Julia Morud

Changes in glycine receptor subunit expression in forebrain regions of the Wistar rat over development.

Glycine receptors (GlyRs) are pentameric membrane proteins in the form of either α-homomers or α-β heteromers. Four out of five subunits; α1-3 and β, have been found in the mammalian brain. Early studies investigating subunit composition and expression patterns of this receptor have proposed a developmental switch from α2 homomers to α1β heteromers as the CNS matures, a conclusion primarily based on results from the spinal cord. However, our previous results indicate that this might not apply to e.g. the forebrain regions. Here we examined alterations in GlyR expression caused by developmental changes in selected brain areas, focusing on reward-related regions. Animals of several ages (P2, P21 and P60) were included to examine potential changes over time. In accordance with previous reports, a switch in expression was observed in the spinal cord. However, the present results indicate that a decrease in α2 subunit expression is not replaced by α1 subunit expression since the generally low levels, and modest increases, of α1 could hardly replace the reduction in α2-mRNA. Instead mRNA measurements indicate that α2 continues to be the dominating α-subunit also in adult animals, usually in combination with high and stable levels of β-subunit expression. This indicates that alterations in GlyR subunit expression are not simply a maturation effect common for the entire CNS, but rather a unique pattern of transition depending on the region at hand.

Genetic Variation in COMT Activity Impacts Learning and Dopamine Release Capacity in the Striatum.

A common genetic polymorphism that results in increased activity of the dopamine regulating enzyme COMT (the COMT Val(158) allele) has been found to associate with poorer cognitive performance and increased susceptibility to develop psychiatric disorders. It is generally assumed that this increase in COMT activity influences cognitive function and psychiatric disease risk by increasing dopamine turnover in cortical synapses, though this cannot be directly measured in humans. Here we explore a novel transgenic mouse model of increased COMT activity, equivalent to the relative increase in activity observed with the human COMT Val(158) allele. By performing an extensive battery of behavioral tests, we found that COMT overexpressing mice (COMT-OE mice) exhibit cognitive deficits selectively in the domains that are affected by the COMT Val(158) allele, stimulus-response learning and working memory, functionally validating our model of increased COMT activity. Although we detected no changes in the level of markers for dopamine synthesis and dopamine transport, we found that COMT-OE mice display an increase in dopamine release capacity in the striatum. This result suggests that increased COMT activity may not only affect dopamine signaling by enhancing synaptic clearance in the cortex, but may also cause changes in presynaptic dopamine function in the striatum. These changes may underlie the behavioral deficits observed in the mice and might also play a role in the cognitive deficits and increased psychiatric disease risk associated with genetic variation in COMT activity in humans.

Ethanol impairment of spontaneous alternation behaviour and associated changes in medial prefrontal glutamatergic gene expression precede putative markers of dependence.

Cognitive impairments are observable in over half of cases with alcoholism, deficits in spatial working memory being particularly common. Previously we observed that rats make more alternation errors in a Y-maze test of spontaneous alternation behaviour/spatial working memory after 5-day intermittent ethanol. Here we used qPCR to quantify changes in gene expression accompanying this behavioural impairment. Male Wistar rats were treated with either saline or ethanol (1 or 2.5g/kg) for 5days followed by 2 drug-free days. Brains were dissected after Y-maze analysis and RNA was extracted from the medial prefrontal cortex, hippocampus and nucleus accumbens. Using the Qiagen GABA & Glutamate PCR array we measured changes in these two neurotransmitter systems. A dose of 1g/kg ethanol did not affect spontaneous alternation behaviour or any other behavioural variable. 2.5g/kg significantly decreased % correct alternations (p=0.028) without affecting total distance (p=0.54) and increased time in the choice area (p=0.023) at the Y-maze centre, indicating a possible impairment in decision-making. In the medial prefrontal cortex, 2.5g/kg ethanol decreased mRNA expression of brain-derived neurotrophic factor, NMDA NR2A subunit, mGluR8 receptor, Homer1, the glutamate transporters SLC1a1 and SLC1a6 and Srr. In the nucleus accumbens this dose did not affect mRNA expression of the dopamine D1 or D2 receptors but did upregulate the GABA transporter GAT-3. Even if only correlational, these data suggest that gene expression changes in the medial prefrontal cortex and associated cognitive impairment occur before adaptation of the dopaminergic system and, presumably, drug dependence.

Nicotine produces chronic behavioral sensitization with changes in accumbal neurotransmission and increased sensitivity to re-exposure.

Tobacco use is often associated with long‐term addiction as well as high risk of relapse following cessation. This is suggestive of persistent neural adaptations, but little is known about the long‐lasting effects of nicotine on neural circuits. In order to investigate the long‐term effects of nicotine exposure, Wistar rats were treated for 3 weeks with nicotine (0.36 mg/kg), and the duration of behavioral and neurophysiological adaptations was evaluated 7 months later. We found that increased drug‐induced locomotion persisted 7 months after the initial behavioral sensitization. In vitro analysis of synaptic activity in the core and shell of the nucleus accumbens (nAc) revealed a decrease in input/output function in both regions of nicotine‐treated rats as compared to vehicle‐treated control rats. In addition, administration of the dopamine D2 receptor agonist quinpirole (5 μM) significantly increased evoked population spike amplitude in the nAc shell of nicotine‐treated rats as compared to vehicle‐treated control rats. To test whether nicotine exposure creates long‐lasting malleable circuits, animals were re‐exposed to nicotine 7 months after the initial exposure. This treatment revealed an increased sensitivity to nicotine among animals previously exposed to nicotine, with higher nicotine‐induced locomotion responses than observed initially. In vitro electrophysiological recordings in re‐exposed rats detected an increased sensitivity to dopamine D2 receptor activation. These results suggest that nicotine produces persistent neural adaptations that make the system sensitive and receptive to future nicotine re‐exposure.

Alterations in ethanol‐induced accumbal transmission after acute and long‐term zinc depletion

Alcoholism is subject to extensive research, but the role of changes in metabolism caused by alcohol consumption has been poorly investigated. Zinc (Zn2+) deficiency is a common metabolic aberration among alcoholics and Zn2+ influences the function of ligand‐gated ion channels, known pharmacological targets of ethanol (EtOH). Here, we investigate whether manipulation of extracellular levels of Zn2+ modulates EtOH‐induced increases of dopamine (DA) output, as measured by in vivo microdialysis in the rat, and whether voluntary EtOH consumption is altered by Zn2+ deficiency. Our findings show that the Zn2+‐chelating agent tricine slowly raises DA levels when perfused in the nucleus accumbens (nAc), whereas the more potent Zn2+ chelator TPEN reduces DA levels. We also show that pre‐treatment with either tricine or TPEN blocks the EtOH‐induced DA elevation. Chronic Zn2+ deficiency induced by a Zn2+‐free diet did not affect EtOH consumption, but excitatory transmission, assessed by striatal field‐potential recordings in the nAc shell, was significantly modulated both by Zn2+‐free diet and by EtOH consumption, as compared with the EtOH naïve controls. The present study indicates that Zn2+ influences EtOHs interaction with the brain reward system, possibly by interfering with glycine receptor and GABAA receptor function. This also implies that Zn2+ deficiency among alcoholics may be important to correct in order to normalize important aspects of brain function.

Temporal Rewiring of Striatal Circuits Initiated by Nicotine.

Drug addiction has been conceptualized as maladaptive recruitment of integrative circuits coursing through the striatum, facilitating drug-seeking and drug-taking behavior. The aim of this study was to define temporal neuroadaptations in striatal subregions initiated by 3 weeks of intermittent nicotine exposure followed by protracted abstinence. Enhanced rearing activity was assessed in motor activity boxes as a measurement of behavioral change induced by nicotine (0.36 mg/kg), whereas electrophysiological field potential recordings were performed to evaluate treatment effects on neuronal activity. Dopamine receptor mRNA expression was quantified by qPCR, and nicotine-induced dopamine release was measured in striatal subregions using in vivo microdialysis. Golgi staining was performed to assess nicotine-induced changes in spine density of medium spiny neurons. The data presented here show that a brief period of nicotine exposure followed by abstinence leads to temporal changes in synaptic efficacy, dopamine receptor expression, and spine density in a subregion-specific manner. Nicotine may thus initiate a reorganization of striatal circuits that continues to develop despite protracted abstinence. We also show that the response to nicotine is modulated in previously exposed rats even after 6 months of abstinence. The data presented here suggests that, even though not self-administered, nicotine may produce progressive neuronal alterations in brain regions associated with goal-directed and habitual performance, which might contribute to the development of compulsive drug seeking and the increased vulnerability to relapse, which are hallmarks of drug addiction.

Involvement of lateral septum in alcohol's dopamine-elevating effect in the rat

Drugs of abuse share the ability to increase extracellular dopamine (DA) levels in the mesolimbic DA system. This effect has been linked to positive and reinforcing experiences of drug consumption and is presumed to be of importance for continued use, as well as for the development of dependence and addiction. Previous rat studies from our lab have implicated a neuronal circuitry involving glycine receptors in nucleus accumbens (nAc) and, secondarily, nicotinic acetylcholine receptors in the ventral tegmental area (VTA) in ethanols (EtOH) DA‐elevating effect. The work presented here, performed in male Wistar rats, suggests that the lateral septum (LS), which has previously been associated with different aspects of EtOH‐related behaviour, is involved as well. In vivo microdialysis methodology demonstrated that blocking the generation of action potentials in LS using tetrodotoxin prevented a DA increase in nAc after accumbal EtOH perfusion. Retrograde tracing and polymerase chain reaction (PCR) were used to identify and characterize cells projecting to VTA from nAc/LS and from LS to nAc. Based on the PCR results, cells projecting from both LS/nAc to anterior VTA and from LS to nAc were mainly GABAergic neurons expressing glycine receptors, and these cells are presumed to be involved in mediating the DA‐elevating effect of EtOH. These results provide further evidence implicating LS in the reinforcing effects of EtOH. Additional studies are needed to investigate LS involvement in EtOH consumption behaviour and its potential role in the development of dependence and addiction.

Transcriptional profiling of the rat nucleus accumbens after modest or high alcohol exposure.

Alcohol use disorder is a chronic relapsing brain disorder and a global health issue. Prolonged high alcohol consumption increases the risk for dependence development, a complex state that includes progressive alterations in brain function. The molecular mechanisms behind these changes remain to be fully disclosed, but several genes show altered expression in various regions of the rat brain even after modest alcohol exposure. The present study utilizes whole-transcriptome sequencing (RNA-seq) to investigate expression changes in the brain nucleus accumbens (NAc), an area of particular interest in addictive disorders, of alcohol consuming rats. The impact on gene expression after eight weeks of moderate voluntary alcohol consumption or voluntary consumption combined with forced excessive exposure was explored in two separate experiments. The results point to a lack of strong and consistent expression alterations in the NAc after alcohol exposure, suggesting that transcriptional effects of alcohol are weak or transient, or occur primarily in brain regions other than NAc.

Progressive modulation of accumbal neurotransmission and anxiety-like behavior following protracted nicotine withdrawal

ABSTRACT Due to the highly addictive properties of nicotine, a low percentage of users successfully maintain cessation for longer periods of time. This might be linked to neuroadaptations elicited by the drug, and understanding progressive changes in neuronal function might provide critical insight into nicotine addiction. We have previously shown that neurotransmission in the nucleus accumbens (nAc), a key brain region with respect to drug reinforcement and relapse, is suppressed for as long as seven months after a brief period of nicotine treatment. Studies were therefore performed to define the temporal properties of these effects, and to assess behavioral correlates to altered neurotransmission. Ex vivo electrophysiology revealed progressive depression of synaptic efficacy in the nAc of rats previously receiving nicotine. In addition, following three months of nicotine withdrawal, the responses to GABAA receptor modulating drugs were blunted together with downregulation of several GABAA receptor subunits. In correlation to reduced accumbal neurotransmission, a reduced anxiety‐like behavior; assessed in the elevated plus‐maze and marble burying tests, were identified in animals pre‐treated with nicotine. Lastly, to test the causal relationship between suppressed excitability in the nAc and reduced anxiety‐like behavior, rats received local administration of diazepam in the nAc while monitoring behavioral effects on the elevated plus‐maze. These results show that nicotine produces long‐lasting changes in the GABAergic system, which are observed first after extended withdrawal. Our data also suggest that nicotine produces a progressive suppression of accumbal excitability, which could result in behavioral alterations that may have implications for further drug intake. HIGHLIGHTSProtracted nicotine abstinence induces progressive changes in accumbal neurotransmission.Temporal‐dependent changes were detected in GABAA neurotransmission and gene expression.Seven months of abstinence appears to reverse GABAA receptor signaling properties.Changes in neurotransmission correlates with altered anxiety‐like behavior.Anxiety‐like behaviors can be manipulated by altering GABAergic transmission.

Acute and chronic modulation of striatal endocannabinoid-mediated plasticity by nicotine: Nicotine modulates eCB-LTD

The endocannabinoid (eCB) system modulates several phenomena related to addictive behaviors, and drug‐induced changes in eCB signaling have been postulated to be important mediators of physiological and pathological reward‐related synaptic plasticity. Here, we studied eCB‐mediated long‐term depression (eCB‐LTD) in the dorsolateral striatum, a brain region critical for acquisition of habitual and automatic behavior. We report that nicotine differentially affects ex vivo eCB signaling depending on previous exposure in vivo. In the nicotine‐naïve brain, nicotine facilitates eCB‐signaling and LTD, whereas tolerance develops to this facilitating effect after subchronic exposure in vivo. In the end, a progressive impairment of eCB‐induced LTD is established after protracted withdrawal from nicotine. Endocannabinoid‐LTD is reinstated 6 months after the last drug injection, but a brief period of nicotine re‐exposure is sufficient to yet again impair eCB‐signaling. LTD induced by the cannabinoid 1 receptor agonist WIN55,212‐2 is not affected, suggesting that nicotine modulates eCB production or release. Nicotine‐induced facilitation of eCB‐LTD is occluded by the dopamine D2 receptor agonist quinpirole, and by the muscarinic acetylcholine receptor antagonist scopolamine. In addition, the same compounds restore eCB‐LTD during protracted withdrawal. Nicotine may thus modulate eCB‐signaling by affecting dopaminergic and cholinergic neurotransmission in a long‐lasting manner. Overall, the data presented here suggest that nicotine facilitates eCB‐LTD in the initial phase, which putatively could promote neurophysiological and behavioral adaptations to the drug. Protracted withdrawal, however, impairs eCB‐LTD, which may influence or affect the ability to maintain cessation.