Chemical Targeting of Rhodol Voltage Sensitive Dyes to Dopaminergic Neurons

Optical imaging of changes in membrane potential of living cells can be achieved by the means of fluorescent voltage sensitive dyes (VSDs). A particularly challenging task is to efficiently deliver these highly lipophilic probes to specific neuronal subpopulations in brain tissue. We have tackled this task by designing a solubilizing, hydrophilic polymer platform that carries a high-affinity ligand for a membrane protein marker of interest and a fluorescent VSD. Here, we disclose an improved design of polymer supported probes for chemical, non-genetic targeting of voltage sensors to axons natively expressing the dopamine transporter in ex vivo mouse brain tissue. We first show that for negatively charged rhodol VSDs functioning on the photoinduced electron transfer principle, poly(ethylene glycol) (PEG) as a carrier enables targeting with higher selectivity than the polysaccharide dextran in HEK cell culture. In the same experimental setting, we also demonstrate that incorporation of an azetidine ring in the rhodol chromophore substantially increases the brightness and voltage sensitivity of the respective VSD. We show that the superior properties of the optimized sensor are transferable to recording of electrically evoked activity from dopaminergic axons in mouse striatal slices after averaging of multiple trials. Finally, we suggest the next milestones for the field to achieve single-scan recordings with non-genetically targeted VSDs in native brain tissue.

Long-Term Depression of Striatal DA Release Induced by mGluRs via Sustained Hyperactivity of Local Cholinergic Interneurons

The cellular mechanisms regulating dopamine (DA) release in the striatum have attracted much interest in recent years. By in vitro amperometric recordings in mouse striatal slices, we show that a brief (5 min) exposure to the metabotropic glutamate receptor agonist DHPG (50 μM) induces a profound depression of synaptic DA release, lasting over 1 h from DHPG washout. This long-term depression is sensitive to glycine, which preferentially inhibits local cholinergic interneurons, as well as to drugs acting on nicotinic acetylcholine receptors and to the pharmacological depletion of released acetylcholine. The same DHPG treatment induces a parallel long-lasting enhancement in the tonic firing of presumed striatal cholinergic interneurons, measured with multi-electrode array recordings. When DHPG is bilaterally infused in vivo in the mouse striatum, treated mice display an anxiety-like behavior. Our results demonstrate that metabotropic glutamate receptors stimulation gives rise to a prolonged depression of the striatal dopaminergic transmission, through a sustained enhancement of released acetylcholine, due to the parallel long-lasting potentiation of striatal cholinergic interneurons firing. This plastic interplay between dopamine, acetylcholine, and glutamate in the dorsal striatum may be involved in anxiety-like behavior typical of several neuropsychiatric disorders.

Dopamine release in nucleus accumbens is under tonic inhibition by adenosine A1 receptors regulated by astrocytic ENT1 and dysregulated by ethanol

Striatal adenosine A1 receptor (A1R) activation can inhibit dopamine release. A1Rs on other striatal neurons are activated by an adenosine tone that is limited by equilibrative nucleoside transporter 1 (ENT1) that is enriched on astrocytes and is ethanol-sensitive. We explored whether dopamine release in nucleus accumbens core is under tonic inhibition by A1Rs, and is regulated by astrocytic ENT1 and ethanol. In ex vivo striatal slices from male and female mice, A1R agonists inhibited dopamine release evoked electrically or optogenetically and detected using fast-scan cyclic voltammetry, most strongly for lower stimulation frequencies and pulse numbers, thereby enhancing the activity-dependent contrast of dopamine release. Conversely, A1R antagonists reduced activity-dependent contrast but enhanced evoked dopamine release levels, even for single optogenetic pulses indicating an underlying tonic inhibition. The ENT1 inhibitor NBTI reduced dopamine release and promoted A1R-mediated inhibition, and conversely, virally-mediated astrocytic overexpression of ENT1 enhanced dopamine release and relieved A1R-mediated inhibition. By imaging the genetically encoded fluorescent adenosine sensor GRAB-Ado, we identified a striatal extracellular adenosine tone that was elevated by the ENT1 inhibitor and sensitive to gliotoxin fluorocitrate. Finally, we identified that ethanol (50 mM) promoted A1R-mediated inhibition of dopamine release, through diminishing adenosine uptake via ENT1. Together, these data reveal that dopamine output dynamics are gated by a striatal adenosine tone, limiting amplitude but promoting contrast, regulated by ENT1, and promoted by ethanol. These data add to the diverse mechanisms through which ethanol modulates striatal dopamine, and to emerging datasets supporting astrocytic transporters as important regulators of striatal function.

Tonic engagement of nicotinic receptors bidirectionally controls striatal spiny projection neuron spike timing

Striatal spiny projection neurons (SPNs) transform convergent excitatory corticostriatal inputs into an inhibitory signal that shapes basal ganglia output. This process is fine-tuned by striatal GABAergic interneurons (GINs), which receive overlapping cortical inputs and mediate rapid corticostriatal feedforward inhibition of SPNs. Adding another level of control, cholinergic interneurons (CINs), which are also vigorously activated by corticostriatal excitation, can 1) disynaptically inhibit SPNs by activating α4β2 nicotinic acetylcholine receptors (nAChRs) on various GINs and 2) directly modulate corticostriatal synaptic strength via pre-synaptic α7 nAChR receptors. Measurements of the disynaptic inhibitory pathway, however, indicate that it is too slow to compete with direct GIN-mediated feed-forward inhibition. Moreover, functional nAChRs are also present on populations of GINs that do not respond to phasic activation of CINs, such as parvalbumin-positive fast-spiking interneurons (PV-FSIs), making the overall role of nAChRs in shaping striatal synaptic integration unclear. Using acute striatal slices we show that upon synchronous optogenetic activation of corticostriatal projections, blockade of α7 nAChRs delayed SPN spikes, whereas blockade of α4β2 nAChRs advanced SPN spikes and increased postsynaptic depolarizations. The nAChR-dependent inhibition was mediated by downstream GABA release, and data suggest that the GABA source was not limited to GINs that respond to phasic CIN activation. In particular, the observed spike-advancement caused by nAChR blockade was associated with a diminished frequency of spontaneous inhibitory postsynaptic currents in SPNs, and a parallel hyperpolarization of PV-FSIs. Taken together, we describe opposing roles for tonic (as opposed to phasic) engagement of nAChRs in striatal function. We conclude that tonic activation of nAChRs by CINs both sharpens the temporal fidelity of corticostriatal signaling via pre-synaptic α7 nAChRs and maintains a GABAergic brake on cortically-driven striatal output, processes that may shape SPN spike timing, striatal processing and synaptic plasticity.

Cannabinoid Receptor Type 1 Regulates Drug Reward Behavior via Glutamate Decarboxylase 67 Transcription

Interaction of cannabinoid receptor type 1 (CB1) and GABAergic neuronal activity is involved in drug abuse-related behavior. However, its role in drug-dependent Pavlovian conditioning is not well understood. In this study, we aimed to evaluate the effects of a CB1 agonist, JWH-210, on the development of conditioned place preference (CPP)-induced by methamphetamine (METH). Pretreatment with a synthetic cannabinoid, JWH-210 (CB1 agonist), increased METH-induced CPP score and METH-induced dopamine release in acute striatal slices. Interestingly, CB1 was expressed in glutamate decarboxylase 67 (GAD67) positive cells, and overexpression of CB1 increased GAD67 expression, while CB1 knockdown reduced GAD67 expression in vivo and in vitro. GAD67 is known as an enzyme involved in the synthesis of GABA. CB1 knockdown in the mice striatum increased METH-induced CPP. When GAD67 decreased in the mice striatum, mRNA level of CB1 did not change, suggesting that CB1 can regulate GAD67 expression. GAD67 knockdown in the mouse striatum augmented apomorphine (dopamine receptor D2 agonist)–induced climbing behavior and METH-induced CPP score. Moreover, in the human brain, mRNA level of GAD67 was found to be decreased in drug users. Therefore, we suggest that CB1 potentiates METH-induced CPP through inhibitory GABAergic regulation of dopaminergic neuronal activity.

Multimodal Detection of Dopamine by Sniffer Cells Expressing Genetically Encoded Fluorescence Sensors

Dopamine serves an important role in supporting both locomotor control and higher brain functions such as motivation and learning. Dopaminergic dysfunction is implicated in an equally multidimensional spectrum of neurological and neuropsychiatric diseases. Extracellular dopamine levels are known to be tightly controlled by presynaptic dopamine transporters (DAT), which is also a main target of psychostimulants. Still, detailed data on dopamine dynamics in space and time is needed to fully understand how dopamine signals are encoded and translated into cellular and behavioral responses, and to uncover the pathological effects of dopamine-related diseases. The recently developed genetically encoded fluorescent dopamine sensors enable unprecedented monitoring of dopamine dynamics and have changed the field of in vivo dopamine recording. However, the potential of these sensors to be used for in vitro and ex vivo assays remains unexplored. Here, we demonstrate a generalizable blueprint for making dopamine 'sniffer' cells for multimodal detection of dopamine in vitro and ex vivo. We generated sniffer cell lines with inducible expression of six different dopamine sensors and performed a head-to-head comparison of sensor properties to guide users in sensor selection. In proof-of-principle experiments, we show how the sniffer cells can be applied to measure release of endogenous dopamine from cultured neurons and striatal slices, and for determining total dopamine content in striatal tissue. Furthermore, we use the sniffer cells to quantify DAT-mediated dopamine uptake, and AMPH-induced and constitutive dopamine efflux as a radiotracer free, high-throughput alternative to electrochemical- and radiotracer-based assays. Importantly, the sniffer cells framework can readily be applied to other transmitter systems for which the list of genetically encoded fluorescent sensors is rapidly growing.

Striatal Dopamine Transporter Function Is Facilitated by Converging Biology of α-Synuclein and Cholesterol

Striatal dopamine transporters (DAT) powerfully regulate dopamine signaling, and can contribute risk to degeneration in Parkinson’s disease (PD). DATs can interact with the neuronal protein α-synuclein, which is associated with the etiology and molecular pathology of idiopathic and familial PD. Here, we tested whether DAT function in governing dopamine (DA) uptake and release is modified in a human-α-synuclein-overexpressing (SNCA-OVX) transgenic mouse model of early PD. Using fast-scan cyclic voltammetry (FCV) in ex vivo acute striatal slices to detect DA release, and biochemical assays, we show that several aspects of DAT function are promoted in SNCA-OVX mice. Compared to background control α-synuclein-null mice (Snca-null), the SNCA-OVX mice have elevated DA uptake rates, and more pronounced effects of DAT inhibitors on evoked extracellular DA concentrations ([DA]o) and on short-term plasticity (STP) in DA release, indicating DATs play a greater role in limiting DA release and in driving STP. We found that DAT membrane levels and radioligand binding sites correlated with α-synuclein level. Furthermore, DAT function in Snca-null and SNCA-OVX mice could also be promoted by applying cholesterol, and using Tof-SIMS we found genotype-differences in striatal lipids, with lower striatal cholesterol in SNCA-OVX mice. An inhibitor of cholesterol efflux transporter ABCA1 or a cholesterol chelator in SNCA-OVX mice reduced the effects of DAT-inhibitors on evoked [DA]o. Together these data indicate that human α-synuclein in a mouse model of PD promotes striatal DAT function, in a manner supported by extracellular cholesterol, suggesting converging biology of α-synuclein and cholesterol that regulates DAT function and could impact DA function and PD pathophysiology.

Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2-autoreceptor activity

SUMMARYThe nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show by application of several single-molecule sensitive super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the dopamine transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and by DA D2-autoreceptor activation in manner dependent on Ca2+-influx via N-type voltage-gated Ca2+-channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2-autoreceptor, syntaxin-1 and clathrin nanodomains. By demonstrating that nanoscopic reorganizations with putative major impact on transmitter homeostasis can take place in dopaminergic varicosities, the data have important implications for understanding modulatory neurotransmitter physiology.

RGS4 negatively modulates Nociceptin/Orphanin FQ opioid receptor signaling: implication for L-Dopa induced dyskinesia

Background and purpose: Regulator of G-protein signal 4 (RGS4) is a signal transduction protein that accelerates intrinsic GTPase activity of Gαi/o and Gαq subunits, suppressing GPCR signaling. Here we investigate whether RGS4 modulates nociceptin/orphanin FQ opioid (NOP) receptor signaling and whether this modulation has relevance for L-Dopa induced dyskinesia. Experimental approach: HEK293T cells transfected with NOP, NOP/RGS4 or NOP/RGS19 were challenged with N/OFQ and the small molecule NOP agonist AT-403, using D1-stimulated cAMP levels as a readout. Primary rat striatal neurons and adult mouse striatal slices were challenged with N/OFQ or AT-403 in the presence of the RGS4 inhibitor, CCG-203920, and D1-stimulated cAMP or pERK responses were monitored. In vivo, CCG-203920 was co-administered with AT-403 and levodopa to 6-hydroxydopamine hemilesioned rats, and dyskinetic movements, striatal biochemical correlates of dyskinesia (pERK and pGluR1 levels) and striatal RGS4 levels were measured. Key results: RGS4 expression reduced NOFQ and AT-403 potency and efficacy in HEK293T cells. CCG-203920 increased N/OFQ potency in primary rat striatal neurons, and potentiated AT-403 response in mouse striatal slices. CCG-203920 enhanced AT-403 mediated inhibition of dyskinesia and its biochemical correlates, without compromising its motor-improving effects. Unilateral dopamine depletion caused bilateral reduction of RGS4 levels which was reversed by levodopa. Levodopa acutely upregulated RGS4 in the lesioned striatum. Conclusions and Implications: RGS4 physiologically inhibits NOP receptor signaling and an RGS4 inhibitor enhances NOP responses. Furthermore, an RGS4 inhibitor improved the antidyskinetic potential of NOP receptor agonists, mitigating the effects of upregulation of striatal RGS4 levels occurring during dyskinesia expression.