Knockdown of Tlr3 in dorsal striatum reduces ethanol consumption and acute functional tolerance in male mice.

Systemic activation of toll-like receptor 3 (TLR3) signaling using poly(I:C), a TLR3 agonist, drives ethanol consumption in several rodent models, while global knockout of Tlr3 reduces drinking in C57BL/6J male mice. To determine if brain TLR3 pathways are involved in drinking behavior, we used CRISPR/Cas9 genome editing to generate a Tlr3 floxed (Tlr3F/F) mouse line. After sequence confirmation and functional validation of Tlr3 brain transcripts, we injected Tlr3F/F male mice with an adeno-associated virus expressing Cre recombinase (AAV5-CMV-Cre-GFP) to knockdown Tlr3 in the medial prefrontal cortex, nucleus accumbens, or dorsal striatum (DS). Only Tlr3 knockdown in the DS decreased two-bottle choice, every-other-day (2BC-EOD) ethanol consumption. DS-specific deletion of Tlr3 also increased intoxication and prevented acute functional tolerance to ethanol. In contrast, poly(I:C)-induced activation of TLR3 signaling decreased intoxication in male C57BL/6J mice, consistent with its ability to increase 2BC-EOD ethanol consumption in these mice. We also found that TLR3 was highly colocalized with DS neurons. AAV5-Cre transfection occurred predominantly in neurons, but there was minimal transfection in astrocytes and microglia. Collectively, our previous and current studies show that activating or inhibiting TLR3 signaling produces opposite effects on acute responses to ethanol and on ethanol consumption. While previous studies, however, used global knockout or systemic TLR3 activation (which alter peripheral and brain innate immune responses), the current results provide new evidence that brain TLR3 signaling regulates ethanol drinking. We propose that activation of TLR3 signaling in DS neurons increases ethanol consumption and that a striatal TLR3 pathway is a potential target to reduce excessive drinking.

Cell-type specific changes in PKC-delta neurons of the central amygdala during alcohol withdrawal.

The central amygdala (CeA) contains a diverse population of cells, including multiple subtypes of GABAergic neurons, along with glia and epithelial cells. Specific CeA cell types have been shown to affect alcohol consumption in animal models of dependence and may be involved in negative affect during alcohol withdrawal. We used single-nuclei RNA sequencing to determine cell-type specificity of differential gene expression in the CeA induced by alcohol withdrawal. Cells within the CeA were classified using unbiased clustering analyses and identified based on the expression of known marker genes. Differential gene expression analysis was performed on each identified CeA cell-type. It revealed differential gene expression in astrocytes and GABAergic neurons associated with alcohol withdrawal. GABAergic neurons were further subclassified into 13 clusters of cells. Analyzing transcriptomic responses in these subclusters revealed that alcohol exposure induced multiple differentially expressed genes in one subtype of CeA GABAergic neurons, the protein kinase C delta (PKCδ) expressing neurons. These results suggest that PKCδ neurons in the CeA may be uniquely sensitive to the effects of alcohol exposure and identify a novel population of cells in CeA associated with alcohol withdrawal.

Medial Prefrontal Cortical Dopamine Responses During Operant Self-Administration of Sweetened Ethanol.

BACKGROUND: Medial prefrontal cortex (mPFC) dysfunction is present in heavy alcohol consumers. Dopamine signaling in mPFC is associated with executive functioning and affects drinking behavior; however, direct measurement of extracellular mPFC dopamine during appetitive and consummatory ethanol (EtOH) self-administration behavior has not been reported. METHODS: We used in vivo microdialysis in freely behaving, adult, male, Long Evans rats to determine extracellular dopamine concentration in the mPFC during operant self-administration of an EtOH-plus-sucrose or sucrose solution. The model separated appetitive/seeking from consummatory phases of the operant session. Dopamine was also monitored in an untrained handling control group, and dialysate EtOH was measured in the EtOH-drinking group. RESULTS: Home cage baseline dopamine was lower in rats that experienced a week of drinking sweetened EtOH compared with sucrose-drinking and handling controls. Transfer into the operant chamber and the initiation of consumption stimulated a relatively higher change in dopamine over baseline in the sweetened EtOH group compared with sucrose and handling controls. However, all groups show a dopamine response during transfer into the operant chamber, and the sucrose group had a relatively higher change in dopamine over baseline during initiation of consumption compared with handling controls. The time courses of dopamine and EtOH in the mPFC differ in the EtOH-consuming rats. CONCLUSIONS: Differences in extracellular mPFC dopamine between EtOH drinkers compared with control groups suggest that mPFC dopamine is involved in the mechanism of operant self-administration of sweetened EtOH and sucrose. Furthermore, the increase in dopamine during consumption is consistent with a role of mPFC dopamine in reward prediction.

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC).

Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer's disease, and autism spectrum disorder-neurological disorders that exhibit elevated mTORC1 activity. Through a protein-protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson's disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states.

Inhibitory Gating of Input Comparison in the CA1 Microcircuit.

Spatial and temporal features of synaptic inputs engage integration mechanisms on multiple scales, including presynaptic release sites, postsynaptic dendrites, and networks of inhibitory interneurons. Here we investigate how these mechanisms cooperate to filter synaptic input in hippocampal area CA1. Dendritic recordings from CA1 pyramidal neurons reveal that proximal inputs from CA3 as well as distal inputs from entorhinal cortex layer III (ECIII) sum sublinearly or linearly at low firing rates due to feedforward inhibition, but sum supralinearly at high firing rates due to synaptic facilitation, producing a high-pass filter. However, during ECIII and CA3 input comparison, supralinear dendritic integration is dynamically balanced by feedforward and feedback inhibition, resulting in suppression of dendritic complex spiking. We find that a particular subpopulation of CA1 interneurons expressing neuropeptide Y (NPY) contributes prominently to this dynamic filter by integrating both ECIII and CA3 input pathways and potently inhibiting CA1 pyramidal neuron dendrites.

Local Integration Accounts for Weak Selectivity of Mouse Neocortical Parvalbumin Interneurons.

Dissecting the functional roles of excitatory and inhibitory neurons in cortical circuits is a fundamental goal in neuroscience. Of particular interest are their roles in emergent cortical computations such as binocular integration in primary visual cortex (V1). We measured the binocular response selectivity of genetically defined subpopulations of excitatory and inhibitory neurons. Parvalbumin (PV+) interneurons received strong inputs from both eyes but lacked selectivity for binocular disparity. Because broad selectivity could result from heterogeneous synaptic input from neighboring neurons, we examined how individual PV+ interneuron selectivity compared to that of the local neuronal network, which is primarily composed of excitatory neurons. PV+ neurons showed functional similarity to neighboring neuronal populations over spatial distances resembling measurements of synaptic connectivity. On the other hand, excitatory neurons expressing CaMKIIα displayed no such functional similarity with the neighboring population. Our findings suggest that broad selectivity of PV+ interneurons results from nonspecific integration within local networks. VIDEO ABSTRACT.

Intravenous ethanol increases extracellular dopamine in the medial prefrontal cortex of the Long-Evans rat.

BACKGROUND: Ethanol (EtOH) affects prefrontal cortex functional roles such as decision making, working memory, and behavioral control. Yet, the pharmacological effect of EtOH on dopamine, a neuromodulator in the medial prefrontal cortex (mPFC), is unclear. Past studies exploring this topic produced conflicting outcomes; however, a handful of factors (temporal resolution, method of drug administration, estrous cycle) possibly contributed to these discrepancies. We sought to mitigate these factors in order to elucidate EtOH's pharmacological effects on mPFC dopamine in Long-Evans rats. METHODS: We administered experimental solutions via an intravenous (iv), handling-free route, monitored dopamine in the mPFC via microdialysis (10-minute samples), and used male rats to avoid estrous cycle/EtOH interactions. First, we rapidly (approximately 2.7 ml/min) or slowly (approximately 0.6 ml/min) administered 1.0 g/kg EtOH and saline infusions, showing that the experimental methods did not contribute to dopamine changes. Then, a cumulative dosing protocol was used to administer 0.25, 0.75, 1.50, and 2.25 g/kg iv EtOH doses to evaluate dose-response. Finally, we monitored dialysate EtOH levels during an oral EtOH self-administration session to compare the dialysate EtOH levels achieved during the pharmacological experiments to those seen during self-administration. RESULTS: IV administration of a rapid or slow 1.0 g/kg EtOH infusion resulted in similar significant 55 ± 9 and 63 ± 15% peak dialysate dopamine increases, respectively. The 0.25, 0.75, 1.50, and 2.25 g/kg EtOH doses produced a nonsignificant 17 ± 5% and significant 36 ± 15, 68 ± 19, and 86 ± 20% peak dialysate dopamine increases, respectively. Self-administration dialysate EtOH concentrations fell within the range of concentrations noted during the EtOH dose-response curve. CONCLUSIONS: These experiments show that, using experimental methods that minimize possibly confounding factors, acute iv EtOH increases extracellular dopamine in the mPFC in a dose-dependent manner, thereby clarifying EtOH's pharmacological effects on the mesocortical dopamine system.

Microdialysis of ethanol during operant ethanol self-administration and ethanol determination by gas chromatography.

Operant self-administration methods are commonly used to study the behavioral and pharmacological effects of many drugs of abuse, including ethanol. However, ethanol is typically self-administered orally, rather than intravenously like many other drugs of abuse. The pharmacokinetics of orally administered drugs are more complex than intravenously administered drugs. Because understanding the relationship between the pharmacological and behavioral effects of ethanol requires knowledge of the time course of ethanol reaching the brain during and after drinking, we use in vivo microdialysis and gas chromatography with flame ionization detection to monitor brain dialysate ethanol concentrations over time. Combined microdialysis-behavioral experiments involve the use of several techniques. In this article, stereotaxic surgery, behavioral training and microdialysis, which can be adapted to test a multitude of self-administration and neurochemical centered hypotheses, are included only to illustrate how they relate to the subsequent phases of sample collection and dialysate ethanol analysis. Dialysate ethanol concentration analysis via gas chromatography with flame-ionization detection, which is specific to ethanol studies, is described in detail. Data produced by these methods reveal the pattern of ethanol reaching the brain during the self-administration procedure, and when paired with neurochemical analysis of the same dialysate samples, allows conclusions to be made regarding the pharmacological and behavioral effects of ethanol.