Progenitor Cells Play a Role in Reinstatement of Ethanol Seeking in Adult Male and Female Ethanol Dependent Rats.

Female and male glial fibrillary acidic protein-thymidine kinase (GFAP-TK) transgenic rats were made ethanol dependent via a six-week chronic intermittent ethanol vapor (CIE) and ethanol drinking (ED) procedure. During the last week of CIE, a subset of male and female TK rats was fed valcyte to ablate dividing progenitor cells and continued the diet until the end of this study. Following week six, all CIE rats experienced two weeks of forced abstinence from CIE-ED, after which they experienced relapse to drinking, extinction, and reinstatement of ethanol seeking sessions. CIE increased ED in female and male rats, with females having higher ethanol consumption during CIE and relapse sessions compared with males. In both sexes, valcyte reduced the levels of Ki-67-labeled progenitor cells in the subgranular zone of the dentate gyrus and did not alter the levels in the medial prefrontal cortex (mPFC). Valcyte increased ED during relapse, increased lever responses during extinction and, interestingly, enhanced latency to extinguish ethanol-seeking behaviors in males. Valcyte reduced the reinstatement of ethanol-seeking behaviors triggered by ethanol cues in females and males. Reduced seeking by valcyte was associated with the normalization of cytokines and chemokines in plasma isolated from trunk blood, indicating a role for progenitor cells in peripheral inflammatory responses. Reduced seeking by valcyte was associated with increases in tight junction protein claudin-5 and oligodendrogenesis in the dentate gyrus and reduction in microglial activity in the dentate gyrus and mPFC in females and males, demonstrating a role for progenitor cells in the dentate gyrus in dependence-induced endothelial and microglial dysfunction. These data suggest that progenitor cells born during withdrawal and abstinence from CIE in the dentate gyrus are aberrant and could play a role in strengthening ethanol memories triggered by ethanol cues via central and peripheral immune responses.

Neural progenitor cells derived from lithium responsive and non-responsive bipolar disorder patients exhibit distinct sensitivity to cell death following methamphetamine.

Bipolar disorder (BD) is characterized by manic and depressive mood episodes and loss of brain gray matter. Lithium has antimanic and neuroprotective properties, but only 30% BD patients respond to lithium pharmacotherapy. Dopamine signaling has been implicated in BD and may contribute to lithium response. Methamphetamine (METH) stimulates dopamine release and models the clinical features of mania but has never been used to study cell death in BD patient neurons. We used BD patient derived neuronal progenitor cells (NPCs) to determine whether the vulnerability to cell death differed in samples from lithium responder (Li-R) and non-responder (Li-NR) BD patients and healthy controls following METH exposure in vitro. We hypothesized that NPCs from Li-R and Li-NR would differ in vulnerability to METH, dopamine signaling and neuroprotection from lithium. Following METH, NPCs from controls and Li-NR showed significantly greater cell loss compared to Li-R. Pre-treatment of NPCs with the D1 dopamine receptor antagonist SCH 23390 reversed the neurotoxic effects of METH. In Li-R NPCs, expression of phosho-ERK1/2 was significantly increased. In Li-NR NPCs, phospho-AKT, D1 and D2 dopamine receptor proteins were significantly increased. Pre-treatment of NPCs with lithium before METH reversed the neurotoxic effects of METH in control NPCs, whereas Li-NR showed less protective benefit. Li-R cells showed decreased levels of cell death after METH and comparatively high viability, and lithium treatment did not increase viability any further. This novel NPC model of mania reveals differences in cell death that could help identify mechanisms of lithium response in BD.

Caveolin-1 Expression in the Dorsal Striatum Drives Methamphetamine Addiction-Like Behavior.

Dopamine D1 receptor (D1R) function is regulated by membrane/lipid raft-resident protein caveolin-1 (Cav1). We examined whether altered expression of Cav1 in the dorsal striatum would affect self-administration of methamphetamine, an indirect agonist at the D1Rs. A lentiviral construct expressing Cav1 (LV-Cav1) or containing a short hairpin RNA against Cav1 (LV-shCav1) was used to overexpress or knock down Cav1 expression respectively, in the dorsal striatum. Under a fixed-ratio schedule, LV-Cav1 enhanced and LV-shCav1 reduced responding for methamphetamine in an extended access paradigm compared to LV-GFP controls. LV-Cav1 and LV-shCav1 also produced an upward and downward shift in a dose-response paradigm, generating a drug vulnerable/resistant phenotype. LV-Cav1 and LV-shCav1 did not alter responding for sucrose. Under a progressive-ratio schedule, LV-shCav1 generally reduced positive-reinforcing effects of methamphetamine and sucrose as seen by reduced breakpoints. Western blotting confirmed enhanced Cav1 expression in LV-Cav1 rats and reduced Cav1 expression in LV-shCav1 rats. Electrophysiological findings in LV-GFP rats demonstrated an absence of high-frequency stimulation (HFS)-induced long-term potentiation (LTP) in the dorsal striatum after extended access methamphetamine self-administration, indicating methamphetamine-induced occlusion of plasticity. LV-Cav1 prevented methamphetamine-induced plasticity via increasing phosphorylation of calcium calmodulin kinase II, suggesting a mechanism for addiction vulnerability. LV-shCav1 produced a marked deficit in the ability of HFS to produce LTP and, therefore, extended access methamphetamine was unable to alter striatal plasticity, indicating a mechanism for resistance to addiction-like behavior. Our results demonstrate that Cav1 expression and knockdown driven striatal plasticity assist with modulating addiction to drug and nondrug rewards, and inspire new strategies to reduce psychostimulant addiction.

Sexually dimorphic prelimbic cortex mechanisms play a role in alcohol dependence: protection by endostatin.

Angiogenesis or proliferation of endothelial cells plays a role in brain microenvironment homeostasis. Previously we have shown enhanced expression of markers of angiogenesis in the medial prefrontal cortex during abstinence in an animal model of ethanol dependence induced by chronic intermittent ethanol vapor (CIE) and ethanol drinking (ED) procedure. Here we report that systemic injections of the angiogenesis inhibitor endostatin reduced relapse to drinking behavior in female CIE-ED rats without affecting relapse to drinking in male CIE-ED rats, and female and male nondependent ED rats. Endostatin did not alter relapse to sucrose drinking in both sexes. Endostatin reduced expression of platelet endothelial cell adhesion molecule-1 (PECAM-1) in all groups; however, rescued expression of tight junction protein claudin-5 in the prelimbic cortex (PLC) of female CIE-ED rats. In both sexes, CIE-ED enhanced microglial activation in the PLC and this was selectively prevented by endostatin in female CIE-ED rats. Endostatin prevented CIE-ED-induced enhanced NF-kB activity and expression and Fos expression in females and did not alter reduced Fos expression in males. Analysis of synaptic processes within the PLC revealed sexually dimorphic adaptations, with CIE-ED reducing synaptic transmission and altering synaptic plasticity in the PLC in females, and increasing synaptic transmission in males. Endostatin prevented the neuroadaptations in the PLC in females via enhancing phosphorylation of CaMKII, without affecting the neuroadaptations in males. Our multifaceted approach is the first to link PLC endothelial cell damage to the behavioral, neuroimmune, and synaptic changes associated with relapse to ethanol drinking in female subjects, and provides a new therapeutic strategy to reduce relapse in dependent subjects.

Chronic ethanol exposure differentially alters neuronal function in the medial prefrontal cortex and dentate gyrus.

Alterations in the function of prefrontal cortex (PFC) and hippocampus have been implicated in underlying the relapse to alcohol seeking behaviors in humans and animal models of moderate to severe alcohol use disorders (AUD). Here we used chronic intermittent ethanol vapor exposure (CIE), 21d protracted abstinence following CIE (21d AB), and re-exposure to one vapor session during protracted abstinence (re-exposure) to evaluate the effects of chronic ethanol exposure on basal synaptic function, neuronal excitability and expression of key synaptic proteins that play a role in neuronal excitability in the medial PFC (mPFC) and dentate gyrus (DG). CIE consistently enhanced excitability of layer 2/3 pyramidal neurons in the mPFC and granule cell neurons in the DG. In the DG, this effect persisted during 21d AB. Re-exposure did not enhance excitability, suggesting resistance to vapor-induced effects. Analysis of action potential kinetics revealed that altered afterhyperpolarization, rise time and decay time constants are associated with the altered excitability during CIE, 21d AB and re-exposure. Molecular adaptations that may underlie increases in neuronal excitability under these different conditions were identified. Quantitative polymerase chain reaction of large-conductance potassium (BK) channel subunit mRNA in PFC and DG tissue homogenates did not show altered expression patterns of BK subunits. Western blotting demonstrates enhanced phosphorylation of Ca2⁺/calmodulin-dependent protein kinase II (CaMKII), and reduced phosphorylation of glutamate receptor GluN2A/2B subunits. These results suggest a novel relationship between activity of CaMKII and GluN receptors in the mPFC and DG, and neuronal excitability in these brain regions in the context of moderate to severe AUD.

SCH23390 Reduces Methamphetamine Self-Administration and Prevents Methamphetamine-Induced Striatal LTD.

Extended-access methamphetamine self-administration results in unregulated intake of the drug; however, the role of dorsal striatal dopamine D1-like receptors (D1Rs) in the reinforcing properties of methamphetamine under extended-access conditions is unclear. Acute (ex vivo) and chronic (in vivo) methamphetamine exposure induces neuroplastic changes in the dorsal striatum, a critical region implicated in instrumental learning. For example, methamphetamine exposure alters high-frequency stimulation (HFS)-induced long-term depression in the dorsal striatum; however, the effect of methamphetamine on HFS-induced long-term potentiation (LTP) in the dorsal striatum is unknown. In the current study, dorsal striatal infusion of SCH23390, a D1R antagonist, prior to extended-access methamphetamine self-administration reduced methamphetamine addiction-like behavior. Reduced behavior was associated with reduced expression of PSD-95 in the dorsal striatum. Electrophysiological findings demonstrate that superfusion of methamphetamine reduced basal synaptic transmission and HFS-induced LTP in dorsal striatal slices, and SCH23390 prevented this effect. These results suggest that alterations in synaptic transmission and synaptic plasticity induced by acute methamphetamine via D1Rs could assist with methamphetamine-induced modification of corticostriatal circuits underlying the learning of goal-directed instrumental actions and formation of habits, mediating escalation of methamphetamine self-administration and methamphetamine addiction-like behavior.

A Novel Sulfonamide, 4-FS, Reduces Ethanol Drinking and Physical Withdrawal Associated With Ethanol Dependence.

Carbonic anhydrase (CA) is abundant in glial cells in the brain and CA type II isoform (CA II) activity in the hippocampus plays an important role in buffering extracellular pH transients produced by neural activity. Chronic ethanol exposure results in respiratory and metabolic acidosis, producing shifts in extracellular pH in the brain and body. These neurophysiological changes by ethanol are hypothesized to contribute to the continued drinking behavior and physical withdrawal behavior in subjects consuming ethanol chronically. We explored whether chronic ethanol self-administration (ethanol drinking, 10% v/v; ED) without or under the influence of chronic intermittent ethanol vapor (CIE-ED) experience alters the expression of CA II in the hippocampus. Postmortem hippocampal tissue analyses demonstrated that CA II levels were enhanced in the hilus region of the hippocampus in ED and CIE-ED rats. We used a novel molecule-4-fluoro-N-(4-sulfamoylphenyl) benzenesulfonamide (4-FS)-a selective CA II inhibitor, to determine whether CA II plays a role in ethanol self-administration in ED and CIE-ED rats and physical withdrawal behavior in CIE-ED rats. 4-FS (20 mg/kg, i.p.) reduced ethanol self-administration in ED rats and physical withdrawal behavior in CIE-ED rats. Postmortem hippocampal tissue analyses demonstrated that 4-FS reduced CA II expression in ED and CIE-ED rats to control levels. In parallel, 4-FS enhanced GABAA receptor expression, reduced ratio of glutamatergic GluN2A/2B receptors and enhanced the expression of Fos, a marker of neuronal activation in the ventral hippocampus in ED rats. These findings suggest that 4-FS enhanced GABAergic transmission and increased activity of neurons of inhibitory phenotypes. Taken together, these findings support the role of CA II in assisting with negative affective behaviors associated with moderate to severe alcohol use disorders (AUD) and that CA II inhibitors are a potential therapeutic target to reduce continued drinking and somatic withdrawal symptoms associated with moderate to severe AUD.