJR-2403. Chronic DHβE (500 ng), a α₄β₂ selective nAChR antagonist, was microinfused 10 min before the administration of either nicotine (5 ng) or RJR-2403 (750 ng) (Fig. 7A). This dose of DHβE was selected on the basis of findings from previous studies in our laboratory (Smith and Dar, 2006). DHβE pretreatment virtually abolished both repeated nicotine- or RJR-2403-induced attenuation of acute Δ⁹-THC-induced ataxia. Conversely, we evaluated the effect of DHβE administration given 10 min after nicotine or RJR-2403 microinfusion (i.e., after nicotine or RJR-2403 microinfusion instead of before nicotine or RJR-2403) on acute intracerebellar Δ⁹-THC-induced ataxia. DHβE, when administered after nicotine or RJR-2403 microinfusion, completely failed to antagonize either nicotine- or RJR-2403-induced attenuation of acute intracerebellar Δ⁹-THC-induced ataxia. Chronic intracerebellar microinfusion of DHβE alone did not alter acute intracerebellar Δ⁹-THC ataxia. The AUC analysis (Fig. 7B) demonstrates that when nicotine or RJR-2403 microinfusion precedes DHβE, AUC levels are dramatically reduced. However, when DHβE is administered before nicotine or RJR-2403, AUC values are significantly increased, approaching that of the aCSF +Δ⁹-THC control, indicating a significant increase in cerebellar ataxia. Drug treatments from Fig. 7A are shown over the appropriate bar with which the treatment corresponds. Figure 8 shows the typical sagittal section of mouse cerebellum after repeated drug microinfusion. The location of the guide cannula can be seen within the superficial layers of the culmen of the anterior lobe of the cerebellum.

Effect of Repeated Intracerebellar Nicotine or RJR-2403 in the Presence and Absence of Acute Intracerebellar Δ⁹-THC on Cerebellar Nitric Oxide Levels.Figure 9 illustrates the effect of repeated nicotine (5 ng), repeated RJR-2403 (750 ng), acute Δ⁹-THC alone (20 μg), or the combination treatments of “nicotine +Δ⁹-THC” or “RJR-2403 +Δ⁹-THC” on NO_x levels in mouse cerebellar tissue using the fluorometric DAN method. Contralateral cerebellar tissue served as the control. The administration of acute Δ⁹-THC alone decreased cerebellar NO_x levels by 25% compared with the control. Chronic nicotine or RJR-2403 alone increased cerebellar NO_x levels by 32 and 29%, respectively, compared with the control. Although Δ⁹-THC decreased cerebellar NO_x levels, repeated pretreatment with either nicotine or RJR-2403 before administration of acute Δ⁹-THC antagonized the Δ⁹-THC-induced decrease in NO_x levels by 36 and 38%, respectively.

Discussion

The attenuation of acute Δ⁹-THC ataxia by repeated intracerebellar nicotine suggested the interesting development of a behavioral cross-tolerance between nicotine and Δ⁹-THC. Acute Δ⁹-THC was administered ∼16 h after the last intracerebellar nicotine microinfusion; the selection of the 16-h time was to ensure elimination and metabolism of nicotine because its half-life in brain is ∼50 min (Ghosheh et al., 1999).

Once cross-tolerance was observed between nicotine and Δ⁹-THC, the α₄β₂ nAChR subtype agonist, RJR-2403 replaced nicotine to elucidate whether activation of the α₄β₂ nAChR subtype could elicit a cross-tolerance to acute Δ⁹-THC ataxia similar to that with nicotine. After repeated microinfusion of RJR-2403, observation of nearly complete abolition of Δ⁹-THC ataxia indicated that 1) repeated RJR-2403, like nicotine, results in the development of cross-tolerance to acute Δ⁹-THC ataxia and 2) cerebellar the α₄β₂ nAChR subtype participates in the development of tolerance to Δ⁹-THC. Thus, the cross-tolerance between repeated nicotine and acute Δ⁹-THC initially observed, in reality, was via participation of the α₄β₂ nAChR subtype. Again, RJR-2403, with even a shorter half-life than nicotine (Studenov et al., 2001), is not expected to be present at the time (16 h) of acute Δ⁹-THC microinfusion.

The cross-tolerance between RJR-2403 and Δ⁹-THC was rapid in onset and developed within 48 h. As the duration of repeated RJR-2403 treatment increased to a maximum of 7 days, there was a corresponding increase in the intensity of cross-tolerance to acute Δ⁹-THC ataxia. The intensity of cross-tolerance, however, was optimal after a 5-day instead of the anticipated 7-day RJR-2403 treatment. Overall, there was nearly a parallel increase in the intensity of the cross-tolerance to acute cerebellar Δ⁹-THC ataxia with increasing dose and duration of RJR-2403 treatment (i.e., the response was dose- and time-dependent). Significant cross-tolerance was detectable up to a maximum of 36 h after the last intracerebellar RJR-2403 microinfusion. Thus, even after nearly a full 1½ days after the last intracerebellar RJR-2403 microinfusion, significant cross-tolerance to cerebellar Δ⁹-THC ataxia was still detectable.

The results of the study also indicated the critical importance of the initial activation of the nAChR as well as the α₄β₂ nAChR subtype in the development of tolerance during repeated nicotine or RJR-2403 treatment. Hexamethonium markedly antagonized nicotine- or RJR-2403-induced attenuation of cerebellar Δ⁹-THC ataxia only when it was microinfused before nicotine or RJR-2403 administration. This demonstrates the critical role of initial activation of nAChRs and the downstream signaling cascade in the development of cross-tolerance between nicotine or RJR-2403 and Δ⁹-THC. The data similarly support the fact that activation of cerebellar nAChRs and the α₄β₂ nAChR subtype was essential during repeated nicotine or RJR-2403 administration to produce attenuation of acute Δ⁹-THC ataxia (cross-tolerance). Likewise, DHβE, given before but not after repeated nicotine or RJR-2403, antagonized nicotine- or RJR-2403-induced attenuation of Δ⁹-THC ataxia, further confirming the 1) importance of initial activation of the nAChR and its α₄β₂ subtype and 2) role of cerebellar α₄β₂ nAChRs in the cross-tolerance between nicotine or RJR-2403 and Δ⁹-THC. The initial activation of the α₄β₂ nAChR subtype with probable subsequent α₄β₂ nAChR downstream signaling pathway seems to be a critical step in the interaction between nicotine or RJR-2403 and Δ⁹-THC. Furthermore, repeated intracerebellar DHβE did not alter Δ⁹-THC ataxia, thereby ruling out any tonic influence of the α₄β₂ nAChR subtype.

Finally, cerebellar tissue NO_x levels after repeated intracerebellar nicotine or RJR-2403 alone as well as in the presence of acute Δ⁹-THC were measured. A marked decrease in NO_x levels was observed after acute Δ⁹-THC treatment in agreement with others (Lévénés et al., 1998; Hillard et al., 1999) and functionally correlated with Δ⁹-THC-induced ataxia. This result demonstrates an inverse functional relationship between acute Δ⁹-THC ataxia and the cerebellar NO_x concentration. Nicotine has been reported to oppose the cannabinoid-induced decrease in nitric oxide concentration (Smith et al., 1998; Pogun et al., 2000). Repeated treatment with nicotine or RJR-2403 alone markedly enhanced the cerebellar NO_x levels (Fig. 9), but no change in the normal coordination was detected (Figs. 2 and 3, respectively). However, microinfusion of acute Δ⁹-THC into these (nicotine- or RJR-2403-treated) animals resulted in a significant reduction in NO_x concentration, which was still comparable with the basal (contralateral) NO_x level. Thus, the ability of Δ⁹-THC to decrease NO_x, below the control (basal) level, was blunted by repeated treatment with nicotine or RJR-2403. Again, the NO_x data are consistent with Rotorod results because repeated treatment with nicotine or RJR-2403 virtually abolished the Δ⁹-THC-induced ataxia (Figs. 2 and 3). The lack of tolerance to acute Δ⁹-THC-induced ataxia after single nicotine or RJR-2403 treatment could be due to no significant increase in NO_x within 24 h. By 48 h and twice administration of nicotine or RJR-2403, the concentration of NO_x may have reached a significant level to attenuate Δ⁹-THC ataxia. Likewise, it took nearly 36 h for the cross-tolerance to disappear, a period that might have been required for the elevated NO_x to return to control level, which could subsequently be dropped below the basal level to produce Δ⁹-THC-evoked ataxia. Both the Rotorod and the NO_x data correlate excellently and suggest the importance of cerebellar NO_x in Δ⁹-THC-induced ataxia and in nicotine-Δ⁹-THC interaction. The NO_x data also provide further support to our hypothesis that the nicotine-Δ⁹-THC interaction mainly involves participation of the nAChR α₄β₂ subtype. This suggestion is also consistent with our recent report in which acute nicotine or RJR-2403 attenuated Δ⁹-THC-induced ataxia through cerebellar nitric oxide participation (Smith and Dar, 2007). It is not unusual to implicate nitric oxide in the development of tolerance to Δ⁹-THC because nitric oxide does play a role in the development of ethanol tolerance (Wazlawik and Morato, 2003). An absence of a change in NO_x levels in the contralateral tissue indicated that the influence of intracerebellar nicotine or RJR-2403 was only ipsilateral, reaffirming the fact that drug microinfusion remained confined to the tissue immediately surrounding the microinfusion site (Meng and Dar, 1996).

The possibility that desensitization of the cerebellar α₄β₂ nAChRs due to repeated nicotine or RJR-2403 treatment could have confounded the results was also considered. Both the Rotorod and NO_x data indicate activation and not desensitization of the nAChR and its subtype α₄β₂ because repeated treatment with 1) nicotine or RJR-2403 significantly and dose dependently attenuated Δ⁹-THC ataxia (Figs. 2 and 3), whereas with desensitization the usual cerebellar ataxia would occur and 2) nicotinic antagonist drugs, hexamethonium or DHβE, were able to block cross-tolerance between nicotine or RJR-2403 and Δ⁹-THC ataxia (Figs. 6 and 7). The overall functional consequence of receptor blockade by antagonist or desensitization would be the same. The significant increase in the cerebellar NO_x due to repeated nicotine or RJR-2403 treatment alone (Fig. 9) provides additional support against nAChR desensitization. Desensitized nonfunctional nAChRs would not be able to increase the production of NO_x. The data also show that repeated nicotine or RJR-2403 did not desensitize CB₁ either because Δ⁹-THC was able to lower the elevated NO_x levels in nicotine- or RJR-2403-pretreated animals (Fig. 9; nicotine versus NIC/THC and RJR-2403 versus RJR/THC). However, it should be stated that most likely desensitization did occur during the 7-day nicotine or RJR-2403 treatment because the degree of cross-tolerance was lesser compared with that for the 5-day treatment.

The ability of repeated microinfusion of nicotine or RJR-2403, dispersed only to a limited cerebellar cortical tissue, to virtually abolish acute Δ⁹-THC ataxia was an interesting finding. Microinfusion of the cerebellar ataxic dose of Δ⁹-THC failed to alter normal motor coordination when microinfusion was outside of the culmen further supporting the functional significance of this site. Thus, a relatively selective site within the cerebellar cortex was found to be functionally important in the mediation of the nicotine or RJR-2403 and Δ⁹-THC motor behavioral interaction and the development of cross tolerance between these psychoactive drugs.

In summary, on the basis of our data there exists a pivotal inverse functional relationship between Δ⁹-THC-induced ataxia and cerebellar NO_x concentration. We observed ataxia after ICB Δ⁹-THC microinfusion because it decreased the cerebellar NO_x below the basal control level. Administration (ICB) of RJR or nicotine alone raised the cerebellar NO_x level much above the basal level that did not permit any change in normal motor coordination. Microinfusion of Δ⁹-THC to animals pretreated with RJR or nicotine was not able to produce ataxia because the ability of Δ⁹-THC to decrease the cerebellar NO_x below the basal level was blunted.