Cholinergic deficits selectively boost cortical intratelencephalic control of striatum in male Huntington's disease model mice.

Huntington's disease (HD) is a progressive, neurodegenerative disease caused by a CAG triplet expansion in huntingtin. Although corticostriatal dysfunction has long been implicated in HD, the determinants and pathway specificity of this pathophysiology are not fully understood. Here, using a male zQ175+/- knock-in mouse model of HD we carry out optogenetic interrogation of intratelencephalic and pyramidal tract synapses with principal striatal spiny projection neurons (SPNs). These studies reveal that the connectivity of intratelencephalic, but not pyramidal tract, neurons with direct and indirect pathway SPNs increased in early symptomatic zQ175+/- HD mice. This enhancement was attributable to reduced pre-synaptic inhibitory control of intratelencephalic terminals by striatal cholinergic interneurons. Lowering mutant huntingtin selectively in striatal cholinergic interneurons with a virally-delivered zinc finger repressor protein normalized striatal acetylcholine release and intratelencephalic functional connectivity, revealing a node in the network underlying corticostriatal pathophysiology in a HD mouse model.

α-Synuclein pathology disrupts mitochondrial function in dopaminergic and cholinergic neurons at-risk in Parkinson's disease.

Background: Pathological accumulation of aggregated α-synuclein (aSYN) is a common feature of Parkinson's disease (PD). However, the mechanisms by which intracellular aSYN pathology contributes to dysfunction and degeneration of neurons in the brain are still unclear. A potentially relevant target of aSYN is the mitochondrion. To test this hypothesis, genetic and physiological methods were used to monitor mitochondrial function in substantia nigra pars compacta (SNc) dopaminergic and pedunculopontine nucleus (PPN) cholinergic neurons after stereotaxic injection of aSYN pre-formed fibrils (PFFs) into the mouse brain. Methods: aSYN PPFs were stereotaxically injected into the SNc or PPN of mice. Twelve weeks later, mice were studied using a combination of approaches, including immunocytochemical analysis, cell- type specific transcriptomic profiling, electron microscopy, electrophysiology and two-photon-laser- scanning microscopy of genetically encoded sensors for bioenergetic and redox status. Results: In addition to inducing a significant neuronal loss, SNc injection of PFFs induced the formation of intracellular, phosphorylated aSYN aggregates selectively in dopaminergic neurons. In these neurons, PFF-exposure decreased mitochondrial gene expression, reduced the number of mitochondria, increased oxidant stress, and profoundly disrupted mitochondrial adenosine triphosphate production. Consistent with an aSYN-induced bioenergetic deficit, the autonomous spiking of dopaminergic neurons slowed or stopped. PFFs also up-regulated lysosomal gene expression and increased lysosomal abundance, leading to the formation of Lewy-like inclusions. Similar changes were observed in PPN cholinergic neurons following aSYN PFF exposure. Conclusions: Taken together, our findings suggest that disruption of mitochondrial function, and the subsequent bioenergetic deficit, is a proximal step in the cascade of events induced by aSYN pathology leading to dysfunction and degeneration of neurons at-risk in PD.

Ca2+ channels couple spiking to mitochondrial metabolism in substantia nigra dopaminergic neurons.

How do neurons match generation of adenosine triphosphate by mitochondria to the bioenergetic demands of regenerative activity? Although the subject of speculation, this coupling is still poorly understood, particularly in neurons that are tonically active. To help fill this gap, pacemaking substantia nigra dopaminergic neurons were studied using a combination of optical, electrophysiological, and molecular approaches. In these neurons, spike-activated calcium (Ca2+) entry through Cav1 channels triggered Ca2+ release from the endoplasmic reticulum, which stimulated mitochondrial oxidative phosphorylation through two complementary Ca2+-dependent mechanisms: one mediated by the mitochondrial uniporter and another by the malate-aspartate shuttle. Disrupting either mechanism impaired the ability of dopaminergic neurons to sustain spike activity. While this feedforward control helps dopaminergic neurons meet the bioenergetic demands associated with sustained spiking, it is also responsible for their elevated oxidant stress and possibly to their decline with aging and disease.

Acute and protracted abstinence from methamphetamine bidirectionally changes intrinsic excitability of indirect pathway spiny projection neurons in the dorsomedial striatum.

Methamphetamine (meth) is an addictive psychostimulant and illicit use presents significant personal and socioeconomic harm. Behavioral studies support the involvement of the dorsal striatum in drug-seeking but stimulant induced dysfunction in this region is understudied. The dorsal striatum can be subdivided into the dorsomedial (DMS) and dorsolateral (DLS) striatum with the DMS implicated in goal-directed and DLS in habitual behaviors; both regions are primarily composed of GABAergic direct (dSPNs) and indirect pathway (iSPNs) spiny projection neurons. To examine the effect of repeated meth on SPNs, mice were administered meth (2 mg/kg) for ten consecutive days and intrinsic excitability, dendritic excitability, and spine density were examined. DMS iSPN intrinsic excitability was increased at 1 day but decreased at 21 days of abstinence. In contrast, DMS dSPN intrinsic excitability was unchanged at either timepoint. Dendritic excitability and spine densities were unaltered in DMS iSPNs and dSPNs at 1 and 21 days of abstinence. The effect of repeated meth on iSPN excitability was specific to the DMS; DLS iSPN intrinsic excitability, dendritic excitability, and spine density were unchanged at 1 and 21 days of abstinence. These findings point toward DMS iSPN dysfunction in meth use disorders with differential dysfunction dependent on abstinence duration.

Dysregulation of the Basal Ganglia Indirect Pathway in Early Symptomatic Q175 Huntington's Disease Mice.

The debilitating psychomotor symptoms of Huntington's disease (HD) are linked partly to degeneration of the basal ganglia indirect pathway. At early symptomatic stages, before major cell loss, indirect pathway neurons exhibit numerous cellular and synaptic changes in HD and its models. However, the impact of these alterations on circuit activity remains poorly understood. To address this gap, optogenetic- and reporter-guided electrophysiological interrogation was used in early symptomatic male and female Q175 HD mice. D2 dopamine receptor-expressing striatal projection neurons (D2-SPNs) were hypoactive during synchronous cortical slow-wave activity, consistent with known reductions in dendritic excitability and cortical input strength. Downstream prototypic parvalbumin-expressing external globus pallidus (PV+ GPe) neurons discharged at 2-3 times their normal rate, even during periods of D2-SPN inactivity, arguing that defective striatopallidal inhibition was not the only cause of their hyperactivity. Indeed, PV+ GPe neurons also exhibited abnormally elevated autonomous firing ex vivo Optogenetic inhibition of PV+ GPe neurons in vivo partially and fully ameliorated the abnormal hypoactivity of postsynaptic subthalamic nucleus (STN) and putative PV- GPe neurons, respectively. In contrast to STN neurons whose autonomous firing is impaired in HD mice, putative PV- GPe neuron activity was unaffected ex vivo, implying that excessive inhibition was responsible for their hypoactivity in vivo Together with previous studies, these data demonstrate that (1) indirect pathway nuclei are dysregulated in Q175 mice through changes in presynaptic activity and/or intrinsic cellular and synaptic properties; and (2) prototypic PV+ GPe neuron hyperactivity and excessive target inhibition are prominent features of early HD pathophysiology.SIGNIFICANCE STATEMENT The early symptoms of Huntington's disease (HD) are linked to degenerative changes in the action-suppressing indirect pathway of the basal ganglia. Consistent with this linkage, the intrinsic properties of cells in this pathway exhibit complex alterations in HD and its models. However, the impact of these changes on activity is poorly understood. Using electrophysiological and optogenetic approaches, we demonstrate that the indirect pathway is highly dysregulated in early symptomatic HD mice through changes in upstream activity and/or intrinsic properties. Furthermore, we reveal that hyperactivity of external globus pallidus neurons and excessive inhibition of their targets are key features of early HD pathophysiology. Together, these findings could help to inform the development and targeting of viral-based, gene therapeutic approaches for HD.

Disruption of mitochondrial complex I induces progressive parkinsonism.

Loss of functional mitochondrial complex I (MCI) in the dopaminergic neurons of the substantia nigra is a hallmark of Parkinson's disease1. Yet, whether this change contributes to Parkinson's disease pathogenesis is unclear2. Here we used intersectional genetics to disrupt the function of MCI in mouse dopaminergic neurons. Disruption of MCI induced a Warburg-like shift in metabolism that enabled neuronal survival, but triggered a progressive loss of the dopaminergic phenotype that was first evident in nigrostriatal axons. This axonal deficit was accompanied by motor learning and fine motor deficits, but not by clear levodopa-responsive parkinsonism-which emerged only after the later loss of dopamine release in the substantia nigra. Thus, MCI dysfunction alone is sufficient to cause progressive, human-like parkinsonism in which the loss of nigral dopamine release makes a critical contribution to motor dysfunction, contrary to the current Parkinson's disease paradigm3,4.

The role of intra-articular neuronal CCR2 receptors in knee joint pain associated with experimental osteoarthritis in mice.

BACKGROUND: C-C chemokine receptor 2 (CCR2) signaling plays a key role in pain associated with experimental murine osteoarthritis (OA) after destabilization of the medial meniscus (DMM). Here, we aimed to assess if CCR2 expressed by intra-articular sensory neurons contributes to knee hyperalgesia in the early stages of the model. METHODS: DMM surgery was performed in the right knee of 10-week-old male wild-type (WT), Ccr2 null, or Ccr2RFP C57BL/6 mice. Knee hyperalgesia was measured using a Pressure Application Measurement device. CCR2 receptor antagonist (CCR2RA) was injected systemically (i.p.) or intra-articularly (i.a.) at different times after DMM to test its ability to reverse knee hyperalgesia. In vivo Ca2+ imaging of the dorsal root ganglion (DRG) was performed to assess sensory neuron responses to CCL2 injected into the knee joint cavity. CCL2 protein in the knee was measured by ELISA. Ccr2RFP mice and immunohistochemical staining for the pan-neuronal marker, protein gene product 9.5 (PGP9.5), or the sensory neuron marker, calcitonin gene-related peptide (CGRP), were used to visualize the location of CCR2 on intra-articular afferents. RESULTS: WT, but not Ccr2 null, mice displayed knee hyperalgesia 2-16 weeks after DMM. CCR2RA administered i.p. alleviated established hyperalgesia in WT mice 4 and 8 weeks after surgery. Intra-articular injection of CCL2 excited sensory neurons in the L4-DRG, as determined by in vivo calcium imaging; responses to CCL2 increased in mice 20 weeks after DMM. CCL2, but not vehicle, injected i.a. rapidly caused transient knee hyperalgesia in naïve WT, but not Ccr2 null, mice. Intra-articular CCR2RA injection also alleviated established hyperalgesia in WT mice 4 and 7 weeks after surgery. CCL2 protein was elevated in the knees of both WT and Ccr2 null mice 4 weeks after surgery. Co-expression of CCR2 and PGP9.5 as well as CCR2 and CGRP was observed in the lateral synovium of naïve mice; co-expression was also observed in the medial compartment of knees 8 weeks after DMM. CONCLUSIONS: The findings suggest that CCL2-CCR2 signaling locally in the joint contributes to knee hyperalgesia in experimental OA, and it is in part mediated through direct stimulation of CCR2 expressed by intra-articular sensory afferents.

Dysregulation of external globus pallidus-subthalamic nucleus network dynamics in parkinsonian mice during cortical slow-wave activity and activation.

KEY POINTS: Reciprocally connected GABAergic external globus pallidus (GPe) and glutamatergic subthalamic nucleus (STN) neurons form a key network within the basal ganglia. In Parkinson's disease and its models, abnormal rates and patterns of GPe-STN network activity are linked to motor dysfunction. Using cell class-specific optogenetic identification and inhibition during cortical slow-wave activity and activation, we report that, in dopamine-depleted mice, (1) D2 dopamine receptor expressing striatal projection neurons (D2-SPNs) discharge at higher rates, especially during cortical activation, (2) prototypic parvalbumin-expressing GPe neurons are excessively patterned by D2-SPNs even though their autonomous activity is upregulated, (3) despite being disinhibited, STN neurons are not hyperactive, and (4) STN activity opposes striatopallidal patterning. These data argue that in parkinsonian mice abnormal, temporally offset prototypic GPe and STN neuron firing results in part from increased striatopallidal transmission and that compensatory plasticity limits STN hyperactivity and cortical entrainment. ABSTRACT: Reciprocally connected GABAergic external globus pallidus (GPe) and glutamatergic subthalamic nucleus (STN) neurons form a key, centrally positioned network within the basal ganglia. In Parkinson's disease and its models, abnormal rates and patterns of GPe-STN network activity are linked to motor dysfunction. Following the loss of dopamine, the activities of GPe and STN neurons become more temporally offset and strongly correlated with cortical oscillations below 40 Hz. Previous studies utilized cortical slow-wave activity and/or cortical activation (ACT) under anaesthesia to probe the mechanisms underlying the normal and pathological patterning of basal ganglia activity. Here, we combined this approach with in vivo optogenetic inhibition to identify and interrupt the activity of D2 dopamine receptor-expressing striatal projection neurons (D2-SPNs), parvalbumin-expressing prototypic GPe (PV GPe) neurons, and STN neurons. We found that, in dopamine-depleted mice, (1) the firing rate of D2-SPNs was elevated, especially during cortical ACT, (2) abnormal phasic suppression of PV GPe neuron activity was ameliorated by optogenetic inhibition of coincident D2-SPN activity, (3) autonomous PV GPe neuron firing ex vivo was upregulated, presumably through homeostatic mechanisms, (4) STN neurons were not hyperactive, despite being disinhibited, (5) optogenetic inhibition of the STN exacerbated abnormal GPe activity, and (6) exaggerated beta band activity was not present in the cortex or GPe-STN network. Together with recent studies, these data suggest that in dopamine-depleted mice abnormally correlated and temporally offset PV GPe and STN neuron activity is generated in part by elevated striatopallidal transmission, while compensatory plasticity prevents STN hyperactivity and limits cortical entrainment.

Mutant huntingtin enhances activation of dendritic Kv4 K+ channels in striatal spiny projection neurons.

Huntington's disease (HD) is initially characterized by an inability to suppress unwanted movements, a deficit attributable to impaired synaptic activation of striatal indirect pathway spiny projection neurons (iSPNs). To better understand the mechanisms underlying this deficit, striatal neurons in ex vivo brain slices from mouse genetic models of HD were studied using electrophysiological, optical and biochemical approaches. Distal dendrites of iSPNs from symptomatic HD mice were hypoexcitable, a change that was attributable to increased association of dendritic Kv4 potassium channels with auxiliary KChIP subunits. This association was negatively modulated by TrkB receptor signaling. Dendritic excitability of HD iSPNs was rescued by knocking-down expression of Kv4 channels, by disrupting KChIP binding, by restoring TrkB receptor signaling or by lowering mutant-Htt (mHtt) levels with a zinc finger protein. Collectively, these studies demonstrate that mHtt induces reversible alterations in the dendritic excitability of iSPNs that could contribute to the motor symptoms of HD.

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine.

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

Cholinergic Interneurons Amplify Thalamostriatal Excitation of Striatal Indirect Pathway Neurons in Parkinson's Disease Models.

The motor symptoms of Parkinson's disease (PD) are thought to stem from an imbalance in the activity of striatal direct- and indirect-pathway spiny projection neurons (SPNs). Disease-induced alterations in the activity of networks controlling SPNs could contribute to this imbalance. One of these networks is anchored by the parafascicular nucleus (PFn) of the thalamus. To determine the role of the PFn in striatal PD pathophysiology, optogenetic, chemogenetic, and electrophysiological tools were used in ex vivo slices from transgenic mice with region-specific Cre recombinase expression. These studies revealed that in parkinsonian mice, the functional connectivity of PFn neurons with indirect pathway SPNs (iSPNs) was selectively enhanced by cholinergic interneurons acting through presynaptic nicotinic acetylcholine receptors (nAChRs) on PFn terminals. Attenuating this network adaptation by chemogenetic or genetic strategies alleviated motor-learning deficits in parkinsonian mice, pointing to a potential new therapeutic strategy for PD patients.

Nicotinic Cholinergic Receptors in VTA Glutamate Neurons Modulate Excitatory Transmission.

Ventral tegmental area (VTA) glutamate neurons are important components of reward circuitry, but whether they are subject to cholinergic modulation is unknown. To study this, we used molecular, physiological, and photostimulation techniques to examine nicotinic acetylcholine receptors (nAChRs) in VTA glutamate neurons. Cells in the medial VTA, where glutamate neurons are enriched, are responsive to acetylcholine (ACh) released from cholinergic axons. VTA VGLUT2+ neurons express mRNA and protein subunits known to comprise heteromeric nAChRs. Electrophysiology, coupled with two-photon microscopy and laser flash photolysis of photoactivatable nicotine, was used to demonstrate nAChR functional activity in the somatodendritic subcellular compartment of VTA VGLUT2+ neurons. Finally, optogenetic isolation of intrinsic VTA glutamatergic microcircuits along with gene-editing techniques demonstrated that nicotine potently modulates excitatory transmission within the VTA via heteromeric nAChRs. These results indicate that VTA glutamate neurons are modulated by cholinergic mechanisms and participate in the cascade of physiological responses to nicotine exposure.

Photoactivatable drugs for nicotinic optopharmacology.

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

Pedunculopontine glutamatergic neurons control spike patterning in substantia nigra dopaminergic neurons.

Burst spiking in substantia nigra pars compacta (SNc) dopaminergic neurons is a key signaling event in the circuitry controlling goal-directed behavior. It is widely believed that this spiking mode depends upon an interaction between synaptic activation of N-methyl-D-aspartate receptors (NMDARs) and intrinsic oscillatory mechanisms. However, the role of specific neural networks in burst generation has not been defined. To begin filling this gap, SNc glutamatergic synapses arising from pedunculopotine nucleus (PPN) neurons were characterized using optical and electrophysiological approaches. These synapses were localized exclusively on the soma and proximal dendrites, placing them in a good location to influence spike generation. Indeed, optogenetic stimulation of PPN axons reliably evoked spiking in SNc dopaminergic neurons. Moreover, burst stimulation of PPN axons was faithfully followed, even in the presence of NMDAR antagonists. Thus, PPN-evoked burst spiking of SNc dopaminergic neurons in vivo may not only be extrinsically triggered, but extrinsically patterned as well.

Early dysfunction and progressive degeneration of the subthalamic nucleus in mouse models of Huntington's disease.

The subthalamic nucleus (STN) is an element of cortico-basal ganglia-thalamo-cortical circuitry critical for action suppression. In Huntington's disease (HD) action suppression is impaired, resembling the effects of STN lesioning or inactivation. To explore this potential linkage, the STN was studied in BAC transgenic and Q175 knock-in mouse models of HD. At <2 and 6 months of age autonomous STN activity was impaired due to activation of KATP channels. STN neurons exhibited prolonged NMDA receptor-mediated synaptic currents, caused by a deficit in glutamate uptake, and elevated mitochondrial oxidant stress, which was ameliorated by NMDA receptor antagonism. STN activity was rescued by NMDA receptor antagonism or the break down of hydrogen peroxide. At 12 months of age approximately 30% of STN neurons had been lost, as in HD. Together, these data argue that dysfunction within the STN is an early feature of HD that may contribute to its expression and course.

Convergent cortical innervation of striatal projection neurons.

Anatomical studies have led to the assertion that intratelencephalic and pyramidal tract cortical neurons innervate different striatal projection neurons. To test this hypothesis, we measured the responses of mouse striatal neurons to optogenetic activation of intratelencephalic and pyramidal tract axons. Contrary to expectation, direct and indirect pathway striatal spiny projection neurons responded to both intratelencephalic and pyramidal tract activation, arguing that these cortical networks innervate both striatal projection neurons.

An autism-associated variant of Epac2 reveals a role for Ras/Epac2 signaling in controlling basal dendrite maintenance in mice.

The architecture of dendritic arbors determines circuit connectivity, receptive fields, and computational properties of neurons, and dendritic structure is impaired in several psychiatric disorders. While apical and basal dendritic compartments of pyramidal neurons are functionally specialized and differentially regulated, little is known about mechanisms that selectively maintain basal dendrites. Here we identified a role for the Ras/Epac2 pathway in maintaining basal dendrite complexity of cortical neurons. Epac2 is a guanine nucleotide exchange factor (GEF) for the Ras-like small GTPase Rap, and it is highly enriched in the adult mouse brain. We found that in vivo Epac2 knockdown in layer 2/3 cortical neurons via in utero electroporation reduced basal dendritic architecture, and that Epac2 knockdown in mature cortical neurons in vitro mimicked this effect. Overexpression of an Epac2 rare coding variant, found in human subjects diagnosed with autism, also impaired basal dendritic morphology. This mutation disrupted Epac2's interaction with Ras, and inhibition of Ras selectively interfered with basal dendrite maintenance. Finally, we observed that components of the Ras/Epac2/Rap pathway exhibited differential abundance in the basal versus apical dendritic compartments. These findings define a role for Epac2 in enabling crosstalk between Ras and Rap signaling in maintaining basal dendrite complexity, and exemplify how rare coding variants, in addition to their disease relevance, can provide insight into cellular mechanisms relevant for brain connectivity.

MEF-2 regulates activity-dependent spine loss in striatopallidal medium spiny neurons.

Striatal dopamine depletion profoundly reduces the density of spines and corticostriatal glutamatergic synapses formed on D(2) dopamine receptor expressing striatopallidal medium spiny neurons, leaving D(1) receptor expressing striatonigral medium spiny neurons relatively intact. Because D(2) dopamine receptors diminish the excitability of striatopallidal MSNs, the pruning of synapses could be a form of homeostatic plasticity aimed at restoring activity into a preferred range. To characterize the homeostatic mechanisms controlling synapse density in striatal medium spiny neurons, striatum from transgenic mice expressing a D(2) receptor reporter construct was co-cultured with wild-type cerebral cortex. Sustained depolarization of these co-cultures induced a profound pruning of glutamatergic synapses and spines in striatopallidal medium spiny neurons. This pruning was dependent upon Ca(2+) entry through Cav1.2 L-type Ca(2+) channels, activation of the Ca(2+)-dependent protein phosphatase calcineurin and up-regulation of myocyte enhancer factor 2 (MEF2) transcriptional activity. Depolarization and MEF2 up-regulation increased the expression of two genes linked to synaptic remodeling-Nur77 and Arc. Taken together, these studies establish a translational framework within which striatal adaptations linked to the symptoms of Parkinson's disease can be explored.

Chapter 16. Two-photon microscopy and multidimensional analysis of cell dynamics.

Two-photon (2P) microscopy is a high-resolution imaging technique that was initially applied by neurobiologists and developmental cell biologists but has subsequently been broadly adapted by immunologists. The value of 2P microscopy is that it affords an unparalleled view of single-cell spatiotemporal dynamics deep within intact tissues and organs. As the technology develops and new transgenic mice and fluorescent probes become available, 2P microscopy will serve as an increasingly valuable tool for assessing cell function and probing molecular mechanisms. Here we discuss the technical aspects related to 2P microscope design, explain in detail various tissue imaging preparations, and walk the reader through the often daunting process of analyzing multidimensional data sets and presenting the experimental results.