- Email: [email protected]
Systemic administration of WIN 55,212-2 increases norepinephrine release in the rat frontal cortex
Brain Research 1046 (2005) 45 – 54 www.elsevier.com/locate/brainres
Research report
Systemic administration of WIN 55,212-2 increases norepinephrine release in the rat frontal cortex V.C. Oropezaa,*, M.E. Pagea,b, E.J. Van Bockstaelea a
Department of Neurosurgery, Farber Institute for Neurosciences, Thomas Jefferson University, 900 Walnut Street, Suite 400, Philadelphia, PA 19107, USA b Department of Neurobiology and Anatomy, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA Accepted 15 March 2005 Available online 28 April 2005
Abstract Cannabinoid agonists modulate a variety of behavioral functions by activating cannabinoid receptors that are widely distributed throughout the central nervous system. In the present study, norepinephrine efflux was assessed in the frontal cortex of rats that received a systemic administration of the cannabinoid agonist, WIN 55,212-2. The synthetic cannabinoid agonist dose-dependently increased the release of norepinephrine in this brain region. Pretreatment with the cannabinoid receptor antagonist, SR 141716A, blocked the increase in norepinephrine release. To identify sites of cellular activation, immunocytochemical detection of c-Fos was combined with detection of the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), in the brainstem nucleus locus coeruleus (LC), a region that is the sole source of norepinephrine to the frontal cortex. Systemic administration of WIN 55,212-2 significantly increased the number of c-Fos immunoreactive cells within TH-containing neurons in the LC compared to vehicle-treated rats. Pretreatment with SR 141716A inhibited the WIN 55,212-2 induced c-Fos expression, while the antagonist alone did not affect c-Fos expression. Taken together, these data indicate that systemically administered cannabinoid agonists stimulate norepinephrine release in the frontal cortex by activating noradrenergic neurons in the coeruleo-frontal cortex pathway. These effects may partially underlie changes in attention, arousal and anxiety observed following exposure to cannabis-based drugs. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, Modulators, transporters, and receptors Topic: Catecholamines Keywords: Cannabinoid; CB1 receptor; Locus coeruleus; Norepinephrine
1. Introduction The psychoactive ingredient in Cannabis sativa, D9-tetrahydrocannabinol (THC), produces characteristic psychotropic responses in humans [25]. Exogenous cannabinoids are also known to alter a wide array of functions in animals such as nociception, thermal regulation and motor activity (for review, see [18]). These effects are mediated by actions of agonists on cannabinoid receptors (CB1) that are widely distributed throughout the central nervous system [7,11,14,24,40]. Though much is known about the central effects of exogenously applied cannabinoids, the neural * Corresponding author. Fax: +1 215 955 4949. E-mail address: [email protected] (V.C. Oropeza). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.03.036
circuits and cellular mechanisms involved in mediating these effects remain to be elucidated. A potential site that may be affected by cannabinoidbased substances is the locus coeruleus (LC), a brainstem region involved in regulating cortical arousal, attention and anxiety [3,4,12,34,45,50 – 52], behaviors known to be altered by cannabinoid administration (for review, see [25]. Many studies have demonstrated that cannabinoids impact on central noradrenergic function. Bonnin et al. [6] found that prenatal exposure to D9-THC, the psychoactive component of marijuana, increased the expression of the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) in neurons during early fetal brain development. Moreover, Hernandez-Tristan et al. [16] showed that in vitro incubation of cultured fetal mesencephalic neurons with
46
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
D9-THC increased the activity of TH and this increase was reversed by SR 141716A, a specific antagonist for the cannabinoid CB1 receptor. Kataoka et al. [21] found that pretreatment with 6-hydroxydopamine (6-OHDA) or lesions of the LC significantly reduce the cataleptogenic effect of D9-THC. Additionally, central noradrenergic systems have been implicated in the hypothermic activity of D9-THC [37]. Previous studies have shown deficits in attention and arousal following the administration of cannabinoid-based substances [32,49]. THC impairs the capacity to discriminate time intervals and space distances, vigilance, memory and cognitive performance [8]. In order to study the role of the endogenous cannabinoid system in the modulation of the noradrenergic system, Tzavara et al. systemically administered SR 141716A and measured NE levels in various brain regions using microdialysis coupled to highpressure liquid chromatography (HPLC). Interestingly, they found that administration of SR 141716A increases norepinephrine outflow in the rat anterior hypothalamus and the frontal cortex [42,43] suggesting a tonic inhibitory role of endogenous cannabinoids on norepinephrine. To further elucidate the role of cannabinoids in the modulation of the noradrenergic system, the synthetic cannabinoid receptor agonist WIN 55,212-2 was administered systemically and changes in NE extracellular efflux in the rat frontal cortex were assessed using in vivo microdialysis followed by HPLC coupled with electrochemical detection (ED). In an effort to verify the involvement of the noradrenergic system in mediating the effects of cannabinoids, immunohistochemical detection of the immediate early gene c-Fos (a marker of increased neuronal activation [27]) was combined with detection of the catecholamine synthesizing enzyme, TH.
2. Methods Male Sprague – Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 200– 250 g were housed three per cage and maintained at a constant temperature of 22 -C on a 12:12 h light:dark cycle (07:00 on and 19:00 off) with unrestricted access to food and water. Animal surgeries were conducted in accordance with the guidelines established by the NIH Guide for Care and Use of Laboratory Animals and approved by both the Drexel University College of Medicine and Thomas Jefferson University Institutional Animal Care and Use Committees. 2.1. Microdialysis and high-pressure liquid chromatography After an acclimation period of approximately 1 week, rats were anesthetized with 8% chloral hydrate (400 mg/kg) and placed in a stereotaxic apparatus with the skull flat. A small burr hole was made in the skull centered at 3.2 mm anterior and T0.7 mm lateral to bregma. The dura was removed, and the microdialysis probe was slowly lowered
5.0 mm from the brain surface into the infralimbic and prelimbic areas of the frontal cortex (plates 8– 10 [30]) and secured with skull screws and dental acrylic. The inlet of the probe was connected to a fluid swivel (Instech Laboratories, Plymouth Meeting, PA), and the rat was placed into a cylindrical plexiglas container covered with bedding. Food and water were freely accessible. Artificial cerebrospinal fluid (aCSF: 174 mM NaCl, 1.7 mM CaCl2, 0.9 mM Mg Cl2 and 4 mM KCl) was continuously perfused through the probe at a rate of 1.5 Al/min by a microliter infusion pump (Harvard Pump F11_ VPF Dual Syringe, Harvard Apparatus, Holliston, MA). Rats were allowed to recover overnight. Approximately 18 h following surgery, dialysate samples were collected every 20 min. After collecting dialysate samples for at least 2 h to establish stable baseline levels, the cannabinoid agonist WIN 55,212-2 (Sigma, St. Louis, MO) was administered to the rats by intraperitoneal injection. Dilutions of the drug were prepared immediately prior to the start of each experiment by dissolving in deionized water containing 0.4% Tween (Sigma Chemical, St. Louis, MO). The doses administered were 0.3, 1.0, 3.0 and 15.0 mg/kg. Dialysate samples were collected for 3 h post-injection and stored at 80 -C for subsequent analysis by HPLC-ED. A control group was treated with vehicle only (water containing 0.4% Tween). In a separate experiment, the ability of the cannabinoid receptor antagonist, SR 141716A (received as a gift from NIDA) to block the actions of WIN 55,212-2 on NE efflux was tested. SR 141716A (0.2 mg/kg) was dissolved in deionized water containing 0.4% Tween and was administered 30 min prior to WIN 55,212-2 (15 mg/ kg). The control group for this experiment received SR 141716A followed by a vehicle injection 30 min later. At the conclusion of the experiment, rats were deeply anesthetized, and 2% pontamine sky blue dye (Alfa Aesar, Ward Hill, MA) was infused through the probe to mark its location. The rats were transcardially perfused with 10% formalin (Fisher Scientific, Pittsburgh, PA), decapitated and the brains removed for subsequent histological verification of probe placement. The data were not included if the placement was outside the infralimbic and prelimbic areas of the frontal cortex. 2.2. Dialysis probe construction and calibration Vertical concentric microdialysis probes were used. A piece of fused silica (Polymicro Technologies, Phoenix, AZ) was inserted through PE 10 tubing and semipermeable membrane made from hollow rayon fibers with a 224 Am o.d. and 35,000 MW cutoff was fixed over the fused silica and into the PE 10 tubing with epoxy. The open end of the dialysis fiber was sealed with a 0.5 mm epoxy plug, and 2 mm of the top of the membrane was coated with epoxy leaving an active membrane length of 3 mm for exchange across the membrane. The in vitro recovery rate was determined by placing the probe in a beaker of aCSF containing a known concentration of NE standard. The
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
concentration of NE in the dialysate was compared to the amount in the bath. Probes that did not correspond to an acceptable range of recovery (12 – 24%) were eliminated. Because the diffusion properties of neurochemicals in brain tissue are likely different from in vivo conditions, reported dialysate values were not corrected for recovery of the probe. 2.3. Quantification of norepinephrine levels The amount of NE in the dialysate samples was determined with HPLC-ED. Dialysate samples (15 Al) were injected into the HPLC system using an autosampler (ESA Inc, Chelmsford, MA). The HPLC consists of an ESA solvent delivery module and a Velosep RP-18 column (100 3.2 mm, 3 Am, Varian Chromatography, Palo Alto, CA) or an MD 150 2 column (ESA Inc., Chelmsford, MA). The mobile phase consisted of 60 mM sodium phosphate buffer (pH 4.2) with 100 AM ethylenediamine tetraacetic acid (EDTA, Fisher Chemicals, Pittsburgh, PA), 1.5 mM sodium octyl-sulfate (Fisher Chemicals, Pittsburgh, PA) and 3.5% (v/v) methanol. The flow rate through the system was 700 Al/min (Velosep RP-18 column) or 300 Al/min (MD 150 2 column). The detection system consisted of an ESA Coulochem II electrochemical detector with either three electrodes placed in series; the conditioning electrode set at +220 mV, the applied potential of the second electrode set at 150 mV and the third electrode set at +220 mV or with a high sensitivity amperometric analytical cell (5041; ESA Inc., Chelmsford, MA) with an applied potential of +220 mV. Peak heights were measured and compared to the peak heights of a 10 8 M standard calibrated daily. The detection limit, defined as the sample amount producing a peak height that is twice the height of the background noise, was approximately 0.5 pg of NE.
3. Data analysis The baseline value against which drug administration was compared to was derived from the average of three samples just prior to manipulation. The neurochemical data were expressed as the mean T SEM. The overall effect of treatment of WIN 55,212-2 on monoamine efflux in the frontal cortex was analyzed using 2-way analysis of variance (ANOVA) with repeated measures over time ( P < 0.05). The absolute amount of neurotransmitter measured in dialysates (pg/ sample) was used as the dependent variable for assessment of within group effects. Basal values plus the next five samples post drug injection were used in the analysis. All statistics were performed using StatView software (JMP, Cary, NC). 3.1. Drug treatment for c-Fos experiments Male Sprague – Dawley rats (Harlan Sprague – Dawley, Indianapolis, IN; 225 – 250 g) were treated with 1.0, 3.0 or
47
15.0 mg/kg of WIN 55,212-2 (n = 5 per dose) systemically. The drug was made freshly everyday by dissolving it in deionized water containing 0.4% Tween (Sigma, St. Louis, MO). A control group of animals (n = 5) was treated with vehicle (deionized water containing 0.4% Tween). A second control group was pretreated with the cannabinoid antagonist SR 141716A (n = 5) at a concentration of 0.2 mg/kg 30 min prior to WIN 55,212-2 administration. A final group of rats was pretreated with 0.2 mg/kg of SR 141716A (n = 5) followed 30 min later by a systemic vehicle injection. 3.2. Tissue fixation Ninety minutes following WIN 55,212-2 administration, rats were anesthetized using sodium pentobarbital (60 mg/kg) and transcardially perfused through the ascending aorta with heparin (1000 units/ml) followed by acrolein (Electron Microscopy Sciences, Hatfield, PA) (3.75% in 2% paraformaldehyde) and finally with 2% paraformaldehyde (Electron Microscopy Sciences) in 0.1 M phosphate buffer (PB, pH 7.4). The brain was removed and 40-Am thick coronal sections were cut through the rostrocaudal extent of the rostral pons (plates 55 – 58 from the Paxinos and Watson rat brain atlas [30]) using a Vibratome Series 1000 and collected into chilled 0.1 M PB. 3.3. Immunocytochemistry Single labeling for c-Fos was performed to determine whether systemic administration of WIN 55,212-2 activates LC neurons. The presence of c-Fos protein was visualized as an immunoperoxidase signal within the cell nucleus. Dual immunofluorescence for TH and c-Fos was used to determine if cells expressing c-Fos were catecholaminergic. Free-floating sections were treated with a 1% sodium borohydride solution for 30 min. They were then rinsed with 0.1 M PB and later washed in 0.1 M tris saline buffer (TS) for 10 min, three times. The tissue was then blocked in 0.5% bovine serum albumin (BSA) in TS for 1 h and then washed for 10 min, three times. The sections were incubated overnight in a rabbit polyclonal antibody for c-Fos (1:5000; Oncogene, Research Triangle Park, NC) in 1% BSA in TS. For dual labeling, sections were also incubated with a mouse antibody directed against TH (1:1000; Immunostar Corp. Hudson, WI). The sections were then washed in 0.1 M TS for 10 min, three times. For single labeling, sections were incubated in a secondary antibody cocktail containing a biotin-conjugated goat anti-rabbit IgG (1:400, Jackson ImmunoResearch, West Grove, PA) in 0.1 M TS containing 0.1% BSA and 0.25% Triton-X 100 for 30 min. Dual labeling tissue was incubated in a secondary antibody cocktail containing fluorescein isothiocyanate (FITC) donkey anti-mouse IgG (1:200, Jackson ImmunoResearch, West Grove, PA) and tetramethyl rhodamine isothiocyanate (TRITC) donkey anti-rabbit (1:200, Jackson ImmunoResearch) in 0.1 M TS containing 0.1% BSA and 0.25%
48
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
Triton-X 100 for 2 h. The single label sections were then incubated in an avidin– biotin complex solution in 0.1 M TS (Vector Labs, Burlingame, CA) for 30 min and then washed. c-Fos was visualized by a 6 min reaction in 22 mg of 3,3Vdiaminobenzidine (DAB) (Sigma, St. Louis, MO) and 10 Al of 30% H2O2 in 100 ml of 0.1 M TS. The sections were then mounted onto gelatin-coated slides, passed through a dehydration series of ethanol finishing with two xylene washes and coverslipped using a DPX (fluorescence) or Permount solution (Aldrich Chemicals, St. Louis, MO). The analysis of c-Fos staining was performed using a microscope (Leica, Wetzlar, Germany) equipped for brightfield and darkfield microscopy. The microscope was fitted with a 35-mm camera system for photography and a CCD camera for digital image capture and subsequent computer analysis, which was carried out using SPOT software. The distribution of c-Fos-positive nuclei was plotted onto schematics obtained from the Swanson atlas [38]. Adjacent Nissl-stained sections were used to define the nuclear and regional cytoarchitectural subdivisions of the brain regions of interest according to Swanson [38] and Paxinos and Watson atlas [30]. The number of peroxidase labeled nuclei within the confines of cell groups of interest was counted and visualized under high magnification in complete series of coronal sections, corrected for double-counting errors using Abercrombie’s correction coefficient [10]. Where appropriate, post-hoc comparisons were made using Fisher’s PLSD test, and statistical significance was determined at P < 0.05.
4. Results 4.1. Effect of WIN 55,212-2 on basal norepinephrine efflux Extracellular NE levels were monitored using in vivo microdialysis and HPLC-ED. WIN 55,212-2 (0.3 mg/kg, 1.0 mg/kg; 3.0 mg/kg; and 15.0 mg/kg) was administered by intraperitoneal injection, and NE efflux in the frontal cortex following drug exposure was assessed. The effects of acute administration of the cannabinoid agonist WIN 55,212-2 are shown in Fig. 1. An overall ANOVA revealed a significant effect of treatment, time and interaction ( F Dose(3,21) = 3.48, P < 0.05, F Time(5,105) = 9.9, P < 0.0001 and F Int(15,105) = 3.2, P < 0.001). Post hoc analysis showed significantly greater NE output at 20 and 40 min post-injection (*Dunnett’s; P < 0.05). At a dose of 15.0 mg/kg, WIN 55,212-2 caused an increase in the extracellular levels of NE in the frontal cortex compared to baseline levels and vehicle administration by 103 T 19.6% from 1.45 pg/15Al to 2.94 pg/15Al ( F[5,25] = 7.67, P < 0.0001, n = 6, one-way ANOVA with repeated measures). The lower dose of WIN 55,212-2 administered (3.0 mg/kg) also caused a significant increase in NE efflux of 45.5 T 7.3% from 1.10 pg/15Al to 1.60 pg/15Al, ( F[5,25] = 6.27, P < 0.001; n = 6, one-way ANOVA with repeated
Fig. 1. The effect of increasing doses of WIN 55,212-2 (1.0 mg/kg [open circles (n = 7); baseline = 1.19 T 0.11 pg/15 Al]; 3.0 mg/kg [gray triangles (n = 6); baseline = 1.1 T 0.7 pg/15.0 Al]; 15 mg/kg [black squares (n = 6); baseline = 1.34 T 0.24 pg/15.0 Al]) on extracellular norepinephrine levels in the rat frontal cortex. Open squares (n = 6) indicate vehicle-treated rats. Administration of WIN 55,212-2 (3.0 mg/kg and 15 mg/kg) elicited significant increases in NE release F[5,25] = 6.27 (P < 0.001) and F[5,25] = 7.67 (P < 0.001) one-way ANOVA with repeated measures, respectively. Treatment with the 1.0 mg/kg of the cannabinoid agonist did not show a significant increase in norepinephrine release (F[8,40] = 0.28, (P = 0.9222) one-way ANOVA with repeated measures). An overall ANOVA revealed a significant effect of treatment, time and interaction (F Dose(3,21) = 3.48, P < 0.05, F Time(5,105) = 9.9, P < 0.0001 and F Int(15,105) = 3.2, P < 0.001). Post hoc analysis showed significantly greater norepinephrine output at 20 and 40 min post-injection (*Dunnett’s; P < 0.05). Arrow indicates time of injection.
measures). Treatment with a concentration of 1.0 mg/kg of WIN 55,212-2 showed a non-significant increase in NE levels by 12.1 T 10.0% from 1.24 pg/15Al to 1.39 pg/15Al ( F[8,40] = 0.28, P = 0.9222; n = 9, one-way ANOVA with repeated measures). Several rats (n = 4) were also treated with a concentration of 0.3 mg/kg of WIN 55,212-2. Similarly, this group did not show a statistically significant change in NE efflux in the frontal cortex compared to control animals (mean baseline was 1.51 T 0.09 pg/15Al). Vehicle treatment did not elicit any change in NE release ( F[3,15] = 1.38, P = 0.29; n = 4, one-way ANOVA with repeated measures). These data suggest that administration of the synthetic cannabinoid agonist WIN 55,212-2 causes a dose-dependent increase in NE efflux in the frontal cortex. 4.2. Effect of SR 141716A on basal and WIN 55,212-2-evoked norepinephrine efflux in frontal cortex To determine whether the effects of WIN 55,212-2 on NE release in the frontal cortex were mediated through modulation of CB1 receptors, a CB1 receptor antagonist, SR 1417164A, was used to block the receptor. The increase in extracellular NE release elicited by WIN 55,212-2 was prevented by systemically pretreating the animals 30 min prior to WIN 55,212-2 injection with 0.2 mg/kg of SR
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
1417164A (Fig. 2). An overall ANOVA revealed no significant effect of treatment (F[1,12] = 0.059; P = 0.8) but a significant effect of time (F[5,60] = 6.3; P < 0.0001) and a significant time by treatment interaction (F[5,60] = 3.03; P < 0.02). To determine whether SR 141716A had any effects on NE release under basal conditions, the cannabinoid receptor antagonist was administered followed 30 min later by a vehicle injection. SR 141716A had no significant effect on extracellular NE release in the rat frontal cortex (F[7,5] = 1.5, P = 0.21; one-way ANOVA with repeated measures, Fig. 3). A 2-way ANOVA with repeated measures comparing 4 tests groups (15 mg/kg WIN 55,212-2, pretreatment with antagonist followed by drug treatment, pretreatment with antagonist followed by vehicle and vehicle alone) revealed a significant effect of time and interaction (F Treatment(3,22) = 1.889, P = 0.16, F Time(22,110) = 6.904 P < 0.0001 and F Int(15,110) = 2.20, P = 0.05). A representative example of the placement of the dialysis probe in the frontal cortex is shown in Fig. 4. 4.3. WIN 55,212-2 induces c-Fos expression in the LC Systemic administration of WIN 55,212-2 caused a significant increase in c-Fos expression in the LC (Fig. 5). The mean number of c-Fos immunoreactive profiles per section in the LC after administration of WIN 55,212-2 at a dose of 15 mg/kg was 77 T 10 (n = 100 sections from five rats) versus 34 T 2 in rats that were treated with 3 mg/kg of the drug (n = 100 sections from five rats) (Fig. 6). In contrast, c-Fos expression was minimal in the LC in handled, untreated rats (14 T 8 c-Fos immunoreactive profiles; n = 100 sections from five rats). The lowest dose
Fig. 2. The increase in extracellular norepinephrine release elicited by WIN 55,212-2 is prevented by prior pretreatment with SR 141716A, a CB1 receptor antagonist. An overall ANOVA revealed no significant effect of treatment (F[1,12] = 0.059; P = 0.8) but a significant effect of time (F[5,60] = 6.3; P < 0.0001) and a significant time by treatment interaction (F[5,60] = 3.03; P < 0.02). Values represent the mean T SEM. Black squares show animals that were given 15 mg/kg of WIN 55,212-2 (n = 6, same group as Fig. 1). Animals pretreated with 0.2 mg/kg of SR 141716A prior to WIN 55,212-2 are shown as gray circles (n = 8). The arrow on the left indicates injection of cannabinoid antagonist SR 141716A, the arrow on the right designates timing of WIN 55,212-2 injection.
49
Fig. 3. A one-way ANOVA revealed no significant effect when treating animals with 0.2 mg/kg of SR 141716A followed by a vehicle injection (F[7,5] = 1.5, P = 0.21). An overall ANOVA revealed no significant effect of treatment (F[1,14] = 1.5; P = 0.24) or time by treatment interaction (F[5,70] = 0.84; P = 0.53) but a significant effect of time (F[5,70] = 2.81; P < 0.05) and a significant. Values represent the mean T SEM. Animals pretreated with 0.2 mg/kg of SR 141716A prior to WIN 55,212-2 are shown as circles (n = 8). Animals treated with 0.2 mg/kg of SR 141716A prior to a saline injection are shown by squares (n = 8). The arrow on the left indicates injection of cannabinoid antagonist SR 141716A, the arrow on the right designates timing of WIN 55,212-2 or vehicle injection.
used in the microdialysis experiments (1.0 mg/kg) was also tested, but the number of cells that expressed c-Fos 60 min post drug administration was the same as the number obtained in the control animals (16 T 5 c-Fos immunoreactive profiles; n = 100 sections from five rats). There was a statistically significant increase in the number of c-Fos labeled cells between the 15 mg/kg and 3 mg/kg doses of WIN 55,212-2 and control animals (P < 0.0001 and P < 0.01 respectively). The difference between the number of cells being activated by the 15.0 mg/kg dose and the 3.0 mg/ kg dose was also statistically significant (P < 0.01).
Fig. 4. Brightfield photomicrograph of a coronal section counterstained with Neutral Red showing histological verification of a microdialysis probe placement in the frontal cortex of a representative experiment. Black arrows indicate the position of the implanted probe. Arrows indicate dorsal and medial orientation of the tissue section. Scale bar = 500 Am.
50
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
Fig. 5. Brightfield photomicrographs of coronal sections through the LC showing immunoperoxidase labeling for c-Fos following a 90 min injection of WIN 55,212-2 (A: 15.0 mg/kg; B: 3.0 mg/kg). A significant increase in c-Fos expression was detected in the LC of rats receiving a systemic administration of WIN 55,212-2 compared to vehicle-treated rats. Arrows indicate dorsal and lateral orientation of tissue. scp: superior cerebellar peduncle, IV: fourth ventricle. Scale bar = 250 Am.
To determine whether the increase in c-Fos expression elicited by drug administration was due to activation of the cannabinoid CB1 receptor, the animals were pretreated with SR 141716A. Pretreatment with the antagonist inhibited the increase in c-Fos expression induced by WIN 55,212-2 (18 T 3 c-Fos immunoreactive profiles; n = 100 sections from five rats). The difference in the number of c-Fos labeled nuclei in sections from rats treated with 15 mg/kg and 3 mg/kg of WIN 55,212-2 and that from animals that were pretreated with 0.2 mg/kg of SR 141716A prior to administration of WIN 55,212-2 (15 mg/kg) was statistically significant (P < 0.0001 and P < 0.01 respectively).
The number of c-Fos labeled nuclei in the LC after pretreatment with the cannabinoid antagonist alone (18 T 3 c-Fos immunoreactive profiles; n = 100 sections from five rats) or antagonist followed by vehicle (21 T 2 c-Fos immunoreactive profiles; n = 100 sections from five rats) was not statistically different from the number of cells labeled after vehicle treatment alone (14 T 8 c-Fos immunoreactive profiles; n = 100 sections from five rats). To identify whether WIN 55,212-2 induced c-Fos in noradrenergic neurons, sections were dually labeled for c-Fos and TH. Dual immunofluorescence microscopy indicated that a significant number of TH containing
Fig. 6. Bar graph showing the mean number of c-Fos-positive nuclei in the LC per section in response to different treatments. veh: vehicle, high D: High dose (15.0 mg/kg), Low dose: (3.0 mg/kg), SR + WIN: antagonist (SR 141716A) injection (0.2 mg/kg) followed 30 min later by agonist (WIN 55,212-2; 15 mg/kg), SR + Veh: antagonist injection of SR 141716A (0.2 mg/kg) followed by vehicle injection. Statistical analysis revealed a significant difference between the high dose compared to control (*P < 0.0001), between the low dose and control (^P < 0.01) and between the low and the high doses (#P < 0.01).
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
51
Fig. 7. Photomicrographs showing immunofluorescence labeling of c-Fos and TH in coronal sections through the LC following an injection of WIN 55,212-2 (15 mg/kg). Paired rats were treated either with an intraperitoneal injection of saline or WIN 55,212-2. c-Fos is labeled with a tetramethyl rhodamine isothiocyanate tagged secondary antibody (red) and TH is labeled with a fluorescein isothiocyanate tagged secondary antibody (green). Panel (C) shows merged images. The asterisk denotes a blood vessel that can be seen in all images. Arrows indicate dorsal and lateral orientation of the tissue section. scp: superior cerebellar peduncle, IV: fourth ventricle. Scale bar = 250 Am.
neurons exhibited c-Fos immunoreactivity following cannabinoid agonist administration (Fig. 7). 4.4. Behavioral effects of WIN 55,212-2 In response to the systemic administration of WIN 55,212-2 at a dose of 15.0 mg/kg, rats adopted a supine position for approximately 60 min post-injection. At a dose of 3 mg/kg, the animals exhibited a lower amount of locomotion. At the lower doses (0.3 and 1.0 mg/kg), the animals had an increase in grooming and sniffing the air compared to controls. The effect, however, only lasted for the first two samples. The systemic administration of SR 141716A at a dose of 0.3 mg/kg did not produce overt behavioral effects.
5. Discussion The precise pharmacological mechanisms and neuroanatomical sites mediating the effect of cannabinoids on cognitive function remain elusive. Because the CB1 receptor is widely distributed on multiple modulatory neurotransmitter systems, it is likely that the behavioral effects elicited by cannabinoids are dependent on multiple neural and transmitter systems. The present study demonstrates that systemic administration of the cannabinoid receptor agonist WIN 55,212-2 stimulates NE efflux in the rat frontal cortex in a dose-dependent fashion. The enhancement of cortical NE is mediated via the cannabinoid CB1 receptor as NE efflux is blocked by pretreatment with the cannabinoid receptor antagonist SR 141716A. In addition, the present study demonstrates that systemic administration of WIN 55,212-2 induces c-Fos expression in noradrenergic neurons of the LC. Cognitive and emotional impairments associated with cannabis-based drug usage may be partially associated with alterations in noradrenergic transmission in the LC-frontal cortex pathway. Modulation of the LC-frontal cortex pathway may contribute to changes in attention, cognition and anxiety commonly observed following THC exposure as this circuit is involved in modulating these behaviors [3,20,44]. Activity of noradrenergic neurons can modulate the level
of cortical arousal and attention to internal and external stimuli and the development of affective disorders such as anxiety and depression [2,3,12]. Previous studies have revealed a linear relationship between LC cellular activation and NE efflux, and thus modulation of LC activity is thought to have global consequences on forebrain physiology [5]. Thus, activation of this neural pathway causing changes in NE efflux may partially underlie the physiologic effects of cannabinoids. Our findings are interesting in light of previous studies that demonstrated an inhibitory effect of THC and synthetic cannabinoid agonists on noradrenergic function. Treatment with THC resulted in decreases in NE in the hippocampus [16]. This decrease correlated to poor performance in the radial arm maze behavioral test [16]. Schlicker et al. [36] also reported inhibition of NE release in the hippocampus following synthetic cannabinoid agonist administration. Our results, however, show an increase in NE efflux in the frontal cortex in response to cannabinoid administration. This discrepancy is surprising given that both the hippocampus and frontal cortex receive noradrenergic inputs from the relatively homogenous nucleus, locus coeruleus; it would be expected that NE efflux in response to cannabinoid administration would be consistent in both regions. The difference in noradrenergic response could be due to a difference in methodologies and/or species used in the different studies. Schlicker et al. [36] performed their studies in human and guinea-pig tissue, whereas our studies were performed in the male Sprague – Dawley rat. The studies performed by this group were also done in vitro, whereas our studies were performed in vivo. Additionally, the opposing effects in NE efflux in response to cannabinoid administration between the hippocampus and the frontal cortex may represent regional differences in the distribution of cannabinoid receptors resulting in different neural circuits being activated.CB1 receptors have been localized to presynaptic axon terminals in the hippocampal formation [17] and presynaptically as well as on postsynaptic cortical interneurons in the frontal cortex [11,15,24]. The distribution of CB1 receptors associated with LC neurons that give rise to noradrenergic projections to the hippocampus and the frontal cortex is also unknown. Future studies are required to determine whether differences in NE efflux in these
52
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
regions arise from modulation of CB1 receptors that are differentially localized to subcellular profiles. Consistent with the results by Schlicker et al. [36] and Hernandez-Tristan et al. [16], studies performed by Tzavara et al. found that SR 141716A increased NE efflux in the frontal cortex and the hypothalamus [42,43]. Our data show no effect on NE efflux in the frontal cortex in response to administration of 0.2 mg/kg of the CB1 receptor antagonist SR 141716A and an increase in NE efflux in response to cannabinoid agonist administration. The dose of the antagonist SR 141716A that was administered by Tzavara et al. [42] in the studies performed involving the frontal cortex was five-fold higher than that used in our studies. It has been documented that SR 141716A can act as an inverse agonist at higher doses [26]. Thus, at the doses used by Tzavara et al. [42], the compound could have been mimicking the pharmacological effects of WIN 55,212-2 that were used in the present study. Moreover, the strain of rats used in both studies was also different. Tzavara et al. [42] used male Wistar rats, whereas, as stated above, male Sprague – Dawley rats were used in this study. Differences in noradrenergic innervation seen in different rat strains have been documented in the literature [9]. These differences in NE innervation between the two rat species could contribute to the discrepant results between the two studies. The studies in which Tzavara et al. [43] found an increase in NE efflux in the hypothalamus in response to SR 141716A were performed using doses comparable to the ones used in the present study. However, the differences in NE efflux seen in both regions could be due to differences in the source of NE input into the two brain regions. The source of NE into the frontal cortex is the LC, whereas the primary source of NE to the hypothalamus is the nucleus of the solitary tract [33]. Variability in effects of cannabinoids on neurotransmitter release can be found in the literature. In vivo microdialysis studies have shown that cannabinoid agonists significantly increase extracellular dopamine [23,39] and glutamate levels while GABA levels are decreased in the frontal cortex [31]. Conflicting reports have arisen with respect to the effect of cannabinoid agonists on acetylcholine release. In vitro studies have shown a decrease in acetylcholine release following cannabinoid exposure [13]. Subsequent in vivo microdialysis experiments, however, showed an enhancement of cortical and hippocampal acetylcholine levels following systemic administration via an indwelling i.p. catheter [1]. Discrepancies in such studies can potentially be attributed to different routes of drug administration, anesthesia used for implanting the dialysis probe and the type of probe utilized (transverse vs. concentric). However, agonists applied by reverse dialysis directly into the frontal cortex caused no change in acetylcholine release, although intrastriatal infusions of cannabinoid agonists decreased acetylcholine efflux [49]. These data suggest that cannabinoid agonists potentiate acetylcholine release in the frontal cortex by activating cannabinoid receptors in brain regions other than the frontal cortex [49]. As stated above, it is yet to
be determined whether local infusion of cannabinoid agonists directly into the frontal cortex alters NE release there. The noradrenergic system is known to be involved in the regulation of stress and anxiety [44]. A large body of evidence also indicates the participation of cannabinoid receptors in the regulation of anxiety-like behaviors. Acute administration of cannabinoids may cause anxiogenic responses in humans [53]. Animal studies have revealed that cannabinoids are capable of causing both anxiogenic and anxiolytic responses depending on the dose administered and the familiarity of the animal to the environment [35]. Others have found, however, that blockade of the CB1 receptor with SR 141716A induces an anxiety-like response in both the elevated plus maze and the defensive withdrawal test [28]. Other studies have shown that inhibition of FAAH, the enzyme involved in the hydrolysis of anandamide, causes anxiolytic responses in validated animal models of anxiety [22]. To date, no clear hypothesis has been made as to the anatomical and biochemical involvement of cannabinoids in the modulation of anxiety. In light of the data presented however, it seems possible that the noradrenergic system might be one of the neural substrates involved in the modulation of anxiety by cannabinoids. In the present study, c-Fos expression was used to examine potential sites of cellular activation with respect to LC neurons. Expression of c-Fos is increased as a result of somatodendritic depolarization and is therefore a useful marker for detecting neuronal activation [27]. The present study indicates that noradrenergic neurons in the LC express c-Fos following synthetic cannabinoid agonist administration and that this activation is prevented by pretreatment with the CB1 receptor antagonist SR 141716A suggesting a CB1 receptor-mediated effect. Our data are consistent with Patel et al. [29] who showed that increases in c-Fos expression in A10 dopaminergic neurons following CP55940, another cannabinoid agonist, administration result from excitatory noradrenergic transmission [29]. Although the presence of c-Fos in noradrenergic neurons is suggestive of modulation of CB1 receptors on LC neurons, these data require careful interpretation. Using light microscopy, CB1 receptor density in the LC is low [40]. However, a more detailed analysis of the distribution of CB1 receptors in the LC using immunoelectron microscopy is warranted to more carefully address this issue. It is conceivable that increased expression of c-Fos in the LC neurons following synthetic cannabinoid agonist administration may involve activation of excitatory medullary or limbic circuits [45 – 47]. Future studies are needed to examine whether systemic administration of cannabinoid agonists directly impacts on CB1 receptors that are located to noradrenergic somatodendritic processes in the LC or whether the induction of c-Fos is mediated by actions on afferent nuclei to the LC. As c-Fos and Jun form a transcription factor complex that regulates gene expression, the present study also suggests a
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
potential cellular mechanism whereby cannabinoid agonists may ultimately impact on responsiveness of LC neurons and long-term gene expression. For example, prenatal exposure to THC increases the expression of TH in neurons during early fetal brain development [6]. It is tempting to speculate that chronic use of cannabinoid-based substances may result in long-term adaptations in noradrenergic synthesizing enzymes via activation of c-Fos and its effectors. Future studies are required to further examine whether systemic administration of cannabinoid agonists modulates CB1 receptors that are present on noradrenergic axon terminals in the frontal cortex. CB1 receptors are commonly distributed to presynaptic axon terminals in other brain regions, including the hippocampus [41], and have been associated with peripheral sympathetic nerves [19]. This can be addressed using local infusion of cannabinoid agonists directly into the frontal cortex. In summary, the present study adds to the growing literature supporting heterogeneous effects of cannabinoid agonists on multiple neurotransmitter systems that most likely translate to complex behavioral responses.
Acknowledgments Supported by NIDA DA09082, DA15395 and a Jefferson Intramural Research Grant. V.O. was supported by a Minority Student Supplement to DA09082 from NIDA.
References [1] E. Acquas, A. Pisanu, P. Marrocu, S.R. Goldberg, G. DiChiara, Delta9-tetrahydrocannabinol enhances cortical and hippocampal acetylcholine release in vivo: a microdialysis study, Eur. J. Pharmacol. 419 (2001) 155 – 161. [2] G. Aston-Jones, F.E. Bloom, Activity of norepinephrine-containing locus coeruleus neurons in behaving rats anticipates fluctuations in the sleep – waking cycle, J. Neurosci. 1 (1981) 876 – 886. [3] G. Aston-Jones, S.L. Foote, F.E. Bloom, Anatomy and physiology of locus coeruleus neurons: functional implications, in: M. Ziegler, C.R. Lake (Eds.), Norepinephrine, Front. Clin. Neurosci., vol. 2, Williams and Wilkins, Baltimore, 1984, pp. 92 – 116. [4] G. Aston-Jones, J. Rajkowski, J. Cohen, Role of locus coeruleus in attention and behavioral flexibility, Biol. Psychiatry 46 (1999) 1309 – 1320. [5] C.W. Berridge, E.D. Abercrombie, Relationship between locus coeruleus discharge rates and rates of norepinephrine release within neocortex as assessed by in vivo microdialysis, Neuroscience 93 (1999) 1263 – 1270. [6] A. Bonnin, R. de Miguel, M.L. Hernandez, J.A. Ramos, J.J. Fernandez-Ruiz, The prenatal exposure to delta 9-tetrahydrocannabinol affects the gene expression and the activity of tyrosine hydroxylase during early brain development, Life Sci. 56 (1995) 2177 – 2184. [7] C.S. Breivogel, S.R. Childers, The functional neuroanatomy of brain cannabinoid receptors, Neurobiol. Dis. 5 (1998) 417 – 431. [8] E.A. Carlini, The good and the bad effects of ( )trans-delta-9tetrahydrocannabinol (Delta(9)-THC) on humans, Toxicon 44 (2004) 461 – 467.
53
[9] F.M. Clark, H.K. Proudfit, Anatomical evidence for genetic differences in the innervation of the rat spinal cord by noradrenergic locus coeruleus neurons, Brain Res. 591 (1992) 44 – 53. [10] R.E. Coggeshall, H.A. Lekan, Methods for determining numbers of cells and synapses: a case for more uniform standards of review [published erratum appears in J Comp Neurol 1996 May 20;369(1):162] [see comments], J. Comp. Neurol. 364 (1996) 6 – 15. [11] M. Egertova, M.R. Elphick, Localisation of cannabinoid receptors in the rat brain using antibodies to the intracellular C-terminal tail of CB, J. Comp. Neurol. 422 (2000) 159 – 171. [12] S.L. Foote, F.E. Bloom, G. Aston-Jones, Nucleus locus coeruleus: new evidence of anatomical and physiological specificity, Physiol. Rev. 63 (1983) 844 – 914. [13] G.L. Gessa, M.A. Casu, G. Carta, M.S. Mascia, Cannabinoids decrease acetylcholine release in the medial – prefrontal cortex and hippocampus, reversal by SR 141716A, Eur. J. Pharmacol. 355 (1998) 119 – 124. [14] M. Herkenham, A.B. Lynn, M.D. Little, M.R. Johnson, L.S. Melvin, B.R. de Costa, K.C. Rice, Cannabinoid receptor localization in brain, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 1932 – 1936. [15] M. Herkenham, B.G. Groen, A.B. Lynn, B.R. De Costa, E.K. Richfield, Neuronal localization of cannabinoid receptors and second messengers in mutant mouse cerebellum, Brain Res. 552 (1991) 301 – 310. [16] R. Hernandez-Tristan, C. Arevalo, S. Canals, M.L. Leret, The effects of acute treatment with delta9-THC on exploratory behaviour and memory in the rat, J. Physiol. Biochem. 56 (2000) 17 – 24. [17] A.F. Hoffman, A.C. Riegel, C.R. Lupica, Functional localization of cannabinoid receptors and endogenous cannabinoid production in distinct neuron populations of the hippocampus, Eur. J. Neurosci. 18 (2003) 524 – 534. [18] A.C. Howlett, Pharmacology of cannabinoid receptors, Annu. Rev. Pharmacol. Toxicol. 35 (1995) 607 – 634. [19] E.J. Ishac, L. Jiang, K.D. Lake, K. Varga, M.E. Abood, G. Kunos, Inhibition of exocytotic noradrenaline release by presynaptic cannabinoid CB1 receptors on peripheral sympathetic nerves, Br. J. Pharmacol. 118 (1996) 2023 – 2028. [20] M. Jouvet, Biogenic amines and the states of sleep, Science 163 (1969) 32 – 41. [21] Y. Kataoka, H. Ohta, M. Fujiwara, R. Oishi, S. Ueki, Noradrenergic involvement in catalepsy induced by delta 9-tetrahydrocannabinol, Neuropharmacology 26 (1987) 55 – 60. [22] S. Kathuria, S. Gaetani, D. Fegley, F. Valino, A. Duranti, A. Tontini, M. Mor, G. Tarzia, G. La Rana, A. Calignano, A. Giustino, M. Tattoli, M. Palmery, V. Cuomo, D. Piomelli, Modulation of anxiety through blockade of anandamide hydrolysis, Nat. Med. 9 (2003) 76 – 81. [23] D.T. Malone, D.A. Taylor, Modulation by fluoxetine of striatal dopamine release following Delta9-tetrahydrocannabinol: a microdialysis study in conscious rats, Br. J. Pharmacol. 128 (1999) 21 – 26. [24] G. Marsicano, B. Lutz, Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain, Eur. J. Neurosci. 11 (1999) 4213 – 4225. [25] B.R. Martin, L.J. Sim-Selley, D.E. Selley, Signaling pathways involved in the development of cannabinoid tolerance, Trends Pharmacol. Sci. 25 (2004) 325 – 330. [26] J.P. Meschler, D.M. Kraichely, G.H. Wilken, A.C. Howlett, Inverse agonist properties of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A) and 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxylic acid phenylamide (CP-272871) for the CB(1) cannabinoid receptor, Biochem. Pharmacol. 60 (2000) 1315 – 1323. [27] J.I. Morgan, T. Curran, Role of ion flux in the control of c-fos expression, Nature 322 (1986) 552 – 555. [28] M. Navarro, E. Hernandez, R.M. Munoz, I. del Arco, M.A. Villanua, M.R. Carrera, F. Rodriguez de Fonseca, Acute administration of the CB1 cannabinoid receptor antagonist SR 141716A induces anxietylike responses in the rat, NeuroReport 8 (1997) 491 – 496.
54
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
[29] S. Patel, C.J. Hillard, Cannabinoid-induced Fos expression within A10 dopaminergic neurons, Brain Res. 963 (2003) 15 – 25. [30] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986. [31] M. Pistis, L. Ferraro, L. Pira, G. Flore, S. Tanganelli, G.L. Gessa, P. Devoto, Delta(9)-tetrahydrocannabinol decreases extracellular GABA and increases extracellular glutamate and dopamine levels in the rat prefrontal cortex: an in vivo microdialysis study, Brain Res. 948 (2002) 155 – 158. [32] H.G. Pope Jr., A.J. Gruber, J.I. Hudson, M.A. Huestis, D. YurgelunTodd, Neuropsychological performance in long-term cannabis users, Arch. Gen. Psychiatry 58 (2001) 909 – 915. [33] J.A. Ricardo, E.T. Koh, Anatomical evidence of direct projections from the nucleus of the solitary tract to the hypothalamus, amygdala, and other forebrain structures in the rat, Brain Res. 153 (1978) 1 – 26. [34] T.W. Robbins, Cortical noradrenaline, attention and arousal, Psychol. Med. 14 (1984) 13 – 21. [35] F. Rodriguez de Fonseca, P. Rubio, F. Menzaghi, E. Merlo-Pich, J. Rivier, G.F. Koob, M. Navarro, Corticotropin-releasing factor (CRF) antagonist [d-Phe12,Nle21,38,C alpha MeLeu37]CRF attenuates the acute actions of the highly potent cannabinoid receptor agonist HU-210 on defensive-withdrawal behavior in rats, J. Pharmacol. Exp. Ther. 276 (1996) 56 – 64. [36] E. Schlicker, J. Timm, J. Zentner, M. Gothert, Cannabinoid CB1 receptor-mediated inhibition of noradrenaline release in the human and guinea-pig hippocampus, Naunyn-Schmiedeberg’s Arch. Pharmacol. 356 (1997) 583 – 589. [37] P.P. Singh, P.K. Das, Role of catecholamines in the hypothermic activity of cannabis in albino rats, Psychopharmacology (Berlin) 50 (1976) 199 – 204. [38] L.W. Swanson, Brain Maps: Structure of the Rat Brain, Elsevier, New York, 1992. [39] G. Tanda, F.E. Pontieri, G. Di Chiara, Cannabinoid and heroin activation of mesolimbic dopamine transmission by a common mu1 opioid receptor mechanism, Science 276 (1997) 2048 – 2050. [40] K. Tsou, S. Brown, M.C. Sanudo-Pena, K. Mackie, J.M. Walker, Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system, Neuroscience 83 (1998) 393 – 411. [41] W. Twitchell, S. Brown, K. Mackie, Cannabinoids inhibit N- and P/Q-type calcium channels in cultured rat hippocampal neurons, J. Neurophysiol. 78 (1997) 43 – 50. [42] E.T. Tzavara, K.W. Perry, D.E. Rodriguez, F.P. Bymaster, G.G. Nomikos, The cannabinoid CB(1) receptor antagonist SR141716A
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
increases norepinephrine outflow in the rat anterior hypothalamus, Eur. J. Pharmacol. 426 (2001) R3 – R4. E.T. Tzavara, R.J. Davis, K.W. Perry, X. Li, C. Salhoff, F.P. Bymaster, J.M. Witkin, G.G. Nomikos, The CB1 receptor antagonist SR141716A selectively increases monoaminergic neurotransmission in the medial prefrontal cortex: implications for therapeutic actions, Br. J. Pharmacol. 138 (2003) 544 – 553. R.J. Valentino, S.L. Foote, M.E. Page, The locus coeruleus as a site for integrating corticotropin-releasing factor and noradrenergic mediation of stress responses, Ann. N. Y. Acad. Sci. 697 (1993) 173 – 188. E.J. Van Bockstaele, G. Aston-Jones, Widespread autonomic afferents to the nucleus paragigantocellularis in the rostral ventrolateral medulla, in: G. Kunos, J. Ciriello (Eds.), Central Neural Mechanisms in Blood Pressure Regulation, Birkhauser Boston, Inc., 1991. E.J. Van Bockstaele, H. Akaoka, G. Aston-Jones, Brainstem afferents to the rostral (juxtafacial) nucleus paragigantocellularis: integration of exteroceptive and interoceptive sensory inputs in the ventral tegmentum, Brain Res. 603 (1993) 1 – 18. E.J. Van Bockstaele, J. Chan, V.M. Pickel, Input from central nucleus of the amygdala efferents to pericoerulear dendrites, some of which contain tyrosine hydroxylase immunoreactivity, J. Neurosci. Res. 45 (1996) 289 – 302. C.D. Verrico, J.D. Jentsch, L. Dazzi, R.H. Roth, Systemic, but not local, administration of cannabinoid CB1 receptor agonists modulate prefrontal cortical acetylcholine efflux in the rat, Synapse 48 (2003) 178 – 183. C.D. Verrico, J.D. Jentsch, R.H. Roth, J.R. Taylor, Repeated, intermittent delta(9)-tetrahydrocannabinol administration to rats impairs acquisition and performance of a test of visuospatial divided attention, Neuropsychopharmacology 29 (2004) 522 – 529. B.D. Waterhouse, H.C. Moises, H.H. Yeh, D.J. Woodward, Norepinephrine enhancement of inhibitory synaptic mechanisms in cerebellum and cerebral cortex: mediation by beta adrenergic receptors, J. Pharmacol. Exp. Ther. 221 (1982) 495 – 506. B.D. Waterhouse, C.S. Lin, R.A. Burne, D.J. Woodward, The distribution of neocortical projection neurons in the locus coeruleus, J. Comp. Neurol. 217 (1983) 418 – 431. D.J. Woodward, H.C. Moises, B.D. Waterhouse, B.J. Hoffer, R. Freedman, Modulatory actions of norepinephrine in the central nervous system, Fed. Proc. 38 (1979) 2109 – 2116. A.W. Zuardi, I. Shirakawa, E. Finkelfarb, I.G. Karniol, Action of cannabidiol on the anxiety and other effects produced by delta 9-THC in normal subjects, Psychopharmacology (Berlin) 76 (1982) 245 – 250.
Research report
Systemic administration of WIN 55,212-2 increases norepinephrine release in the rat frontal cortex V.C. Oropezaa,*, M.E. Pagea,b, E.J. Van Bockstaelea a
Department of Neurosurgery, Farber Institute for Neurosciences, Thomas Jefferson University, 900 Walnut Street, Suite 400, Philadelphia, PA 19107, USA b Department of Neurobiology and Anatomy, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA Accepted 15 March 2005 Available online 28 April 2005
Abstract Cannabinoid agonists modulate a variety of behavioral functions by activating cannabinoid receptors that are widely distributed throughout the central nervous system. In the present study, norepinephrine efflux was assessed in the frontal cortex of rats that received a systemic administration of the cannabinoid agonist, WIN 55,212-2. The synthetic cannabinoid agonist dose-dependently increased the release of norepinephrine in this brain region. Pretreatment with the cannabinoid receptor antagonist, SR 141716A, blocked the increase in norepinephrine release. To identify sites of cellular activation, immunocytochemical detection of c-Fos was combined with detection of the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH), in the brainstem nucleus locus coeruleus (LC), a region that is the sole source of norepinephrine to the frontal cortex. Systemic administration of WIN 55,212-2 significantly increased the number of c-Fos immunoreactive cells within TH-containing neurons in the LC compared to vehicle-treated rats. Pretreatment with SR 141716A inhibited the WIN 55,212-2 induced c-Fos expression, while the antagonist alone did not affect c-Fos expression. Taken together, these data indicate that systemically administered cannabinoid agonists stimulate norepinephrine release in the frontal cortex by activating noradrenergic neurons in the coeruleo-frontal cortex pathway. These effects may partially underlie changes in attention, arousal and anxiety observed following exposure to cannabis-based drugs. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, Modulators, transporters, and receptors Topic: Catecholamines Keywords: Cannabinoid; CB1 receptor; Locus coeruleus; Norepinephrine
1. Introduction The psychoactive ingredient in Cannabis sativa, D9-tetrahydrocannabinol (THC), produces characteristic psychotropic responses in humans [25]. Exogenous cannabinoids are also known to alter a wide array of functions in animals such as nociception, thermal regulation and motor activity (for review, see [18]). These effects are mediated by actions of agonists on cannabinoid receptors (CB1) that are widely distributed throughout the central nervous system [7,11,14,24,40]. Though much is known about the central effects of exogenously applied cannabinoids, the neural * Corresponding author. Fax: +1 215 955 4949. E-mail address: [email protected] (V.C. Oropeza). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.03.036
circuits and cellular mechanisms involved in mediating these effects remain to be elucidated. A potential site that may be affected by cannabinoidbased substances is the locus coeruleus (LC), a brainstem region involved in regulating cortical arousal, attention and anxiety [3,4,12,34,45,50 – 52], behaviors known to be altered by cannabinoid administration (for review, see [25]. Many studies have demonstrated that cannabinoids impact on central noradrenergic function. Bonnin et al. [6] found that prenatal exposure to D9-THC, the psychoactive component of marijuana, increased the expression of the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) in neurons during early fetal brain development. Moreover, Hernandez-Tristan et al. [16] showed that in vitro incubation of cultured fetal mesencephalic neurons with
46
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
D9-THC increased the activity of TH and this increase was reversed by SR 141716A, a specific antagonist for the cannabinoid CB1 receptor. Kataoka et al. [21] found that pretreatment with 6-hydroxydopamine (6-OHDA) or lesions of the LC significantly reduce the cataleptogenic effect of D9-THC. Additionally, central noradrenergic systems have been implicated in the hypothermic activity of D9-THC [37]. Previous studies have shown deficits in attention and arousal following the administration of cannabinoid-based substances [32,49]. THC impairs the capacity to discriminate time intervals and space distances, vigilance, memory and cognitive performance [8]. In order to study the role of the endogenous cannabinoid system in the modulation of the noradrenergic system, Tzavara et al. systemically administered SR 141716A and measured NE levels in various brain regions using microdialysis coupled to highpressure liquid chromatography (HPLC). Interestingly, they found that administration of SR 141716A increases norepinephrine outflow in the rat anterior hypothalamus and the frontal cortex [42,43] suggesting a tonic inhibitory role of endogenous cannabinoids on norepinephrine. To further elucidate the role of cannabinoids in the modulation of the noradrenergic system, the synthetic cannabinoid receptor agonist WIN 55,212-2 was administered systemically and changes in NE extracellular efflux in the rat frontal cortex were assessed using in vivo microdialysis followed by HPLC coupled with electrochemical detection (ED). In an effort to verify the involvement of the noradrenergic system in mediating the effects of cannabinoids, immunohistochemical detection of the immediate early gene c-Fos (a marker of increased neuronal activation [27]) was combined with detection of the catecholamine synthesizing enzyme, TH.
2. Methods Male Sprague – Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 200– 250 g were housed three per cage and maintained at a constant temperature of 22 -C on a 12:12 h light:dark cycle (07:00 on and 19:00 off) with unrestricted access to food and water. Animal surgeries were conducted in accordance with the guidelines established by the NIH Guide for Care and Use of Laboratory Animals and approved by both the Drexel University College of Medicine and Thomas Jefferson University Institutional Animal Care and Use Committees. 2.1. Microdialysis and high-pressure liquid chromatography After an acclimation period of approximately 1 week, rats were anesthetized with 8% chloral hydrate (400 mg/kg) and placed in a stereotaxic apparatus with the skull flat. A small burr hole was made in the skull centered at 3.2 mm anterior and T0.7 mm lateral to bregma. The dura was removed, and the microdialysis probe was slowly lowered
5.0 mm from the brain surface into the infralimbic and prelimbic areas of the frontal cortex (plates 8– 10 [30]) and secured with skull screws and dental acrylic. The inlet of the probe was connected to a fluid swivel (Instech Laboratories, Plymouth Meeting, PA), and the rat was placed into a cylindrical plexiglas container covered with bedding. Food and water were freely accessible. Artificial cerebrospinal fluid (aCSF: 174 mM NaCl, 1.7 mM CaCl2, 0.9 mM Mg Cl2 and 4 mM KCl) was continuously perfused through the probe at a rate of 1.5 Al/min by a microliter infusion pump (Harvard Pump F11_ VPF Dual Syringe, Harvard Apparatus, Holliston, MA). Rats were allowed to recover overnight. Approximately 18 h following surgery, dialysate samples were collected every 20 min. After collecting dialysate samples for at least 2 h to establish stable baseline levels, the cannabinoid agonist WIN 55,212-2 (Sigma, St. Louis, MO) was administered to the rats by intraperitoneal injection. Dilutions of the drug were prepared immediately prior to the start of each experiment by dissolving in deionized water containing 0.4% Tween (Sigma Chemical, St. Louis, MO). The doses administered were 0.3, 1.0, 3.0 and 15.0 mg/kg. Dialysate samples were collected for 3 h post-injection and stored at 80 -C for subsequent analysis by HPLC-ED. A control group was treated with vehicle only (water containing 0.4% Tween). In a separate experiment, the ability of the cannabinoid receptor antagonist, SR 141716A (received as a gift from NIDA) to block the actions of WIN 55,212-2 on NE efflux was tested. SR 141716A (0.2 mg/kg) was dissolved in deionized water containing 0.4% Tween and was administered 30 min prior to WIN 55,212-2 (15 mg/ kg). The control group for this experiment received SR 141716A followed by a vehicle injection 30 min later. At the conclusion of the experiment, rats were deeply anesthetized, and 2% pontamine sky blue dye (Alfa Aesar, Ward Hill, MA) was infused through the probe to mark its location. The rats were transcardially perfused with 10% formalin (Fisher Scientific, Pittsburgh, PA), decapitated and the brains removed for subsequent histological verification of probe placement. The data were not included if the placement was outside the infralimbic and prelimbic areas of the frontal cortex. 2.2. Dialysis probe construction and calibration Vertical concentric microdialysis probes were used. A piece of fused silica (Polymicro Technologies, Phoenix, AZ) was inserted through PE 10 tubing and semipermeable membrane made from hollow rayon fibers with a 224 Am o.d. and 35,000 MW cutoff was fixed over the fused silica and into the PE 10 tubing with epoxy. The open end of the dialysis fiber was sealed with a 0.5 mm epoxy plug, and 2 mm of the top of the membrane was coated with epoxy leaving an active membrane length of 3 mm for exchange across the membrane. The in vitro recovery rate was determined by placing the probe in a beaker of aCSF containing a known concentration of NE standard. The
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
concentration of NE in the dialysate was compared to the amount in the bath. Probes that did not correspond to an acceptable range of recovery (12 – 24%) were eliminated. Because the diffusion properties of neurochemicals in brain tissue are likely different from in vivo conditions, reported dialysate values were not corrected for recovery of the probe. 2.3. Quantification of norepinephrine levels The amount of NE in the dialysate samples was determined with HPLC-ED. Dialysate samples (15 Al) were injected into the HPLC system using an autosampler (ESA Inc, Chelmsford, MA). The HPLC consists of an ESA solvent delivery module and a Velosep RP-18 column (100 3.2 mm, 3 Am, Varian Chromatography, Palo Alto, CA) or an MD 150 2 column (ESA Inc., Chelmsford, MA). The mobile phase consisted of 60 mM sodium phosphate buffer (pH 4.2) with 100 AM ethylenediamine tetraacetic acid (EDTA, Fisher Chemicals, Pittsburgh, PA), 1.5 mM sodium octyl-sulfate (Fisher Chemicals, Pittsburgh, PA) and 3.5% (v/v) methanol. The flow rate through the system was 700 Al/min (Velosep RP-18 column) or 300 Al/min (MD 150 2 column). The detection system consisted of an ESA Coulochem II electrochemical detector with either three electrodes placed in series; the conditioning electrode set at +220 mV, the applied potential of the second electrode set at 150 mV and the third electrode set at +220 mV or with a high sensitivity amperometric analytical cell (5041; ESA Inc., Chelmsford, MA) with an applied potential of +220 mV. Peak heights were measured and compared to the peak heights of a 10 8 M standard calibrated daily. The detection limit, defined as the sample amount producing a peak height that is twice the height of the background noise, was approximately 0.5 pg of NE.
3. Data analysis The baseline value against which drug administration was compared to was derived from the average of three samples just prior to manipulation. The neurochemical data were expressed as the mean T SEM. The overall effect of treatment of WIN 55,212-2 on monoamine efflux in the frontal cortex was analyzed using 2-way analysis of variance (ANOVA) with repeated measures over time ( P < 0.05). The absolute amount of neurotransmitter measured in dialysates (pg/ sample) was used as the dependent variable for assessment of within group effects. Basal values plus the next five samples post drug injection were used in the analysis. All statistics were performed using StatView software (JMP, Cary, NC). 3.1. Drug treatment for c-Fos experiments Male Sprague – Dawley rats (Harlan Sprague – Dawley, Indianapolis, IN; 225 – 250 g) were treated with 1.0, 3.0 or
47
15.0 mg/kg of WIN 55,212-2 (n = 5 per dose) systemically. The drug was made freshly everyday by dissolving it in deionized water containing 0.4% Tween (Sigma, St. Louis, MO). A control group of animals (n = 5) was treated with vehicle (deionized water containing 0.4% Tween). A second control group was pretreated with the cannabinoid antagonist SR 141716A (n = 5) at a concentration of 0.2 mg/kg 30 min prior to WIN 55,212-2 administration. A final group of rats was pretreated with 0.2 mg/kg of SR 141716A (n = 5) followed 30 min later by a systemic vehicle injection. 3.2. Tissue fixation Ninety minutes following WIN 55,212-2 administration, rats were anesthetized using sodium pentobarbital (60 mg/kg) and transcardially perfused through the ascending aorta with heparin (1000 units/ml) followed by acrolein (Electron Microscopy Sciences, Hatfield, PA) (3.75% in 2% paraformaldehyde) and finally with 2% paraformaldehyde (Electron Microscopy Sciences) in 0.1 M phosphate buffer (PB, pH 7.4). The brain was removed and 40-Am thick coronal sections were cut through the rostrocaudal extent of the rostral pons (plates 55 – 58 from the Paxinos and Watson rat brain atlas [30]) using a Vibratome Series 1000 and collected into chilled 0.1 M PB. 3.3. Immunocytochemistry Single labeling for c-Fos was performed to determine whether systemic administration of WIN 55,212-2 activates LC neurons. The presence of c-Fos protein was visualized as an immunoperoxidase signal within the cell nucleus. Dual immunofluorescence for TH and c-Fos was used to determine if cells expressing c-Fos were catecholaminergic. Free-floating sections were treated with a 1% sodium borohydride solution for 30 min. They were then rinsed with 0.1 M PB and later washed in 0.1 M tris saline buffer (TS) for 10 min, three times. The tissue was then blocked in 0.5% bovine serum albumin (BSA) in TS for 1 h and then washed for 10 min, three times. The sections were incubated overnight in a rabbit polyclonal antibody for c-Fos (1:5000; Oncogene, Research Triangle Park, NC) in 1% BSA in TS. For dual labeling, sections were also incubated with a mouse antibody directed against TH (1:1000; Immunostar Corp. Hudson, WI). The sections were then washed in 0.1 M TS for 10 min, three times. For single labeling, sections were incubated in a secondary antibody cocktail containing a biotin-conjugated goat anti-rabbit IgG (1:400, Jackson ImmunoResearch, West Grove, PA) in 0.1 M TS containing 0.1% BSA and 0.25% Triton-X 100 for 30 min. Dual labeling tissue was incubated in a secondary antibody cocktail containing fluorescein isothiocyanate (FITC) donkey anti-mouse IgG (1:200, Jackson ImmunoResearch, West Grove, PA) and tetramethyl rhodamine isothiocyanate (TRITC) donkey anti-rabbit (1:200, Jackson ImmunoResearch) in 0.1 M TS containing 0.1% BSA and 0.25%
48
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
Triton-X 100 for 2 h. The single label sections were then incubated in an avidin– biotin complex solution in 0.1 M TS (Vector Labs, Burlingame, CA) for 30 min and then washed. c-Fos was visualized by a 6 min reaction in 22 mg of 3,3Vdiaminobenzidine (DAB) (Sigma, St. Louis, MO) and 10 Al of 30% H2O2 in 100 ml of 0.1 M TS. The sections were then mounted onto gelatin-coated slides, passed through a dehydration series of ethanol finishing with two xylene washes and coverslipped using a DPX (fluorescence) or Permount solution (Aldrich Chemicals, St. Louis, MO). The analysis of c-Fos staining was performed using a microscope (Leica, Wetzlar, Germany) equipped for brightfield and darkfield microscopy. The microscope was fitted with a 35-mm camera system for photography and a CCD camera for digital image capture and subsequent computer analysis, which was carried out using SPOT software. The distribution of c-Fos-positive nuclei was plotted onto schematics obtained from the Swanson atlas [38]. Adjacent Nissl-stained sections were used to define the nuclear and regional cytoarchitectural subdivisions of the brain regions of interest according to Swanson [38] and Paxinos and Watson atlas [30]. The number of peroxidase labeled nuclei within the confines of cell groups of interest was counted and visualized under high magnification in complete series of coronal sections, corrected for double-counting errors using Abercrombie’s correction coefficient [10]. Where appropriate, post-hoc comparisons were made using Fisher’s PLSD test, and statistical significance was determined at P < 0.05.
4. Results 4.1. Effect of WIN 55,212-2 on basal norepinephrine efflux Extracellular NE levels were monitored using in vivo microdialysis and HPLC-ED. WIN 55,212-2 (0.3 mg/kg, 1.0 mg/kg; 3.0 mg/kg; and 15.0 mg/kg) was administered by intraperitoneal injection, and NE efflux in the frontal cortex following drug exposure was assessed. The effects of acute administration of the cannabinoid agonist WIN 55,212-2 are shown in Fig. 1. An overall ANOVA revealed a significant effect of treatment, time and interaction ( F Dose(3,21) = 3.48, P < 0.05, F Time(5,105) = 9.9, P < 0.0001 and F Int(15,105) = 3.2, P < 0.001). Post hoc analysis showed significantly greater NE output at 20 and 40 min post-injection (*Dunnett’s; P < 0.05). At a dose of 15.0 mg/kg, WIN 55,212-2 caused an increase in the extracellular levels of NE in the frontal cortex compared to baseline levels and vehicle administration by 103 T 19.6% from 1.45 pg/15Al to 2.94 pg/15Al ( F[5,25] = 7.67, P < 0.0001, n = 6, one-way ANOVA with repeated measures). The lower dose of WIN 55,212-2 administered (3.0 mg/kg) also caused a significant increase in NE efflux of 45.5 T 7.3% from 1.10 pg/15Al to 1.60 pg/15Al, ( F[5,25] = 6.27, P < 0.001; n = 6, one-way ANOVA with repeated
Fig. 1. The effect of increasing doses of WIN 55,212-2 (1.0 mg/kg [open circles (n = 7); baseline = 1.19 T 0.11 pg/15 Al]; 3.0 mg/kg [gray triangles (n = 6); baseline = 1.1 T 0.7 pg/15.0 Al]; 15 mg/kg [black squares (n = 6); baseline = 1.34 T 0.24 pg/15.0 Al]) on extracellular norepinephrine levels in the rat frontal cortex. Open squares (n = 6) indicate vehicle-treated rats. Administration of WIN 55,212-2 (3.0 mg/kg and 15 mg/kg) elicited significant increases in NE release F[5,25] = 6.27 (P < 0.001) and F[5,25] = 7.67 (P < 0.001) one-way ANOVA with repeated measures, respectively. Treatment with the 1.0 mg/kg of the cannabinoid agonist did not show a significant increase in norepinephrine release (F[8,40] = 0.28, (P = 0.9222) one-way ANOVA with repeated measures). An overall ANOVA revealed a significant effect of treatment, time and interaction (F Dose(3,21) = 3.48, P < 0.05, F Time(5,105) = 9.9, P < 0.0001 and F Int(15,105) = 3.2, P < 0.001). Post hoc analysis showed significantly greater norepinephrine output at 20 and 40 min post-injection (*Dunnett’s; P < 0.05). Arrow indicates time of injection.
measures). Treatment with a concentration of 1.0 mg/kg of WIN 55,212-2 showed a non-significant increase in NE levels by 12.1 T 10.0% from 1.24 pg/15Al to 1.39 pg/15Al ( F[8,40] = 0.28, P = 0.9222; n = 9, one-way ANOVA with repeated measures). Several rats (n = 4) were also treated with a concentration of 0.3 mg/kg of WIN 55,212-2. Similarly, this group did not show a statistically significant change in NE efflux in the frontal cortex compared to control animals (mean baseline was 1.51 T 0.09 pg/15Al). Vehicle treatment did not elicit any change in NE release ( F[3,15] = 1.38, P = 0.29; n = 4, one-way ANOVA with repeated measures). These data suggest that administration of the synthetic cannabinoid agonist WIN 55,212-2 causes a dose-dependent increase in NE efflux in the frontal cortex. 4.2. Effect of SR 141716A on basal and WIN 55,212-2-evoked norepinephrine efflux in frontal cortex To determine whether the effects of WIN 55,212-2 on NE release in the frontal cortex were mediated through modulation of CB1 receptors, a CB1 receptor antagonist, SR 1417164A, was used to block the receptor. The increase in extracellular NE release elicited by WIN 55,212-2 was prevented by systemically pretreating the animals 30 min prior to WIN 55,212-2 injection with 0.2 mg/kg of SR
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
1417164A (Fig. 2). An overall ANOVA revealed no significant effect of treatment (F[1,12] = 0.059; P = 0.8) but a significant effect of time (F[5,60] = 6.3; P < 0.0001) and a significant time by treatment interaction (F[5,60] = 3.03; P < 0.02). To determine whether SR 141716A had any effects on NE release under basal conditions, the cannabinoid receptor antagonist was administered followed 30 min later by a vehicle injection. SR 141716A had no significant effect on extracellular NE release in the rat frontal cortex (F[7,5] = 1.5, P = 0.21; one-way ANOVA with repeated measures, Fig. 3). A 2-way ANOVA with repeated measures comparing 4 tests groups (15 mg/kg WIN 55,212-2, pretreatment with antagonist followed by drug treatment, pretreatment with antagonist followed by vehicle and vehicle alone) revealed a significant effect of time and interaction (F Treatment(3,22) = 1.889, P = 0.16, F Time(22,110) = 6.904 P < 0.0001 and F Int(15,110) = 2.20, P = 0.05). A representative example of the placement of the dialysis probe in the frontal cortex is shown in Fig. 4. 4.3. WIN 55,212-2 induces c-Fos expression in the LC Systemic administration of WIN 55,212-2 caused a significant increase in c-Fos expression in the LC (Fig. 5). The mean number of c-Fos immunoreactive profiles per section in the LC after administration of WIN 55,212-2 at a dose of 15 mg/kg was 77 T 10 (n = 100 sections from five rats) versus 34 T 2 in rats that were treated with 3 mg/kg of the drug (n = 100 sections from five rats) (Fig. 6). In contrast, c-Fos expression was minimal in the LC in handled, untreated rats (14 T 8 c-Fos immunoreactive profiles; n = 100 sections from five rats). The lowest dose
Fig. 2. The increase in extracellular norepinephrine release elicited by WIN 55,212-2 is prevented by prior pretreatment with SR 141716A, a CB1 receptor antagonist. An overall ANOVA revealed no significant effect of treatment (F[1,12] = 0.059; P = 0.8) but a significant effect of time (F[5,60] = 6.3; P < 0.0001) and a significant time by treatment interaction (F[5,60] = 3.03; P < 0.02). Values represent the mean T SEM. Black squares show animals that were given 15 mg/kg of WIN 55,212-2 (n = 6, same group as Fig. 1). Animals pretreated with 0.2 mg/kg of SR 141716A prior to WIN 55,212-2 are shown as gray circles (n = 8). The arrow on the left indicates injection of cannabinoid antagonist SR 141716A, the arrow on the right designates timing of WIN 55,212-2 injection.
49
Fig. 3. A one-way ANOVA revealed no significant effect when treating animals with 0.2 mg/kg of SR 141716A followed by a vehicle injection (F[7,5] = 1.5, P = 0.21). An overall ANOVA revealed no significant effect of treatment (F[1,14] = 1.5; P = 0.24) or time by treatment interaction (F[5,70] = 0.84; P = 0.53) but a significant effect of time (F[5,70] = 2.81; P < 0.05) and a significant. Values represent the mean T SEM. Animals pretreated with 0.2 mg/kg of SR 141716A prior to WIN 55,212-2 are shown as circles (n = 8). Animals treated with 0.2 mg/kg of SR 141716A prior to a saline injection are shown by squares (n = 8). The arrow on the left indicates injection of cannabinoid antagonist SR 141716A, the arrow on the right designates timing of WIN 55,212-2 or vehicle injection.
used in the microdialysis experiments (1.0 mg/kg) was also tested, but the number of cells that expressed c-Fos 60 min post drug administration was the same as the number obtained in the control animals (16 T 5 c-Fos immunoreactive profiles; n = 100 sections from five rats). There was a statistically significant increase in the number of c-Fos labeled cells between the 15 mg/kg and 3 mg/kg doses of WIN 55,212-2 and control animals (P < 0.0001 and P < 0.01 respectively). The difference between the number of cells being activated by the 15.0 mg/kg dose and the 3.0 mg/ kg dose was also statistically significant (P < 0.01).
Fig. 4. Brightfield photomicrograph of a coronal section counterstained with Neutral Red showing histological verification of a microdialysis probe placement in the frontal cortex of a representative experiment. Black arrows indicate the position of the implanted probe. Arrows indicate dorsal and medial orientation of the tissue section. Scale bar = 500 Am.
50
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
Fig. 5. Brightfield photomicrographs of coronal sections through the LC showing immunoperoxidase labeling for c-Fos following a 90 min injection of WIN 55,212-2 (A: 15.0 mg/kg; B: 3.0 mg/kg). A significant increase in c-Fos expression was detected in the LC of rats receiving a systemic administration of WIN 55,212-2 compared to vehicle-treated rats. Arrows indicate dorsal and lateral orientation of tissue. scp: superior cerebellar peduncle, IV: fourth ventricle. Scale bar = 250 Am.
To determine whether the increase in c-Fos expression elicited by drug administration was due to activation of the cannabinoid CB1 receptor, the animals were pretreated with SR 141716A. Pretreatment with the antagonist inhibited the increase in c-Fos expression induced by WIN 55,212-2 (18 T 3 c-Fos immunoreactive profiles; n = 100 sections from five rats). The difference in the number of c-Fos labeled nuclei in sections from rats treated with 15 mg/kg and 3 mg/kg of WIN 55,212-2 and that from animals that were pretreated with 0.2 mg/kg of SR 141716A prior to administration of WIN 55,212-2 (15 mg/kg) was statistically significant (P < 0.0001 and P < 0.01 respectively).
The number of c-Fos labeled nuclei in the LC after pretreatment with the cannabinoid antagonist alone (18 T 3 c-Fos immunoreactive profiles; n = 100 sections from five rats) or antagonist followed by vehicle (21 T 2 c-Fos immunoreactive profiles; n = 100 sections from five rats) was not statistically different from the number of cells labeled after vehicle treatment alone (14 T 8 c-Fos immunoreactive profiles; n = 100 sections from five rats). To identify whether WIN 55,212-2 induced c-Fos in noradrenergic neurons, sections were dually labeled for c-Fos and TH. Dual immunofluorescence microscopy indicated that a significant number of TH containing
Fig. 6. Bar graph showing the mean number of c-Fos-positive nuclei in the LC per section in response to different treatments. veh: vehicle, high D: High dose (15.0 mg/kg), Low dose: (3.0 mg/kg), SR + WIN: antagonist (SR 141716A) injection (0.2 mg/kg) followed 30 min later by agonist (WIN 55,212-2; 15 mg/kg), SR + Veh: antagonist injection of SR 141716A (0.2 mg/kg) followed by vehicle injection. Statistical analysis revealed a significant difference between the high dose compared to control (*P < 0.0001), between the low dose and control (^P < 0.01) and between the low and the high doses (#P < 0.01).
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
51
Fig. 7. Photomicrographs showing immunofluorescence labeling of c-Fos and TH in coronal sections through the LC following an injection of WIN 55,212-2 (15 mg/kg). Paired rats were treated either with an intraperitoneal injection of saline or WIN 55,212-2. c-Fos is labeled with a tetramethyl rhodamine isothiocyanate tagged secondary antibody (red) and TH is labeled with a fluorescein isothiocyanate tagged secondary antibody (green). Panel (C) shows merged images. The asterisk denotes a blood vessel that can be seen in all images. Arrows indicate dorsal and lateral orientation of the tissue section. scp: superior cerebellar peduncle, IV: fourth ventricle. Scale bar = 250 Am.
neurons exhibited c-Fos immunoreactivity following cannabinoid agonist administration (Fig. 7). 4.4. Behavioral effects of WIN 55,212-2 In response to the systemic administration of WIN 55,212-2 at a dose of 15.0 mg/kg, rats adopted a supine position for approximately 60 min post-injection. At a dose of 3 mg/kg, the animals exhibited a lower amount of locomotion. At the lower doses (0.3 and 1.0 mg/kg), the animals had an increase in grooming and sniffing the air compared to controls. The effect, however, only lasted for the first two samples. The systemic administration of SR 141716A at a dose of 0.3 mg/kg did not produce overt behavioral effects.
5. Discussion The precise pharmacological mechanisms and neuroanatomical sites mediating the effect of cannabinoids on cognitive function remain elusive. Because the CB1 receptor is widely distributed on multiple modulatory neurotransmitter systems, it is likely that the behavioral effects elicited by cannabinoids are dependent on multiple neural and transmitter systems. The present study demonstrates that systemic administration of the cannabinoid receptor agonist WIN 55,212-2 stimulates NE efflux in the rat frontal cortex in a dose-dependent fashion. The enhancement of cortical NE is mediated via the cannabinoid CB1 receptor as NE efflux is blocked by pretreatment with the cannabinoid receptor antagonist SR 141716A. In addition, the present study demonstrates that systemic administration of WIN 55,212-2 induces c-Fos expression in noradrenergic neurons of the LC. Cognitive and emotional impairments associated with cannabis-based drug usage may be partially associated with alterations in noradrenergic transmission in the LC-frontal cortex pathway. Modulation of the LC-frontal cortex pathway may contribute to changes in attention, cognition and anxiety commonly observed following THC exposure as this circuit is involved in modulating these behaviors [3,20,44]. Activity of noradrenergic neurons can modulate the level
of cortical arousal and attention to internal and external stimuli and the development of affective disorders such as anxiety and depression [2,3,12]. Previous studies have revealed a linear relationship between LC cellular activation and NE efflux, and thus modulation of LC activity is thought to have global consequences on forebrain physiology [5]. Thus, activation of this neural pathway causing changes in NE efflux may partially underlie the physiologic effects of cannabinoids. Our findings are interesting in light of previous studies that demonstrated an inhibitory effect of THC and synthetic cannabinoid agonists on noradrenergic function. Treatment with THC resulted in decreases in NE in the hippocampus [16]. This decrease correlated to poor performance in the radial arm maze behavioral test [16]. Schlicker et al. [36] also reported inhibition of NE release in the hippocampus following synthetic cannabinoid agonist administration. Our results, however, show an increase in NE efflux in the frontal cortex in response to cannabinoid administration. This discrepancy is surprising given that both the hippocampus and frontal cortex receive noradrenergic inputs from the relatively homogenous nucleus, locus coeruleus; it would be expected that NE efflux in response to cannabinoid administration would be consistent in both regions. The difference in noradrenergic response could be due to a difference in methodologies and/or species used in the different studies. Schlicker et al. [36] performed their studies in human and guinea-pig tissue, whereas our studies were performed in the male Sprague – Dawley rat. The studies performed by this group were also done in vitro, whereas our studies were performed in vivo. Additionally, the opposing effects in NE efflux in response to cannabinoid administration between the hippocampus and the frontal cortex may represent regional differences in the distribution of cannabinoid receptors resulting in different neural circuits being activated.CB1 receptors have been localized to presynaptic axon terminals in the hippocampal formation [17] and presynaptically as well as on postsynaptic cortical interneurons in the frontal cortex [11,15,24]. The distribution of CB1 receptors associated with LC neurons that give rise to noradrenergic projections to the hippocampus and the frontal cortex is also unknown. Future studies are required to determine whether differences in NE efflux in these
52
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
regions arise from modulation of CB1 receptors that are differentially localized to subcellular profiles. Consistent with the results by Schlicker et al. [36] and Hernandez-Tristan et al. [16], studies performed by Tzavara et al. found that SR 141716A increased NE efflux in the frontal cortex and the hypothalamus [42,43]. Our data show no effect on NE efflux in the frontal cortex in response to administration of 0.2 mg/kg of the CB1 receptor antagonist SR 141716A and an increase in NE efflux in response to cannabinoid agonist administration. The dose of the antagonist SR 141716A that was administered by Tzavara et al. [42] in the studies performed involving the frontal cortex was five-fold higher than that used in our studies. It has been documented that SR 141716A can act as an inverse agonist at higher doses [26]. Thus, at the doses used by Tzavara et al. [42], the compound could have been mimicking the pharmacological effects of WIN 55,212-2 that were used in the present study. Moreover, the strain of rats used in both studies was also different. Tzavara et al. [42] used male Wistar rats, whereas, as stated above, male Sprague – Dawley rats were used in this study. Differences in noradrenergic innervation seen in different rat strains have been documented in the literature [9]. These differences in NE innervation between the two rat species could contribute to the discrepant results between the two studies. The studies in which Tzavara et al. [43] found an increase in NE efflux in the hypothalamus in response to SR 141716A were performed using doses comparable to the ones used in the present study. However, the differences in NE efflux seen in both regions could be due to differences in the source of NE input into the two brain regions. The source of NE into the frontal cortex is the LC, whereas the primary source of NE to the hypothalamus is the nucleus of the solitary tract [33]. Variability in effects of cannabinoids on neurotransmitter release can be found in the literature. In vivo microdialysis studies have shown that cannabinoid agonists significantly increase extracellular dopamine [23,39] and glutamate levels while GABA levels are decreased in the frontal cortex [31]. Conflicting reports have arisen with respect to the effect of cannabinoid agonists on acetylcholine release. In vitro studies have shown a decrease in acetylcholine release following cannabinoid exposure [13]. Subsequent in vivo microdialysis experiments, however, showed an enhancement of cortical and hippocampal acetylcholine levels following systemic administration via an indwelling i.p. catheter [1]. Discrepancies in such studies can potentially be attributed to different routes of drug administration, anesthesia used for implanting the dialysis probe and the type of probe utilized (transverse vs. concentric). However, agonists applied by reverse dialysis directly into the frontal cortex caused no change in acetylcholine release, although intrastriatal infusions of cannabinoid agonists decreased acetylcholine efflux [49]. These data suggest that cannabinoid agonists potentiate acetylcholine release in the frontal cortex by activating cannabinoid receptors in brain regions other than the frontal cortex [49]. As stated above, it is yet to
be determined whether local infusion of cannabinoid agonists directly into the frontal cortex alters NE release there. The noradrenergic system is known to be involved in the regulation of stress and anxiety [44]. A large body of evidence also indicates the participation of cannabinoid receptors in the regulation of anxiety-like behaviors. Acute administration of cannabinoids may cause anxiogenic responses in humans [53]. Animal studies have revealed that cannabinoids are capable of causing both anxiogenic and anxiolytic responses depending on the dose administered and the familiarity of the animal to the environment [35]. Others have found, however, that blockade of the CB1 receptor with SR 141716A induces an anxiety-like response in both the elevated plus maze and the defensive withdrawal test [28]. Other studies have shown that inhibition of FAAH, the enzyme involved in the hydrolysis of anandamide, causes anxiolytic responses in validated animal models of anxiety [22]. To date, no clear hypothesis has been made as to the anatomical and biochemical involvement of cannabinoids in the modulation of anxiety. In light of the data presented however, it seems possible that the noradrenergic system might be one of the neural substrates involved in the modulation of anxiety by cannabinoids. In the present study, c-Fos expression was used to examine potential sites of cellular activation with respect to LC neurons. Expression of c-Fos is increased as a result of somatodendritic depolarization and is therefore a useful marker for detecting neuronal activation [27]. The present study indicates that noradrenergic neurons in the LC express c-Fos following synthetic cannabinoid agonist administration and that this activation is prevented by pretreatment with the CB1 receptor antagonist SR 141716A suggesting a CB1 receptor-mediated effect. Our data are consistent with Patel et al. [29] who showed that increases in c-Fos expression in A10 dopaminergic neurons following CP55940, another cannabinoid agonist, administration result from excitatory noradrenergic transmission [29]. Although the presence of c-Fos in noradrenergic neurons is suggestive of modulation of CB1 receptors on LC neurons, these data require careful interpretation. Using light microscopy, CB1 receptor density in the LC is low [40]. However, a more detailed analysis of the distribution of CB1 receptors in the LC using immunoelectron microscopy is warranted to more carefully address this issue. It is conceivable that increased expression of c-Fos in the LC neurons following synthetic cannabinoid agonist administration may involve activation of excitatory medullary or limbic circuits [45 – 47]. Future studies are needed to examine whether systemic administration of cannabinoid agonists directly impacts on CB1 receptors that are located to noradrenergic somatodendritic processes in the LC or whether the induction of c-Fos is mediated by actions on afferent nuclei to the LC. As c-Fos and Jun form a transcription factor complex that regulates gene expression, the present study also suggests a
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
potential cellular mechanism whereby cannabinoid agonists may ultimately impact on responsiveness of LC neurons and long-term gene expression. For example, prenatal exposure to THC increases the expression of TH in neurons during early fetal brain development [6]. It is tempting to speculate that chronic use of cannabinoid-based substances may result in long-term adaptations in noradrenergic synthesizing enzymes via activation of c-Fos and its effectors. Future studies are required to further examine whether systemic administration of cannabinoid agonists modulates CB1 receptors that are present on noradrenergic axon terminals in the frontal cortex. CB1 receptors are commonly distributed to presynaptic axon terminals in other brain regions, including the hippocampus [41], and have been associated with peripheral sympathetic nerves [19]. This can be addressed using local infusion of cannabinoid agonists directly into the frontal cortex. In summary, the present study adds to the growing literature supporting heterogeneous effects of cannabinoid agonists on multiple neurotransmitter systems that most likely translate to complex behavioral responses.
Acknowledgments Supported by NIDA DA09082, DA15395 and a Jefferson Intramural Research Grant. V.O. was supported by a Minority Student Supplement to DA09082 from NIDA.
References [1] E. Acquas, A. Pisanu, P. Marrocu, S.R. Goldberg, G. DiChiara, Delta9-tetrahydrocannabinol enhances cortical and hippocampal acetylcholine release in vivo: a microdialysis study, Eur. J. Pharmacol. 419 (2001) 155 – 161. [2] G. Aston-Jones, F.E. Bloom, Activity of norepinephrine-containing locus coeruleus neurons in behaving rats anticipates fluctuations in the sleep – waking cycle, J. Neurosci. 1 (1981) 876 – 886. [3] G. Aston-Jones, S.L. Foote, F.E. Bloom, Anatomy and physiology of locus coeruleus neurons: functional implications, in: M. Ziegler, C.R. Lake (Eds.), Norepinephrine, Front. Clin. Neurosci., vol. 2, Williams and Wilkins, Baltimore, 1984, pp. 92 – 116. [4] G. Aston-Jones, J. Rajkowski, J. Cohen, Role of locus coeruleus in attention and behavioral flexibility, Biol. Psychiatry 46 (1999) 1309 – 1320. [5] C.W. Berridge, E.D. Abercrombie, Relationship between locus coeruleus discharge rates and rates of norepinephrine release within neocortex as assessed by in vivo microdialysis, Neuroscience 93 (1999) 1263 – 1270. [6] A. Bonnin, R. de Miguel, M.L. Hernandez, J.A. Ramos, J.J. Fernandez-Ruiz, The prenatal exposure to delta 9-tetrahydrocannabinol affects the gene expression and the activity of tyrosine hydroxylase during early brain development, Life Sci. 56 (1995) 2177 – 2184. [7] C.S. Breivogel, S.R. Childers, The functional neuroanatomy of brain cannabinoid receptors, Neurobiol. Dis. 5 (1998) 417 – 431. [8] E.A. Carlini, The good and the bad effects of ( )trans-delta-9tetrahydrocannabinol (Delta(9)-THC) on humans, Toxicon 44 (2004) 461 – 467.
53
[9] F.M. Clark, H.K. Proudfit, Anatomical evidence for genetic differences in the innervation of the rat spinal cord by noradrenergic locus coeruleus neurons, Brain Res. 591 (1992) 44 – 53. [10] R.E. Coggeshall, H.A. Lekan, Methods for determining numbers of cells and synapses: a case for more uniform standards of review [published erratum appears in J Comp Neurol 1996 May 20;369(1):162] [see comments], J. Comp. Neurol. 364 (1996) 6 – 15. [11] M. Egertova, M.R. Elphick, Localisation of cannabinoid receptors in the rat brain using antibodies to the intracellular C-terminal tail of CB, J. Comp. Neurol. 422 (2000) 159 – 171. [12] S.L. Foote, F.E. Bloom, G. Aston-Jones, Nucleus locus coeruleus: new evidence of anatomical and physiological specificity, Physiol. Rev. 63 (1983) 844 – 914. [13] G.L. Gessa, M.A. Casu, G. Carta, M.S. Mascia, Cannabinoids decrease acetylcholine release in the medial – prefrontal cortex and hippocampus, reversal by SR 141716A, Eur. J. Pharmacol. 355 (1998) 119 – 124. [14] M. Herkenham, A.B. Lynn, M.D. Little, M.R. Johnson, L.S. Melvin, B.R. de Costa, K.C. Rice, Cannabinoid receptor localization in brain, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 1932 – 1936. [15] M. Herkenham, B.G. Groen, A.B. Lynn, B.R. De Costa, E.K. Richfield, Neuronal localization of cannabinoid receptors and second messengers in mutant mouse cerebellum, Brain Res. 552 (1991) 301 – 310. [16] R. Hernandez-Tristan, C. Arevalo, S. Canals, M.L. Leret, The effects of acute treatment with delta9-THC on exploratory behaviour and memory in the rat, J. Physiol. Biochem. 56 (2000) 17 – 24. [17] A.F. Hoffman, A.C. Riegel, C.R. Lupica, Functional localization of cannabinoid receptors and endogenous cannabinoid production in distinct neuron populations of the hippocampus, Eur. J. Neurosci. 18 (2003) 524 – 534. [18] A.C. Howlett, Pharmacology of cannabinoid receptors, Annu. Rev. Pharmacol. Toxicol. 35 (1995) 607 – 634. [19] E.J. Ishac, L. Jiang, K.D. Lake, K. Varga, M.E. Abood, G. Kunos, Inhibition of exocytotic noradrenaline release by presynaptic cannabinoid CB1 receptors on peripheral sympathetic nerves, Br. J. Pharmacol. 118 (1996) 2023 – 2028. [20] M. Jouvet, Biogenic amines and the states of sleep, Science 163 (1969) 32 – 41. [21] Y. Kataoka, H. Ohta, M. Fujiwara, R. Oishi, S. Ueki, Noradrenergic involvement in catalepsy induced by delta 9-tetrahydrocannabinol, Neuropharmacology 26 (1987) 55 – 60. [22] S. Kathuria, S. Gaetani, D. Fegley, F. Valino, A. Duranti, A. Tontini, M. Mor, G. Tarzia, G. La Rana, A. Calignano, A. Giustino, M. Tattoli, M. Palmery, V. Cuomo, D. Piomelli, Modulation of anxiety through blockade of anandamide hydrolysis, Nat. Med. 9 (2003) 76 – 81. [23] D.T. Malone, D.A. Taylor, Modulation by fluoxetine of striatal dopamine release following Delta9-tetrahydrocannabinol: a microdialysis study in conscious rats, Br. J. Pharmacol. 128 (1999) 21 – 26. [24] G. Marsicano, B. Lutz, Expression of the cannabinoid receptor CB1 in distinct neuronal subpopulations in the adult mouse forebrain, Eur. J. Neurosci. 11 (1999) 4213 – 4225. [25] B.R. Martin, L.J. Sim-Selley, D.E. Selley, Signaling pathways involved in the development of cannabinoid tolerance, Trends Pharmacol. Sci. 25 (2004) 325 – 330. [26] J.P. Meschler, D.M. Kraichely, G.H. Wilken, A.C. Howlett, Inverse agonist properties of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716A) and 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1H-pyrazole-3-carboxylic acid phenylamide (CP-272871) for the CB(1) cannabinoid receptor, Biochem. Pharmacol. 60 (2000) 1315 – 1323. [27] J.I. Morgan, T. Curran, Role of ion flux in the control of c-fos expression, Nature 322 (1986) 552 – 555. [28] M. Navarro, E. Hernandez, R.M. Munoz, I. del Arco, M.A. Villanua, M.R. Carrera, F. Rodriguez de Fonseca, Acute administration of the CB1 cannabinoid receptor antagonist SR 141716A induces anxietylike responses in the rat, NeuroReport 8 (1997) 491 – 496.
54
V.C. Oropeza et al. / Brain Research 1046 (2005) 45 – 54
[29] S. Patel, C.J. Hillard, Cannabinoid-induced Fos expression within A10 dopaminergic neurons, Brain Res. 963 (2003) 15 – 25. [30] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986. [31] M. Pistis, L. Ferraro, L. Pira, G. Flore, S. Tanganelli, G.L. Gessa, P. Devoto, Delta(9)-tetrahydrocannabinol decreases extracellular GABA and increases extracellular glutamate and dopamine levels in the rat prefrontal cortex: an in vivo microdialysis study, Brain Res. 948 (2002) 155 – 158. [32] H.G. Pope Jr., A.J. Gruber, J.I. Hudson, M.A. Huestis, D. YurgelunTodd, Neuropsychological performance in long-term cannabis users, Arch. Gen. Psychiatry 58 (2001) 909 – 915. [33] J.A. Ricardo, E.T. Koh, Anatomical evidence of direct projections from the nucleus of the solitary tract to the hypothalamus, amygdala, and other forebrain structures in the rat, Brain Res. 153 (1978) 1 – 26. [34] T.W. Robbins, Cortical noradrenaline, attention and arousal, Psychol. Med. 14 (1984) 13 – 21. [35] F. Rodriguez de Fonseca, P. Rubio, F. Menzaghi, E. Merlo-Pich, J. Rivier, G.F. Koob, M. Navarro, Corticotropin-releasing factor (CRF) antagonist [d-Phe12,Nle21,38,C alpha MeLeu37]CRF attenuates the acute actions of the highly potent cannabinoid receptor agonist HU-210 on defensive-withdrawal behavior in rats, J. Pharmacol. Exp. Ther. 276 (1996) 56 – 64. [36] E. Schlicker, J. Timm, J. Zentner, M. Gothert, Cannabinoid CB1 receptor-mediated inhibition of noradrenaline release in the human and guinea-pig hippocampus, Naunyn-Schmiedeberg’s Arch. Pharmacol. 356 (1997) 583 – 589. [37] P.P. Singh, P.K. Das, Role of catecholamines in the hypothermic activity of cannabis in albino rats, Psychopharmacology (Berlin) 50 (1976) 199 – 204. [38] L.W. Swanson, Brain Maps: Structure of the Rat Brain, Elsevier, New York, 1992. [39] G. Tanda, F.E. Pontieri, G. Di Chiara, Cannabinoid and heroin activation of mesolimbic dopamine transmission by a common mu1 opioid receptor mechanism, Science 276 (1997) 2048 – 2050. [40] K. Tsou, S. Brown, M.C. Sanudo-Pena, K. Mackie, J.M. Walker, Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system, Neuroscience 83 (1998) 393 – 411. [41] W. Twitchell, S. Brown, K. Mackie, Cannabinoids inhibit N- and P/Q-type calcium channels in cultured rat hippocampal neurons, J. Neurophysiol. 78 (1997) 43 – 50. [42] E.T. Tzavara, K.W. Perry, D.E. Rodriguez, F.P. Bymaster, G.G. Nomikos, The cannabinoid CB(1) receptor antagonist SR141716A
[43]
[44]
[45]
[46]
[47]
[48]
[49]
[50]
[51]
[52]
[53]
increases norepinephrine outflow in the rat anterior hypothalamus, Eur. J. Pharmacol. 426 (2001) R3 – R4. E.T. Tzavara, R.J. Davis, K.W. Perry, X. Li, C. Salhoff, F.P. Bymaster, J.M. Witkin, G.G. Nomikos, The CB1 receptor antagonist SR141716A selectively increases monoaminergic neurotransmission in the medial prefrontal cortex: implications for therapeutic actions, Br. J. Pharmacol. 138 (2003) 544 – 553. R.J. Valentino, S.L. Foote, M.E. Page, The locus coeruleus as a site for integrating corticotropin-releasing factor and noradrenergic mediation of stress responses, Ann. N. Y. Acad. Sci. 697 (1993) 173 – 188. E.J. Van Bockstaele, G. Aston-Jones, Widespread autonomic afferents to the nucleus paragigantocellularis in the rostral ventrolateral medulla, in: G. Kunos, J. Ciriello (Eds.), Central Neural Mechanisms in Blood Pressure Regulation, Birkhauser Boston, Inc., 1991. E.J. Van Bockstaele, H. Akaoka, G. Aston-Jones, Brainstem afferents to the rostral (juxtafacial) nucleus paragigantocellularis: integration of exteroceptive and interoceptive sensory inputs in the ventral tegmentum, Brain Res. 603 (1993) 1 – 18. E.J. Van Bockstaele, J. Chan, V.M. Pickel, Input from central nucleus of the amygdala efferents to pericoerulear dendrites, some of which contain tyrosine hydroxylase immunoreactivity, J. Neurosci. Res. 45 (1996) 289 – 302. C.D. Verrico, J.D. Jentsch, L. Dazzi, R.H. Roth, Systemic, but not local, administration of cannabinoid CB1 receptor agonists modulate prefrontal cortical acetylcholine efflux in the rat, Synapse 48 (2003) 178 – 183. C.D. Verrico, J.D. Jentsch, R.H. Roth, J.R. Taylor, Repeated, intermittent delta(9)-tetrahydrocannabinol administration to rats impairs acquisition and performance of a test of visuospatial divided attention, Neuropsychopharmacology 29 (2004) 522 – 529. B.D. Waterhouse, H.C. Moises, H.H. Yeh, D.J. Woodward, Norepinephrine enhancement of inhibitory synaptic mechanisms in cerebellum and cerebral cortex: mediation by beta adrenergic receptors, J. Pharmacol. Exp. Ther. 221 (1982) 495 – 506. B.D. Waterhouse, C.S. Lin, R.A. Burne, D.J. Woodward, The distribution of neocortical projection neurons in the locus coeruleus, J. Comp. Neurol. 217 (1983) 418 – 431. D.J. Woodward, H.C. Moises, B.D. Waterhouse, B.J. Hoffer, R. Freedman, Modulatory actions of norepinephrine in the central nervous system, Fed. Proc. 38 (1979) 2109 – 2116. A.W. Zuardi, I. Shirakawa, E. Finkelfarb, I.G. Karniol, Action of cannabidiol on the anxiety and other effects produced by delta 9-THC in normal subjects, Psychopharmacology (Berlin) 76 (1982) 245 – 250.