Effect of Δ9-tetrahydrocannabinol on phosphorylated CREB in rat cerebellum: An immunohistochemical study

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<ul><li><p>rch</p><p>nol</p><p>u</p><p>ann</p><p>derful friend and scientist who died in September 2003.</p><p>D9-THC exposure. This might be a mechanism by which D9-THC interferes with motor and cognitive functions.</p><p>evidence has also implicated the cerebellum in diverse higher</p><p>ents with cerebellar</p><p>Brain Research 1048 (200D 2005 Elsevier B.V. All rights reserved.</p><p>Theme: Neural basis of behavior</p><p>Topic: Drugs of abuse: opioids and others</p><p>Keywords: Cannabinoids; CREB; Cerebellum; Immunohistochemistry</p><p>1. Introduction</p><p>Cannabinoid receptors CB1 are expressed at a very high</p><p>density in the cerebellum, an area of the brain implicated in</p><p>motor coordination, so it is not surprising that in humans</p><p>cannabinoids such as D9-tetrahydrocannabinol (D9-THC),the principal psychoactive component of marijuana, have</p><p>complex effects on psychomotor function. In mice, direct</p><p>injection of synthetic cannabinoids into the cerebellum</p><p>produces motor impairments in the rotorod test, and these</p><p>deficits are no longer seen in animals that have cerebellar</p><p>injections of an antisense oligonucleotide directed to a</p><p>sequence coding for CB1 receptors [11]. Although it has</p><p>long been known in clinical neurology as much as in</p><p>experimental neuroscience that the cerebellum is essential</p><p>for the co-ordination of movement, a growing body ofHer work on CREB and cannabinoids was the basis of our study.</p><p>Abstract</p><p>Several converging lines of evidence indicate that drugs of abuse may exert their long-term effects on the central nervous system by</p><p>modulating signaling pathways controlling gene expression. Cannabinoids produce, beside locomotor effects, cognitive impairment through</p><p>central CB1 cannabinoid receptors. Data clearly indicate that the cerebellum, an area enriched with CB1 receptors, has a role not only in</p><p>motor function but also in cognition. This immunohistochemical study examines the effect of D9-tetrahydrocannabinol (D9-THC), theprincipal psychoactive component of marijuana, on the levels of phosphorylated CREB (p-CREB) in the rat cerebellum. Acute treatments</p><p>with D9-THC at doses of 5 or 10 mg/kg induced a significant increase of p-CREB in the granule cell layer of the cerebellum, an effectblocked by the CB1 receptor antagonist SR 141716A. Following chronic D9-THC administration (10 mg/kg/day for 4 weeks), the density ofp-CREB was markedly attenuated compared to controls, and this attenuation persisted 3 weeks after withdrawal from D9-THC.</p><p>These data provide evidence for the involvement of cerebellar granule cells in the adaptive changes occurring during acute and chronicDedicated to the memory of Anna Porcella, a wonRaymond Mongeau , Luca Pani</p><p>aNeuroscienze PharmaNess S.C.A.R.L., Via Palabanda, 9 09125 Cagliari, ItalybInstitute of Neurogenetics and Neuropharmacology, C.N.R., Cagliari, Italy</p><p>Accepted 13 April 2005</p><p>Available online 23 May 2005Resea</p><p>Effect of D9-tetrahydrocannabirat cerebellum: An imm</p><p>Maria Antonietta Casua,*, Carla Pisua, Angela S0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.</p><p>doi:10.1016/j.brainres.2005.04.053</p><p>* Corresponding author. Fax: +39 70 924 2206.</p><p>E-mail address: mantonietta.casu@pharmaness.it (M.A. Casu).report</p><p>on phosphorylated CREB in</p><p>nohistochemical study</p><p>aa, Simone Tambaroa, Gabriele Pinna Spadaa,a a,b</p><p>5) 41 47</p><p>www.elsevier.com/locate/brainrescognitive functions. For example, patidiseases have impaired spatial cognition, executive dysfunc-</p><p>tions with difficulties in planning, abstraction and working</p></li><li><p>immunohistochemistry. For the immunohistochemistry,</p><p>controls were performed by subtracting the primary</p><p>Reseamemory [29]. These observations raise the possibility that the</p><p>cerebellar mechanisms implicated in learning and memory</p><p>might also be relevant in the mechanism of action of</p><p>cannabinoids. The intake of marijuana induces, beside</p><p>locomotor effects such as hypolocomotion, ataxia and</p><p>catalepsy, clear cognitive impairments [32]. Prolonged</p><p>marijuana usage disrupts short-term memory, working</p><p>memory, attentional skills and memory retrieval [4]. Admin-</p><p>istration of cannabis extracts also causes long-lasting</p><p>memory deficits in rodents, and similar deficits are produced</p><p>by either D9-THC, endogenous cannabinoids or differentsynthetic CB1 cannabinoid receptor agonists [6,18,19,21,31].</p><p>Several of the molecular and cellular adaptations involved</p><p>in addiction are believed to be also implicated in learning and</p><p>memory. Of particular interest is the activation of the cAMP</p><p>pathway and CREB-mediated transcription which have been</p><p>related to learning and long-term potentiation of synaptic</p><p>transmission [35]. Numerous CNS processes, including</p><p>neurotransmitter synthesis, gene expression and cellular</p><p>proliferation, are controlled by neurotransmitters acting</p><p>through second messenger systems that phosphorylate the</p><p>transcription factor CREB (cyclic AMP response element-</p><p>binding protein). CREB is an ubiquitously expressed protein</p><p>regulated by several intracellular pathways that binds to</p><p>specific DNA sequences (named CREs or cAMP-response</p><p>elements) in the regulatory regions of target genes [24]. The</p><p>transcriptional activity of these dimers is stimulated upon</p><p>phosphorylation of CREB at Ser133 by several protein</p><p>kinases, including protein kinase A, Ca 2+/calmodulin-kinase</p><p>II and IVand several kinases in themitogen-activated protein-</p><p>kinase cascade (MAPK). Thus, CREB represents a site of</p><p>convergence where diverse signaling pathways and their</p><p>associated stimuli produce plasticity by altering gene</p><p>expression [30].</p><p>Cannabinoids may exert their effects on brain and</p><p>behavior by modulating signaling pathways controlling</p><p>gene expression. Accordingly, studies have demonstrated</p><p>that D9-THC induces the expression of the immediate-earlygene c-fos [20]. Furthermore, acute administration of D9-THC in rats induces a progressive and transient activation of</p><p>extracellular signal-regulated kinase (ERK) in the dorsal</p><p>striatum, nucleus accumbens and hippocampus [10,33]. It</p><p>has recently been reported that acute D9-THC increasescomponents of the ERK pathway (ERK, p-CREB and c-fos)</p><p>in the rat cerebellum, while repeated treatment with D9-THCprevents this acute effect of D9-THC [28].</p><p>Given that learning, memory and drug addiction share</p><p>some intracellular signaling cascade events dependent on</p><p>the activation of CREB [25] and the relationships existing</p><p>between cognitive deficits, the cerebellum and the adverse</p><p>effects of cannabinoids, it was important to better under-</p><p>stand the effect of D9-THC on CREB in the rat cerebellum.It was still unknown where in the cerebellum an acute D9-THC-induced response might be displayed. Furthermore, it</p><p>9</p><p>M.A. Casu et al. / Brain42was crucial to know whether chronic D -THC would alterlevels of activated CREB a day or several weeks after theantibody in the procedure. The staining was performed</p><p>as previously reported [8]: after rinsing in phosphate-</p><p>buffered saline with 0.2% Triton X-100 (PBS + T),</p><p>sections were incubated with 0.3% of H2O2 in PBS and,</p><p>after extensive washing, with a blocking solution contain-last administration of D9-THC. To this aim, we performedan immunohistochemical study on the cerebellum of acutely</p><p>or chronically treated rats using an antibody raised against</p><p>phosphorylated CREB (p-CREB), the activated form that</p><p>binds to DNA.</p><p>2. Materials and methods</p><p>2.1. Animals</p><p>Adult male SpragueDawley albino rats (Charles River,</p><p>Italy) were housed in groups of 5 in standard plastic cages</p><p>with water and food ad libitum. The animal facility was</p><p>under a 12:12 h lightdark cycle, constant temperature of</p><p>22 T 2 -C and relative humidity of 60%. All experimentalprotocols were performed in strict accordance with the E.C.</p><p>regulation for care and use of experimental animals (CEE</p><p>No. 86/609).</p><p>The appropriate concentration of D9-THC (purchased asa 10 mg/ml in ethanol solution, from Sigma, St. Louis,</p><p>Missouri, USA) was prepared by evaporating the alcohol</p><p>with nitrogen and emulsifying the residue with Cremophor,</p><p>ethanol and saline (1:1:18). For the acute treatment, rats</p><p>received vehicle (n = 10) or D9-THC at the doses of 2.5 (n =10), 5 (n = 10) and 10 mg/kg i.p. (n = 10) and were</p><p>sacrificed 90 min after for immunohistochemistry. A</p><p>separate group of rats (n = 5) received an acute injection</p><p>of SR 141716A (1 mg/kg) 15 min before the treatment with</p><p>D9-THC or its vehicle. For chronic treatments, rats (n = 12)were injected intraperitoneally with D9-THC once a day for4 weeks at a dose of 10 mg/kg and were sacrificed 12 h (n =</p><p>6) or 3 weeks after (n = 6). Control animals (n = 6) were</p><p>given vehicle for the same time.</p><p>D9-THC-treated animals presented typical behavioralcannabinoid effects such as a reduction in spontaneous</p><p>locomotor activity.</p><p>2.2. Immunohistochemistry</p><p>Rats were perfused transcardially 90 min after D9-THCtreatment with 4% paraformaldehyde in 0.2 M phosphate</p><p>buffer (PB). The brains were subsequently cryoprotected</p><p>overnight with a solution of 30% sucrose in 0.1 M PB at 4</p><p>-C. Alternate sagittal sections of 40 Am were cut at sledge(Microm HM 400 R). Adjacent sections were processed</p><p>for Nissl staining (cresyl violet from Sigma) or p-CREB</p><p>rch 1048 (2005) 4147ing 1% BSA and 20% normal goat serum in PBS + T to</p><p>reduce background.</p></li><li><p>found in the granular layer of the paramedian lobule, which</p><p>contains numerous densely packed small granule cell</p><p>ReseaSections were incubated overnight at 4 -C with a rabbitanti-P-CREB antibody (1:500; Cell Signaling Technology).</p><p>After rinsing, sections were incubated with an anti-rabbit</p><p>biotinylated IgG (1:200; Vector, Burlingame, CA, USA) for</p><p>1 h followed by an avidinbiotin complex (1:400;</p><p>Vectastain ABC kit, Vector) for an additional hour. After</p><p>washing, sections were exposed to 3,3V-diaminobenzidine(0.06% in PBS) containing 1% cobalt chloride and 1%</p><p>nickel ammonium sulfate for 15 min. Immunostaining was</p><p>developed by adding 5 Al H2O2 (0.1% in PBS) to each 500Al of 3,3V-diaminobenzidine. After washing in PBS + T, allsections were mounted on gelatin-coated glass slides, air-</p><p>dried, dehydrated in ascending concentrations of ethanol,</p><p>cleared with xylene and coverslipped with Entellan. The</p><p>mounted sections were examined under a BX-60 Olympus</p><p>light-microscope (Olympus Optical, GmbH, Hamburg,</p><p>Germany) at 20 and 40.</p><p>2.3. Quantitative analysis</p><p>The analysis was performed in the granular layer of the</p><p>cerebellar cortex between bregma coordinates 0 and 3.4 mm</p><p>away from the midline [26] corresponding to the para-</p><p>median lobule as the most uniform staining was found in</p><p>this area. The analysis was carried out using an image</p><p>analysis system (KS 300; Karl Zeiss Vision GmbH,</p><p>Hallbergmoos, Germany) by choosing for each section</p><p>three randomly chosen fields, in at least ten alternate</p><p>sections for each animal, and by measuring the percentage</p><p>of the area occupied by p-CREB staining with respect to a</p><p>2000 Am2 standardized area (% IM area percentage). Grayvalues, measured in immunostaining negative areas of the</p><p>sections, were subtracted as background from the resulting</p><p>binary picture. All data were expressed as mean T SEM andanalyzed using a one-way analysis of variance (ANOVA).</p><p>When a significant interaction (P &lt; 0.05) was found, the</p><p>NewmanKeuls post-hoc test was used.</p><p>2.4. Protein extraction and Western blot analysis</p><p>Rats were killed, and the cerebella were rapidly removed,</p><p>dissected and placed on an ice-cold plate. Total cell protein</p><p>extract and Western blot were performed as previously</p><p>described with some modifications [9]. In brief, tissue</p><p>samples were homogenized at 4 -C by an homogenizersystem (GlasCol, Terre Hante, IN, USA) in 100 Al of 20mM HEPES buffer (pH 7.9) containing in mM: NaCl, 125,</p><p>MgCl2, 5; glycerol, 12%; ethylenediaminetetracetic acid</p><p>(EDTA), 0.2; Nonidet P-40, 0.1%; dithithreitol (DTT), 5;</p><p>phenilmethylsulphonil fluoride (PMSF), 0.5; leupeptin, 0.5</p><p>Ag/ml; and pepstatin A 0.7 Ag/ml. The extracts were thencentrifuged at 15,000g (at 4 -C) for 20 min, and theresulting supernatant was collected as total cell extracts. An</p><p>aliquot was analyzed for protein concentration by using a</p><p>M.A. Casu et al. / BrainProtein assay kit II (Bio-Rad Laboratories, Hercules, CA,</p><p>USA), and the rest was frozen at 80 -C until assayed.neurons, while very few stained cells were found in the</p><p>molecular layer. No positive staining was present in the</p><p>omission control (without the primary antibody; Fig. 1C).</p><p>This staining typology was present in all lobules of the</p><p>cerebellar cortex. Adjacent sections were stained with Nissl</p><p>to examine the complete neuronal population, and based on</p><p>this analysis, it was clear that only background levels of p-</p><p>CREB-IM were found in areas such as the Purkinje cell</p><p>layer (Figs. 1DE). There were no apparent changes in the</p><p>distribution patterns in any of the acutely treated groups.</p><p>However, a marked increase of p-CREB-IM was observed</p><p>in the granular layer of rats acutely treated with D9-THC(Fig. 2). The administration of D9-THC at doses of 5 and 10mg/kg i.p. produced an almost 50% increase of p-CREB-IM</p><p>(one-way ANOVA F3,36 = 21.77, P &lt; 0.01). The lower dose</p><p>of D9-THC (2.5 mg/kg) did not modify the density of p-CREB-IM (Fig. 2E). The increase of p-CREB-IM after the</p><p>acute treatment with D9-THC was completely blocked bypretreatment with the CB1 receptor antagonist SR 141716A</p><p>(Fig. 2F), suggesting that it is mediated by CB1 receptorAliquots of cerebellar extracts containing 50 Ag of totalprotein were separated by sodium dodecyl sulfate-poly-</p><p>acrylamide gels (SDS PAGE) and transferred to nitrocellu-</p><p>lose membranes (Bio-Rad). Blots were blocked with 5%</p><p>nonfat dry milk in TBST (0.1% Tween 20 in Tris borate</p><p>saline) and probed with a specific antibody against Phospho</p><p>MAPK 1/2 (Cell signaling Technology, Beverly, MA) with a</p><p>1:1000 dilution in 1% BSA TBST. After washing in TBST,</p><p>blots were probed with a horseradish-peroxidase-conjugated</p><p>antibody with a 1:2000 dilution in TBST plus 5% milk and,</p><p>after washing in TBST, chemiluminescence was detected by</p><p>West Pico chemiluminescent substrate (Pierce, Rockford,</p><p>IL). Immunoreactive bands were visualized with a Fuji Las</p><p>1000 image analyzer (Raytest Isotopenmessgerate GmbH,</p><p>Straubenhartd, Germany), and the optical density of</p><p>immunoreactive bands was measured using a specific</p><p>software (AIDA 2.11, Raytest Isotopenmessgerate GmbH,</p><p>Straubenhartd, Germany). Students t test was performed as</p><p>a statistical analysis using GraphPad Prism program (San</p><p>Diego, CA, USA).</p><p>3. Results</p><p>The purpose of the present study was to examine whether</p><p>acute treatment (2.5, 5 and 10 mg/kg) or chronic treatment</p><p>(10 mg/kg/day 28 days) with D9-THC affects p-CREBimmunostaining (p-CREB-IM) in the rat cerebellum and, if</p><p>it does, to determine where in the cerebellar cortex this</p><p>effect of D9-THC occurs.As shown in Figs. 1AB, in the cerebellar cortex of</p><p>untreated rats, intense p-CREB immunoreactive cells were</p><p>rch 1048 (2005) 4147 43activation. Note that SR 141716A at a dose of 1 mg/kg did</p><p>not have any effect by itself on p-CREB-IM (Fig. 2F). Since</p></li><li><p>ellar c</p><p>atter</p><p>nular layer (C). Notice, at high magnification, the p-CREB positive granule cells</p><p>stained with Nissl (DE). Scale bars A...</p></li></ul>


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