Dennis R. Sparta

Excitatory transmission from the amygdala to nucleus accumbens facilitates reward seeking

The basolateral amygdala (BLA) has a crucial role in emotional learning irrespective of valence. The BLA projection to the nucleus accumbens (NAc) is thought to modulate cue-triggered motivated behaviours, but our understanding of the interaction between these two brain regions has been limited by the inability to manipulate neural-circuit elements of this pathway selectively during behaviour. To circumvent this limitation, we used in vivo optogenetic stimulation or inhibition of glutamatergic fibres from the BLA to the NAc, coupled with intracranial pharmacology and ex vivo electrophysiology. Here we show that optical stimulation of the pathway from the BLA to the NAc in mice reinforces behavioural responding to earn additional optical stimulation of these synaptic inputs. Optical stimulation of these glutamatergic fibres required intra-NAc dopamine D1-type receptor signalling, but not D2-type receptor signalling. Brief optical inhibition of fibres from the BLA to the NAc reduced cue-evoked intake of sucrose, demonstrating an important role of this specific pathway in controlling naturally occurring reward-related behaviour. Moreover, although optical stimulation of glutamatergic fibres from the medial prefrontal cortex to the NAc also elicited reliable excitatory synaptic responses, optical self-stimulation behaviour was not observed by activation of this pathway. These data indicate that whereas the BLA is important for processing both positive and negative affect, the glutamatergic pathway from the BLA to the NAc, in conjunction with dopamine signalling in the NAc, promotes motivated behavioural responding. Thus, optogenetic manipulation of anatomically distinct synaptic inputs to the NAc reveals functionally distinct properties of these inputs in controlling reward-seeking behaviours.

Distinct extended amygdala circuits for divergent motivational states

The co-morbidity of anxiety and dysfunctional reward processing in illnesses such as addiction and depression suggests that common neural circuitry contributes to these disparate neuropsychiatric symptoms. The extended amygdala, including the bed nucleus of the stria terminalis (BNST), modulates fear and anxiety, but also projects to the ventral tegmental area (VTA), a region implicated in reward and aversion, thus providing a candidate neural substrate for integrating diverse emotional states. However, the precise functional connectivity between distinct BNST projection neurons and their postsynaptic targets in the VTA, as well as the role of this circuit in controlling motivational states, have not been described. Here we record and manipulate the activity of genetically and neurochemically identified VTA-projecting BNST neurons in freely behaving mice. Collectively, aversive stimuli exposure produced heterogeneous firing patterns in VTA-projecting BNST neurons. By contrast, in vivo optically identified glutamatergic projection neurons displayed a net enhancement of activity to aversive stimuli, whereas the firing rate of identified GABAergic (γ-aminobutyric acid-containing) projection neurons was suppressed. Channelrhodopsin-2-assisted circuit mapping revealed that both BNST glutamatergic and GABAergic projections preferentially innervate postsynaptic non-dopaminergic VTA neurons, thus providing a mechanistic framework for in vivo circuit perturbations. In vivo photostimulation of BNST glutamatergic projections resulted in aversive and anxiogenic behavioural phenotypes. Conversely, activation of BNST GABAergic projections produced rewarding and anxiolytic phenotypes, which were also recapitulated by direct inhibition of VTA GABAergic neurons. These data demonstrate that functionally opposing BNST to VTA circuits regulate rewarding and aversive motivational states, and may serve as a crucial circuit node for bidirectionally normalizing maladaptive behaviours.

Construction of implantable optical fibers for long-term optogenetic manipulation of neural circuits

In vivo optogenetic strategies have redefined our ability to assay how neural circuits govern behavior. Although acutely implanted optical fibers have previously been used in such studies, long-term control over neuronal activity has been largely unachievable. Here we describe a method to construct implantable optical fibers to readily manipulate neural circuit elements with minimal tissue damage or change in light output over time (weeks to months). Implanted optical fibers readily interface with in vivo electrophysiological arrays or electrochemical detection electrodes. The procedure described here, from implant construction to the start of behavioral experimentation, can be completed in approximately 2–6 weeks. Successful use of implantable optical fibers will allow for long-term control of mammalian neural circuits in vivo, which is integral to the study of the neurobiology of behavior.

Motivation for Alcohol Becomes Resistant to Quinine Adulteration After 3 to 4 Months of Intermittent Alcohol Self-Administration

BACKGROUND Continued consumption of alcohol despite deleterious consequences is a hallmark of alcoholism and represents a critical challenge to therapeutic intervention. Previous rat studies showed that enduring alcohol self-administration despite pairing alcohol with normally aversive stimuli was only observed after very long-term intake (>8 months). Aversion-resistant alcohol intake has been previously interpreted to indicate pathological or compulsive motivation to consume alcohol. However, given the time required to model compulsive alcohol seeking in previous studies, there is considerable interest in developing more efficient and quantitative rodent models of aversion-resistant alcohol self-administration. METHODS Outbred Wistar rats underwent 3 to 4 months or approximately 1.5 months of intermittent, home-cage, two-bottle access (IAA) to 20% alcohol (v/v) or water. Then, after brief operant training, the effect of the bitter-tasting quinine (0.1 g/l) on the motivation to seek alcohol was quantified via progressive ratio (PR). Motivation for quinine-adulterated 2% sucrose under PR was assayed in a separate cohort of 3 to 4 months IAA rats. The effects of quinine on home-cage alcohol consumption in IAA rats and rats with continuous access to alcohol were also examined. Finally, a dose-response for quinine taste preference in IAA and continuous-access animals was determined. RESULTS Motivation for alcohol after 3 to 4 months IAA, measured using an operant PR procedure, was not altered by adulteration of alcohol with 0.1 g/l quinine. In contrast, after 3 to 4 months of IAA, motivation for sucrose under PR was significantly reduced by adulteration of sucrose with 0.1 g/l quinine. In addition, motivation for alcohol after only approximately 1.5 months IAA was significantly reduced by adulteration of alcohol with 0.1 g/l quinine. Furthermore, home-cage alcohol intake by IAA rats was insensitive to quinine at concentrations (0.01, 0.03 g/l) that significantly reduced alcohol drinking in animals with continuous access to alcohol. Finally, no changes in quinine taste preference after 3 to 4 months IAA or continuous access to alcohol were observed. CONCLUSIONS We have developed a novel and technically simple hybrid operant/IAA model in which quinine-resistant motivation for alcohol is evident after an experimentally tractable period of time (3 to 4 months vs. 8 months). Quinine dramatically reduced sucrose and water intake by IAA rats, indicating that continued responding for alcohol in IAA rats despite adulteration with the normally aversive quinine might reflect maladaptive or compulsive motivation for alcohol. This model could facilitate identification of novel therapeutic interventions for pathological alcohol seeking in humans.

Alcoholism and obesity: overlapping neuropeptide pathways?

Ethanol is a caloric compound, and ethanol drinking and food intake are both appetitive and consummatory behaviors. Furthermore, both ethanol and food have rewarding properties. It is therefore possible that overlapping central pathways are involved with uncontrolled eating and excessive ethanol consumption. A growing list of peptides has been shown to regulate food intake and/or energy homeostasis. Peptides such as the melanocortins, corticotropin releasing factor, and cholecystokinin promote reductions of food intake while others such as galanin and neuropeptide Y stimulate feeding. The present review highlights research aimed at determining if ingestive peptides also regulate voluntary ethanol intake, with an emphasis on the melanocortins and neuropeptide Y. It is suggested that research directed at ingestive peptides may expand our understanding of the neurobiological mechanisms that drive ethanol self-administration, and may reveal new therapeutic candidates for treating alcohol abuse and alcoholism.

A role for neuropeptide Y in neurobiological responses to ethanol and drugs of abuse

In recent years, evidence has emerged suggesting that neuropeptide Y (NPY) is involved with neurobiological responses to ethanol and other drugs of abuse. Here, we provide an overview of physiological, pharmacological, and genetic research showing that: (A) administration of ethanol, as well as ethanol withdrawal, alter central NPY expression, (B) NPY modulates ethanol consumption under certain conditions, and (C) NPY signaling modulates the sedative effects of several drugs, including ethanol, sodium pentobarbital, and ketamine. Evidence suggesting possible mechanism(s) by which NPY signaling modulates ethanol consumption are considered. It is suggested that NPY may influence ethanol consumption by regulating basal levels of anxiety, by modulating the sedative effects of ethanol, and/or by modulating ethanols rewarding properties.

Effects of Food Availability and Administration of Orexigenic and Anorectic Agents on Elevated Ethanol Drinking Associated With Drinking in the Dark Procedures

BACKGROUND Drinking in the dark (DID) procedures have recently been developed to induce high levels of ethanol drinking in C57BL/6J mice, which result in blood ethanol concentrations reaching levels that have measurable affects on physiology and/or behavior. The present study determined if increased ethanol drinking associated with DID procedures may be motivated by caloric need rather than by the postingestive pharmacological effects of ethanol. To this end, food availability was manipulated or mice were given peripheral administration of orexigenic or anorectic agents during DID procedures. METHODS C57BL/6J had 2-hours of access to the 20% (v/v) ethanol solution beginning 3-hours into the dark cycle on days 1 to 3, and 4-hours of access to the ethanol bottle on day 4 of DID procedures. In Experiment 1, the effects of food deprivation on ethanol consumption during DID procedures was assessed. In Experiments 2 and 3, mice were given intraperitoneal (i.p.) injection of the orexigenic peptide ghrelin (0, 10 or 30 mg/kg) or the anorectic protein leptin (0 or 20 microg/g), respectively, before access to ethanol on day 4 of DID procedures. In Experiment 4, hourly consumption of food and a 0.05% saccharin solution were assessed over a period of hours that included those used with DID procedures. RESULTS Consistent with previous research, mice achieved blood ethanol concentrations (BECs) that ranged between 100 and 150 mg% on day 4 of DID experiments. Neither food deprivation nor administration of orexigenic or anorectic compounds significantly altered ethanol drinking with DID procedures. Interestingly, mice exhibited their highest level of food and saccharin solution consumption during hours that overlapped with DID procedures. CONCLUSIONS The present observations are inconsistent with the hypothesis that C57BL/6J mice consume large amounts of ethanol during DID procedures in order to satisfy a caloric need.

Optogenetic strategies to investigate neural circuitry engaged by stress

Optogenetic techniques have given researchers unprecedented access to the function of discrete neural circuit elements and have been instrumental in the identification of novel brain pathways that become dysregulated in neuropsychiatric diseases. For example, stress is integrally linked to the manifestation and pathophysiology of neuropsychiatric illness, including anxiety, addiction and depression. Due to the heterogeneous populations of genetically and neurochemically distinct neurons in areas such as the bed nucleus of the stria terminalis (BNST), as well as their substantial number of projections, our understanding of how neural circuits become disturbed after stress has been limited. Using optogenetic tools, we are now able to selectively isolate distinct neural circuits that contribute to these disorders and perturb these circuits in vivo, which in turn may lead to the normalization of maladaptive behavior. This review will focus on current optogenetic strategies to identify, manipulate, and record from discrete neural circuit elements in vivo as well as highlight recent optogenetic studies that have been utilized to parcel out BNST function.

Strain Specific Synaptic Modifications on Ventral Tegmental Area Dopamine Neurons After Ethanol Exposure

BACKGROUND Genetic factors and previous alcohol experience influence alcohol consumption in both humans and rodents. Specifically, a prior experience with ethanol increases ethanol intake in both ethanol-preferring C57BL/6 (C57) and ethanol non-preferring DBA/2 (DBA) mice. Whereas the ventral tegmental area (VTA) importantly regulates dopamine levels and ethanol intake, it is unknown whether ethanol experience differentially alters synaptic properties of VTA dopamine neurons in ethanol-preferring and non-preferring mice. METHODS The properties of excitatory and inhibitory inputs and the ability to elicit long-term potentiation (LTP) were assessed with whole-cell patch-clamp recordings in VTA dopamine neurons from C57 and DBA mice 24 hours after a single ethanol (2 g/kg, IP) or equivalent saline injection. RESULTS Ethanol exposure increased gamma-aminobutyric acid (GABA) release onto VTA dopamine neurons in DBA mice, as previously observed in C57 mice. However, a single ethanol exposure reduced alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) function and LTP in VTA dopamine neurons from DBA but not C57 mice. CONCLUSIONS A single ethanol exposure selectively reduced glutamate receptor function in VTA dopamine neurons from the ethanol non-preferring DBA strain but enhanced GABA signaling in both C57 and DBA strains. These results support the notion that VTA dopamine neurons are a central target of ethanol-induced neural plasticity, which could contribute to ethanol consumption. Furthermore, these findings highlight the possible need for specialized therapeutic interventions for alcoholism based on individual intrinsic differences.

Inhibition of projections from the basolateral amygdala to the entorhinal cortex disrupts the acquisition of contextual fear

The development of excessive fear and/or stress responses to environmental cues such as contexts associated with a traumatic event is a hallmark of post-traumatic stress disorder (PTSD). The basolateral amygdala (BLA) has been implicated as a key structure mediating contextual fear conditioning. In addition, the hippocampus has an integral role in the encoding and processing of contexts associated with strong, salient stimuli such as fear. Given that both the BLA and hippocampus play an important role in the regulation of contextual fear conditioning, examining the functional connectivity between these two structures may elucidate a role for this pathway in the development of PTSD. Here, we used optogenetic strategies to demonstrate that the BLA sends a strong glutamatergic projection to the hippocampal formation through the entorhinal cortex (EC). Next, we photoinhibited glutamatergic fibers from the BLA terminating in the EC during the acquisition or expression of contextual fear conditioning. In mice that received optical inhibition of the BLA-to-EC pathway during the acquisition session, we observed a significant decrease in freezing behavior in a context re-exposure session. In contrast, we observed no differences in freezing behavior in mice that were only photoinhibited during the context re-exposure session. These data demonstrate an important role for the BLA-to-EC glutamatergic pathway in the acquisition of contextual fear conditioning.