Oxidative Stress Research on Huntington's Disease Neurons Using Genetically Encoded Biosensors

Many neurodegenerative diseases, including Huntington’s disease (HD), are associated with oxidative stress in the neurons of the brain. Genetically encoded biosensorsare useful for studying these processesin vitro. Human cell cultures expressing the biosensors can serve as a cell model for developing and testing effective agents that reduce oxidative stress. In this work, transgenes encoding biosensors of glutathione oxidative potential(Grx1-roGFP2) with cytoplasmic and mitochondrial localization were introduced into human induced pluripotent stem cells of a healthy donor and an HD patient using CRISPR/Cas9-mediated genome editing. The cells were subsequently differentiated into medium spiny neurons of the striatum. The expression of the biosensors was detected in the iPSCs, neuronal precursors and mature neurons.The obtained cells could be used to study the redox potential of glutathione in HD neurons and to screen for new drug compounds aimed at reducing oxidative stress. Keywords: genetically encoded biosensors, Huntington’s disease, induced pluripotent stem cells, medium spiny neurons, oxidative stress, glutathione, Grx1-roGFP2

Error-related Signaling in Nucleus Accumbens D2 Receptor-expressing Neurons Guides Avoidance-based Goal-directed Behavior

Learnt associations between environmental cues and the outcomes they predict (cue-outcome associations) play a major role in behavioral control, guiding not only which responses we should perform, but also which we should avoid, in order to achieve a specific goal. The encoding of such cue-outcome associations, as well as the performance of cue-guided goal-directed behavior, is thought to involve dopamine D1 and D2 receptor-expressing medium spiny neurons (D1-/D2-MSNs) of the nucleus accumbens (NAc). Here, using a visual discrimination task in mice, we assessed the role of NAc D1-/D2-MSNs in cue-guided goal-directed avoidance of inappropriate responding. Cell-type specific neuronal silencing and in-vivo imaging revealed NAc D2-MSNs to selectively contribute to cue-guided avoidance behavior, with activation of NAc D2-MSNs following response error playing an important role in optimizing future goal-directed behavior. Our findings indicate that error-signaling by NAc D2-MSNs underlies the ability to use environmental cues to avoid inappropriate behavior.

Cell-Type-Specific Adaptions in Striatal Medium-Sized Spiny Neurons and Their Roles in Behavioral Responses to Drugs of Abuse

Drug addiction is defined as a compulsive pattern of drug-seeking- and taking- behavior, with recurrent episodes of abstinence and relapse, and a loss of control despite negative consequences. Addictive drugs promote reinforcement by increasing dopamine in the mesocorticolimbic system, which alters excitatory glutamate transmission within the reward circuitry, thereby hijacking reward processing. Within the reward circuitry, the striatum is a key target structure of drugs of abuse since it is at the crossroad of converging glutamate inputs from limbic, thalamic and cortical regions, encoding components of drug-associated stimuli and environment, and dopamine that mediates reward prediction error and incentive values. These signals are integrated by medium-sized spiny neurons (MSN), which receive glutamate and dopamine axons converging onto their dendritic spines. MSN primarily form two mostly distinct populations based on the expression of either DA-D1 (D1R) or DA-D2 (D2R) receptors. While a classical view is that the two MSN populations act in parallel, playing antagonistic functional roles, the picture seems much more complex. Herein, we review recent studies, based on the use of cell-type-specific manipulations, demonstrating that dopamine differentially modulates dendritic spine density and synapse formation, as well as glutamate transmission, at specific inputs projecting onto D1R-MSN and D2R-MSN to shape persistent pathological behavioral in response to drugs of abuse. We also discuss the identification of distinct molecular events underlying the detrimental interplay between dopamine and glutamate signaling in D1R-MSN and D2R-MSN and highlight the relevance of such cell-type-specific molecular studies for the development of innovative strategies with potential therapeutic value for addiction. Because drug addiction is highly prevalent in patients with other psychiatric disorders when compared to the general population, we last discuss the hypothesis that shared cellular and molecular adaptations within common circuits could explain the co-occurrence of addiction and depression. We will therefore conclude this review by examining how the nucleus accumbens (NAc) could constitute a key interface between addiction and depression.

Pyk2 Stabilizes Striatal Medium Spiny Neuron Structure and Striatal-Dependent Action

In day-to-day life, we often choose between pursuing familiar behaviors that have been rewarded in the past or adjusting behaviors when new strategies might be more fruitful. The dorsomedial striatum (DMS) is indispensable for flexibly arbitrating between old and new behavioral strategies. The way in which DMS neurons host stable connections necessary for sustained flexibility is still being defined. An entry point to addressing this question may be the structural scaffolds on DMS neurons that house synaptic connections. We find that the non-receptor tyrosine kinase Proline-rich tyrosine kinase 2 (Pyk2) stabilizes both dendrites and spines on striatal medium spiny neurons, such that Pyk2 loss causes dendrite arbor and spine loss. Viral-mediated Pyk2 silencing in the DMS obstructs the ability of mice to arbitrate between rewarded and non-rewarded behaviors. Meanwhile, the overexpression of Pyk2 or the closely related focal adhesion kinase (FAK) enhances this ability. Finally, experiments using combinatorial viral vector strategies suggest that flexible, Pyk2-dependent action involves inputs from the medial prefrontal cortex (mPFC), but not the ventrolateral orbitofrontal cortex (OFC). Thus, Pyk2 stabilizes the striatal medium spiny neuron structure, likely providing substrates for inputs, and supports the capacity of mice to arbitrate between novel and familiar behaviors, including via interactions with the medial-prefrontal cortex.

Input-specific control of interneuron numbers in nascent striatal networks

The assembly of functional neuronal circuits requires appropriate numbers of distinct classes of neurons, but the mechanisms through which their relative proportions are established remain poorly defined. Investigating the mouse striatum, here we found that the two most prominent subtypes of striatal interneurons, parvalbumin-expressing (PV+) GABAergic and cholinergic (ChAT+) interneurons, undergo extensive programmed cell death between the first and second postnatal weeks. Remarkably, the survival of PV+ and ChAT+ interneurons is regulated by distinct mechanisms mediated by their specific afferent connectivity. While long-range cortical inputs control PV+ interneuron survival, ChAT+ interneuron survival is regulated by local input from the medium spiny neurons. Our results identify input-specific circuit mechanisms that operate during the period of programmed cell death to establish the final number of interneurons in nascent striatal networks.

Striatal compartmentalization and clustering of different subtypes of astrocytes is altered in the zQ175 Huntington′s disease mouse model

Huntington′s disease (HD) is a devastating neurodegenerative disease that primarily affects the striatum, a brain region that controls movement and some forms of cognition. Dysfunction and loss of medium spiny neurons of the striatum is accompanied by astrogliosis (increased astrocyte density and pathology). For decades, astrocytes were considered a homogeneous cell type, but recent transcriptomic analyses revealed astrocytes are a heterogeneous population classified into multiple subtypes depending on the expression of different gene markers. Here, we studied whether three different striatal astrocyte subtypes expressing glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), or S100 calcium-binding protein B (S100B) are differentially altered in HD. We conducted a comparative immunofluorescence analysis in the striatum of WT and the heterozygous zQ175 HD mouse model and found that the expression and abundance of GFAP+ and S100B+ astrocytes increased in zQ175 mice, while GS+ astrocytes showed no alteration. We then explored whether there was a differential spatial distribution of any of these subtypes within the striatum. We developed a systematic brain compartmentalization approach and found that while GS+ and S100B+ astrocytes were more homogeneously distributed throughout the striatum in zQ175 mice, GFAP+ astrocytes preferentially accumulated in the dorsomedial and dorsolateral striatum, which are regions associated with goal-directed and habitual behaviors. Additionally, GFAP+ astrocytes in zQ175 mice showed increased clustering, a parameter that indicates increased proximity and that is associated with localized inflammation and/or neurodegeneration. Our data suggest a differential susceptibility in both increased density and striatal compartmentalization of different subtypes of astrocytes in zQ175. These results highlight new potential implications for our understanding of astrocyte pathology in HD.

A distinct D1-MSN subpopulation down-regulates dopamine to promote negative emotional state

Dopamine (DA) level in the nucleus accumbens (NAc) is critical for reward and aversion encoding. DA released from the ventral mesencephalon (VM) DAergic neurons increases the excitability of VM-projecting D1-dopamine receptor-expressing medium spiny neurons (D1-MSNs) in the NAc to enhance DA release and augment rewards. However, how such a DA positive feedback loop is regulated to maintain DA homeostasis and reward-aversion balance remains elusive. Here we report that the ventral pallidum (VP) projection of NAc D1-MSNs (D1NAc-VP) is inhibited by rewarding stimuli and activated by aversive stimuli. In contrast to the VM projection of D1-MSN (D1NAc-VM), activation of D1NAc-VP projection induces aversion, but not reward. D1NAc-VP MSNs are distinct from the D1NAc-VM MSNs, which exhibit conventional functions of D1-MSNs. Activation of D1NAc-VP projection stimulates VM GABAergic transmission, inhibits VM DAergic neurons, and reduces DA release into the NAc. Thus, D1NAc-VP and D1NAc-VM MSNs cooperatively control NAc dopamine balance and reward-aversion states.

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.