miRNA-mediated control of gephyrin synthesis drives sustained inhibitory synaptic plasticity.

Activity-dependent protein synthesis is crucial for long-lasting forms of synaptic plasticity. However, our understanding of translational mechanisms controlling GABAergic synapses is limited. One distinct form of inhibitory long-term potentiation (iLTP) enhances postsynaptic clusters of GABAARs and the primary inhibitory scaffold, gephyrin, to promote sustained synaptic strengthening. While we previously found that persistent iLTP requires mRNA translation, the mechanisms controlling plasticity-induced gephyrin translation remain unknown. We identify miR153 as a novel regulator of Gphn mRNA translation which controls gephyrin protein levels and synaptic clustering, ultimately impacting inhibitory synaptic structure and function. iLTP induction downregulates miR153, reversing its translational suppression of Gphn mRNA and promoting de novo gephyrin protein synthesis and synaptic clustering during iLTP. Finally, we find that reduced miR153 expression during iLTP is driven by an excitation-transcription coupling pathway involving calcineurin, NFAT and HDACs, which also controls the miRNA-dependent upregulation of GABAARs. Together, we delineate a miRNA-dependent post-transcriptional mechanism that controls the expression of the key synaptic scaffold, gephyrin, and may converge with parallel miRNA pathways to coordinate gene upregulation to maintain inhibitory synaptic plasticity.

A chemogenetic approach for dopamine imaging with tunable sensitivity.

Genetically-encoded dopamine (DA) sensors enable high-resolution imaging of DA release, but their ability to detect a wide range of extracellular DA levels, especially tonic versus phasic DA release, is limited by their intrinsic affinity. Here we show that a human-selective dopamine receptor positive allosteric modulator (PAM) can be used to boost sensor affinity on-demand. The PAM enhances DA detection sensitivity across experimental preparations (in vitro, ex vivo and in vivo) via one-photon or two-photon imaging. In vivo photometry-based detection of optogenetically-evoked DA release revealed that DETQ administration produces a stable 31 minutes window of potentiation without effects on animal behavior. The use of the PAM revealed region-specific and metabolic state-dependent differences in tonic DA levels and enhanced single-trial detection of behavior-evoked phasic DA release in cortex and striatum. Our chemogenetic strategy can potently and flexibly tune DA imaging sensitivity and reveal multi-modal (tonic/phasic) DA signaling across preparations and imaging approaches.

Salience signaling and stimulus scaling of ventral tegmental area glutamate neuron subtypes.

Ventral tegmental area (VTA) glutamatergic neurons participate in reward, aversion, drug-seeking, and stress. Subsets of VTA VGluT2+ neurons are capable of co-transmitting glutamate and GABA (VGluT2+VGaT+ neurons), transmitting glutamate without GABA (VGluT2+VGaT- neurons), or co-transmitting glutamate and dopamine (VGluT2+TH+ neurons), but whether these molecularly distinct subpopulations show behavior-related differences is not wholly understood. We identified that neuronal activity of each VGluT2+ subpopulation is sensitive to reward value but signaled this in different ways. The phasic maximum activity of VGluT2+VGaT+ neurons increased with sucrose concentration, whereas VGluT2+VGaT- neurons increased maximum and sustained activity with sucrose concentration, and VGluT2+TH+ neurons increased sustained but not maximum activity with sucrose concentration. Additionally, VGluT2+ subpopulations signaled consummatory preferences in different ways. VGluT2+VGaT- neurons and VGluT2+TH+ neurons showed a signaling preference for a behaviorally-preferred fat reward over sucrose, but in temporally-distinct ways. In contrast, VGluT2+VGaT+ neurons uniquely signaled a less behaviorally-preferred sucrose reward compared with fat. Further experiments suggested that VGluT2+VGaT+ consummatory reward-related activity was related to sweetness, partially modulated by hunger state, and not dependent on caloric content or behavioral preference. All VGluT2+ subtypes increased neuronal activity following aversive stimuli but VGluT2+VGaT+ neurons uniquely scaled their magnitude and sustained activity with footshock intensity. Optogenetic activation of VGluT2+VGaT+ neurons during low intensity footshock enhanced fear-related behavior without inducing place preference or aversion. We interpret these data such that VTA glutamatergic subpopulations signal different elements of rewarding and aversive experiences and highlight the unique role of VTA VGluT2+VGaT+ neurons in enhancing the salience of behavioral experiences.

Adaptor protein-3 produces synaptic vesicles that release phasic dopamine.

The burst firing of midbrain dopamine neurons releases a phasic dopamine signal that mediates reinforcement learning. At many synapses, however, high firing rates deplete synaptic vesicles (SVs), resulting in synaptic depression that limits release. What accounts for the increased release of dopamine by stimulation at high frequency? We find that adaptor protein-3 (AP-3) and its coat protein VPS41 promote axonal dopamine release by targeting vesicular monoamine transporter VMAT2 to the axon rather than dendrites. AP-3 and VPS41 also produce SVs that respond preferentially to high-frequency stimulation, independent of their role in axonal polarity. In addition, conditional inactivation of VPS41 in dopamine neurons impairs reinforcement learning, and this involves a defect in the frequency dependence of release rather than the amount of dopamine released. Thus, AP-3 and VPS41 promote the axonal polarity of dopamine release but enable learning by producing a distinct population of SVs tuned specifically to high firing frequency that confers the phasic release of dopamine.

Alterations in neurotransmitter co-release in Parkinson's disease.

Parkinson's disease is a neurological disorder characterized by degeneration of midbrain dopamine neurons, which results in numerous adaptations in basal ganglia circuits. Research over the past twenty-five years has identified that midbrain dopamine neurons of the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) co-release multiple other transmitters including glutamate and GABA, in addition to their canonical transmitter, dopamine. This review summarizes previous work characterizing neurotransmitter co-release from dopamine neurons, work examining potential changes in co-release dynamics that result in animal models of Parkinson's disease, and future opportunities for determining how dysfunction in co-release may contribute to circuit dysfunction in Parkinson's disease.

Adaptor Protein-3 Produces Synaptic Vesicles that Release Phasic Dopamine.

The burst firing of midbrain dopamine neurons releases a phasic dopamine signal that mediates reinforcement learning. At many synapses, however, high firing rates deplete synaptic vesicles (SVs), resulting in synaptic depression that limits release. What accounts for the increased release of dopamine by stimulation at high frequency? We find that adaptor protein-3 (AP-3) and its coat protein VPS41 promote axonal dopamine release by targeting vesicular monoamine transporter VMAT2 to the axon rather than dendrites. AP-3 and VPS41 also produce SVs that respond preferentially to high frequency stimulation, independent of their role in axonal polarity. In addition, conditional inactivation of VPS41 in dopamine neurons impairs reinforcement learning, and this involves a defect in the frequency dependence of release rather than the amount of dopamine released. Thus, AP-3 and VPS41 promote the axonal polarity of dopamine release but enable learning by producing a novel population of SVs tuned specifically to high firing frequency that confers the phasic release of dopamine.

Gait Abnormalities and Aberrant D2 Receptor Expression and Signaling in Mice Carrying the Human Pathogenic Mutation DRD2I212F.

A dopamine D2 receptor mutation was recently identified in a family with a novel hyperkinetic movement disorder. That allelic variant D2-I212F is a constitutively active and G protein-biased receptor. We now describe mice engineered using CRISPR-Cas9-mediated gene editing technology to carry the D2-I212F variant. Drd2I212F mice exhibited gait abnormalities resembling those in other mouse models of chorea and/or dystonia and had striatal D2 receptor expression that was decreased approximately 30% per Drd2I212F allele. Electrically evoked inhibitory postsynaptic conductances in midbrain dopamine neurons and striatum from Drd2I212F mice, caused by G protein activation of potassium channels, exhibited slow kinetics (e.g., approximately four- to sixfold slower decay) compared with Drd2 +/+ mice. Current decay initiated by photolytic release of the D2 antagonist sulpiride from CyHQ-sulpiride was also ∼fourfold slower in midbrain slices from Drd2I212F mice than Drd2 +/+ mice. Furthermore, in contrast to Drd2 +/+ mice, in which dopamine is several-fold more potent at neurons in the nucleus accumbens than in the dorsal striatum, reflecting activation of Gα o versus Gα i, dopamine had similar potencies in those two brain regions of Drd2I212F mice. Repeated cocaine treatment, which decreases dopamine potency in the nucleus accumbens of Drd2 +/+ mice, had no effect on dopamine potency in Drd2 I212F mice. The results demonstrate the pathogenicity of the D2-I212F mutation and the utility of this mouse model for investigating the role of pathogenic DRD2 variants in early-onset hyperkinetic movement disorders. SIGNIFICANCE STATEMENT: The first dopamine receptor mutation to cause a movement disorder, D2-I212F, was recently identified. The mutation makes receptor activation of G protein-mediated signaling more efficient. To confirm the pathogenesis of D2-I212F, this study reports that mice carrying this mutation have gait abnormalities consistent with the clinical phenotype. The mutation also profoundly alters D2 receptor expression and function in vivo. This mouse model will be useful for further characterization of the mutant receptor and for evaluation of potential therapeutic drugs.

miRNA-mediated control of gephyrin synthesis drives sustained inhibitory synaptic plasticity.

Activity-dependent protein synthesis is crucial for many long-lasting forms of synaptic plasticity. However, our understanding of the translational mechanisms controlling inhibitory synapses is limited. One distinct form of inhibitory long-term potentiation (iLTP) enhances postsynaptic clusters of GABAARs and the primary inhibitory scaffold, gephyrin, to promote sustained synaptic strengthening. While we previously found that persistent iLTP requires mRNA translation, the precise mechanisms controlling gephyrin translation during this process remain unknown. Here, we identify miR153 as a novel regulator of Gphn mRNA translation which controls gephyrin protein levels and synaptic clustering, ultimately impacting GABAergic synaptic structure and function. We find that iLTP induction downregulates miR153, reversing its translational suppression of Gphn mRNA and allowing for increased de novo gephyrin protein synthesis and synaptic clustering during iLTP. Finally, we find that reduced miR153 expression during iLTP is driven by an excitation-transcription coupling pathway involving calcineurin, NFAT and HDACs, which also controls the miRNA-dependent upregulation of GABAARs. Overall, this work delineates a miRNA-dependent post-transcriptional mechanism that controls the expression of the key synaptic scaffold, gephyrin, and may converge with parallel miRNA pathways to coordinate gene upregulation to maintain inhibitory synaptic plasticity.

Properties and modulation of excitatory inputs to the locus coeruleus.

Excitatory inputs drive burst firing of locus coeruleus (LC) noradrenaline (NA) neurons in response to a variety of stimuli. Though a small number of glutamatergic LC afferents have been investigated, the overall landscape of these excitatory inputs is largely unknown. The current study used an optogenetic approach to isolate three glutamatergic afferents: the prefrontal cortex (PFC), lateral hypothalamus (LH) and periaqueductal grey (PAG). AAV5-DIO-ChR2 was injected into each region in male and female CaMKII-Cre mice and the properties of excitatory inputs on LC-NA cells were measured. Notably we found differences among these inputs. First, the pattern of axonal innervation differed between inputs such that LH afferents were concentrated in the posterior portion of the LC-NA somatic region while PFC afferents were denser in the medial dendritic region. Second, basal intrinsic properties varied for afferents, with LH inputs having the highest connectivity and the largest amplitude excitatory postsynaptic currents while PAG inputs had the lowest initial release probability. Third, while orexin and oxytocin had minimal effects on any input, dynorphin strongly inhibited excitatory inputs originating from the LH and PAG, and corticotrophin releasing factor (CRF) selectively inhibited inputs from the PAG. Overall, these results demonstrate that individual afferents to the LC have differing properties, which may contribute to the modularity of the LC and its ability to mediate various behavioural outcomes. KEY POINTS: Excitatory inputs to the locus coeruleus (LC) are important for driving noradrenaline neuron activity and downstream behaviours in response to salient stimuli, but little is known about the functional properties of different glutamate inputs that innervate these neurons We used a virus-mediated optogenetic approach to compare glutamate afferents from the prefrontal cortex (PFC), the lateral hypothalamus (LH) and the periaqueductal grey (PAG). While PFC was predicted to make synaptic inputs, we found that the LH and PAG also drove robust excitatory events in LC noradrenaline neurons. The strength, kinetics, and short-term plasticity of each input differed as did the extent of neuromodulation by both dynorphin and corticotrophin releasing factor. Thus each input displayed a unique set of basal properties and modulation by peptides. This characterization is an important step in deciphering the heterogeneity of the LC.

Divergent properties and independent regulation of striatal dopamine and GABA co-transmission.

Substantia nigra pars compacta (SNc) dopamine neurons play a key role in regulating the activity of striatal circuits within the basal ganglia. In addition to dopamine, these neurons release several other transmitters, including the major inhibitory neurotransmitter γ-aminobutyric acid (GABA). Both dopamine and GABA are loaded into SNc synaptic vesicles by the vesicular monoamine transporter 2 (VMAT2), and co-release of GABA provides strong inhibition to the striatum by directly inhibiting striatal medium spiny projection neurons (MSNs) through activation of GABAA receptors. Here, we found that despite both dopamine and GABA being co-packaged by VMAT2, the properties of transmission, including Ca2+ sensitivity, release probability, and requirement of active zone scaffolding proteins, differ between the two transmitters. Moreover, the extent by which presynaptic neuromodulators inhibit co-transmission also varied. Differences in modulation and the mechanisms controlling release allow for independent regulation of dopamine and GABA signals despite both being loaded via similar mechanisms.

Cocaine shifts dopamine D2 receptor sensitivity to gate conditioned behaviors.

Cocaine addiction is a chronic, relapsing disorder characterized by maladaptation in the brain mesolimbic and nigrostriatal dopamine system. Although changes in the properties of D2-receptor-expressing medium spiny neurons (D2-MSNs) and connected striatal circuits following cocaine treatment are known, the contributions of altered D2-receptor (D2R) function in mediating the rewarding properties of cocaine remain unclear. Here, we describe how a 7-day exposure to cocaine alters dopamine signaling by selectively reducing the sensitivity, but not the expression, of nucleus accumbens D2-MSN D2Rs via an alteration in the relative expression and coupling of G protein subunits. This cocaine-induced reduction of D2R sensitivity facilitated the development of the rewarding effects of cocaine as blocking the reduction in G protein expression was sufficient to prevent cocaine-induced behavioral adaptations. These findings identify an initial maladaptive change in sensitivity by which mesolimbic dopamine signals are encoded by D2Rs following cocaine exposure.

Extinction blunts paraventricular thalamic contributions to heroin relapse.

Here, we use optogenetics and chemogenetics to investigate the contribution of the paraventricular thalamus (PVT) to nucleus accumbens (NAc) pathway in aversion and heroin relapse in two different heroin self-administration models in rats. In one model, rats undergo forced abstinence in the home cage prior to relapse testing, and in the other, they undergo extinction training, a procedure that is likened to cognitive behavioral therapy. We find that the PVT→NAc pathway is both sufficient and necessary to drive aversion and heroin seeking after abstinence, but not extinction. The ability of extinction to reduce this pathway's contribution to heroin relapse is accompanied by a loss of synaptic plasticity in PVT inputs onto a specific subset of NAc neurons. Thus, extinction may exert therapeutic reductions in opioid seeking by altering synaptic plasticity within the PVT→NAc pathway, resulting in reduced aversion during opioid withdrawal as well as reduced relapse propensity.

Loss of nigral excitation of cholinergic interneurons contributes to parkinsonian motor impairments.

Progressive loss of dopamine inputs in Parkinson's disease leads to imbalances in coordinated signaling of dopamine and acetylcholine (ACh) in the striatum, which is thought to contribute to parkinsonian motor symptoms. As reciprocal interactions between dopamine inputs and cholinergic interneurons (ChIs) control striatal dopamine and ACh transmission, we examined how partial dopamine depletion in an early-stage mouse model for Parkinson's disease alters nigral regulation of cholinergic activity. We found region-specific alterations in how remaining dopamine inputs regulate cholinergic excitability that differ between the dorsomedial (DMS) and dorsolateral (DLS) striatum. Specifically, we found that dopamine depletion downregulates metabotropic glutamate receptors (mGluR1) on DLS ChIs at synapses where dopamine inputs co-release glutamate, abolishing the ability of dopamine inputs to drive burst firing. This loss underlies parkinsonian motor impairments, as viral rescue of mGluR1 signaling in DLS ChIs was sufficient to restore circuit function and attenuate motor deficits in early-stage parkinsonian mice.

Opioid-Induced Adaptations of cAMP Dynamics in the Nucleus Accumbens.

To investigate how opioid exposure alters dopamine (DA) responses in medium spiny neurons (MSNs), Muntean et al. used a novel cAMP sensor to track cAMP dynamics and report a coordinated effort of adaptations in D1- and D2-MSNs to integrate DA inputs and shift signaling strengths in various states of opioid dependence.

Cholinergic Interneurons Provide a Link to Balance Excitation across Striatal Output Neurons.

D1-MSNs and D2-MSNs mediate output from the accumbens. How activity of one regulates the other is poorly understood. In this issue of Neuron, Francis et al. (2019) show that D1-MSN firing induces D2-MSN LTP via the recruitment of cholinergic interneurons.

Cholinergic Transmission at Muscarinic Synapses in the Striatum Is Driven Equally by Cortical and Thalamic Inputs.

The release of acetylcholine from cholinergic interneurons (ChIs) directly modulates striatal output via muscarinic receptors on medium spiny neurons (MSNs). While thalamic inputs provide strong excitatory input to ChIs, cortical inputs primarily regulate MSN firing. Here, we found that, while thalamic inputs do drive ChI firing, a subset of ChIs responds robustly to stimulation of cortical inputs as well. To examine how input-evoked changes in ChI firing patterns drive acetylcholine release at cholinergic synapses onto MSNs, muscarinic M4-receptor-mediated synaptic events were measured in MSNs overexpressing G-protein gated potassium channels (GIRK2). Stimulation of both cortical and thalamic inputs was sufficient to equally drive muscarinic synaptic events in MSNs, resulting from the broad synaptic innervation of the stimulus-activated ChI population across many MSNs. Taken together, this indicates an underappreciated role for the extensive cholinergic network, in which small populations of ChIs can drive substantial changes in post-synaptic receptor activity across the striatum.

Synaptic Vesicle Recycling Pathway Determines Neurotransmitter Content and Release Properties.

In contrast to temporal coding by synaptically acting neurotransmitters such as glutamate, neuromodulators such as monoamines signal changes in firing rate. The two modes of signaling have been thought to reflect differences in release by different cells. We now find that midbrain dopamine neurons release glutamate and dopamine with different properties that reflect storage in different synaptic vesicles. The vesicles differ in release probability, coupling to presynaptic Ca2+ channels and frequency dependence. Although previous work has attributed variation in these properties to differences in location or cytoskeletal association of synaptic vesicles, the release of different transmitters shows that intrinsic differences in vesicle identity drive different modes of release. Indeed, dopamine but not glutamate vesicles depend on the adaptor protein AP-3, revealing an unrecognized linkage between the pathway of synaptic vesicle recycling and the properties of exocytosis. Storage of the two transmitters in different vesicles enables the transmission of distinct signals.

A Photoactivatable Botulinum Neurotoxin for Inducible Control of Neurotransmission.

Regulated secretion is critical for diverse biological processes ranging from immune and endocrine signaling to synaptic transmission. Botulinum and tetanus neurotoxins, which specifically proteolyze vesicle fusion proteins involved in regulated secretion, have been widely used as experimental tools to block these processes. Genetic expression of these toxins in the nervous system has been a powerful approach for disrupting neurotransmitter release within defined circuitry, but their current utility in the brain and elsewhere remains limited by lack of spatial and temporal control. Here we engineered botulinum neurotoxin B so that it can be activated with blue light. We demonstrate the utility of this approach for inducibly disrupting excitatory neurotransmission, providing a first-in-class optogenetic tool for persistent, light-triggered synaptic inhibition. In addition to blocking neurotransmitter release, this approach will have broad utility for conditionally disrupting regulated secretion of diverse bioactive molecules, including neuropeptides, neuromodulators, hormones, and immune molecules. VIDEO ABSTRACT.

Dopamine Cells Differentially Regulate Striatal Cholinergic Transmission across Regions through Corelease of Dopamine and Glutamate.

The balance of dopamine and acetylcholine in the dorsal striatum is critical for motor and learning functions. Midbrain dopamine cells and local cholinergic interneurons (ChIs) densely innervate the striatum and have strong reciprocal actions on each other. Although dopamine inputs regulate ChIs, the functional consequences of dopamine neuron activity across dorsal striatal regions is poorly understood. Here, we find that midbrain dopamine neurons drive pauses in the firing of dorsomedial ChIs but robust bursts in dorsolateral ChIs. Pauses are mediated by dopamine D2 receptors, while bursts are driven by glutamate corelease and activation of a mGluR-mediated excitatory conductance. We find the frequency of muscarinic cholinergic transmission to medium spiny neurons is greater in the dorsomedial striatum. This regional variation in transmission is moderated by the different actions of dopamine and glutamate corelease. These results delineate a mechanism by which dopamine inputs maintain consistent levels of cholinergic activity across the dorsal striatum.