Neuroscience Letters 591 (2015) 65–68
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
␣CaMKII autophosphorylation mediates neuronal activation in the hippocampal dentate gyrus after alcohol and cocaine in mice Isabella Schöpf a , Alanna C. Easton b , Jalal Solati c,d , Yulia Golub c , Johannes Kornhuber a , K. Peter Giese e , Christian P. Müller a,b,∗ a
Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany MRC Social, Genetic and Developmental Psychiatry Research Centre, Institute of Psychiatry, King’s College London, De Crespigny Park, London SE5 8AF, UK c Department of Child and Adolescent Mental Health, University Clinic Erlangen, Schwabachanlage 6 and 11, 91054 Erlangen, Germany d Department of Biology, Faculty of Science, Karaj branch, Islamic Azad University, Karaj, Iran e Centre for the Cellular Basis of Behavior, MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King’s College London, James Black Centre, 125 Coldharbour Lane, London SE5 8AF, UK b
h i g h l i g h t s • ˛CaMKII is invoved in the establishment of drug addiction. • Alcohol and cocaine increase c-fos expression in the dentate gyrus of mice. • No increase in c-fos expression in ␣CaMKII autophosphorylation deficient mice.
a r t i c l e
i n f o
Article history: Received 19 January 2015 Received in revised form 12 February 2015 Accepted 13 February 2015 Available online 17 February 2015 Keywords: Hippocampus ␣caMKIIT286A C-fos Immunohistochemistry
a b s t r a c t Psychoactive drug-induced cellular activation is a key mechanism to promote neuronal plasticity and addiction. Alpha Ca2+ /calmodulin-dependent protein kinase II (␣CaMKII) and its autophosphorylation play a key role in the development of drug use associated behaviours. It has been suggested that ␣CaMKII autophosphorylation is necessary for drug-induced neuronal activation in the mesolimbic system. Here, we show an alcohol- and cocaine-induced increase in c-fos expression in the hippocampal dentate gyrus, which is absent in ␣CaMKIIT286A autophosphorylation deficient mice. These findings may suggest a role in hippocampal ␣CaMKII autophosphorylation in the acute neuroplastic effects of alcohol and cocaine. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The systematic use of psychoactive drugs is a wide spread phenomenon on a world wide scale [19]. The establishment of drug use behaviours is a systematic process, which requires multiple types of learning and memory [15,18,24]. Drugs of abuse, like cocaine and alcohol, can develop addiction in a significant number of people by modifying neuronal circuits and synaptic transmission [23]. The alpha Ca2+ /calmodulin-dependent protein kinase II (␣CaMKII) plays a key role in the establishment of drug use associ-
∗ Corresponding author at: Section of Addiction Medicine, Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University Erlangen-Nuremberg, Schwabachanlage 6, Erlangen, 91054, Germany. Tel.: +49 9131 85 36896. E-mail address: [email protected] (C.P. Müller). http://dx.doi.org/10.1016/j.neulet.2015.02.031 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
ated behaviours [17]. Especially its ability to switch to autonomous activity upon autophosphorylation at threonine-286 is very important for the speed of how a new behavior is learnt and at neuronal level for the induction of long-term potentiation (LTP) [1,4,13,14]. There is considerable evidence that ␣CaMKII autophosphorylation also contributes to the speed in which the reinforcing action of alcohol and cocaine is established in humans [5–7]. Previous work has revealed that one single cocaine exposure could induce neuronal activation and LTP in the ventral tegmental area [21]. We asked whether similar effects could also be seen in the hippocampal dentate gyrus (DG), which has been found to be one of the few brain regions where adult neurogenesis occurs [8] and a crucial structure for drug-related behaviour and drug memories [18,22]. We hypothesized that a single exposure to either cocaine or alcohol can induce neuronal activation in the DG, which should be under the control of ␣CaMKII autophosphorylation.
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2. Material and methods All experiments were undertaken in accordance with the UK Home Office Animals (Experimental Procedures) Act 1986. ␣CaMKII autophosphorylation deficient male and female ␣CaMKIIT286A mice (MT) and their wild-type (WT) littermates [10] were studied in gender-balanced design (WT: n = 14; MT: n = 14) [5–7]. Mutants were generated using a gene targeting strategy which utilizes a replacement vector containing the point mutation (threonine-286 changed to alanine; T286A) within the autoinhibitory domain [10]. This missense mutation enables a selective blockage of CaMKII autophosphorylation but does not affect the Ca2+ dependent activity [10]. Mice were individually housed in Tecniplast cages (32 cm × 16 cm × 14 cm), using Litaspen sawdust and nesting materials (Sizzlenest, Datsand, Manchester UK). Water and food was supplied ad libitum. Animals were kept on a 12:12 h light/dark cycle form the outset (lights on at 7:00 am). For experimental procedures mice were moved from the home cage to a temporary cage and alcohol (2 g/kg), cocaine (15 mg/kg) or saline were i.p. (vinj = 10 ml/kg) injected. Corresponding to the peak transmitter response seen in vivo, c-fos activity was measured 70 min after administration [7]. Subsequently, mice were sacrificed under isoflurane narcosis and transcardially perfused with 0.1 M PBS for 10 min and then fixed with 4% paraformaldehyde (PFA) solution for a further 10 min (flow rate 4 ml/min). The brain was removed and left in 4% PFA solution overnight at 4 ◦ C. Brains were then transferred to a 30% saccharose solution and stored at 4 ◦ C until brains were fully submerged. Brains were then snap frozen in isopentane at −60 ◦ C and stored at −80 ◦ C until the whole brain was cut into 40 m coronal sections by cryosectioning. All sections were collected and then stored at −20 ◦ C in an anti-freezing solution until processed for immunohistochemical staining. The floating coronal sections were incubated with an anti-c-Fos rabbit polyclonal antibody (1:30.000, Calbiochem, Germany) for 20 h. c-Fos immunoreactive cells were visualized using a biotinylated donkey anti-rabbit secondary antibody (1:500, Santa Cruz, Germany) and the avidin-biotin complex (ABC-Elite kit rabbit, Vector Laboratories, Germany [3]). The number of c-Fos-immunoreactive cells in the DG was determined using stereological quantification. The examined regions were defined according to the stereotaxic coordinates. Stereological quantification of the c-Fos positive cells was carried out strictly blind to the experimental conditions with the optical fractionator estimating total numbers of c-Fos positive cells. After histological processing the sections had a mounted section thickness of 30 m, a fixed distance of 2 m and an optical dissector height of 26 m, measured with an electronic microtator attached to the microscope. All counting procedures and measurements of reference volumes were conducted on a light microscope (Nikon Eclipse 80i) equipped with a semiautomatic stereology system (Stereoinvestigator, Version 8.27, MicroBrightField, Colchester, Vermont, USA). C-Fos-positive cells were counted within a 70 × 70 m counting frame, which was spaced in a 90 × 90 m counting grid. Positive cells were counted, if their nucleus came into focus. Positive cells, which intersected the uppermost focal plane (exclusion plane) or the lateral exclusion boundaries of the counting frame were not counted. The total counts of positive c-Fos cells were multiplied by the ratio of reference volume to sampling volume in order to obtain the estimated number of cFos-positive cells for each structure [6]. Numbers of c-fos positive cells were quantified separately in the left and right DG (Fig. 1). As we did not see significant differences between hemispheres and genders in this study, data were collapsed for analysis. All quantitative data were expressed as mean ± SEM. One hemisphere of one animal could not be analyzed. For the data analysis, oneway ANOVA was used followed by Bonferroni-corrected t-tests.
Fig. 1. Localization of the scored region of interest for quantifying c-fos activation (between lines). Hippocampal dentate gyrus was analysed on coronal slices between −1.70 mm and −2.8 mm relative to bregma (a). Low amplification image of the localization of the sample photomicrographs shown below (b).
The software SPSS 22.0 was applied with a significance level of p < 0.05. 3. Results We found a significant increase in c-fos expression in the DG in WT mice after alcohol and cocaine treatment compared to saline treatment (F2,25 = 12.036, p < 0.001; Fig. 2). We did not detect any significant difference in c-fos expression in ␣CaMKIIT286A mice after the various treatments (p > 0.05). Pairwise comparisons showed significant differences in c-fos activation between ␣CaMKIIT286A mice and WT mice in the DG after alcohol (t = 5.539, p < 0.002) and cocaine treatment (t = 4.587, p < 0.002), but not after saline treatment (p > 0.05). 4. Discussion It was shown for the psychoactive drugs, alcohol and cocaine, that they induce neuronal activation by influencing various receptor systems in the limbic brain [16]. A recent study has demonstrated that already a single exposure to cocaine increases c-fos expression in the nucleus accumbens (NAcc) in WT, but not in ␣CaMKIIT286A mice [7]. Here, we show that alcohol and cocaine can induce c-fos expression also in the DG. To determine the upstream effects, which led to NAcc activation, we investigated the neuronal activity in the DG under the same conditions. There is an important glutamatergic projection from the hippocampus to the NAcc [9,11]. It was suggested that this connection plays a role for adjusting the dopaminergic tone and mediating psychomotor stimulating effects of drugs in the Nacc [9]. In the present study, we found reduced cfos activation in the DG after alcohol treatment in ␣CaMKIIT286A mice. c-fos, as an immediate early gene, is a marker for neuronal activation after drug consumption [20]. These findings are in line with a recent study [6] that observed a decreased dopamine (DA) response in ␣CaMKIIT286A mice after acute alcohol administration in the NAcc. A likely mechanism to explain this decrease could be the lack of activating impulses from the hippocampus to release
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Fig. 2. ␣CaMKII autophosphorylation-deficient mice show an reduced increase in c-fos expression after single alcohol (2 g/kg, i.p.) or cocaine treatment (15 mg/kg, i.p.) in the hippocampal dentate gyrus compared to the wild-type (mean ± SEM) (***p < 0.001) (a,b). c-fos labelling in wild-type (WT; c,e,g) and ␣CaMKIIT286A mice (d,f,h) after different treatments. Photomicrographs show c-fos positive cells (black) of the dentate gyrus (scale: 80 × 80 m).
DA in the Nacc. At present it is unclear whether the lack of c-fos activation in ␣CaMKIIT286A mice is only characteristic for alcohol and cocaine, or whether it translates to other psychoactive drugs as well, which had been shown to require CaMKII phosphorylation. Several studies have demonstrated that cocaine leads to an acute increase in DA levels in the NAcc [2,7]. In ␣CaMKIIT286A mice, the peak DA response after cocaine i.p. treatment was blurred. The mutant mice also showed a significant lack of cocaine induced c-fos activation in the NAcc [7]. To investigate possible up-stream effects, we quantified neuronal activation after cocaine treatment in the DG and found increased activation in WT animals which was absent in ␣CaMKIIT286A mice. This may suggest an attenuated hippocampal input to the Nacc ␣CaMKIIT286A mice after cocaine treatment. This may contribute to the observed lack of Nacc c-fos activation after cocaine and also to the delayed establishment of behaviours indicative of the reinforcing action of cocaine [12].
5. Conclusions Altogether, the present study suggests that ␣CaMKII autophosphorylation is a crucial mechanism to generate neuronal activation in the hippocampal DG after alcohol and cocaine administration. This may have important downstream effects with the hippocampal projections to the NAcc being important to establish acute drug-induced locomotor and reinforcing effects. Conflict of interest Authors declare no conflict of interest. Acknowledgements We thank Jörg Distler for his expert technical assistance and Dr. Fabio Canneva for support. This work was supported by funding from the Interdisciplinary Center for Clinical Research
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Erlangen (Project E13). The present work was performed in (partial) fulfilment of the requirements for obtaining the degree “Dr. med.” (I.S.). References [1] S.F. Cooke, J. Wu, F. Plattner, M. Errington, M. Rowan, et al., Autophosphorylation of alphaCaMKII is not a general requirement for NMDA receptor-dependent LTP in the adult mouse, J. Physiol. 574 (2006) 805–818. [2] G. Di Chiara, A. Imperato, Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats, Proc. Natl. Acad. Sci. USA 85 (1988) 5274–5278. [3] M. Dragunow, R. Faull, The use of c-fos as a metabolic marker in neuronal pathway tracing, J. Neurosci. Meth. 29 (1989) 261–265. [4] A.C. Easton, A. Lourdusamy, E. Loth, R. Torro, K.P. Giese, J. Kornhuber, et al., CaMKII polymorphisms predict working memory performance in humans, Mol. Psychiatry 18 (2012) 850–852. [5] A.C. Easton, W. Lucchesi, K. Mizuno, C. Fernandes, G. Schumann, K.P. Giese, et al., alphaCaMKII autophosphorylation controls the establishment of alcohol-induces conditioned place preference in mice, Behav. Brain. Res. 252 (2013) 72–76. [6] A.C. Easton, W. Lucchesi, A. Lourdusamy, B. Lenz, J. Solati, Y. Golub, et al., alphaCaMKII autophosphorylation controls the establishment of alcohol drinking behavior, Neuropsychopharmacology 38 (2013) 1636–1647. [7] A.C. Easton, A. Lourdusamy, M. Havranek, K. Mizuno, J. Solati, Y. Golub, T.-K. Clarke, et al., alphaCaMKII controls the establishment of cocaine’s reinforcing effects in mice and humans, Transl. Psychiatry. 4 (2014) e457. [8] A.J. Eisch, C.D. Mandyam, Drug dependence and addiction, II: adult neurogeneses and drug abuse, Am. J. Psychiatry 161 (2004) 426. [9] B.J. Everitt, T.W. Robbins, Neural systems of reinforcement for drug addiction: from actions to habits to compulsion, Nat. Neurosci. 8 (2005) 1481–1489. [10] K.P. Giese, N.B. Fedorov, R.K. Filipkowski, A.J. Silva, Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning, Science 279 (1998) 870–873.
[11] Y. Goto, P. O’ Donnell, Synchronous activity in the hippocampus and nucleus accumbens in vivo, J. Neurosci. 21 (2001), RC131 1–5. [12] J.P. Huston, M.A. de Souza Silva, B. Topic, C.P. Müller, What’s conditioned in conditioned place preference? Trends Pharmacol. Sci. 34 (3) (2013) 162–166. [13] E.E. Irvine, J. Vernon, K.P. Giese, AlphaCaMKII autophosphorylation contributes to rapid learning but is not necessary for memory, Nat. Neurosci. 8 (2005) 411–412. [14] E.E. Irvine, L.S. von Hertzen, F. Plattner, K.P. Giese, alphaCaMKII autophosphorylation: a fast track to memory, Trends Neurosci. 29 (2006) 459–465. [15] A.E. Kelley, Memory and addiction: shared neural circuitry and molecular mechanisms, Neuron 44 (2004) 161–179. [16] G.F. Koob, P.P. Sanna, F.E. Bloom, Neuroscience of addiction, Neuron 21 (1998) 467–476. [17] C.Y. Li, X. Mao, L. Wei, Genes and (common) pathways underlying drug addiction, PLoS Comput. Biol. 4 (2008) e2. [18] C.P. Müller, Episodic memories and their relevance for psychoactive drug use and addiction, Front. Behav. Neurosci. 7 (34) (2013) 1–13. [19] C.P. Müller, G. Schumann, Drugs as an instrument: a new framework for non-addictive psychoactive drug use, Behav. Brain Sci. 34 (6) (2011) 293–310. [20] H.A. Robertson, M.L. Paul, R. Moratalla, A.M. Graybiel, Expression of the immediate early gene c-fos in basal ganglia: induction by dopaminergic drugs, Can. J. Neurol. Sci. 18 (1991) 380–383. [21] M.A. Ungless, J.L. Whistler, R.C. Malenka, A. Bonci, Single cocaine exposure in vivo induces long-term potentiation in dopamine neurons, Nature 411 (2001) 583–587. [22] S.R. Vorel, X. Liu, R.J. Hayes, J.A. Spector, E.L. Gardner, Relapse to cocaine-seeking after hippocampal theta burst stimulation, Science 292 (2001) 1175–1178. [23] F.A. Wagner, J.C. Anthony, From first drug use to drug dependence: developmental periods of risk for dependence upon marijuana, cocaine, and alcohol, Neuropsychopharmacology 26 (2002) 479–488. [24] N.M. White, Addictive drugs as reinforcers: multiple partial actions on memory systems, Addiction 91 (1996) 921–949.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
␣CaMKII autophosphorylation mediates neuronal activation in the hippocampal dentate gyrus after alcohol and cocaine in mice Isabella Schöpf a , Alanna C. Easton b , Jalal Solati c,d , Yulia Golub c , Johannes Kornhuber a , K. Peter Giese e , Christian P. Müller a,b,∗ a
Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nuremberg, Schwabachanlage 6, 91054 Erlangen, Germany MRC Social, Genetic and Developmental Psychiatry Research Centre, Institute of Psychiatry, King’s College London, De Crespigny Park, London SE5 8AF, UK c Department of Child and Adolescent Mental Health, University Clinic Erlangen, Schwabachanlage 6 and 11, 91054 Erlangen, Germany d Department of Biology, Faculty of Science, Karaj branch, Islamic Azad University, Karaj, Iran e Centre for the Cellular Basis of Behavior, MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King’s College London, James Black Centre, 125 Coldharbour Lane, London SE5 8AF, UK b
h i g h l i g h t s • ˛CaMKII is invoved in the establishment of drug addiction. • Alcohol and cocaine increase c-fos expression in the dentate gyrus of mice. • No increase in c-fos expression in ␣CaMKII autophosphorylation deficient mice.
a r t i c l e
i n f o
Article history: Received 19 January 2015 Received in revised form 12 February 2015 Accepted 13 February 2015 Available online 17 February 2015 Keywords: Hippocampus ␣caMKIIT286A C-fos Immunohistochemistry
a b s t r a c t Psychoactive drug-induced cellular activation is a key mechanism to promote neuronal plasticity and addiction. Alpha Ca2+ /calmodulin-dependent protein kinase II (␣CaMKII) and its autophosphorylation play a key role in the development of drug use associated behaviours. It has been suggested that ␣CaMKII autophosphorylation is necessary for drug-induced neuronal activation in the mesolimbic system. Here, we show an alcohol- and cocaine-induced increase in c-fos expression in the hippocampal dentate gyrus, which is absent in ␣CaMKIIT286A autophosphorylation deficient mice. These findings may suggest a role in hippocampal ␣CaMKII autophosphorylation in the acute neuroplastic effects of alcohol and cocaine. © 2015 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The systematic use of psychoactive drugs is a wide spread phenomenon on a world wide scale [19]. The establishment of drug use behaviours is a systematic process, which requires multiple types of learning and memory [15,18,24]. Drugs of abuse, like cocaine and alcohol, can develop addiction in a significant number of people by modifying neuronal circuits and synaptic transmission [23]. The alpha Ca2+ /calmodulin-dependent protein kinase II (␣CaMKII) plays a key role in the establishment of drug use associ-
∗ Corresponding author at: Section of Addiction Medicine, Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University Erlangen-Nuremberg, Schwabachanlage 6, Erlangen, 91054, Germany. Tel.: +49 9131 85 36896. E-mail address: [email protected] (C.P. Müller). http://dx.doi.org/10.1016/j.neulet.2015.02.031 0304-3940/© 2015 Elsevier Ireland Ltd. All rights reserved.
ated behaviours [17]. Especially its ability to switch to autonomous activity upon autophosphorylation at threonine-286 is very important for the speed of how a new behavior is learnt and at neuronal level for the induction of long-term potentiation (LTP) [1,4,13,14]. There is considerable evidence that ␣CaMKII autophosphorylation also contributes to the speed in which the reinforcing action of alcohol and cocaine is established in humans [5–7]. Previous work has revealed that one single cocaine exposure could induce neuronal activation and LTP in the ventral tegmental area [21]. We asked whether similar effects could also be seen in the hippocampal dentate gyrus (DG), which has been found to be one of the few brain regions where adult neurogenesis occurs [8] and a crucial structure for drug-related behaviour and drug memories [18,22]. We hypothesized that a single exposure to either cocaine or alcohol can induce neuronal activation in the DG, which should be under the control of ␣CaMKII autophosphorylation.
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2. Material and methods All experiments were undertaken in accordance with the UK Home Office Animals (Experimental Procedures) Act 1986. ␣CaMKII autophosphorylation deficient male and female ␣CaMKIIT286A mice (MT) and their wild-type (WT) littermates [10] were studied in gender-balanced design (WT: n = 14; MT: n = 14) [5–7]. Mutants were generated using a gene targeting strategy which utilizes a replacement vector containing the point mutation (threonine-286 changed to alanine; T286A) within the autoinhibitory domain [10]. This missense mutation enables a selective blockage of CaMKII autophosphorylation but does not affect the Ca2+ dependent activity [10]. Mice were individually housed in Tecniplast cages (32 cm × 16 cm × 14 cm), using Litaspen sawdust and nesting materials (Sizzlenest, Datsand, Manchester UK). Water and food was supplied ad libitum. Animals were kept on a 12:12 h light/dark cycle form the outset (lights on at 7:00 am). For experimental procedures mice were moved from the home cage to a temporary cage and alcohol (2 g/kg), cocaine (15 mg/kg) or saline were i.p. (vinj = 10 ml/kg) injected. Corresponding to the peak transmitter response seen in vivo, c-fos activity was measured 70 min after administration [7]. Subsequently, mice were sacrificed under isoflurane narcosis and transcardially perfused with 0.1 M PBS for 10 min and then fixed with 4% paraformaldehyde (PFA) solution for a further 10 min (flow rate 4 ml/min). The brain was removed and left in 4% PFA solution overnight at 4 ◦ C. Brains were then transferred to a 30% saccharose solution and stored at 4 ◦ C until brains were fully submerged. Brains were then snap frozen in isopentane at −60 ◦ C and stored at −80 ◦ C until the whole brain was cut into 40 m coronal sections by cryosectioning. All sections were collected and then stored at −20 ◦ C in an anti-freezing solution until processed for immunohistochemical staining. The floating coronal sections were incubated with an anti-c-Fos rabbit polyclonal antibody (1:30.000, Calbiochem, Germany) for 20 h. c-Fos immunoreactive cells were visualized using a biotinylated donkey anti-rabbit secondary antibody (1:500, Santa Cruz, Germany) and the avidin-biotin complex (ABC-Elite kit rabbit, Vector Laboratories, Germany [3]). The number of c-Fos-immunoreactive cells in the DG was determined using stereological quantification. The examined regions were defined according to the stereotaxic coordinates. Stereological quantification of the c-Fos positive cells was carried out strictly blind to the experimental conditions with the optical fractionator estimating total numbers of c-Fos positive cells. After histological processing the sections had a mounted section thickness of 30 m, a fixed distance of 2 m and an optical dissector height of 26 m, measured with an electronic microtator attached to the microscope. All counting procedures and measurements of reference volumes were conducted on a light microscope (Nikon Eclipse 80i) equipped with a semiautomatic stereology system (Stereoinvestigator, Version 8.27, MicroBrightField, Colchester, Vermont, USA). C-Fos-positive cells were counted within a 70 × 70 m counting frame, which was spaced in a 90 × 90 m counting grid. Positive cells were counted, if their nucleus came into focus. Positive cells, which intersected the uppermost focal plane (exclusion plane) or the lateral exclusion boundaries of the counting frame were not counted. The total counts of positive c-Fos cells were multiplied by the ratio of reference volume to sampling volume in order to obtain the estimated number of cFos-positive cells for each structure [6]. Numbers of c-fos positive cells were quantified separately in the left and right DG (Fig. 1). As we did not see significant differences between hemispheres and genders in this study, data were collapsed for analysis. All quantitative data were expressed as mean ± SEM. One hemisphere of one animal could not be analyzed. For the data analysis, oneway ANOVA was used followed by Bonferroni-corrected t-tests.
Fig. 1. Localization of the scored region of interest for quantifying c-fos activation (between lines). Hippocampal dentate gyrus was analysed on coronal slices between −1.70 mm and −2.8 mm relative to bregma (a). Low amplification image of the localization of the sample photomicrographs shown below (b).
The software SPSS 22.0 was applied with a significance level of p < 0.05. 3. Results We found a significant increase in c-fos expression in the DG in WT mice after alcohol and cocaine treatment compared to saline treatment (F2,25 = 12.036, p < 0.001; Fig. 2). We did not detect any significant difference in c-fos expression in ␣CaMKIIT286A mice after the various treatments (p > 0.05). Pairwise comparisons showed significant differences in c-fos activation between ␣CaMKIIT286A mice and WT mice in the DG after alcohol (t = 5.539, p < 0.002) and cocaine treatment (t = 4.587, p < 0.002), but not after saline treatment (p > 0.05). 4. Discussion It was shown for the psychoactive drugs, alcohol and cocaine, that they induce neuronal activation by influencing various receptor systems in the limbic brain [16]. A recent study has demonstrated that already a single exposure to cocaine increases c-fos expression in the nucleus accumbens (NAcc) in WT, but not in ␣CaMKIIT286A mice [7]. Here, we show that alcohol and cocaine can induce c-fos expression also in the DG. To determine the upstream effects, which led to NAcc activation, we investigated the neuronal activity in the DG under the same conditions. There is an important glutamatergic projection from the hippocampus to the NAcc [9,11]. It was suggested that this connection plays a role for adjusting the dopaminergic tone and mediating psychomotor stimulating effects of drugs in the Nacc [9]. In the present study, we found reduced cfos activation in the DG after alcohol treatment in ␣CaMKIIT286A mice. c-fos, as an immediate early gene, is a marker for neuronal activation after drug consumption [20]. These findings are in line with a recent study [6] that observed a decreased dopamine (DA) response in ␣CaMKIIT286A mice after acute alcohol administration in the NAcc. A likely mechanism to explain this decrease could be the lack of activating impulses from the hippocampus to release
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Fig. 2. ␣CaMKII autophosphorylation-deficient mice show an reduced increase in c-fos expression after single alcohol (2 g/kg, i.p.) or cocaine treatment (15 mg/kg, i.p.) in the hippocampal dentate gyrus compared to the wild-type (mean ± SEM) (***p < 0.001) (a,b). c-fos labelling in wild-type (WT; c,e,g) and ␣CaMKIIT286A mice (d,f,h) after different treatments. Photomicrographs show c-fos positive cells (black) of the dentate gyrus (scale: 80 × 80 m).
DA in the Nacc. At present it is unclear whether the lack of c-fos activation in ␣CaMKIIT286A mice is only characteristic for alcohol and cocaine, or whether it translates to other psychoactive drugs as well, which had been shown to require CaMKII phosphorylation. Several studies have demonstrated that cocaine leads to an acute increase in DA levels in the NAcc [2,7]. In ␣CaMKIIT286A mice, the peak DA response after cocaine i.p. treatment was blurred. The mutant mice also showed a significant lack of cocaine induced c-fos activation in the NAcc [7]. To investigate possible up-stream effects, we quantified neuronal activation after cocaine treatment in the DG and found increased activation in WT animals which was absent in ␣CaMKIIT286A mice. This may suggest an attenuated hippocampal input to the Nacc ␣CaMKIIT286A mice after cocaine treatment. This may contribute to the observed lack of Nacc c-fos activation after cocaine and also to the delayed establishment of behaviours indicative of the reinforcing action of cocaine [12].
5. Conclusions Altogether, the present study suggests that ␣CaMKII autophosphorylation is a crucial mechanism to generate neuronal activation in the hippocampal DG after alcohol and cocaine administration. This may have important downstream effects with the hippocampal projections to the NAcc being important to establish acute drug-induced locomotor and reinforcing effects. Conflict of interest Authors declare no conflict of interest. Acknowledgements We thank Jörg Distler for his expert technical assistance and Dr. Fabio Canneva for support. This work was supported by funding from the Interdisciplinary Center for Clinical Research
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