Enhancement of behavioral sensitization, anxiety-like behavior, and hippocampal and frontal cortical CREB levels following cocaine abstinence in mice exposed to cocaine during adolescence

Adolescence has been linked to greater risk-taking and novelty-seeking behavior and a higher prevalence of drug abuse and risk of relapse. Decreases in cyclic adenosine monophosphate response element binding protein (CREB) and phosphorylated CREB (pCREB) have been reported after repeated cocaine administration in animal models. We compared the behavioral effects of cocaine and abstinence in adolescent and adult mice and investigated possible age-related differences in CREB and pCREB levels. Adolescent and adult male Swiss mice received one daily injection of saline or cocaine (10 mg/kg, i.p.) for 8 days. On day 9, the mice received a saline injection to evaluate possible environmental conditioning. After 9 days of withdrawal, the mice were tested in the elevated plus maze to evaluate anxiety-like behavior. Twelve days after the last saline/cocaine injection, the mice received a challenge injection of either cocaine or saline, and locomotor activity was assessed. One hour after the last injection, the brains were extracted, and CREB and pCREB levels were evaluated using Western blot in the prefrontal cortex (PFC) and hippocampus. The cocaine-pretreated mice during adolescence exhibited a greater magnitude of the expression of behavioral sensitization and greater cocaine withdrawal-induced anxiety-like behavior compared with the control group. Significant increases in CREB levels in the PFC and hippocampus and pCREB in the hippocampus were observed in cocaine-abstinent animals compared with the animals treated with cocaine in adulthood. Interestingly, significant negative correlations were observed between cocaine sensitization and CREB levels in both regions. These results suggest that the behavioral and neurochemical consequences of psychoactive substances in a still-developing nervous system can be more severe than in an already mature nervous systemFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Effects of cocaine treatment in behavioral parameters and in the expression of CREB/pCREB and BDNF in adolescent and adult mice.

A fase da adolescência difere da fase adulta em parâmetros comportamentais e neuroquímicos. Alterações do fator de transcrição CREB, da neurotrofina BDNF e seu receptor TrkB têm sido encontradas no circuito dopaminérgico-mesolímbico com o uso de cocaína. Este estudo avalia os efeitos da administração de cocaína em camundongos adolescentes e adultos na sensibilização psicomotora, no comportamento tipo ansiedade, nos níveis protéicos de CREB/pCREB, BDNF e TrkB e na expressão gênica de BDNF e TrkB no córtex pré-frontal (CPF) e hipocampo desses animais. Os adolescentes mostraram-se mais suscetíveis aos efeitos da droga, com maior ativação locomotora, maior efeito de condicionamento ao ambiente, maior nível de ansiedade induzido pela droga e alterações neuroquímicas distintas dos adultos (diminuição dos níveis de CREB com o tratamento repetido e aumento na abstinência em CPF e hipocampo; aumento de RNAm de BDNF após tratamento agudo, repetido e abstinência em CPF e diminuição no hipocampo), sugerindo maior vulnerabilidade desse grupo no desenvolvimento da dependência.The adolescence differs from adulthood in behavioral and neurochemichal aspects. Changes in the activity of the transcriptional factor CREB, neurotrofin BDNF and its receptor TrkB have been found in the mesolimbic-dopaminergic circuitry following the use of cocaine. This study evaluates the effects of cocaine administration in adolescent and adult mice in the locomotor sensitization and in the anxiety-like behavior. The study also accesses the protein levels of CREB/pCREB, BDNF and TrkB, and gene expression of BDNF and TrkB in the prefrontal cortex and hippocampus of these animals. In a general way, adolescents were more susceptible to drug effects, with increased levels of locomotor activity, environmental conditioning, anxiety-like behavior and greater neurochemichal alterations, suggesting an increased risk in the development of drug dependence

Study of the pathways of CREB activation in cocaine-induced neuroplasticity in adolescent and adult mice.

A fase da adolescência difere da adulta em parâmetros comportamentais e neurológicos. A proteína de ligação responsiva ao AMPc (CREB) é um fator de transcrição regulado por três vias principais: PKA, CaMK e ERK, as quais apresentam atividade alterada em resposta à cocaína. Neste trabalho, foram avaliadas as atividades dessas vias, bem como os níveis de um de seus genes alvo (prodinorfina), do inibidor endógeno de CRE (Icer) e da proteína ativadora de CREB (CBP) em resposta à administração de cocaína, além dos comportamentos induzidos pelo psicoestimulante. Os adolescentes apresentaram maior sensibilização psicomotora, atividade exploratória e comportamento tipo ansiedade em comparação aos adultos. Os adolescentes exibiram aumento da atividade da via da PKA e da expressão de Icer, CBP e Pdyn; os adultos apresentaram diminuição de CaMKs, GluR1 e Icer. Essas diferentes respostas neuroadaptativas podem ajudar na compreensão dos mecanismos ontogênicos envolvidos na dependência.The adolescence differs from adulthood in behavioral and neurochemichal aspects. The cAMP responsive binding protein (CREB) is a transcription factor regulated by three main pathways in the mesolimbic-dopaminergic circuit: PKA, CaMK and ERK, which are modulated by cocaine. This study evaluated the activity of these pathways, as well as the levels of one of its main target genes (prodynorphin), endogenous CRE inhibitor (Icer) and CREB binding protein (CBP) in response to cocaine administration, besides the behaviors induced by the psychostimulant. Adolescents presented greater locomotor activation, psychomotor sensitization, exploratory activity and anxiety-like behavior compared to adults. Adolescents exhibited increased PKA pathway activity and expression of Icer, CBP, and Pdyn; adults showed decreased levels of CaMKs, GluR1 and Icer. These different neuroadaptive responses may help understanding the ontogenic mechanisms involved in addiction

Enhancement of Behavioral Sensitization, Anxiety-Like Behavior, and Hippocampal and Frontal Cortical CREB Levels Following Cocaine Abstinence in Mice Exposed to Cocaine during Adolescence

Adolescence has been linked to greater risk-taking and novelty-seeking behavior and a higher prevalence of drug abuse and risk of relapse. Decreases in cyclic adenosine monophosphate response element binding protein (CREB) and phosphorylated CREB (pCREB) have been reported after repeated cocaine administration in animal models. We compared the behavioral effects of cocaine and abstinence in adolescent and adult mice and investigated possible age-related differences in CREB and pCREB levels. Adolescent and adult male Swiss mice received one daily injection of saline or cocaine (10 mg/kg, i.p.) for 8 days. On day 9, the mice received a saline injection to evaluate possible environmental conditioning. After 9 days of withdrawal, the mice were tested in the elevated plus maze to evaluate anxiety-like behavior. Twelve days after the last saline/cocaine injection, the mice received a challenge injection of either cocaine or saline, and locomotor activity was assessed. One hour after the last injection, the brains were extracted, and CREB and pCREB levels were evaluated using Western blot in the prefrontal cortex (PFC) and hippocampus. The cocaine-pretreated mice during adolescence exhibited a greater magnitude of the expression of behavioral sensitization and greater cocaine withdrawal-induced anxiety-like behavior compared with the control group. Significant increases in CREB levels in the PFC and hippocampus and pCREB in the hippocampus were observed in cocaine-abstinent animals compared with the animals treated with cocaine in adulthood. Interestingly, significant negative correlations were observed between cocaine sensitization and CREB levels in both regions. These results suggest that the behavioral and neurochemical consequences of psychoactive substances in a still-developing nervous system can be more severe than in an already mature nervous system. © 2013 Valzachi et al

Total locomotor activity in the open field test.

<p>(<b>A</b>) Total locomotor activity on the habituation days and during repeated saline or cocaine injection. Total locomotor activity in mice on H2 was lower than on H1 in both age groups (⁺<i>p</i> < 0.01, <i>vs</i>. H1; <i>n</i> = 50 mice, adolescent group; <i>n</i> = 48 mice, adult group). Both adolescent and adult mice treated with cocaine exhibited greater locomotor activity on D8 than on D1 (⁺<i>p</i> < 0.01, <i>vs</i>. D1; <i>n</i> = 25, saline adolescent [Sal adol]; <i>n</i> = 25, cocaine adolescent [Coc adol], acute cocaine; <i>n</i> = 23, saline adult [Sal adult]; <i>n</i> = 25, cocaine adult [Coc adult]). (<b>B</b>) Effects of saline injection on total locomotor activity on D9 after repeated injections of saline or cocaine. Animals pretreated with cocaine exhibited greater locomotor activity than animals pretreated with saline (^#<i>p</i> < 0.01, <i>vs</i>. saline). Adolescents exhibited greater increases in activity than adult mice (*<i>p</i> < 0.05, <i>vs</i>. adults). (<b>C</b>) Effect of a challenge injection of saline or cocaine on total locomotor activity on D20. Acute and repeated cocaine induced more locomotor activity in adolescents than in adults (*<i>p</i> < 0.05, <i>vs</i>. adults, age × challenge injection interaction). Mice treated with acute cocaine exhibited greater locomotor activity compared with the control groups, regardless of age (^#<i>p</i> < 0.01, <i>vs</i>. control, pretreatment × challenge injection interaction). Repeated cocaine administration produced greater locomotor activity compared with the acute cocaine group, regardless of age (^●<i>p</i> < 0.05, <i>vs</i>. acute, pretreatment × challenge injection interaction). Adolescent groups: control (<i>n</i> = 12), acute cocaine (<i>n</i> = 13), abstinence (<i>n</i> = 11), repeated cocaine (<i>n</i> = 14). Adult groups: control (<i>n</i> = 11), acute cocaine (<i>n</i> = 12), abstinence (<i>n</i> = 12), repeated cocaine (<i>n</i> = 13). The data are expressed as mean ± SEM.</p

Anxiety-like behavior.

<p>(<b>A</b>) Central locomotor activity after saline or cocaine injections. Central locomotion was higher on D8 than on D1 in all of the groups (⁺<i>p</i> < 0.01). Cocaine-treated adolescent mice exhibited lower central locomotion than their saline-treated counterparts (^#<i>p</i> < 0.01). Saline-treated adolescent mice exhibited greater central locomotor activity compared with all of the other groups (^&<i>p</i> < 0.05, <i>vs</i>. cocaine adolescent group and saline and cocaine adult groups). (<b>B</b>) Time spent in the open arms of the elevated plus maze on Day 18. Saline-pretreated adolescent mice spent more time in the open arms compared with all of the other groups (⁺<i>p</i> < 0.05, <i>vs</i>. cocaine adolescent group and saline and cocaine adult groups). Cocaine-pretreated adolescent and adult mice spent less time in the open arms compared with their respective saline-pretreated control groups (^#<i>p</i> < 0.05, <i>vs</i>. saline; <i>n</i> = 25, saline adolescent [Sal adol]; <i>n</i> = 25, cocaine adolescent [Coc adol], acute cocaine; <i>n</i> = 23, saline adult [Sal adult]; <i>n</i> = 25, cocaine adult [Coc adult]). The data are expressed as mean ± SEM.</p

Correlations between sensitization and CREB and pCREB levels.

<p>A significant negative correlation was observed between cocaine-induced sensitization and (<b>A</b>) CREB levels and (<b>B</b>) pCREB levels in the PFC and (<b>C</b>) pCREB in the hippocampus in adolescent mice. No relationship was observed between sensitization and CREB or pCREB levels in adult mice. </p

CREB and pCREB Western blot.

<p>(<b>A</b>) CREB protein levels in the PFC. Acute cocaine increased CREB levels (^#<i>p</i> < 0.05, vs. control group; pretreatment × challenge interaction), whereas repeated cocaine decreased CREB levels (^●<i>p</i> < 0.05, vs. acute group). Abstinence in mice pretreated with cocaine during adolescence increased CREB levels compared with all of the other groups (*<i>p</i> < 0.05, <i>vs</i>. all groups, considering pretreatment and age). (<b>B</b>) pCREB protein levels in the PFC. The acute cocaine groups exhibited higher pCREB levels compared with their respective control groups (^#<i>p</i> < 0.05, <i>vs</i>. control), whereas the repeated and abstinence groups displayed lower pCREB levels compared with their respective acute groups, both in adolescent and adult mice (^●<i>p</i> < 0.05, repeated <i>vs</i>. acute and abstinence <i>vs</i>. acute). (<b>C</b>) CREB protein levels in the hippocampus. In adolescents, repeated cocaine decreased CREB levels compared with the control group (^●<i>p</i> < 0.01, <i>vs</i>. control group). Abstinence increased CREB protein levels compared with the saline and repeated groups (*<i>p</i> < 0.01). (<b>D</b>) pCREB protein levels in the hippocampus. Repeated cocaine decreased pCREB levels compared with the acute and abstinence groups (^●<i>p</i> < 0.05). pCREB levels in the abstinence group were higher than in the repeated group (*<i>p</i> < 0.05). Adolescent groups: control (<i>n</i> = 7 for all groups, except for the pCREB-Hippocampus, which was <i>n</i> = 6), acute cocaine (<i>n</i> = 7 for all groups), abstinence (<i>n</i> = 7 for all groups), repeated cocaine (<i>n</i> = 8 for all groups, except for CREB and pCREB in the hippocampus, which was <i>n</i> = 7). Adult groups: control (<i>n</i> = 7 for all groups), acute cocaine (<i>n</i> = 7 for all groups), abstinence (<i>n</i> = 7 for all groups), repeated cocaine (<i>n</i> = 8 for all groups, except for pCREB in the PFC, which was <i>n</i> = 7). The data are expressed as mean ± SEM.</p