Glycogen synthase kinase 3 beta alters anxiety-, depression-, and addiction-related behaviors and neuronal activity in the nucleus accumbens shell
Introduction
Mood disorders such as anxiety and depression have high comorbidity with drug addiction in humans (Pettinati et al., 2013). Dysregulation of the reward system may constitute a common molecular mechanism of these neuropsychiatric disorders and therefore understanding complex neuroadaptations common to neuropsychiatric disorders constitutes a promising avenue for therapeutics.
The nucleus accumbens is heavily implicated in the control of emotional behavior and reward (Pontieri et al., 1995, Pliakas et al., 2001, Green et al., 2006, Nestler and Carlezon, 2006, Larson et al., 2011). As part of the ventral striatum, the nucleus accumbens has as its sole output two major populations of medium spiny neurons (MSNs) whose activity is modulated by a population of tonically active interneurons (TANs), which are mostly cholinergic (Lenz and Lobo, 2013). Despite comprising not more than 5% of the total population of neurons in the NAc, TANs play important roles in reward prediction, task attention, memory, addiction, and aversive behaviors (Aosaki et al., 1994, Apicella, 2002, Anagnostaras et al., 2003, Furey et al., 2008, Williams and Adinoff, 2008, Lenz and Lobo, 2013). TANs control MSN activity and are particularly responsive to salient reward-related stimuli (Morris et al., 2004). Early studies have provided evidence for a role of TANs in cocaine addiction with immunotoxin mediated cell ablation resulting in increased sensitivity to cocaine (Hikida et al., 2001) and preventing behavioral abnormalities associated with cocaine induced by centrally active acetylcholinesterase inhibitors in the NAc (Hikida et al., 2003). More recently, studies using optogenetics confirmed that selective inhibition of TANs results in suppression of cocaine induced behaviors (Witten et al., 2010), further confirming the pivotal role of these cells in reward behavior and addiction. At the circuit level, activation of TANs has been shown to elicit both fast glutamatergic transmission (Higley et al., 2011) and GABAergic inhibition of MSNs, the latter coincident with synchronous cholinergic activation and sufficient to pause MSNs firing (English et al., 2012). These studies suggest a complex role of these cells in the NAc circuit that deserves further investigation.
Intracellular kinase signaling cascades, activated through a variety of mechanisms, have proven important mediators of NAcSh function, and by extension, the etiology of neuropsychiatric disorders. Specifically, the ERK/MAPK, PKA, and PKC signaling cascades have been studied in the NAcSh with success (Self et al., 1998, Schroeder et al., 2008, Ortinski et al., 2015). The AKT/GSK3β pathway has also garnered particular attention for its role in dopamine signaling, the actions of antipsychotic drugs, and even responses to addictive drugs, especially in the nucleus accumbens (Perrine et al., 2008, Beaulieu et al., 2009, Nwaneshiudu and Unterwald, 2010, Beaulieu et al., 2011, Wilkinson et al., 2011, Miller et al., 2014).
GSK3β was originally discovered for its role in glycogen synthesis but has since been implicated in a variety of cellular processes (Wildburger and Laezza, 2012), and dysregulation of this kinase has been implicated in bipolar disorder and neurodegenerative disorders (Grimes and Jope, 2001, Jope, 2011). One of the mechanisms of action of lithium, the commonly prescribed mood stabilizer, is inhibition of GSK3β (Klein and Melton, 1996, Stambolic et al., 1996). Heterozygous GSK3β knockout mice show reductions in depression-like behavior similar to the effects of lithium (O'Brien et al., 2004). Drugs of abuse, especially cocaine, can modulate levels of GSK3β in the NAc (Perrine et al., 2008) and GSK3β is involved in cocaine-induced hyperactivity, cocaine sensitization, cocaine reward memory, and cocaine conditioned place preference (Miller et al., 2009, Miller et al., 2014, Shi et al., 2014). Previous studies indicate that the role of GSK3β is highly dependent on brain region and even cell type as global knockdown may not have the same effects as regional or even cell-type specific knockdown (Latapy et al., 2012, Urs et al., 2012, Zhou et al., 2012). Thus, GSK3β has therapeutic potential for comorbid depression and addiction, but knowledge gaps exist on its brain region specific mechanism of action.
The environmental enrichment manipulation combines novelty, exercise, and social contact to produce robust protective depression and addiction phenotypes (Green et al., 2002, Green et al., 2010). Enrichment increases the ratio of phosphorylated (inactive) to total GSK3β in the hippocampus and cortex (Hu et al., 2013) and environmental enrichment is able to reverse cognitive deficits caused by constitutively active expression of GSK3 in mice (Pardo et al., 2015). Therefore GSK3 may be involved in protecting against depression and addiction phenotypes.
The role of GSK3β in cocaine self-administration, the addiction paradigm with the most face validity, is so far lacking. Additionally, few studies have examined anxiety and depression behaviors along with addiction-related behaviors in the same animals. The current study therefore explores anxiety-like, depression-like, and addiction-related behaviors in the same animals following knockdown of GSK3β in the NAcSh of rats.
In order to analyze the role of GSK3β specifically in the NAcSh in behavior relevant to affective disorders and drug addiction, we designed and constructed a novel adeno-associated viral vector (AAV2) which uses RNA interference to knockdown GSK3β in adult rats and allows for prolonged knockdown of GSK3β in the adult brain. The AAV2 serotype infects neuronal cells in vivo but is not specific to any one neuronal cell type. To provide correlative functional outcomes to the behavioral studies we investigated the role of GSK3β on spontaneous firing and intrinsic excitability of tonically active neurons (TANs), comparing electrophysiological properties of these neurons between GSK3β knockdown vs. control.
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Animals
Male Sprague-Dawley rats (Harlan, Houston, TX) were obtained at 21-days-old (electrophysiology) or 225–250 g (behavior) and maintained in a controlled environment (temperature, 22 °C; relative humidity; 50%; 12 h light/dark cycle, lights on 0600 h) in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) approved colony in standard polycarbonate cages with ad libitum access to food and water except during surgery and behavioral experiments. All surgical procedures
Vector knockdown validation
The hairpin sequence was first validated in vitro by co-transfecting HEK293 cells with a plasmid expressing GSK3β and either the control hairpin plasmid or each of the GSK3β hairpin plasmids and comparing the amount of GSK3β mRNA in the cells. The chosen shRNA construct reduced the amount of GSK3β mRNA by > 90% (Fig. 1A). AAV-shGSK3β was further validated in vivo by injecting AAV-shGSK3β or AAV-shCTRL into the NAcSh and comparing the amount of GSK3β protein expression via Western blot analysis.
Discussion
This study found that GSK3β in the NAcSh of rats modulates addiction-, depression-, and anxiety-related behavior and also causes a reduction in spontaneous activity of TANs attributable to changes in intrinsic excitability. However, it is important to note that with the current state of technology, it is impossible to state for certain that the observed electrophysiological results in TANs directly produced the observed behavioral effects. Regardless, given that GSK3β knockdown increases rather
Funding
This work was supported by the National Institute on Drug Abuse at the National Institutes of Health (grant numbers: R01 DA029091 (TAG), T32 DA007287 (EJC)) and the National Institute of Mental Health (R01 MH095995 (FL)).
Conflict of interest
The authors declare no financial conflicts of interest.
References (78)
- et al.
Localization of dopamine D2 receptors on cholinergic interneurons of the dorsal striatum and nucleus accumbens of the rat
Brain Res.
(2003) Leading tonically active neurons of the striatum from reward detection to context recognition
Trends Neurosci.
(2007)- et al.
Neuromedin U receptor 2 knockdown in the paraventricular nucleus modifies behavioral responses to obesogenic high-fat food and leads to increased body weight
Neuroscience
(2014) - et al.
Cholinergic interneurons of the nucleus accumbens and dorsal striatum are activated by the self-administration of cocaine
Neuroscience
(2003) - et al.
Selective activation of cholinergic interneurons enhances accumbal phasic dopamine release: setting the tone for reward processing
Cell Rep.
(2012) - et al.
Dopamine neurons control striatal cholinergic neurons via regionally heterogeneous dopamine and glutamate signaling
Neuron
(2014) - et al.
Inoculation stress hypothesis of environmental enrichment
Neurosci. Biobehav. Rev.
(2015) - et al.
Contributory role for nornicotine in nicotine neuropharmacology: nornicotine-evoked [3 H] dopamine overflow from rat nucleus accumbens slices
Biochem. Pharmacol.
(2001) - et al.
Environmental enrichment produces a behavioral phenotype mediated by low cyclic adenosine monophosphate response element binding (CREB) activity in the nucleus accumbens
Biol. psychiatry
(2010) - et al.
The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling
Prog. Neurobiol.
(2001)
The Na1.2 channel is regulated by GSK3
Biochimica biophysica acta
Low GSK-3 activity in frontal cortex of schizophrenic patients
Schizophrenia Res.
Cholinergic depletion in the nucleus accumbens: effects on amphetamine response and sensorimotor gating
Prog. Neuro-Psychopharmacology Biol. Psychiatry
Optogenetic insights into striatal function and behavior
Behav. Brain Res.
Cocaine-induced hyperactivity and sensitization are dependent on GSK3
Neuropharmacology
Inhibition of GSK3 attenuates dopamine D1 receptor agonist-induced hyperactivity in mice
Brain Res. Bull.
Coincident but distinct messages of midbrain dopamine and striatal tonically active neurons
Neuron
The mesolimbic dopamine reward circuit in depression
Biol. psychiatry
Cocaine-seeking is associated with PKC-dependent reduction of excitatory signaling in accumbens shell D2 dopamine receptor-expressing neurons
Neuropharmacology
Re-emergence of striatal cholinergic interneurons in movement disorders
Trends Neurosci.
Cue-induced Reinstatement of Alcohol-seeking Behavior Is Associated with Increased ERK1/2 Phosphorylation in Specific Limbic Brain Regions: Blockade by the MGluR5 Antagonist MPEP. in: Neuropharmacology
The fibroblast growth factor 14.voltage-gated sodium channel complex is a new target of glycogen synthase kinase 3 (GSK3)
J. Biol. Chem.
The mechanism of intrinsic amplification of hyperpolarizations and spontaneous bursting in striatal cholinergic interneurons
Neuron
Selective cognitive dysfunction in acetylcholine M1 muscarinic receptor mutant mice
Nat. Neurosci.
Functional status of the serotonin 5-HT2C receptor (5-HT2CR) drives interlocked phenotypes that precipitate relapse-like behaviors in cocaine dependence
Neuropsychopharmacol. official Publ. Am. Coll. Neuropsychopharmacol.
Administration of the D2 dopamine receptor antagonist sulpiride into the shell, but not the core, of the nucleus accumbens attenuates cocaine priming-induced reinstatement of drug seeking
Neuropsychopharmacol. official Publ. Am. Coll. Neuropsychopharmacol.
Responses of tonically active neurons in the primate's striatum undergo systematic changes during behavioral sensorimotor conditioning
J. Neurosci.
Tonically active neurons in the primate striatum and their role in the processing of information about motivationally relevant events
Eur. J. Neurosci.
Glycogen synthase kinase 3beta and Alzheimer's disease: pathophysiological and therapeutic significance
Cell. Mol. life Sci. CMLS
Akt/GSK3 signaling in the action of psychotropic drugs
Annu. Rev. Pharmacol. Toxicol.
Beyond cAMP: the regulation of Akt and GSK3 by dopamine receptors
Front. Mol. Neurosci.
Spontaneous activity of neostriatal cholinergic interneurons in vitro
J. Neurosci.
Involvement of Ih in dopamine modulation of tonic firing in striatal cholinergic interneurons
J. Neurosci.
Convergent evidence for impaired AKT1-GSK3beta signaling in schizophrenia
Nat. Genet.
GABAergic circuits mediate the reinforcement-related signals of striatal cholinergic interneurons
Nat. Neurosci.
Selective effects of cholinergic modulation on task performance during selective attention
Neuropsychopharmacol. official Publ. Am. Coll. Neuropsychopharmacol.
Environmental enrichment decreases intravenous amphetamine self-administration in rats: dose-response functions for fixed- and progressive-ratio schedules
Psychopharmacology
Induction of activating transcription factors (ATFs) ATF2, ATF3, and ATF4 in the nucleus accumbens and their regulation of emotional behavior
J. Neurosci.
Induction of inducible cAMP early repressor expression in nucleus accumbens by stress or amphetamine increases behavioral responses to emotional stimuli
J. Neurosci. official J. Soc. Neurosci.
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