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CB₁ Receptor Antagonism Increases Hippocampal Acetylcholine Release: Site and Mechanism of Action

Eli Lilly and Company, Lilly Corporate Center, Neuroscience Discovery Research, Indianapolis, Indiana (A.D., M.R.W., R.J.D., G.G.N.); and Center for Neuroscience of Coimbra, Institute of Biochemistry, Faculty of Medicine, University of Coimbra, Coimbra, Portugal (A.K., R.J.R., N.R., R.A.C.)

Received April 16, 2006; accepted July 19, 2006

	Abstract

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Evidence indicates that blockade of cannabinoid receptors increases acetylcholine (ACh) release in brain cortical regions. Although it is assumed that this type of effect is mediated through CB₁ receptor (CB₁R) antagonism, several in vitro functional studies recently have suggested non-CB₁R involvement. In addition, neither the precise neuroanatomical site nor the exact mechanisms underlying this effect are known. We thoroughly examined these issues using a combination of systemic and local administration of CB₁R antagonists, different methods of in vivo microdialysis, CB₁R knockout (KO) mice, tissue measurements of ACh, and immunochemistry. First, we showed that systemic injections of the CB₁R antagonists N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR-141716A) and N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) dose-dependently increased hippocampal ACh efflux. Likewise, local hippocampal, but not septal, infusions of SR141716A or AM251 increased hippocampal ACh release. It is noteworthy that the stimulatory effects of systemically administered CB₁R antagonists on hippocampal ACh release were completely abolished in CB₁R KO mice. CB₁R KO mice had similar basal but higher stress-enhanced hippocampal ACh levels compared with wild-type controls. It is interesting that dopamine D₁ receptor antagonism counteracted the stimulatory effect of CB₁R blockade on hippocampal ACh levels. Finally, immunohistochemical methods revealed that a high proportion of CB₁R-positive nerve terminals were found in hippocampus and confirmed the colocalization of CB₁ receptors with cholinergic and dopaminergic nerve terminals. In conclusion, hippocampal ACh release may specifically be controlled through CB₁Rs located on both cholinergic and dopaminergic neuronal projections, and CB₁R antagonism increases hippocampal ACh release, probably through both a direct disinhibition of ACh release and an indirect increase in dopaminergic neurotransmission at the D₁ receptors.

The effect of cannabinoid receptor stimulation on hippocampal ACh efflux has been well documented. On the other hand, limited evidence suggests that cannabinoid receptor antagonism increases ACh release in the hippocampus (Gessa et al., 1998; Tzavara et al., 2003b). Although it is assumed that this effect is mediated through CB₁R, recent studies indicate that cannabinoids may mediate some of their neurochemical/behavioral actions through novel, yet-to-be-identified central cannabinoid receptors (Di Marzo et al., 2000; Haller et al., 2002; Köfalvi et al., 2005). As such, a putative cannabinoid receptor, which is sensitive to the CB₁R antagonist SR141716A (Rimonabant) (Rinaldi-Carmona et al., 1994) but insensitive to the CB₁R antagonist AM251 (Lan et al., 1999), has been shown to inhibit the release of glutamate in the hippocampus (Köfalvi et al., 2003). Thus, an increase in hippocampal ACh levels elicited by both antagonists would indicate that this effect is probably mediated through CB₁R antagonism.

It is also possible that cannabinoid receptor antagonism at a site other than the hippocampus may regulate hippocampal ACh release. One obvious candidate is the septum, because stimulation of septal cannabinoid receptors increases hippocampal ACh release (Tzavara et al., 2003b), and the septum provides the main cholinergic input to the hippocampus (Dutar et al., 1995). Finally, CB₁R antagonism may regulate hippocampal ACh levels through a modulation of the dopaminergic system: blockade of CB₁ receptors increases brain dopamine levels (Tzavara et al., 2003a), and stimulation of dopaminergic receptors affects ACh release (Day and Fibiger, 1994). In particular, increased dopaminergic neurotransmission through D₁ receptors increases whereas through D₂ receptors decreases hippocampal ACh efflux, respectively (Day and Fibiger, 1994). Moreover, stimulation of D₂ and D₁ receptors mediates the inhibitory and stimulatory actions of high and low doses of cannabinoids on hippocampal ACh release, respectively (Nava et al., 2001; Tzavara et al., 2003b). Thus, a facilitation of dopaminergic neurotransmission at D₁ receptors may be involved in a possible stimulatory effect of hippocampal ACh release induced by CB₁R antagonism.

The current study sought to address these issues by conducting a detailed analysis of the site and mechanism of action through which CB₁R blockade modulates hippocampal ACh efflux. For this reason, a combined neurochemical and neuropharmacological approach that included local and systemic administrations, dual and quantitative microdialysis, and studies in CB₁R knockout (KO) mice was used. In addition, a histochemical analysis of the distribution of vesicular acetylcholine transporter (VAChT), dopamine transporter, and CB₁R immunoreactivity with a novel, highly sensitive method (Köfalvi et al., 2005) within the hippocampus was used to examine the neuroanatomical interrelationship of these elements.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Animals. All studies were conducted according to the guidelines set forth by the National Institutes of Health and implemented by the Animal Care and Use Committee of Eli Lilly and Company. We used male Wistar rats (250-300 g; Harlan Sprague-Dawley, Indianapolis, IN), male CB₁R KO, and corresponding wild-type (WT) mice. All animals were housed in a vivarium for at least 7 days before use with food and water available ad libitum. Both CB₁R KO and WT mice were derived from a stock of genotyped animals received from the University of Bonn (Bonn, Germany). CB R KO (CB₁ ^-/-) mice and their homozygous controls (CB ^+/+₁ mice) were developed in C57BL/6J mice by replacing most of the CB₁R coding sequence with a nonreceptor sequence through homologous recombination in MPI2 embryonic stem cells, as described previously by Zimmer et al. (1999). Both KO and WT mice used in the present study were derived from the same newly established breeding colony (by interbreeding of heterozygous mice and subsequent genotype characterization) and were matched for age and weight.

Surgical Procedures
Implantation of Microdialysis Guide Cannulae and Probe Insertions in the Hippocampus. Seven days before being used in microdialysis experiments, rats were anesthetized with a mixture of chloral hydrate and pentobarbital (170 and 36 mg/kg, respectively, in 30% propylene glycol and 14% ethanol), placed in a stereotaxic apparatus, and implanted unilaterally with guide cannulae (Bioanalytical Systems, Inc., West Lafayette, IN) in the hippocampus (coordinates: anteroposterior, -5.2; mediolateral, 5.2; dorsoventral, -3.8) according to the stereotaxic atlas of Paxinos and Watson (1998). Twenty-four hours before testing, a 4-mm concentric microdialysis probe (BAS Bioanalytical Systems, West Lafayette, IN) was inserted through the guide cannula.

Likewise, for mice, 2-mm microdialysis probes (CMA/Microdialysis, Solna, Sweden) were implanted unilaterally in the hippocampus (mediolateral, +3.1; anteroposterior, -3.3; dorsoventral, -4.2, based on the stereotaxic atlas of Franklin and Paxinos, 1997) under anesthesia with a mixture of chloral hydrate and pentobarbital. Animals were given a 48-h recovery period before being used in microdialysis experiments. The correct placement of the probes was verified histologically at the end of each experiment.

In Vivo Microdialysis of Hippocampal ACh Concentrations. Acetylcholine determination in hippocampal dialysates was performed as described previously (Damsma et al., 1988) with some modifications (Tzavara et al., 2003a,b). On the day of the experiment, a modified Ringer's solution (147.0 mM NaCl, 3.0 mM KCl, 1.3 mM CaCl₂, 1.0 mM MgCl₂, 1.0 mM Na₂HPO₄·7H₂O, and 0.2 mM NaH₂PO₄·H₂O, pH 7.25) supplemented with either 0.1 µM (rats) or 0.3 µM (mice) neostigmine was perfused at a rate of 2.4 µl/min (rats) or 1.5 µl/min (mice) in the hippocampus. Samples were collected every 15 min (unless indicated otherwise) and analyzed immediately online with high-performance liquid chromatography coupled to electrochemical detection, with a 150 x 3-mm acetylcholine-3 column (ESA, Inc., Chelmsford, MA) maintained at 35°C. The mobile phase (100 mM disodium hydrogen phosphate, 2 mM 1-octanesulfonic acid, and 50 µl/l of the microbicide reagent MB; ESA, Inc.; pH 8.0 was adjusted with phosphoric acid) was delivered by a high-performance liquid chromatography pump (ESA, Inc.) at 0.4 ml/min. The potentiostat used for electrochemical detection (ESA Coulochem II) was connected with a solid-phase reactor for ACh (ESA Inc.; ACh-SPR) and with an analytical cell with platinum target (ESA 5041). Animals were given a 3-h stabilization period before four baseline samples were collected. Thereafter, animals were systemically injected or locally infused with vehicle or drug, and an additional 6 to 12 samples (see Results) were collected.

Drugs and Experimental Design. In experiment 1, rats that had been implanted previously with microdialysis probes directed at the hippocampus as described were injected intraperitoneally with vehicle (0.9% NaCl containing 2% dimethyl sulfoxide and 2% cremophor EL), SR141716A (1, 3, or 10 mg/kg; synthesized at Lilly Research Laboratories), or AM251 (3 or 10 mg/kg; purchased from Tocris Cookson, Inc., Ellisville, MO). Drugs were suspended in vehicle and injected at a volume of 3 ml/kg. The doses of SR141716A and AM251 were selected on the basis of results of previous studies (Tzavara et al., 2001; Chambers et al., 2004).

In experiment 2, mice (CB₁R KO or WT) that were implanted previously with microdialysis probes as described were injected intraperitoneally with vehicle (see Implantation of Microdialysis Guide Cannulae and Probe Insertions in the Hippocampus), SR141716A (20 mg/kg), or AM251 (10 mg/kg). Drugs were suspended in vehicle and injected at a volume of 10 ml/kg. In preliminary studies, it was determined that in mice, a higher dose of SR141716A but not AM251 was required than in rats to induce robust and reproducible increases in hippocampal ACh levels. This may be due to different metabolic rates or differences in receptor density between species.

In experiment 3, rats were also implanted with guide cannulae and probes in the medial septal area (Moor et al., 1994; Tzavara et al., 2003b), and they were locally infused with SR141716A or AM251 at a rate of 2.4 µl/min in either the hippocampus or the septum for 60 min after the initial basal levels of hippocampal ACh had been established. Drugs (SR141716A or AM251) were dissolved in perfusion solution containing 1% DMSO and 1% cremophor EL at a final concentration of 1 mM. A lower concentration of SR141716A (0.1 mM) has been shown previously not to affect hippocampal ACh efflux (Tzavara et al., 2003b). It should also be noted that although the in vivo recovery of the administered CB₁R antagonists was not determined, this has been shown previously to be <1% for a compound, nicotine, administered locally under similar experimental conditions, such as those used in the present study (Marshall et al., 1997). Neostigmine was omitted from the perfusion solution used for septal perfusions because the probe in the septum was only used for drug delivery (Moor et al., 1994; Tzavara et al., 2003b).

In experiment 4, the basal and stress-induced levels of hippocampal ACh efflux were compared between CB₁R KO and WT mice using different methods of microdialysis (conventional, semiquantitative/low perfusion rate, and the quantitative/zero-net-flux method), tissue level measurements, and an animal model of exposure to stress (predatory odor test). Semiquantitative microdialysis was conducted by perfusing CB₁R KO or WT mice that had been implanted previously with microdialysis probes at a very low perfusion rate (0.08 µl/min) with a perfusion solution that did not contain neostigmine; this method allows for a better estimate of the basal steady-state concentrations of neurotransmitters in the sampled extracellular fluid than conventional microdialysis, because at low perfusion rates, their in vivo recovery reaches high levels (Gerber et al., 2001). After a standard 3-h stabilization period, dialysate was collected until a final volume of 40 µl was reached (8.3 h) and analyzed offline for ACh content. In the zero-net-flux microdialysis method (Day et al., 2001), CB₁R KO or WT mice implanted with microdialysis probes were perfused with a perfusion solution (1.5 µl/min) that did not contain neostigmine but contained known concentrations of ACh (2.5, 5.0, 10, or 25 nM) instead. The premise of the method is that when the concentration of ACh in the perfusion solution ([ACh_in]) is lower than extracellular hippocampal ACh levels, ACh will flow down its concentration gradient into the probe, which increases the ACh concentration in the dialysate [ACh_out]. On the other hand, if [ACh_in] is higher than hippocampal levels, then ACh will flow out of the probe, and [ACh_out] will be less than [ACh_in]. By measuring the point where [ACh_in-out] = 0, we can accurately determine the basal, steady-state concentrations of ACh in the extracellular fluid of the hippocampus. After a 3-h stabilization period, dialysate samples were collected every 20 min for a total of 10 samples and analyzed offline for ACh content.

To measure hippocampal tissue levels of ACh, brains were dissected from CB₁R KO or WT mice after decapitation, and the hippocampi were removed. Hippocampi were placed on a freeze plate, and tissue samples were weighed. Frozen tissue was suspended in a 1.5-ml Eppendorf vial in 0.5 ml of 0.1 N trichloroacetic acid containing 2 µM ethyl-homocholine and sonicated. The resulting solution was left at 0°C for 1 h and centrifuged at 12,000g. Next, the vial was placed in an autosampler (Bio-Rad Laboratories, Hercules, CA), and the supernatant was injected (20 µl). ACh was detected electrochemically with a Bioanalytical Systems LC-4C detector using a platinum electrode at 500-mV potential. Data were subsequently collected and analyzed with EZChrome Elite (Scientific Software, Inc., Pleasanton, CA).

Next, we examined whether CB₁R KO mice had altered evoked (i.e., stress-enhanced) concentrations of hippocampal ACh efflux compared with WT mice. Mice implanted with microdialysis probes directed at the hippocampus were exposed to the predatory odor stress test. After the baseline period, mice were placed in a bucket that contained soiled bedding from rat cages for a period of 60 min, whereas four additional hippocampal dialysate samples were collected. The predatory odor stress test was used to determine the effects of stress on hippocampal ACh in WT and KO mice, because exposure of mice to predatory odors has been shown previously to result in robust increases in cortical/hippocampal ACh efflux (Smith et al., 2005).

In experiment 5, we used a novel, highly sensitive method of quantification to calculate what percentage of cholinergic and dopaminergic nerve terminals in the hippocampus contained CB₁ receptors (see Immunochemical Analysis).

In experiment 6, rats were injected subcutaneously with vehicle (0.9% NaCl) or the D₁ receptor antagonist SCH23390 (0.3 mg/kg; purchased from Tocris) at a volume of 1 ml/kg. These injections occurred 15 min before being injected systemically (i.p.) or infused locally in the hippocampus with SR141716A (10 mg/kg for i.p. injection and 1 mM for local infusion). Local infusions were conducted as described above for experiment 3.

Immunochemical Analysis. Immunochemical analyses were performed as described previously (Köfalvi et al., 2005). In brief, synaptosomes from hippocampi of male Wistar rats were obtained through a discontinuous Percoll gradient, following the procedure described by Díaz-Hernandez et al. (2002) with minor modifications. Hippocampi were homogenized in 0.25 M sucrose and 5 mM TES, pH 7.4. The homogenate was spun for 3 min at 2000g at 4°C, and the resulting supernatant was spun again at 9500g for 13 min. Then the pellets were resuspended in 8 ml of 0.25 M sucrose and 5 mM TES, pH 7.4. Two milliliters of this synaptosomal suspension was placed onto 3 ml of Percoll discontinuous gradients containing 0.32 M sucrose, 1 mM EDTA, 0.25 mM dithiothreitol, and 3, 10, or 23% Percoll, pH 7.4. The gradients were centrifuged at 25,000g for 11 min at 4°C. Synaptosomes were collected between the 10 and 23% Percoll bands and diluted in 15 ml of HEPES-buffered medium (140 mM NaCl, 5 mM KCl, 5 mM NaHCO₃, 1.2 mM NaH₂PO₄, 1 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4). The synaptosomes were placed onto coverslips coated previously with poly(L-lysine), fixed with 4% paraformaldehyde for 15 min, and washed twice with PBS (140 mM NaCl, 3 mM KCl, 20 mM NaH₂PO₄, and 15 mM KH₂PO₄, pH 7.4). Permeabilization was performed in PBS containing 0.2% Triton X-100 for 10 min; afterward, the synaptosomes were incubated in PBS medium containing 3% bovine serum albumin and 5% normal rat serum for 1 h. The synaptosomes were then washed twice with PBS and incubated with rabbit anti-CB₁ receptor and guinea pig antivesicular acetylcholine transporter (anti-VAChT; 1:500; Chemicon International, Temecula, CA), rat antidopamine transporter (anti-DAT; 1:500, Chemicon) or mouse antisynaptophysin (1:200; Sigma, St. Louis, MO) for 1 h at room temperature. The rabbit CB₁R antibody, a generous gift of Dr. Ken Mackie, was raised against glutathione transferase corresponding to the last 15 amino acids of the rat CB₁R (1:3000). No staining with this CB₁R antibody was seen in the CB₁R homozygote null-mutant mouse, obtained from Dr. Catherine Ledent (Université Libre de Bruxelles, Brussels, Belgium) (Köfalvi et al., 2005). All antibodies gave one band in Western analysis of rat hippocampal tissue. The synaptosomes were then washed three times with PBS/bovine serum albumin (3%) and were incubated for 1 h at room temperature with a AlexaFluor-488 (green)-labeled goat antirabbit IgG antibodies (1:200; Molecular Probes, Leiden, The Netherlands) or goat anti-guinea pig or goat anti-rat or goat anti-mouse, all labeled with AlexaFluor-598 (red; 1:200 for all; Molecular Probes). After washing and mounting on slides with Prolong Antifade, the preparations were visualized in a Zeiss Axiovert 200 (Carl Zeiss AG, Jena, Germany) inverted microscope equipped with a cooled charge-coupled device camera and analyzed with MetaFluor 4.0 software (Molecular Devices, Sunnyvale, CA). To test the selectivity of the secondary antibodies, we carried out the same procedure as described above on 2-2 coverslips from each animal but without applying primary antibodies. Under this condition, the secondary antibodies failed to label the synaptosomes, and only 0 to 3 bright spots were observed in each field (nonspecific staining).

Statistics. The microdialysis data were expressed as mean (± S.E.M.) absolute values or multifold changes from baseline, which is the average of the four basal values before vehicle or drug injection; data were also expressed as average changes from baseline over a certain period of time (overall effects). Absolute values or percentage changes were analyzed with a three-, two-, or one-way analysis of variance (ANOVA) with treatment, time, or genotype as variables. Individual time points were analyzed with a one-way ANOVA followed by Bonferroni post hoc tests. A P level of 0.05 was used for statistical significance.

	Results

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Experiment 1
Effects of Systemic Injections of the CB₁R Antagonists SR141716A and AM251 on Hippocampal ACh Efflux. This experiment examined the dose-dependent nature of CB₁R antagonist administration and compared the effects of two slightly different (mainly based on functional responses, see Introduction) CB₁R antagonists, SR141716A and AM251, on hippocampal ACh efflux.

SR141716A and AM251 Dose-Dependently Increase ACh Efflux in the Hippocampus. There were no statistically significant differences in the basal values of ACh among the various experimental groups in this or subsequent experiments with values ranging from 125 to 500 fmol/sample. Figure 1A indicates that SR141716A dose-dependently increases hippocampal ACh efflux. Thus, analysis with a two-way ANOVA revealed a significant interaction (F_{45, 390} = 15.45, P < 0.0001), treatment (F_{3, 390} = 16.17, P < 0.001), and time (F_{15, 390} = 25.42, P < 0.0001) effect. Subsequent analysis at individual time points with a one-way ANOVA indicated that rats injected with 3 or 10 but not 1 mg/kg SR141716A had significantly increased levels of hippocampal ACh. This significant increase persisted for eight samples (120 min) and reached a maximum level of almost 200% above basal values for the highest injected dose. The 3 mg/kg dose of SR141716A resulted in significantly increased hippocampal ACh efflux that persisted for two sample periods (30 min) and reached a maximum of 150% above basal values. Injections of 1 mg/kg increased hippocampal ACh efflux to a maximum of 50% above basal values; this increase failed to reach significance. In all drug-injected groups, ACh efflux followed the same kinetic profile, with the highest increases being reached at the 30-min time point before values diminished over the course of time. Vehicle-injected animals showed an initial increase at the 15-min time point; this reflects the stress-induced increase in hippocampal ACh that results from the injection.

View larger version (39K): [in this window] [in a new window]   Fig. 1. Systemic administration (1, 3, and 10 mg/kg i.p., indicated by arrows) of the CB1 receptor antagonists SR141716A (A) and AM251 (B) dose-dependently increases hippocampal ACh efflux in rats. These stimulatory effects of SR141716A and AM251 (20 and 10 mg/kg i.p., respectively, indicated by arrows) on hippocampal ACh efflux are abolished in CB1 receptor KO mice (C and D, respectively). Data are expressed as mean (± S.E.M., n = 5-8 per group) hippocampal ACh efflux percentage of baseline). *, P < 0.05; **, P < 0.01 versus vehicle-injected rats (A and B) or vehicle-injected WT mice (C and D).

Experiment 2
Effects of Systemic Injections of the CB₁R Antagonists SR141716A and AM251 on Hippocampal ACh Efflux in CB₁R KO or WT Mice. In this experiment, we determined whether the effects of SR141716A and AM251 on hippocampal ACh efflux that were observed in experiment 1 were retained in mice in which the CB₁R had been genetically deleted.

The Stimulatory Effects of Systemic Injections of SR141716A and AM251 on Hippocampal ACh Efflux Are Abolished in CB₁R KO Mice. There were no statistically significant differences in the basal values of ACh among different experimental groups and between CB₁R KO and WT mice with values ranging from 65 to 125 fmol/sample. As can be seen in Fig. 1C, injections of SR141716A increased hippocampal ACh efflux in WT but not KO mice. Thus, a two-way ANOVA yielded a significant interaction (F_{30, 170} = 24.55, P < 0.0001), treatment (F_{3, 170} = 17.09, P < 0.001), and time (F_{10, 170} = 20.37, P < 0.0001) effect. SR141716A caused a significant increase during three sample periods (45 min), which reached a maximum of 200% above basal values at the 45-min time point. Figure 1D shows that the same effect occurs after injections of AM251 in WT or KO mice. Therefore, whereas AM251 increased hippocampal ACh efflux in WT mice, there was no effect in CB₁R KO mice. Thus, a two-way ANOVA resulted in a significant interaction (F_{30, 180} = 17.20, P < 0.0001), treatment (F_{3, 180} = 15.40, P < 0.001), and time (F_{10, 180} = 24.08, P < 0.0001) effect. The increase in hippocampal ACh induced by AM251 only reached significance at one time point (30 min) but reached a maximum value of approximately 200% above basal values.

Experiment 3
Effects of Local Perfusion of CB₁R Antagonists in the Septum or the Hippocampus on Hippocampal ACh Efflux. Next, we wanted to determine through which specific neuroanatomical site in the septohippocampal pathway CB₁R antagonists modulate hippocampal ACh efflux.

Local Hippocampal Infusion of SR141716A or AM251 Increased whereas Septal Infusion Decreased Hippocampal ACh Efflux. As demonstrated in Fig. 2A, infusions of CB₁R antagonists locally in the hippocampus significantly increased hippocampal ACh efflux (F_{2, 69} = 12.15, P < 0.0001). In particular, local infusion of either SR141716A or AM251 significantly increased ACh release in the hippocampus (Fig. 2A) during both the 1-h infusion period (P < 0.05 for SR141716A and p < 0.001 for AM251) and the 1-h postinfusion period (P < 0.001 for AM251). In contrast to the effects observed after systemic injections, local infusion produced a greater increase in hippocampal ACh efflux after infusions of AM251 compared with infusions of SR141716A.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Local infusion of the CB1 receptor antagonists SR141716A and AM251 in the hippocampus (A) but not the septum (B) significantly increases hippocampal ACh efflux during both the 1-h infusion and the 1-h postinfusion periods; septal infusions decreased hippocampal ACh efflux during the 1-h postinfusion period only. Data are expressed as mean (± S.E.M., n = 5-8 per group) overall hippocampal ACh efflux (percentage of baseline). *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus vehicle-infused rats.

Experiment 4
Analysis of Basal and Stress-Induced Hippocampal ACh Levels in CB₁R KO or WT Mice. Based on the data obtained through pharmacological blockade of CB₁R, we sought to investigate whether genetic deletion of CB₁R would affect basal hippocampal ACh efflux and tissue content. Basal levels were quantified using conventional microdialysis, semiquantitative microdialysis, the zero-net-flux method of microdialysis, or tissue level analysis.

CB₁R KO Mice Have Similar Basal but Higher Stress-Enhanced Hippocampal ACh Levels Compared with WT Mice. CB₁R KO mice did not have significantly different basal hippocampal ACh levels compared with WT mice, as assessed using tissue level measurements (Fig. 3A). Semiquantitative dialysis resulted in a slight tendency toward increased ACh efflux in the hippocampus in KO mice, but this tendency did not reach statistical significance (Fig. 3B). The zero-net-flux method of dialysis also failed to reveal significant differences in basal levels between KO and WT mice, with both groups having values of approximately 5 nM (Fig. 3C). However, Fig. 3D shows that CB₁R KO mice did have higher stress-induced levels of hippocampal ACh efflux. Statistical analysis with a two-way ANOVA of the data obtained in the predatory odor stress test revealed a significant interaction (F_{13, 130} = 6.16, P < 0.01) and time (F_{13, 130} = 52.29, P < 0.0001) effect. In particular, CB₁R KO mice had increased ACh levels during the 15- to 45-min time periods (Fig. 3D). This effect reached a maximum of 50% above values obtained with WT mice and approximately 150% above basal values. In WT mice, hippocampal ACh efflux was increased by more than 100% above basal values. For both WT and KO mice, this increase was greatest 30 min after the exposure to predatory odor. There was a second increase in hippocampal ACh that occurred at the 90-min time point. This increase was a direct result of the stress-induced increase associated with removing the animal from the test area (bucket with soiled bedding from rats) and returning it to the microdialysis bowl. An analysis (Student's t test) of the average overall ACh efflux during stress indicated that CB₁R KO mice had significantly higher overall levels compared with WT mice (t_{1, 10} = 2.27, P < 0.05; Fig. 3D).

View larger version (27K): [in this window] [in a new window]   Fig. 3. CB1R KO and WT mice have similar basal hippocampal ACh levels, as determined using tissue content analysis (A), semiquantitative microdialysis (B), and the zero-net-flux microdialysis method (C). However, CB1R KO mice have higher stress-induced levels of hippocampal ACh, as determined using the predatory odor (PO) stress test (D, shaded area). D, inset, the average overall increase in ACh during exposure to the PO stress test over a 1-h period. Data are expressed as mean (± S.E.M., n = 5-8) ACh content (A) or efflux (B-D). *, P < 0.05 versus WT mice.

CB₁ Receptors Are Colocalized with Vesicular Acetylcholine Transporter and Dopamine Transporter in Nerve Terminals of the Rat Hippocampus. The protocol for separation of nerve terminals is designed to exclude contamination by postsynaptic elements. Nonetheless, we stained the nerve terminals for PSD95, a postsynaptic marker protein, and observed no PSD95 positivity in synaptophysin-costained plates of synaptosomes (data not shown), establishing the specificity of the isolation procedure. Figure 4, A and B, illustrates that 7.1% of nerve terminals/varicosities (identified by synaptophysin, 7140 dots counted) display VAChT positivity, which correlates well with a previous finding obtained through electron microscopic analysis (Towart et al., 2003). Similar results were observed with DAT (9.3% of 4310 synaptophysin-positive terminals). Finally, we observed that almost all VAChT-positive nerve terminals (91.1%) colocalize with the CB₁R immunoreactivity (6311 counted; Fig. 4, A and B), and slightly fewer, 60.1% of DAT-positive terminals, express CB₁Rs. This novel finding clearly demonstrates the high density of CB₁Rs in hippocampal cholinergic and dopaminergic nerve terminals, suggesting a substantial endo-cannabinoid control on cholinergic and dopaminergic neurotransmission.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Rat hippocampal cholinergic and dopaminergic nerve terminals are strongly equipped with CB1 receptors. A, representative double-labeling images of antisynaptophysin (marker of all nerve terminals) with anti-VAChT (specific marker of cholinergic nerve terminals), with anti-DAT (specific marker of dopaminergic nerve terminals), and with anti-CB1 receptor, and anti-CB1 receptor with anti-VAChT and with anti-DAT. B, the extent of CB1R, VAChT, and DAT colocalization with synaptophysin (syn, taken as 100%), CB1 receptor colocalization with VAChT (taken as 100%), and with DAT (taken as 100%). Data represent the mean ± S.E.M. of n = 6 to 8 plates from three young adult rats after counting approximately 4 to 7000 terminals for each marker.

D₁ Receptor Antagonism Prevents the Stimulatory Effect of CB₁R Blockade on Hippocampal ACh Efflux. Figure 5A shows that whereas systemic injections of SR141716A resulted in a robust and persistent increase in hippocampal ACh, this increase was completely abolished by a prior subcutaneous injection of SCH23390 at a dose that had no effect on its own. Thereafter, a two-way ANOVA revealed a significant interaction (F_{32, 208} = 19.36, P < 0.0001), treatment (F_{2, 208} = 18.33, P < 0.001), and time (F_{16, 208} = 23.07, P < 0.0001) effect. The increase induced by SR141716A reached significance compared with the animals receiving both compounds at the 30-min time point, and this significant increase persisted for six samples. The maximum increase reached a level of approximately 150% above basal levels and occurred at the 45-min time point. Combined injections of SCH23390 and SR141716A resulted in a slight increase compared with animals injected with SCH23390 and vehicle, but this increase did not reach statistical significance. Animals receiving vehicle injections showed only a small increase in ACh efflux during the first 15 min after injection, as presented under Experiment 1 (data not shown).

View larger version (33K): [in this window] [in a new window]   Fig. 5. The increase in hippocampal ACh efflux induced by either a systemic (10 mg/kg i.p.; A) injection or a local (1 mM; B) perfusion of SR141716A is completely abolished by administration of the dopamine D1 receptor antagonist SCH23390 (0.3 mg/kg, s.c.) 15 min before administration of SR141716A. Data are expressed as mean (± S.E.M., n = 4-7 per group). *, P < 0.05; **, P < 0.01 versus SCH23390 +SR141716A-treated rats.

	Discussion

Top Abstract Materials and Methods Results Discussion Conclusions References

 
The results from the present study suggest that CB₁R antagonism increases the efflux of ACh in the hippocampus through both a direct and an indirect mechanism. The direct mechanism involves CB₁Rs that are located on hippocampal cholinergic nerve terminals and can be invalidated by genetic deletion. These are the cannabinoid receptors that are most likely responsible for the decrease in hippocampal ACh efflux elicited by CB₁R agonists (Tzavara et al., 2003b) and through which endogenous cannabinoids modulate ACh release. Thus, CB₁R antagonists administered systemically or locally in the hippocampus increase ACh release through disinhibition of a primarily inhibitory action through CB₁R overarching any other opposing action that is mediated elsewhere (e.g., in the septum; see Fig. 6). The indirect mechanism, on the other hand, is mediated through CB₁Rs that could be positioned on hippocampal dopaminergic nerve terminals and can also be genetically invalidated; still, an extrahippocampal localization is not excluded from the present study. Stimulation of these CB₁Rs by endogenous or exogenous cannabinomimetics could potentially decrease dopaminergic (DA) release in the hippocampus, and accordingly, CB₁R antagonists could increase ACh release through disinhibition and a stimulatory action of dopamine through D₁ receptor activation. This notion is supported by the fact that the stimulatory effects of CB₁R antagonists administered systemically or locally in the hippocampus on hippocampal ACh efflux can be counteracted by pharmacological blockade of D₁ receptors. Overall, these direct and indirect mechanisms combine to produce a robust and dose-dependent increase in hippocampal ACh efflux after pharmacological blockade of CB₁Rs. The increase in hippocampal ACh efflux induced by CB₁R antagonism could potentially explain why CB₁R inactivation enhances cognitive performance in relevant animal models (Chaperon and Thiebot, 1999).

View larger version (27K): [in this window] [in a new window]   Fig. 6. Schematic diagram of suggested direct and indirect mechanisms by which CB1R antagonism affects ACh efflux in the hippocampus. Antagonism of CB1R can increase hippocampal ACh efflux indirectly through the DA system (D1 receptor-mediated stimulation) (1) and directly through CB1R located on cholinergic nerve terminals (2). Blockade of CB1R in the septum can also have an inhibitory effect on hippocampal ACh efflux through an interaction with GABAb receptors (possibly via an increase in GABA release in the septum) located on septohippocampal cholinergic/GABAergic neurons (3).

The increase in hippocampal ACh release, induced by pharmacological blockade of CB₁Rs, is specifically mediated through CB₁Rs within the hippocampal but not the septal brain region. In fact, CB₁R antagonism in the septum induces a decrease in hippocampal ACh efflux. This is not surprising, given that we have recently reported that CB₁R agonists administered locally in the septum increase hippocampal ACh release (Tzavara et al., 2003a). Thus, endo-cannabinoids acting through CB₁Rs at the level of septum through a yet-unidentified mechanism could stimulate the septohippocampal cholinergic projections eliciting ACh release in the nerve terminal region; accordingly, CB₁R antagonism might exert a blocking effect at the septal brain region and an ensuing decrease in hippocampal ACh. It should be noted that CB₁R is a general marker of GABAergic nerve terminals in the brain (Irving et al., 2000; Freund et al., 2003; Köfalvi et al., 2005), and their blockade can increase brain GABA levels. Facilitation of GABAergic neurotransmission in the septum, in turn, decreases hippocampal ACh efflux (Dutar et al., 1995). In further support of this notion, a recent study indicated that cholinergic cell bodies in the septum that project to the hippocampus express both GABA_b receptors and CB₁Rs (Nyiri et al., 2005). Thus, topical CB₁R antagonism at the level of septum could result in a decrease in hippocampal ACh release through a mechanism that involves stimulation of GABA_b receptors (possibly through an enhanced release of GABA) localized on septohippocampal cholinergic/GABAergic-projecting neurons (Fig. 6).

In contrast to pharmacological blockade, genetic deletion of CB₁Rs did not modulate basal steady-state hippocampal ACh efflux as assessed using different methods of quantification. Thus, it seems that there may be a compensatory mechanism after long-term inactivation of CB₁Rs. This is partially supported by our own recent data, in which pharmacological blockade of CB₁R increased plasma corticosterone levels; still, genetic deletion of CB₁ R did not influence basal corticosterone concentrations, although the stimulatory effects of CB₁R antagonists were completely abolished (Wade et al., 2006). Despite the negative findings on basal hippocampal ACh levels after genetic deletion of CB₁ receptors, CB₁R KO mice had higher stress-induced hippocampal ACh compared with WT mice. Thus, even though there seems to be a compensatory response after long-term inactivation of CB₁ receptors, CB₁R KO mice still have higher hippocampal ACh levels when the hippocampal cholinergic system is actively recruited. To the extent that an increase in hippocampal ACh efflux is associated with an enhanced coping ability and an improvement in cognitive performance (Degroot and Nomikos, 2005), this ACh hyper-responsiveness could explain why the CB₁R KO mice perform better in learning and memory tasks and can even experience "impaired forgetting" (i.e., inability to forget a previously learned response), even if it would be beneficial to the animals to neutralize/forget this response (Reibaud et al., 1999; Varvel and Lichtman, 2002). In addition, it could explain why CB₁R antagonism modulates anxiety levels by enhancing the ability of the animals to perceive aversive stimuli and respond accordingly through active avoidance (Degroot and Nomikos, 2004).

Unlike the bimodal effect seen after the administration of aCB₁R agonist, CB₁R antagonists dose-dependently and uniformly increased hippocampal ACh efflux. In part, this differentiation may result from neuroanatomical specificity of the effects obtained with CB₁R antagonists and agonists, the level of the endogenous endocannabinoid tone, and its disruptions by these compounds. Whereas previous data from our laboratory indicated that CB₁R agonism differentially controls hippocampal ACh release through both the septum and the hippocampus, depending on which dose was used, the uniform effect seen in the current study was specifically controlled through CB₁R antagonism in the hippocampus that seemed to play a protagonist role. Nevertheless, septal perfusions of CB₁R antagonists did suppress hippocampal ACh efflux, an effect which probably involved stimulation of the septal GABAergic system (see above). Thus, CB₁R antagonism at the level of hippocampus seems to override an inhibitory action of CB₁R antagonism at the level of septum, resulting in a prevailing stimulatory action on hippocampal ACh release after systemic administration of CB₁R antagonists.

Because D₁ receptor antagonism completely counteracted the increase in hippocampal ACh efflux that was observed after pharmacological blockade of CB₁Rs, it could be argued that CB₁R antagonism increases hippocampal ACh efflux solely through an indirect mechanism. However, as depicted in Fig. 6 and as demonstrated with septal infusions of SR141716A and AM251, CB₁R antagonism at the level of septum could also have an inhibitory effect on hippocampal ACh efflux. Therefore, if only the indirect mechanism is involved in increased hippocampal ACh efflux observed after inactivation of CB₁R, then removing this mechanism through D₁ receptor blockade should actually result in ACh levels below basal values, because now only the inhibitory mechanism at the level of septum remains operating. However, D₁ receptor blockade simply counteracted the increase in hippocampal ACh efflux induced by CB₁R blockade. Thus, it seems more plausible that a direct mechanism at the level of the hippocampus is also involved, which partially offsets the inhibitory mechanism that remains active once the indirect stimulatory mechanism is removed. This notion is supported by the present data demonstrating a high density of CB₁Rs on hippocampal cholinergic nerve terminals.

Although we and others described previously that CB₁Rs are primarily located on the nerve terminals of cholecystokinin-positive GABAergic interneurons in the rat and human hippocampus (Katona et al., 2000), it is now generally believed that other neuron types also express CB₁Rs (Marsicano et al., 2003). The low levels of CB₁R immunoreactivity in other cell types in electron microscopic assays might be due to accessibility/sensitivity problems. Here, using a much more sensitive method, we report that a relatively high percentage of cholinergic and dopaminergic hippocampal nerve terminals and varicosities are equipped with the CB₁R, indicating an even more widespread and extensive role for the endo-cannabinoid system in the regulation of hippocampal function.

	Conclusions

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Pharmacological antagonism of CB₁Rs increases hippocampal ACh efflux through both a direct mechanism through CB₁Rs only at the level of the hippocampus and an indirect mechanism that involves both CB₁R disinhibition and D₁ receptor stimulation most likely at the level of hippocampus, although other sites cannot be convincingly excluded. The increase in hippocampal ACh levels in response to CB₁R antagonists is abolished after inactivation of either mechanism. It is possible that when combined, both mechanisms can override a demonstrated suppression of hippocampal ACh release induced by blockade of CB₁Rs in the septum. Genetic invalidation of CB₁Rs engenders a compensated normalization of basal steady-state hippocampal ACh levels, although exposure to an aversive stimulus renders the animals more susceptible to mobilization of the cholinergic system and its neurophysiological consequences. This increase in hippocampal ACh efflux probably accounts for the enhanced cognitive effect observed in preclinical models (Chaperon and Thiebot, 1999). In addition, it could account for the modulation of anxiety responses, as described by Degroot and Nomikos (2004). Our study sheds light onto the mechanism of action through which CB₁R antagonism increases hippocampal ACh efflux, which further helps us understand the role of the brain endo-cannabinoid system in regulating cholinergic function in the brain, and, consequently, neuroadaptive, both cognitive and affective, responses of the organism.

	Acknowledgements

 
We are grateful to Ken Mackie for generously supplying us with the antibody.

	Footnotes

 
A.K. was supported by an International Brain Research Organization 2005 Research Fellowship.

ABBREVIATIONS: ACh, acetylcholine; VAChT, vesicular acetylcholine transporter; CB₁R, cannabinoid receptor type 1; SR141716A, N-(piperidin1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride; AM251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; KO, knockout; WT, wild type; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; PBS, phosphate-buffered saline; BSA, bovine serum albumin; ANOVA, analysis of variance; DAT, dopamine transporter; DA, dopaminergic; SCH23390, R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine.

Address correspondence to: Dr. George G. Nomikos, Amgen, Amgen Cambridge Research Center, Neuroscience, One Kendall Square, Building 1000, Cambridge, MA 02139. E-mail: gnomikos{at}amgen.com

	References

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Chambers AP, Sharkey KA, and Koopmans HS (2004) Cannabinoid (CB)₁ receptor antagonist, AM 251, causes a sustained reduction of daily food intake in the rat. Physiol Behav 82: 863-869.[CrossRef][Medline]

Chaperon F and Thiebot MH (1999) Behavioral effects of cannabinoid agents in animals. Crit Rev Neurobiol 13: 243-281.[Medline]

Damsma G, Biessels PT, Westerink BH, De Vries JB, and Horn AS (1988) Differential effects of 4-aminopyridine and 2,4-diaminopyridine on the in vivo release of acetylcholine and dopamine in freely moving rats measured by intrastriatal dialysis. Eur J Pharmacol 145: 15-20.[CrossRef][Medline]

Day JC and Fibiger HC (1994) Dopaminergic regulation of septohippocampal cholinergic neurons. J Neurochem 63: 2086-2092.[Medline]

Day JC, Kornecook TJ, and Quirion R (2001) Application of in vivo microdialysis to the study of cholinergic systems. Methods 23: 21-39.[CrossRef][Medline]

Degroot A and Nomikos GG (2004) Genetic deletion and pharmacological blockade of CB1 receptors modulates anxiety in the shock-probe burying test. Eur J Neurosci 20: 1059-1064.[CrossRef][Medline]

Degroot A and Nomikos GG (2005) Fluoxetine disrupts the integration of anxiety and aversive memories. Neuropsychopharmacology 30: 391-400.[CrossRef][Medline]

Devane WA, Dysarz FA 3rd, Johnson MR, Melvin LS, and Howlett AC (1988) Determination and characterization of a cannabinoid receptor in rat brain. Mol Pharmacol 34: 605-613.[Abstract]

Dìaz-Hernandez M, Pereira MF, Pinter J, Cunha RA, Ribeiro JA, and Miras-Portugal MT (2002) Modulation of the rat hippocampal dinucleotide receptor by adenosine receptor activation. J Pharmacol Exp Ther 301: 441-450.[Abstract/Free Full Text]

Di Marzo V, Breivogel CS, Tao Q, Bridgen DT, Razdan RK, Zimmer AM, Zimmer A, and Martin BR (2000) Levels, metabolism, and pharmacological activity of anandamide in CB₁ cannabinoid receptor knockout mice: evidence for non-CB₁, non-CB₂ receptor-mediated actions of anandamide in mouse brain. J Neurochem 75: 2434-2444.[CrossRef][Medline]

Dutar P, Bassant MH, Senut MC, and Lamour Y (1995) The septohippocampal pathway: structure and function of a central cholinergic system. Physiol Rev 75: 393-427.[Free Full Text]

Franklin KBJ and Paxinos G (1997) The Mouse Brain in Stereotaxic Coordinates. Academic Press Inc., San Diego, CA.

Freund TF, Katona I, and Piomelli D (2003) Role of endogenous cannabinoids in synaptic signaling. Physiol Rev 83: 1017-1066.[Abstract/Free Full Text]

Gerber DJ, Sotnikova TD, Gainetdinov RR, Huang SY, Caron MG, and Tonegawa S (2001) Hyperactivity, elevated dopaminergic transmission, and response to amphetamine in M1 muscarinic acetylcholine receptor-deficient mice. Proc Natl Acad Sci USA 98: 15312-15317.[Abstract/Free Full Text]

Gessa GL, Casu MA, Carta G, and Mascia MS (1998) Cannabinoids decrease acetylcholine release in the medial-prefrontal cortex and hippocampus, reversal by SR 141716A. Eur J Pharmacol 355: 119-124.[CrossRef][Medline]

Haller J, Bakos N, Szirmay M, Ledent C, and Freund TF (2002) The effects of genetic and pharmacological blockade of the CB1 cannabinoid receptor on anxiety. Eur J Neurosci 16: 1395-1398.[CrossRef][Medline]

Howlett AC, Breivogel CS, Childers SR, Deadwyler SA, Hampson RE, and Porrino LJ (2004) Cannabinoid physiology and pharmacology: 30 years of progress. Neuropharmacology 47 (Suppl 1): 345-358.[CrossRef][Medline]

Inui K, Egashira N, Mishima K, Yano A, Matsumoto Y, Hasebe N, Abe K, Hayakawa K, Ikeda T, Iwasaki K, et al. (2004) The serotonin 1A receptor agonist 8-OHDPAT reverses delta 9-tetrahydrocannabinol-induced impairment of spatial memory and reduction of acetylcholine release in the dorsal hippocampus in rats. Neurotox Res 6: 153-158.[Medline]

Irving AJ, Coutts AA, Harvey J, Rae MG, Mackie K, Bewick GS, and Pertwee RG (2000) Functional expression of cell surface cannabinoid CB₁ receptors on presynaptic inhibitory terminals in cultured rat hippocampal neurons. Neuroscience 98: 253-262.[CrossRef][Medline]

Katona I, Sperlágh B, Maglóczky Z, Sántha E, Köfalvi A, Czirják S, Mackie K, Vizi ES, and Freund TF (2000) GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus. Neuroscience 100: 797-804.[CrossRef][Medline]

Köfalvi A, Rodrigues RJ, Ledent C, Mackie K, Vizi ES, Cunha RA, and Sperlágh B (2005) Involvement of cannabinoid receptors in the regulation of neurotransmitter release in the rodent striatum: a combined immunochemical and pharmacological analysis. J Neurosci 25: 2874-2884.[Abstract/Free Full Text]

Köfalvi A, Vizi ES, Ledent C, and Sperlágh B (2003) Cannabinoids inhibit the release of [³H]glutamate from rodent hippocampal synaptosomes via a novel CB₁ receptor-independent action. Eur J Neurosci 18: 1973-1978.[CrossRef][Medline]

Lan R, Liu Q, Fan P, Lin S, Fernando SR, McCallion D, Pertwee R, and Makriyannis A (1999) Structure-activity relationships of pyrazole derivatives as cannabinoid receptor antagonists. J Med Chem 42: 769-776.[CrossRef][Medline]

Marshall DL, Redfern PH, and Wonnacott S (1997) Presynaptic nicotinic modulation of dopamine release in the three ascending pathways studied by in vivo microdialysis: comparison of naive and chronic nicotine-treated rats. J Neurochem 68: 1511-1519.[Medline]

Marsicano G, Goodenough S, Monory K, Hermann H, Eder M, Cannich A, Azad SC, Cascio MG, Gutierrez SO, van der Stelt M, et al. (2003) CB1 cannabinoid receptors and on-demand defense against excitotoxicity. Science (Wash DC) 302: 84-88.[Abstract/Free Full Text]

Moor E, de Boer P, Beldhuis HJ, and Westerink BH (1994) A novel approach for studying septo-hippocampal cholinergic neurons in freely moving rats: a microdialysis study with dual-probe design. Brain Res 648: 32-38.[CrossRef][Medline]

Nava F, Carta G, Colombo G, and Gessa GL (2001) Effects of chronic Delta(9)-tetrahydrocannabinol treatment on hippocampal extracellular acetylcholine concentration and alternation performance in the T-maze. Neuropharmacology 41: 392-399.[CrossRef][Medline]

Nyiri G, Szabadits E, Cserep C, Mackie K, Shigemoto R, and Freund TF (2005) GABAB and CB1 cannabinoid receptor expression identifies two types of septal cholinergic neurons. Eur J Neurosci 21: 3034-3042.[CrossRef][Medline]

Paxinos G and Watson C (1998) The Rat Brain in Stereotaxic Coordinates, 4th ed, Academic Press, San Diego.

Reibaud M, Obinu MC, Ledent C, Parmentier M, Böhme GA, and Imperato A (1999) Enhancement of memory in cannabinoid CB₁ receptor knock-out mice. Eur J Pharmacol 379: R1-R2.[CrossRef][Medline]

Rinaldi-Carmona M, Barth F, Heaulme M, Shire D, Calandra B, Congy C, Martinez S, Maruani J, Neliat G, Caput D, et al. (1994) SR141716A, a potent and selective antagonist of the brain cannabinoid receptor. FEBS Lett 350: 240-244.[CrossRef][Medline]

Smith DG, Davis RJ, Rorick-Kehn L, Morin M, Witkin JM, McKinzie DL, Nomikos GG, and Gehlert DR (2005) Melanin-concentrating hormone-1 receptor modulates neuroendocrine, behavioral, and corticolimbic neurochemical stress responses in mice. Neuropsychopharmacology 31: 1135-1145.

Towart LA, Alves SE, Znamensky V, Hayashi S, McEwen BS, and Milner TA (2003) Subcellular relationships between cholinergic terminals and estrogen receptor-alpha in the dorsal hippocampus. J Comp Neurol 463: 390-401.[CrossRef][Medline]

Tzavara ET, Davis RJ, Perry KW, Li X, Salhoff C, Bymaster FP, Witkin JM, and Nomikos GG (2003a) The CB1 receptor antagonist SR141716A selectively increases monoaminergic neurotransmission in the medial prefrontal cortex: implications for therapeutic actions. Br J Pharmacol 138: 544-553.[CrossRef][Medline]

Tzavara ET, Perry KW, Rodriguez DE, Bymaster FP, and Nomikos GG (2001) The cannabinoid CB(1) receptor antagonist SR141716A increases norepinephrine outflow in the rat anterior hypothalamus. Eur J Pharmacol 426: R3-R4.[CrossRef][Medline]

Tzavara ET, Wade M, and Nomikos GG (2003b) Biphasic effects of cannabinoids on acetylcholine release in the hippocampus: site and mechanism of action. J Neurosci 23: 9374-9384.[Abstract/Free Full Text]

van der Stelt M and Di Marzo V (2003) The endo-cannabinoid system in the basal ganglia and in the mesolimbic reward system: implications for neurological and psychiatric disorders. Eur J Pharmacol 480: 133-150.[CrossRef][Medline]

Varvel SA and Lichtman AH (2002) Evaluation of CB1 receptor knockout mice in the Morris water maze. J Pharmacol Exp Ther 301: 915-924.[Abstract/Free Full Text]

Wade MR, Degroot A, and Nomikos GG (2006) Cannabinoid CBl receptor antagonism modulates plasma corticosterone in rodents. Eur J Pharmacol, in press.

Wade MR and Nomikos GG (2005) Tolerance to the procholinergic action of the D1 receptor full agonist dihydrexidine. Psychopharmacology (Berl) 182: 393-399.[CrossRef][Medline]

Zimmer A, Zimmer AM, Hohmann AG, Herkenham M, and Bonner TI (1999) Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice. Proc Natl Acad Sci USA 96: 5780-5785.[Abstract/Free Full Text]

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