Residual effects of focal brain ischaemia upon cannabinoid CB1 receptor density and functionality in female rats

BR A I N R ES E A RC H 1 3 7 3 ( 2 01 1 ) 1 9 5 –2 01

available at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

Residual effects of focal brain ischaemia upon cannabinoid CB1 receptor density and functionality in female rats Maria Luisa Rojo a , Ingegerd Söderström b , Christopher J. Fowler a,⁎ a

Department of Pharmacology and Clinical Neuroscience, Medicine, Umeå University, SE-901 87 Umeå, Sweden Department of Public Health and Clinical Medicine, Medicine, Umeå University, SE-901 87 Umeå, Sweden

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Ischaemic insult results in short-term changes in cannabinoid-1 (CB1) receptor expression in

Accepted 1 December 2010

the brain, but it is not known whether long-term changes occur, which could potentially

Available online 8 December 2010

mean a change in the intrinsic ability of the brain to withstand new ischaemic episodes. In this study, we have investigated the expression and functionality of CB1 receptors in coronal

Keywords:

brain slices obtained from ovariectomised female rats 46 days after middle cerebral artery

Cannabinoid

occlusion (MCAO). The animals were treated with either 17ß-oestradiol or placebo pellets

Middle cerebral artery occlusion

6 h after MCAO and thereafter housed either in isolated or enriched environments. [3H]

Ischaemia

CP55,940 autoradiography indicated no significant effect of 17ß-oestradiol treatment or

Stroke

housing environment upon CB1 receptor densities. There was, however, a modest but

Receptor reserve

significant decrease in the CB1 receptor density on the ipsilateral side relative to the

[35S]GTPγS autoradiography

contralateral side in the frontal cortex, parietal cortex, CA1–CA3 regions of the

CP55,940

hippocampus, thalamus and hypothalamus. CB1 receptor functionality was assessed by measurement of basal and CP55,940-stimulated [35S]GTPγS autoradiography. In the frontal cortex, parietal cortex, CA1–CA3 regions of the hippocampus and dentate gyrus, a robust stimulation, blocked by the CB1 receptor inverse agonist AM251, was seen. There were no significant changes in the response to CP55,940 with respect either to the 17ß-oestradiol treatment, housing environment or MCAO. Our results reveal that although there are modest long-term decreases in ipsilateral CB1 receptor densities following MCAO in female rats, these decreases do not result in a functional CB1 receptor deficit. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

Cannabinoid CB1 receptors are G-protein coupled receptors present in high concentrations in the brain and which are

involved in a number of physiological and behavioural events such as regulation of appetite, pain, motor function, body temperature as well as the psychotropic effects sought after by recreational users of cannabis (review see Breivogel and

⁎ Corresponding author. Fax: + 46 90 785 2752. E-mail address: [email protected] (C.J. Fowler). Abbreviations: AM251, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1-H-pyrazole-3-carboxamide; CA1–CA3, cornu ammonis 1–cornu ammonis 3 pyramidal cell layer; CB, cannabinoid; CP55,940, (−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl) phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol; Enr, enriched environment; Est, 17ß-oestradiol; GTPγS, guanosine 5′-[γ-thio]triphosphate; Iso, isolated environment; MCAO, middle cerebral artery occlusion; Pl, placebo; WIN55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3(4-morpholinylmethyl)- pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.12.001

196

BR A I N R ES E A RC H 1 3 7 3 ( 2 01 1 ) 1 9 5 –20 1

Sim-Selley, 2009). One important characteristic of CB1 receptors and of their endogenous ligands (“endocannabinoids”) is their plasticity in response to a number of physiological and pathological stressors and their involvement in preserving homeostasis in the face of these stressors (review see Bisogno and Di Marzo, 2007). A case in point is the responsiveness of the cannabinoid system following cerebral insults such as ischaemia or trauma. Early studies showed that cannabinoids were neuroprotective following global and focal cerebral ischaemia, that cerebral insult produced an increase in endocannabinoid levels, and that the outcome of the ischaemic insult (infarct size, neurological deficit) was worse in CB1 receptor knockout mice than in wild-type mice (Nagayama et al., 1999; Hansen et al., 2001; Panikashvili et al., 2001; Parmentier-Batteur et al., 2002). Consistent with the notion that CB1 receptors may be important in maintaining homeostatic function in the face of adversity, a reduced CB1 receptor function, as assessed by measurement of agonist-stimulated [35S]GTPγS binding, has been reported for the frontal cortex in Alzheimer's disease (Ramírez et al., 2005). The decreased density of basal ganglia CB1 receptors in Huntington's disease is an early event (Glass et al., 2000), and in a mouse model of Huntington's disease, the loss of CB1 receptors and the onset of symptoms are both delayed if the animals are housed in an enriched environment, suggesting some form of causality (Glass et al., 2004). Subsequent data indicated that the nature and/or region of damage is of importance with respect to the modulatory role played by the cannabinoid system and as a result, blockade, rather than activation of cannabinoid receptors can produce neuroprotection in some cases (reviews, see Hillard, 2008; Pellegrini-Giampietro et al., 2009; Fowler et al., 2010). Nevertheless, the CB receptor agonist KN38-7271 ((−)-(2R)-3(2-hydroxymethylindanyl-4-oxy)-phenyl-4,4,4-trifluorobutane-1-sulfonate, formally BAY 38-7271, Mauler et al., 2003) has been granted orphan drug designation for the treatment of moderate and severe closed traumatic brain injury and has shown promising results, according to a press release from the manufacturers, in a Phase IIa study in the latter.1 Several studies have demonstrated that CB1 receptor expression, at the mRNA or protein level, is increased after middle cerebral artery occlusion (MCAO) in male animals, although the size of the response is dependent upon the time interval studied, as well as the temperature used during the lesion (Jin et al., 2000; Hayakawa et al., 2007; Zhang et al., 2008; see also Amantea et al., 2007). However, these studies focussed upon early (≤3 days) changes, and it is not known whether there is a change in CB1 receptor expression or function at a later time point after MCAO. This is of importance, since a residually decreased CB1 receptor function might impact upon the ability of the animal to withstand a new ischaemic insult. In the present study, we have investigated CB1 receptor densities and function in coronal brain sections from female ovariectomized rats obtained 46 days

1 http://www.ema.europa.eu/docs/en_GB/document_library/ Orphan_designation/2009/10/WC500005297.pdf; http://www. keyneurotek.de/englisch/pdf/KN387271001%20Auswertung_engl_ final.pdf.

after MCAO. The animals had been supplemented with either 17ß-oestradiol or placebo pellets 6 h after the MCAO, and had thereafter been housed in either isolated or enriched environments. These procedures were found to improve cognitive recovery after the ischaemic insult without impacting the size of the damaged area after MCAO (for the behavioural data for these animals, see Söderström et al., 2009), and thus provide an excellent opportunity to determine if these changes in brain plasticity associated with cognitive responses are mirrored by changes in CB1 receptor expression and function.

2.

Results

2.1.

Infarct volumes and plasma 17ß-oestradiol levels

Infarct volumes and plasma 17ß-oestradiol levels for the animals used in this study have been reported previously (Söderström et al., 2009), but are reproduced here to aid the reader. The infarct volumes on day 46 after MCAO were 9.3 ± 2.5 in enriched environment/17ß-oestradiol treated animals (“Enr/Est”); 12.2 ± 1.5 in isolated environment /17ß-oestradiol treated animals (“Iso/Est”); 11.8 ± 3.3 in enriched environment/ placebo treated animals (“Enr/Pl”); and 10.1 ± 1.7 in isolated environment/placebo treated animals (“Iso/Pl”), (means ± s.e.m., n = 7–8; data as % of the contralateral hemisphere). There was no significant difference between groups. Mean plasma 17ßoestradiol levels on day 46 after MCAO were <10 pg/ml in all groups. In a separate group of animals, the mean plasma 17ß-oestradiol levels increased to a mean peak of 597 pg/ml at 4 h after pellet administration. Levels thereafter declined to 215 pg/ml at 24 h and thereafter decreased to levels similar to those seen in placebo treated animals by 3 weeks (Söderström et al., 2009).

2.2.

[3H]CP55,940 receptor autoradiography

CB1 receptor expression in the coronal sections (3 per animal) was assessed autoradiographically using the agonist ligand [3H]CP55,940. Binding in the presence of an excess of nonradioactive WIN55,212-2, a CB receptor agonist, was used to define non-specific binding. An example of the total [3H] CP55,940 binding, together with an annotation of the regions assessed, is shown in Fig. 1A. Non-specific binding densities were very low (data not shown). The specific binding data for the six regions assessed are shown in Fig. 1B–G. For the parietal cortex, i.e. the region nearest the damaged tissue, care was taken to ensure that clearly affected ipsilateral areas were not included in the analysis. Two-way ANOVA measurements were conducted to assess the effects of the treatments of the animals after the MCAO (where the “treatment” parameter consists of four groups: Enr/Est, Iso/Est, Enr/Pl and Iso/Pl) and where the second arm of the test refers to the “side” (i.e. ipsi- vs. contralateral). Since the ipsi- and contralateral measurements were from the same animals, two-way ANOVA with repeated measures for “side” were used. There was no significant effect of “treatment” in any of the regions tested (p > 0.1 in all cases), and there was no interaction of treatment × side (p > 0.1 in all cases). However, a significant

BR A I N R ES E A RC H 1 3 7 3 ( 2 01 1 ) 1 9 5 –2 01

197

Fig. 1 – CB1 receptor densities as assessed by [3H]CP55,940 autoradiography in the brain of 17ß-oestrogen (Est) and placebo (Pl) treated rats housed in either enriched (Enr) or isolated (Iso) environments following MCAO. Panel A shows an example of an autoradiograph of the total binding for a coronal section from an Iso/Pl animal where the regions quantified in panels B-G are indicated, and where the extent of the damage is shown as a dotted line. The autoradiograph for the non-specific binding was barely visible (not shown). In Panels B-G, the data are specific binding and are means and s.e.m., n = 4–6. Significance was assessed by two-way ANOVA determinations with repeated measures for “side” (ipsi- vs. contralateral). The p values for the “side” are shown in the Figures. In no case were the p values for either treatment or treatment × side significant (p > 0.1 in all cases). *p < 0.05, **p < 0.01, two-tailed one-sample t-test vs 100% for the ipsilateral values expressed as % of the corresponding contralateral values.

effect on the hemispheric side was seen in five out of six regions, the dentate gyrus being the exception (Fig. 1B–G). In order to assess the magnitude of this effect for the different treatments, the ipsilateral values were expressed as % of the corresponding contralateral densities for the regions where a significant ANOVA was seen. For the Iso/Pl group, mean values of 85 ± 5*%, 73 ± 7*%, 94 ± 2*%, 83 ± 6*% and 83 ± 5*% was found for the frontal cortex, parietal cortex, hippocampal CA1–CA3 regions, thalamus and hypothalamus, respectively (means ± s.e.m., n = 6–7; *indicates that the upper 95% confidence interval of the mean <100%). The loss of binding was in general slightly less marked for the other treatments. Thus, for example, for the Iso/Est group, the values for the ipsilateral specific binding as % of the contralateral binding were 94 ± 4, 79 ± 4*, 98 ± 3, 93 ± 3 and 88 ± 5* for the frontal cortex, parietal cortex, hippocampal CA1–CA3 region, thalamus and hypothalamus, respectively (mean ± s.e.m., n = 7–8).

2.3.

[35S]GTPγS autoradiography

CB1 receptor functionality in the coronal sections (3 per animal) was assessed autoradiographically by measuring basal and agonist-stimulated [35S]GTPγS binding. Examples of [35S]GTPγS autoradiographies from coronal slices from the same rat are shown in Fig. 2A. Basal, CP55,940-stimulated and AM251 + CP55,940 autoradiographic densities were quantitated for all

six regions. The difference between basal and CP55,940stimulated [35S]GTPγS binding (“stimulation above basal”) was thereafter determined. Only samples where data were obtained for both hemispheric sides were included in the analyses. The stimulation above the basal level for the thalamic and hypothalamic regions was not sufficiently robust, since in both cases only 1/4 (thalamus) and 2/4 (hypothalamus) of the contralateral treatment groups showed a significant stimulation by CP55,940 (two-tailed one sample t-test vs. a hypothetical value of 0). In consequence, these regions were not further analysed. In the other four regions (Fig. 2B–E), 10 μM CP55,940 elicited a robust increase in binding (in all cases, p < 0.05 except for the contralateral parietal cortex for the Iso/Pl group [p = 0.075], two-tailed one sample t-test vs. a hypothetical value of 0), and the increase was not seen in the presence of the CB1 receptor antagonist/inverse agonist AM251 (10 μM). Thus, for example, in the frontal cortex of the Iso/Pl animals, the basal, CP55,940, AM251+ CP55,940 and stimulation above basal [35S] GTPγS autoradiographic binding densities (nCi/g) for ipsi/ contralateral sides were: 109 ± 29/105 ± 29, 225 ± 40/215 ± 38, 114 ± 26/99 ± 22, and 116 ± 21/110 ± 22, respectively (means ± s.e.m., n = 7). There were no significant differences in the stimulation above basal values between either treatment groups (p > 0.6) or between the ipsi- and contralateral sides for any of the treatment paradigms or brain regions investigated (Fig. 2B–E).

198

3.

BR A I N R ES E A RC H 1 3 7 3 ( 2 01 1 ) 1 9 5 –20 1

Discussion

It is well established that patients who have suffered an ischaemic stroke have a high risk of hospitalisation due to a repeated attack (Roberts et al., 2009). There are a number of factors that play a role in contributing to this distressing statistic, but a long-term loss of CB1 receptors in the brain could be of importance given the protective role played by these receptors in certain forms of ischaemia (ParmentierBatteur et al., 2002; review, see Fowler et al., 2010). Long-term changes in CB1 receptor densities and functionality following an ischaemic insult have not previously been investigated. Therefore, in the present study, we have investigated specific [3H]CP55,940 binding and CP55,940-stimulated [35S]GTPγS binding in female ovariectomised rats 46 days after MCAO. The animals were treated with either 17ß-oestradiol or placebo pellets 6 h after MCAO, and thereafter housed in either isolated or enriched environments. These treatments affect the recovery of cognitive function, but not the infarct size (Söderström et al., 2009), in contrast to studies where 17ßoestradiol is given prior to the neuronal insult, where a decreased infarct size is seen (see e.g. Amantea et al., 2007).

Autoradiographic measurement of specific [3H]CP55,940 binding indicated that with the exception of the dentate gyrus there was a modest, but significant loss of CB1 receptors on the ipsilateral side relative to the contralateral side. Although the effect was most pronounced in the placebo-treated animals housed in individual cages, two-way ANOVA indicated that there was no significant effect of treatment in any of the brain regions investigated, indicating that the altered plasticity produced by 17ß-oestradiol and housing in an enriched environment, which results in an improved cognitive recovery after MCAO (Söderström et al., 2009), is not accompanied by a changed expression of CB1 receptors. There are reports in the literature that brain CB1 receptor expression and functionality is regulated both acutely and in the long-term by oestrogen levels (Rodríguez de Fonseca et al., 1994; Mize and Alper, 2000; Amantea et al., 2007; Riebe et al., 2010). In general most of these studies have investigated short time-frames after acute or repeated oestrogen administration and therefore are not strictly comparable to the present study. In our study, the increased plasma 17ß-oestradiol levels seen at 4 h after pellet implantation returned to baseline levels after 3 weeks (Söderström et al., 2009), i.e. 3 weeks before measurement of CB1 receptor densities.

Fig. 2 – CB1 receptor functionality after MCAO. In panel A, examples of basal and 10 μM CP55,940-stimulated [35S]GTPγS autoradiography are shown for an Iso/Pl animal together with the corresponding autoradiographs for slices incubated with 10 μM AM251 + 10 μM CP55,940 and with 10 μM GTPγS (non-specific binding). In Panels B to E, the whole columns (with upward-pointing s.e.m. bars, n = 5–8) represent the [35S]GTPγS autoradiographic densities for the 10 μM CP55,940-stimulated slices, after subtraction of non-specific binding. The lighter shaded columns (with downward-pointing s.e.m. bars, n = 5–8) enclosed within these columns represent the basal values, after subtraction of non-specific binding. Thus, the part of each column above the basal columns represents the mean difference between basal and stimulated [35S]GTPγS (“stimulation above basal”). Two-way ANOVAs with repeated measures for “side” (ipsi- vs. contralateral) were conduced for the individual stimulation above basal values and the p values for the “side” are shown in the figures. In no case were the p values for either treatment or treatment × side significant (p > 0.2 in all cases).

BR A I N R ES E A RC H 1 3 7 3 ( 2 01 1 ) 1 9 5 –2 01

Whilst the [3H]CP55,940 autoradiography data indicate a long-term disturbance in CB1 receptor expression following MCAO, they do not give any information as to whether this is a homogeneous or heterogeneous effect within a given region. A selective loss of CB1 receptors located on GABAergic interneurons, for example, would be expected to result in a loss of endocannabinoid retrograde control of inhibitory signalling, whereas a selective receptor loss on glutamatergic nerve terminals would produce the opposite result. Such effects of neuronal insult on the different nerve populations have been invoked to explain data showing damaging, rather than protective, effects of the endocannabinoid system in the brain (reviews, see Hillard, 2008; Pellegrini-Giampietro et al., 2009; Fowler et al., 2010). However, a decreased receptor expression, regardless of its cellular localisation, may not affect functionality at all, if the brain region in question shows a large receptor reserve. Such a receptor reserve has been demonstrated for hippocampal CB1 receptors (Gifford et al., 1999; Breivogel and Childers, 2000). Further, an unchanged receptor expression may not reflect a changed functionality if the coupling of the receptor in question to its G-protein is disturbed. This has been shown to occur for frontal cortical CB1 receptors in Alzheimer's disease (Ramírez et al., 2005). We therefore studied the functionality of the CB1 receptors in the coronal slices by measuring the stimulation of [35S]GTPγS binding by the agonist CP55,940. In the four regions where robust stimulation was seen, we found no significant influence upon the increase over basal binding elicited by CP55,940 of either the treatment group or the hemispheric side investigated. This would indicate that the relatively modest changes in CB1 receptor density seen 46 days after MCAO are not sufficient to elicit a functional deficit on the ipsilateral side. In conclusion, the present investigation has indicated that MCAO results in a decreased expression of CB1 receptors at a late time point after the cerebral insult, but that this decreased expression does not give a deficit in receptor functionality, presumably due to the presence of a large CB1 receptor reserve. Thus, these data suggest that a permanent deficit in CB1 receptor function is not likely to be involved in the recurrence after an ischaemic stroke at least not in this experimental animal model of MCAO. Our study illustrates the importance of complementing measurements of receptor density with corresponding measurements of receptor function, particularly when a receptor reserve is present.

4.

Experimental procedures

4.1.

Animals and tissue collection

The animals, experimental procedures and outcomes used (evaluation of infarct volumes, sensorimotor function and cognitive function) have been described previously (Söderström et al., 2009). Briefly, 6- to 7-week-old female Sprague– Dawley rats were acclimatised and thereafter accustomed to handling before being ovariectomised bilaterally under Hypodorm/Dormicum anaesthesia. One week later, MCAO was undertaken on anaesthetised animals using a modified

199

procedure of Longa et al. (1989), with the animals being temperature controlled at 37 °C. In this procedure, the right common carotid artery was exposed, the internal carotid artery was isolated. The middle cerebral artery was then occluded for 90 min using a monofilament nylon suture coated with poly-L-lysine. Six hours after the end of ischaemia, the animals received subcutaneous implants into the neck of either placebo (“Pl”) or 17ß-oestradiol (“Est”, 0.075 mg 60 day release pellets, Innovative Research of America, Sarasota, FL, USA). The animals were then housed either singly in a standard cage (425 × 266 × 185 mm) (“Iso”) or in groups (n = 7–8) in large cages (820 × 610 × 450 cm) containing ladders, wooden tunnels, swings and chains, and elevated horizontal and inclined boards (enriched environment, “Enr”). The animals were killed by decapitation between 08:00 and 09:00 h on day 46 after MCAO, and their brains were collected and immediately frozen at −80 °C. Cryostat coronal sections (10 μm) were then made and mounted on slides for determination of infarct volume (Söderström et al., 2009) and for the autoradiographic studies described here. The experimental procedures were in accordance with European and Swedish legislation, and the experimental protocol was approved by the local animal ethical committee.

4.2. Autoradiographic measurement of CB1 receptor density and functionality CB1 receptor density in the coronal sections was measured using [ 3 H]CP55,940 (specific activity 174.6 Ci.mmol − 1 , Perkin Elmer, Waltham MA, USA) as ligand using the method of Glass et al. (1997) as modified by Mato and Pazos (2004). Briefly, the sections were preincubated in 50 mM Tris HCl buffer, pH 7.4. containing 5% bovine serum albumin (BSA), and then incubated with 3 nM of ligand for 2 h at 37 °C in the same buffer. Non-specific binding was assessed using 10 μM WIN55,212-2 (Tocris Bioscience, Ellisville, MO, USA). After incubation, the sections were washed (2 h, 4 °C, 50 mM TrisHCl buffer, pH 7.4, containing 1% BSA) twice and thereafter dipped briefly in (ice-cold/4 °C) MQ water. After the sections were dried, they were placed in cassettes containing tritium microscale standards (American Radiolabeled Chemicals inc, St. Louis, Mo, USA) and Kodak film (BioMax MR Film, SigmaAldrich Corp, St. Louis, MO USA) and the films were allowed to develop over 3 weeks at 4 °C. CB1 receptor functionality was assessed in the coronal sections by measurement of agonist-stimulated [35S]GTPγS autoradiography using the method of Sim et al. (1996) with the modifications described in Rodríguez-Gaztelumendi et al. (2009). Briefly, the sections were preincubated in “preincubation buffer” (100 mM NaCl, 2 mM GDP, 3 mM MgCl2, 1 mM dithiothreitol, 0.2 mM EGTA, 0.5% BSA and 50 mM Tris HCl, pH 7.7) for 30 min at room temperature and thereafter incubated for 2 h at 25 °C in preincubation buffer containing 10 mU/ml adenosine deaminase and 0.04 nM [35S]GTPγS (nominal specific activity 1250 Ci/mmol, Perkin Elmer) in the absence (basal) and presence of either 10 μM CP55,940 (Tocris Biosciences) to assess agonist-stimulated binding or 10 μM CP55,940 + 10 μM AM250 (Tocris Biosciences) to confirm that the stimulation was mediated by CB1 receptors. Non-specific binding was defined using 10 μM GTPγS. Following incubation,

200

BR A I N R ES E A RC H 1 3 7 3 ( 2 01 1 ) 1 9 5 –20 1

the slices were washed twice in cold 50 mM Tris HCl buffer, pH 7.4 containing 0.1% BSA, dipped in cold MQ water and placed in cassettes with Kodak film (BioMax MR Film) and [14C] microscale standards (GE Healthcare Biosciences, Piscataway, NJ, USA) and allowed to develop for 48 h at 4 °C.

4.3.

Analysis of data

Data analysis was undertaken as described in RodríguezGaztelumendi et al. (2009), whereby the developed films were digitised and the autoradiographic densities were determined by densitometry using the Scion Imaging software (Fredrick, MD, USA) and with the autoradiographic microscales to calibrate the signals. The individual values reported here are means of determinations from three coronal sections per animal. Statistical determinations were undertaken using the statistical package built into the GraphPad Prism computer programme (GraphPad Software Inc, San Diego, CA, USA).

Acknowledgments The authors would like to thank Prof. Tommy Olsson for his useful advice and comments on the manuscript. MLR is a Marie Curie Research Fellow (Contract no. MTKD-CT-2006039039, under the FP6 Transfer of Knowledge scheme). CJF would like to thank the Swedish Research Council (grant no. 12158) and the Research Funds of the Medical Faculty, Umeå University for research support. IS thanks the Swedish Stroke Foundation, Gamla Tjänarinnor Foundation, Åke Wibergs Foundation, King Gustaf V/Queen Victoria Foundation, Stohnes Foundation, Swedish Medical Research Council, and Faculty of Medicine of Umeå University for research support.

REFERENCES

Amantea, D., Spagnuolo, P., Bari, M., Fezza, F., Mazzei, C., Tassorelli, C., Morrone, L., Corosaniti, M., Maccarrone, M., Bagetta, G., 2007. Modulation of the endocannabinoid system by focal brain ischemia in the rat is involved in neuroprotection afforded by 17β-estradiol. FEBS J. 274, 4464–4475. Bisogno, T., Di Marzo, V., 2007. Short- and long-term plasticity of the endocannabinoid system in neuropsychiatric and neurological disorders. Pharmacol. Res. 56, 428–442. Breivogel, C., Childers, S., 2000. Cannabinoid agonist signal transduction in rat brain: comparison of cannabinoid agonists in receptor binding, G-protein activation, and adenylyl cyclase inhibition. J. Pharmacol. Exp. Ther. 295, 328–336. Breivogel, C.S., Sim-Selley, L.J., 2009. Basic neuroanatomy and neuropharmacology of cannabinoids. Int. Rev. Psychiatry 21, 113–121. Fowler, C.J., Rojo, M.L., Rodriguez-Gaztelumendi, A., 2010. Modulation of the endocannabinoid system: neuroprotection or neurotoxicity? Exp. Neurol. 224, 37–47. Gifford, A., Bruneus, M., Gatley, S., Lan, R., Makriyannis, A., Volkow, N., 1999. Large receptor reserve for cannabinoid actions in the central nervous system. J. Pharmacol. Exp. Ther. 288, 478–483.

Glass, M., Dragnow, M., Faull, R., 1997. Cannabinoid receptors in the human brain: a detailed anatomical and quantitative autoradiographic study in the fetal, neonatal and adult human brain. Neuroscience 77, 299–318. Glass, M., Dragunow, M., Faull, R.L.M., 2000. The pattern of neurodegeneration in Huntington's disease: a comparative study of cannabinoid, dopamine, adenosine and GABAA receptor alterations in the human basal ganglia in Huntington's disease. Neuroscience 97, 505–519. Glass, M., van Dellen, A., Blakemore, C., Hannan, A.J., Faull, R.L.M., 2004. Delayed onset of Huntington's disease in mice in an enriched environment correlates with delayed loss of cannabinoid CB1 receptors. Neuroscience 123, 207–212. Hansen, H.H., Schmid, P.C., Bittigau, P., Lastres-Becker, I., Berrendero, F., Manzanares, J., Ikonomidou, C., Schmid, H.H.O., Fernández-Ruiz, J.J., Hansen, H.S., 2001. Anandamide, but not 2-arachidonoylglycerol, accumulates during in vivo neurodegeneration. J. Neurochem. 78, 1415–1427. Hayakawa, K., Mishima, K., Nozako, M., Hazekawa, M., Ogata, A., Fujioka, M., Harada, K., Mishima, S., Orito, K., Egashira, N., Iwasaki, K., Fujiwara, M., 2007. Δ9-tetrahydrocannabinol (Δ9-THC) prevents cerebral infarction via hypothalamic-independent hypothermia. Life Sci. 80, 1466–1471. Hillard, C.J., 2008. Role of cannabinoids and endocannabinoids in cerebral ischemia. Curr. Pharm. Des. 14, 2347–2361. Jin, K.L., Mao, X.O., Goldsmith, P.C., Greenberg, D.A., 2000. CB1 cannabinoid receptor induction in experimental stroke. Ann. Neurol. 48, 257–261. Longa, E.Z., Weinstein, P.R., Carlson, S., Cummins, R., 1989. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20, 84–91. Mato, S., Pazos, A., 2004. Influence of age, postmortem delay and freezing storage period on cannabinoid receptor density and functionality in human brain. Neuropharmacology 46, 716–726. Mauler, F., Hinz, V., Augstein, K.-H., Faßbender, M., Horváth, E., 2003. Neuroprotective and brain edema-reducing efficacy of the novel cannabinoid receptor agonist BAY 38-7271. Brain Res. 989, 99–111. Mize, A.L., Alper, R.H., 2000. Acute and long-term effects of 17ß-estradiol on Gi/o coupled neurotransmitter receptor function in the female rat brain as assessed by agonist-stimulated [35S]GTPγS binding. Brain Res. 859, 326–333. Nagayama, T., Sinor, A.D., Simon, R.P., Chen, J., Graham, S.H., Jin, K., Greenberg, D.A., 1999. Cannabinoids and neuroprotection in global and focal cerebral ischemia and in neuronal cultures. J. Neurosci. 19, 2987–2995. Panikashvili, D., Simeonidou, C., Ben-Shabat, S., Hanuš, L., Breuer, A., Mechoulam, R., Shohami, E., 2001. An endogenous cannabinoid (2-AG) is neuroprotective after brain injury. Nature 413, 527–531. Parmentier-Batteur, S., Jin, K., Mao, X.O., Xie, L., Greenberg, D.A., 2002. Increased severity of stroke in CB1 cannabinoid receptor knock-out mice. J. Neurosci. 22, 9771–9775. Pellegrini-Giampietro, D.E., Mannaioni, G., Bagetta, G., 2009. Post-ischemic brain damage: the endocannabinoid system in the mechanisms of neuronal death. FEBS J. 276, 2–12. Ramírez, B.G., Blazquez, C., Gómez del Pulgar, T., Guzmán, M., de Ceballos, M.L., 2005. Prevention of Alzheimer's disease pathology by cannabinoids: neuroprotection mediated by blockade of microglial activation. J. Neurosci. 25, 1904–1913. Riebe, C.J.N., Hill, M.N., Lee, T.T.Y., Hillard, C.J., Gorzalka, B.B., 2010. Estrogenic regulation of limbic cannabinoid receptor binding. Psychoneuroendocrinology 35, 1265–1269. Roberts, C.S., Gorelick, P.B., Ye, X., Harley, C., Goldberg, G.A., 2009. Additional stroke-related and non-stroke-related cardiovascular costs and hospitalizations in managed-care patients after ischemic stroke. Stroke 40, 1425–1432.

BR A I N R ES E A RC H 1 3 7 3 ( 2 01 1 ) 1 9 5 –2 01

Rodríguez de Fonseca, F., Cebeira, M., Ramos, J.A., Martín, M., Fernández-Ruiz, J.J., 1994. Cannabinoid receptors in rat brain areas: sexual differences, fluctuations during estrous cycle and changes after gonadectomy and sex steroid replacement. Life Sci. 54, 159–170. Rodríguez-Gaztelumendi, A., Rojo, M., Pazos, A., Díaz, A., 2009. Altered CB1 receptor-signaling in prefrontal cortex from an animal model of depression is reversed by chronic fluoxetine. J. Neurochem. 108, 1423–1433. Sim, L.J., Hampson, R.E., Deadwyler, S.A., Childers, S.R., 1996. Effects of chronic treatment with Δ9-tetrahydrocannabinol on

201

cannabinoid-stimulated [35S]GTPγS autoradiography in rat brain. J. Neurosci. 16, 8057–8066. Söderström, I., Strand, M., Ingridsson, A.-C., Nasic, S., Olsson, T., 2009. 17ß-estradiol and enriched environment accelerate cognitive recovery after focal brain ischemia. Eur. J. Neurosci. 29, 1215–1224. Zhang, M., Martin, B.R., Adler, M.W., Razdan, R.K., Ganea, D., Tuma, R.F., 2008. Modulation of the balance between cannabinoid CB1 and CB2 receptor activation during cerebral ischemic/reperfusion injury. Neuroscience 152, 753–760.