Dopamine Receptors Differentially Control Binge Alcohol Drinking-Mediated Synaptic Plasticity of the Core Nucleus Accumbens Direct and Indirect Pathways.

Binge alcohol drinking, a behavior characterized by rapid repeated alcohol intake, is most prevalent in young adults and is a risk factor for excessive alcohol consumption and alcohol dependence. Although the alteration of synaptic plasticity is thought to contribute to this behavior, there is currently little evidence that this is the case. We used drinking in the dark (DID) as a model of binge alcohol drinking to assess its effects on spike timing-dependent plasticity (STDP) in medium spiny neurons (MSNs) of the core nucleus accumbens (NAc) by combining patch-clamp recordings with calcium imaging and optogenetics. After 2 weeks of daily alcohol binges, synaptic plasticity was profoundly altered. STDP in MSNs expressing dopamine D1 receptors shifted from spike-timing-dependent long-term depression (tLTD), the predominant form of plasticity in naive male mice, to spike-timing-dependent long-term potentiation (tLTP) in DID mice, an effect that was totally reversed in the presence of 4 µm SCH23390, a dopamine D1 receptor antagonist. In MSNs presumably expressing dopamine D2 receptors, tLTP, the main form of plasticity in naive mice, was inhibited in DID mice. Interestingly, 1 µm sulpiride, a D2 receptor antagonist, restored tLTP. Although we observed no alterations of AMPA and NMDA receptor properties, we found that the AMPA/NMDA ratio increased at cortical and amygdaloid inputs but not at hippocampal inputs. Also, DID effects on STDP were accompanied by lower dendritic calcium transients. These data suggest that the role of dopamine in mediating the effects of binge alcohol drinking on synaptic plasticity of NAc MSNs differs markedly whether these neurons belong to the direct or indirect pathways.SIGNIFICANCE STATEMENT We examined the relationship between binge alcohol drinking and spike timing-dependent plasticity in nucleus accumbens (NAc) neurons. We found that repeated drinking bouts modulate differently synaptic plasticity in medium spiny neurons of the accumbens direct and indirect pathways. While timing-dependent long-term depression switches to long-term potentiation (LTP) in the former, timing-dependent LTP is inhibited in the latter. These effects are not accompanied by changes in AMPA and NMDA receptor properties at cortical, amygdaloid, and hippocampal synapses. Interestingly, dopamine D1 and D2 receptor antagonists have opposite effects on plasticity. Our data show that whether core NAc medium spiny neurons belong to the direct or indirect pathways determines the form of spike timing-dependent plasticity (STDP), the manner by which STDP responds to binge alcohol drinking, and its sensitivity to dopamine receptor antagonists.

Binge alcohol drinking alters the differential control of cholinergic interneurons over nucleus accumbens D1 and D2 medium spiny neurons.

Animals studies support the notion that striatal cholinergic interneurons (ChIs) play a central role in basal ganglia function by regulating associative learning, reward processing, and motor control. In the nucleus accumbens (NAc), a brain region that mediates rewarding properties of substance abuse, acetylcholine regulates glutamatergic, dopaminergic, and GABAergic neurotransmission in naïve mice. However, it is unclear how ChIs orchestrate the control of these neurotransmitters/modulators to determine the synaptic excitability of medium spiny neurons (MSNs), the only projecting neurons that translate accumbens electrical activity into behavior. Also unknown is the impact of binge alcohol drinking on the regulation of dopamine D1- and D2 receptor-expressing MSNs (D1- and D2-MSNs, respectively) by ChIs. To investigate this question, we optogenetically stimulated ChIs while recording evoked and spontaneous excitatory postsynaptic currents (sEPSCs) in nucleus accumbens core D1- and D2-MSN of ChAT.ChR2.eYFPxDrd1.tdtomato mice. In alcohol-naïve mice, we found that stimulating NAc ChIs decreased sEPSCs frequency in both D1- and D2-MSNs, presumably through a presynaptic mechanism. Interestingly, ChI stimulation decreased MSN synaptic excitability through different mechanisms in D1- vs. D2-MSNs. While decrease of ChI-mediated sEPSCs frequency in D1-MSNs was mediated by dopamine, the same effect in D2-MSNs resulted from a direct control of glutamate release by ChIs. Interestingly, after 2 weeks of binge alcohol drinking, optogenetic stimulation of ChIs enhanced glutamate release in D1-MSNs, while its effect on D2-MSNs remained unchanged. Taken together, these data suggest that cholinergic interneurons could be a key target for regulation of NAc circuitry and for alcohol consumption.

Binge Alcohol Drinking Alters Synaptic Processing of Executive and Emotional Information in Core Nucleus Accumbens Medium Spiny Neurons.

The nucleus accumbens (NAc) is a forebrain region mediating the positive-reinforcing properties of drugs of abuse, including alcohol. It receives glutamatergic projections from multiple forebrain and limbic regions such as the prefrontal cortex (PFCx) and basolateral amygdala (BLA), respectively. However, it is unknown how NAc medium spiny neurons (MSNs) integrate PFCx and BLA inputs, and how this integration is affected by alcohol exposure. Because progress has been hampered by the inability to independently stimulate different pathways, we implemented a dual wavelength optogenetic approach to selectively and independently stimulate PFCx and BLA NAc inputs within the same brain slice. This approach functionally demonstrates that PFCx and BLA inputs synapse onto the same MSNs where they reciprocally inhibit each other pre-synaptically in a strict time-dependent manner. In alcohol-naïve mice, this temporal gating of BLA-inputs by PFCx afferents is stronger than the reverse, revealing that MSNs prioritize high-order executive processes information from the PFCx. Importantly, binge alcohol drinking alters this reciprocal inhibition by unilaterally strengthening BLA inhibition of PFCx inputs. In line with this observation, we demonstrate that in vivo optogenetic stimulation of the BLA, but not PFCx, blocks binge alcohol drinking escalation in mice. Overall, our results identify NAc MSNs as a key integrator of executive and emotional information and show that this integration is dysregulated during binge alcohol drinking.

Continuous and non-invasive thermography of mouse skin accurately describes core body temperature patterns, but not absolute core temperature.

Body temperature is an important physiological parameter in many studies of laboratory mice. Continuous assessment of body temperature has traditionally required surgical implantation of a telemeter, but this invasive procedure adversely impacts animal welfare. Near-infrared thermography provides a non-invasive alternative by continuously measuring the highest temperature on the outside of the body (Tskin), but the reliability of these recordings as a proxy for continuous core body temperature (Tcore) measurements has not been assessed. Here, Tcore (30 s resolution) and Tskin (1 s resolution) were continuously measured for three days in mice exposed to ad libitum and restricted feeding conditions. We subsequently developed an algorithm that optimised the reliability of a Tskin-derived estimate of Tcore. This identified the average of the maximum Tskin per minute over a 30-min interval as the optimal way to estimate Tcore. Subsequent validation analyses did however demonstrate that this Tskin-derived proxy did not provide a reliable estimate of the absolute Tcore due to the high between-animal variability in the relationship between Tskin and Tcore. Conversely, validation showed that Tskin-derived estimates of Tcore reliably describe temporal patterns in physiologically-relevant Tcore changes and provide an excellent measure to perform within-animal comparisons of relative changes in Tcore.

Conditional, inducible gene silencing in dopamine neurons reveals a sex-specific role for Rit2 GTPase in acute cocaine response and striatal function.

Dopamine (DA) signaling is critical for movement, motivation, and addictive behavior. The neuronal GTPase, Rit2, is enriched in DA neurons (DANs), binds directly to the DA transporter (DAT), and is implicated in several DA-related neuropsychiatric disorders. However, it remains unknown whether Rit2 plays a role in either DAergic signaling and/or DA-dependent behaviors. Here we leveraged the TET-OFF system to conditionally silence Rit2 in Pitx3IRES2-tTA mouse DANs. Following DAergic Rit2 knockdown (Rit2-KD), mice displayed an anxiolytic phenotype, with no change in baseline locomotion. Further, males exhibited increased acute cocaine sensitivity, whereas DAergic Rit2-KD suppressed acute cocaine sensitivity in females. DAergic Rit2-KD did not affect presynaptic TH and DAT protein levels in females, nor was TH was affected in males; however, DAT was significantly diminished in males. Paradoxically, despite decreased DAT levels in males, striatal DA uptake was enhanced, but was not due to enhanced DAT surface expression in either dorsal or ventral striatum. Finally, patch recordings in nucleus accumbens (NAcc) medium spiny neurons (MSNs) revealed reciprocal changes in spontaneous EPSP (sEPSP) frequency in male and female D1+ and D2+ MSNs following DAergic Rit2-KD. In males, sEPSP frequency was decreased in D1+, but not D2+, MSNs, whereas in females sEPSP frequency decreased in D2+, but not D1+, MSNs. Moreover, DAergic Rit2-KD abolished the ability of cocaine to reduce sEPSP frequency in D1+, but not D2+, male MSNs. Taken together, our studies are among the first to acheive AAV-mediated, conditional and inducible DAergic knockdown in vivo. Importantly, our results provide the first evidence that DAergic Rit2 expression differentially impacts striatal function and DA-dependent behaviors in males and females.

Luciferase shRNA Presents off-Target Effects on Voltage-Gated Ion Channels in Mouse Hippocampal Pyramidal Neurons.

RNA interference (RNAi) is a straightforward approach to study gene function from the in vitro cellular level to in vivo animal behavior. Although RNAi-mediated gene knockdown has become essentially routine in neuroscience over the past ten years, off-target effects of short hairpin RNAs (shRNAs) should be considered as the proper choice of control shRNA is critical in order to perform meaningful experiments. Luciferase shRNA (shLuc), targeting firefly luciferase, and scrambled shRNAs (shScrs) have been widely used as controls for vertebrate cell research. However, thorough validation of control shRNAs has not been made to date. Here, we performed thorough physiological and morphological studies against control shRNAs in mouse hippocampal CA1 pyramidal neurons. As expected, all control shRNAs exhibited normal basal synaptic transmission and dendritic morphology. However, to our surprise, shLuc exerted severe off-target effects on voltage-gated ion channel function, while the shScr had no detectable changes. These results indicate that thorough validation of shRNA is imperative and, in the absence of such validation, that shScr is the best available negative control for gene knockdown studies.

Responses of Nucleus Tractus Solitarius (NTS) early and late neurons to blood pressure changes in anesthetized F344 rats.

Previously, many different types of NTS barosensitive neurons were identified. However, the time course of NTS barosensitive neuronal activity (NA) in response to arterial pressure (AP) changes, and the relationship of NA-AP changes, have not yet been fully quantified. In this study, we made extracellular recordings of single NTS neurons firing in response to AP elevation induced by occlusion of the descending aorta in anesthetized rats. Our findings were that: 1) Thirty-five neurons (from 46 neurons) increased firing, whereas others neurons either decreased firing upon AP elevation, or were biphasic: first decreased firing upon AP elevation and then increased firing during AP decrease. 2) Fourteen neurons with excitatory responses were activated and rapidly increased their firing during the early phase of AP increase (early neurons); whereas 21 neurons did not increase firing until the mean arterial pressure changes (ΔMAP) reached near/after the peak (late neurons). 3) The early neurons had a significantly higher firing rate than late neurons during AP elevation at a similar rate. 4) Early neuron NA-ΔMAP relationship could be well fitted and characterized by the sigmoid logistic function with the maximal gain of 29.3. 5) The increase of early NA correlated linearly with the initial heart rate (HR) reduction. 6) The late neurons did not contribute to the initial HR reduction. However, the late NA could be well correlated with HR reduction during the late phase. Altogether, our study demonstrated that the NTS excitatory neurons could be grouped into early and late neurons based on their firing patterns. The early neurons could be characterized by the sigmoid logistic function, and different neurons may differently contribute to HR regulation. Importantly, the grouping and quantitative methods used in this study may provide a useful tool for future assessment of functional changes of early and late neurons in disease models.