Optogenetic and transcriptomic interrogation of enhanced muscle function in the paralyzed mouse whisker pad.

The functional state of denervated muscle is a critical factor in the ability to restore movement after injury- or disease-related paralysis. Here we used peripheral optogenetic stimulation and transcriptome profiling in the mouse whisker system to investigate the time course of changes in neuromuscular function following complete unilateral facial nerve transection. While most skeletal muscles rapidly lose functionality after lower motor neuron denervation, optogenetic muscle stimulation of the paralyzed whisker pad revealed sustained increases in the sensitivity, velocity, and amplitude of whisker movements, and reduced fatigability, starting 48 h after denervation. RNA-seq analysis showed distinct regulation of multiple gene families in denervated whisker pad muscles compared with the atrophy-prone soleus, including prominent changes in ion channels and contractile fibers. Together, our results define the unique functional and transcriptomic landscape of denervated facial muscles and have general implications for restoring movement after neuromuscular injury or disease. NEW & NOTEWORTHY Optogenetic activation of muscle can be used to noninvasively induce movements and probe muscle function. We used this technique in mice to investigate changes in whisker movements following facial nerve transection. We found unexpectedly enhanced functional properties of whisker pad muscle following denervation, accompanied by unique transcriptomic changes. Our findings highlight the utility of the mouse whisker pad for investigating the restoration of movement after paralysis.

Inhibitory and excitatory neuromodulation by hydrogen peroxide: translating energetics to information.

Historically, brain neurochemicals have been broadly classified as energetic or informational. However, increasing evidence implicates metabolic substrates and byproducts as signalling agents, which blurs the boundary between energy and information, and suggests the introduction of a new category for 'translational' substances that convey changes in energy state to information. One intriguing example is hydrogen peroxide (H2 O2 ), which is a small, readily diffusible molecule. Produced during mitochondrial respiration, this reactive oxygen species, can mediate dynamic regulation of neuronal activity and transmitter release by activating inhibitory ATP-sensitive K(+) (KATP ) channels, as well as a class of excitatory non-selective cation channels, TRPM2. Studies using ex vivo guinea pig brain slices have revealed that activity-generated H2 O2 can act via KATP channels to inhibit dopamine release in dorsal striatum and dopamine neuron activity in the substantia nigra pars compacta. In sharp contrast, endogenously generated H2 O2 enhances the excitability of GABAergic projection neurons in the dorsal striatum and substantia nigra pars reticulata by activating TRPM2 channels. These studies suggest that the balance of excitation vs. inhibition produced in a given cell by metabolically generated H2 O2 will be dictated by the relative abundance of H2 O2 -sensitive ion channel targets that receive this translational signal.

TRPM2 channels are required for NMDA-induced burst firing and contribute to H(2)O(2)-dependent modulation in substantia nigra pars reticulata GABAergic neurons.

Substantia nigra pars reticulata (SNr) GABAergic neurons are projection neurons that convey output from the basal ganglia to target structures. These neurons exhibit spontaneous regular firing, but also exhibit burst firing in the presence of NMDA or when excitatory glutamatergic input to the SNr is activated. Notably, an increase in burst firing is also seen in Parkinson's disease. Therefore, elucidating conductances that mediate spontaneous activity and changes of firing pattern in these neurons is essential for understanding how the basal ganglia control movement. Using ex vivo slices of guinea pig midbrain, we show that SNr GABAergic neurons express transient receptor potential melastatin 2 (TRPM2) channels that underlie NMDA-induced burst firing. Furthermore, we show that spontaneous firing rate and burst activity are modulated by the reactive oxygen species H(2)O(2) acting via TRPM2 channels. Thus, our results indicate that activation of TRPM2 channels is necessary for burst firing in SNr GABAergic neurons and their responsiveness to modulatory H(2)O(2). These findings have implications not only for normal regulation, but also for Parkinson's disease, which involves excitotoxicity and oxidative stress.

Cell-Type Specific Connectivity of Whisker-Related Sensory and Motor Cortical Input to Dorsal Striatum.

The anterior dorsolateral striatum (DLS) is heavily innervated by convergent excitatory projections from the primary motor (M1) and sensory cortex (S1) and considered an important site of sensorimotor integration. M1 and S1 corticostriatal synapses have functional differences in their connection strength with striatal spiny projection neurons (SPNs) and fast-spiking interneurons (FSIs) in the DLS and, as a result, exert distinct influences on sensory-guided behaviors. In the present study, we tested whether M1 and S1 inputs exhibit differences in the subcellular anatomical distribution of striatal neurons. We injected adeno-associated viral vectors encoding spaghetti monster fluorescent proteins (sm.FPs) into M1 and S1 in male and female mice and used confocal microscopy to generate 3D reconstructions of corticostriatal inputs to single identified SPNs and FSIs obtained through ex vivo patch clamp electrophysiology. We found that M1 and S1 dually innervate SPNs and FSIs; however, there is a consistent bias towards the M1 input in SPNs that is not found in FSIs. In addition, M1 and S1 inputs were distributed similarly across the proximal, medial, and distal regions of SPN and FSI dendrites. Notably, closely localized M1 and S1 clusters of inputs were more prevalent in SPNs than FSIs, suggesting that cortical inputs are integrated through cell-type specific mechanisms. Our results suggest that the stronger functional connectivity from M1 to SPNs compared to S1, as previously observed, is due to a higher quantity of synaptic inputs. Our results have implications for how sensorimotor integration is performed in the striatum through cell-specific differences in corticostriatal connections.

Cell-Type Specific Connectivity of Whisker-Related Sensory and Motor Cortical Input to Dorsal Striatum.

The anterior dorsolateral striatum (DLS) is heavily innervated by convergent excitatory projections from the primary motor (M1) and sensory cortex (S1) and is considered an important site of sensorimotor integration. M1 and S1 corticostriatal synapses have functional differences in the strength of their connections with striatal spiny projection neurons (SPNs) and fast-spiking interneurons (FSIs) in the DLS, and as a result exert an opposing influence on sensory-guided behaviors. In the present study, we tested whether M1 and S1 inputs exhibit differences in the subcellular anatomical distribution onto striatal neurons. We injected adeno-associated viral vectors encoding spaghetti monster fluorescent proteins (sm.FPs) into M1 and S1, and used confocal microscopy to generate 3D reconstructions of corticostriatal inputs to single identified SPNs and FSIs obtained through ex-vivo patch-clamp electrophysiology. We found that SPNs are less innervated by S1 compared to M1, but FSIs receive a similar number of inputs from both M1 and S1. In addition, M1 and S1 inputs were distributed similarly across the proximal, medial, and distal regions of SPNs and FSIs. Notably, clusters of inputs were prevalent in SPNs but not FSIs. Our results suggest that SPNs have stronger functional connectivity to M1 compared to S1 due to a higher density of synaptic inputs. The clustering of M1 and S1 inputs onto SPNs but not FSIs suggest that cortical inputs are integrated through cell-type specific mechanisms and more generally have implications for how sensorimotor integration is performed in the striatum. Significance Statement: The dorsolateral striatum (DLS) is a key brain area involved in sensorimotor integration due to its dense innervation by the primary motor (M1) and sensory cortex (S1). However, the quantity and anatomical distribution of these inputs to the striatal cell population has not been well characterized. In this study we demonstrate that corticostriatal projections from M1 and S1 differentially innervate spiny projection neurons (SPNs) and fast-spiking interneurons (FSIs) in the DLS. S1 inputs innervate SPNs less than M1 and are likely to form synaptic clusters in SPNs but not in FSIs. These findings suggest that sensorimotor integration is partly achieved by differences in the synaptic organization of corticostriatal inputs to local striatal microcircuits.

Activity-dependent somatodendritic dopamine release in the substantia nigra autoinhibits the releasing neuron.

Somatodendritic dopamine (DA) release from midbrain DA neurons activates D2 autoreceptors on these cells to regulate their activity. However, the source of autoregulatory DA remains controversial. Here, we test the hypothesis that D2 autoreceptors on a given DA neuron in the substantia nigra pars compacta (SNc) are activated primarily by DA released from that same cell, rather than from its neighbors. Voltage-clamp recording allows monitoring of evoked D2-receptor-mediated inhibitory currents (D2ICs) in SNc DA neurons as an index of DA release. Single-cell application of antibodies to Na+ channels via the recording pipette decreases spontaneous activity of recorded neurons and attenuates evoked D2ICs; antibodies to SNAP-25, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, also decrease D2IC amplitude. Evoked D2ICs are nearly abolished by the light chain of botulinum neurotoxin A, which cleaves SNAP-25, whereas synaptically activated GABAB-receptor-mediated currents are unaffected. Thus, somatodendritic DA release in the SNc autoinhibits the neuron that releases it.

Mitochondria are the source of hydrogen peroxide for dynamic brain-cell signaling.

Hydrogen peroxide (H(2)O(2)) is emerging as a ubiquitous small-molecule messenger in biology, particularly in the brain, but underlying mechanisms of peroxide signaling remain an open frontier for study. For example, dynamic dopamine transmission in dorsolateral striatum is regulated on a subsecond timescale by glutamate via H(2)O(2) signaling, which activates ATP-sensitive potassium (K(ATP)) channels to inhibit dopamine release. However, the origin of this modulatory H(2)O(2) has been elusive. Here we addressed three possible sources of H(2)O(2) produced for rapid neuronal signaling in striatum: mitochondrial respiration, monoamine oxidase (MAO), and NADPH oxidase (Nox). Evoked dopamine release in guinea-pig striatal slices was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Using direct fluorescence imaging of H(2)O(2) and tissue analysis of ATP, we found that coapplication of rotenone (50 nM), a mitochondrial complex I inhibitor, and succinate (5 mM), a complex II substrate, limited H(2)O(2) production, but maintained tissue ATP content. Strikingly, coapplication of rotenone and succinate also prevented glutamate-dependent regulation of dopamine release, implicating mitochondrial H(2)O(2) in release modulation. In contrast, inhibitors of MAO or Nox had no effect on dopamine release, suggesting a limited role for these metabolic enzymes in rapid H(2)O(2) production in the striatum. These data provide the first demonstration that respiring mitochondria are the primary source of H(2)O(2) generation for dynamic neuronal signaling.

Investigating learning-related neural circuitry with chronic in vivo optical imaging.

Fundamental aspects of brain function, including development, plasticity, learning, and memory, can take place over time scales of days to years. Chronic in vivo imaging of neural activity with cellular resolution is a powerful method for tracking the long-term activity of neural circuits. We review recent advances in our understanding of neural circuit function from diverse brain regions that have been enabled by chronic in vivo cellular imaging. Insight into the neural basis of learning and decision-making, in particular, benefit from the ability to acquire longitudinal data from genetically identified neuronal populations, deep brain areas, and subcellular structures. We propose that combining chronic imaging with further experimental and computational innovations will advance our understanding of the neural circuit mechanisms of brain function.

Basal ganglia control of substantia nigra dopaminergic neurons.

Although substantia nigra dopaminergic neurons are spontaneously active both in vivo and in vitro, this activity does not depend on afferent input as these neurons express an endogenous calcium-dependent oscillatory mechanism sufficient to drive action potential generation. However, afferents to these neurons, a large proportion of them GABAergic and arising from other nuclei in the basal ganglia, play a crucial role in modulating the activity of dopaminergic neurons. In the absence of afferent activity or when in brain slices, dopaminergic neurons fire in a very regular, pacemaker-like mode. Phasic activity in GABAergic, glutamatergic, and cholinergic inputs modulates the pacemaker activity into two other modes. The most common is a random firing pattern in which interspike intervals assume a Poisson-like distribution, and a less common pattern, often in response to a conditioned stimulus or a reward in which the neurons fire bursts of 2-8 spikes time-locked to the stimulus. Typically in vivo, all three firing patterns are observed, intermixed, in single nigrostriatal neurons varying over time. Although the precise mechanism(s) underlying the burst are currently the focus of intensive study, it is obvious that bursting must be triggered by afferent inputs. Most of the afferents to substantia nigra pars compacta dopaminergic neurons comprise monosynaptic inputs from GABAergic projection neurons in the ipsilateral neostriatum, the globus pallidus, and the substantia nigra pars reticulata. A smaller fraction of the basal ganglia inputs, something less than 30%, are glutamatergic and arise principally from the ipsilateral subthalamic nucleus and pedunculopontine nucleus. The pedunculopontine nucleus also sends a cholinergic input to nigral dopaminergic neurons. The GABAergic pars reticulata projection neurons also receive inputs from all of these sources, in some cases relaying them disynaptically to the dopaminergic neurons, thereby playing a particularly significant role in setting and/or modulating the firing pattern of the nigrostriatal neurons.

Opposing Influence of Sensory and Motor Cortical Input on Striatal Circuitry and Choice Behavior.

The striatum is the main input nucleus of the basal ganglia and is a key site of sensorimotor integration. While the striatum receives extensive excitatory afferents from the cerebral cortex, the influence of different cortical areas on striatal circuitry and behavior is unknown. Here, we find that corticostriatal inputs from whisker-related primary somatosensory (S1) and motor (M1) cortex differentially innervate projection neurons and interneurons in the dorsal striatum and exert opposing effects on sensory-guided behavior. Optogenetic stimulation of S1-corticostriatal afferents in ex vivo recordings produced larger postsynaptic potentials in striatal parvalbumin (PV)-expressing interneurons than D1- or D2-expressing spiny projection neurons (SPNs), an effect not observed for M1-corticostriatal afferents. Critically, in vivo optogenetic stimulation of S1-corticostriatal afferents produced task-specific behavioral inhibition, which was bidirectionally modulated by striatal PV interneurons. Optogenetic stimulation of M1 afferents produced the opposite behavioral effect. Thus, our results suggest opposing roles for sensory and motor cortex in behavioral choice via distinct influences on striatal circuitry.

A calcium-activated nonselective cation conductance underlies the plateau potential in rat substantia nigra GABAergic neurons.

Plateau potentials can be elicited in nigral GABAergic neurons by injection of 500 ms depolarizing current pulses from hyperpolarized holding potentials in whole-cell recordings in vitro. In approximately one-third of these neurons, plateau potentials were observed under control conditions and could be elicited in the remaining neurons after blocking potassium conductances. Application of the L-type calcium channel agonist Bay K 8644 or activation of NMDA receptors enhanced plateau potentials observed under control conditions and caused a plateau to be elicited in neurons not exhibiting it previously. The plateau potential was abolished in calcium-free buffer, as well as by nickel or cadmium. The L-type calcium channel blockers nimodipine and nifedipine abolished the plateau potential observed under control conditions but did not affect plateaus unmasked by tetraethylammonium. Plateau potentials observed under control conditions as well as those observed in the presence of Bay K 8644, NMDA, or tetraethylammonium were abolished in low-sodium buffer and by the calcium-activated nonselective cation conductance blocker flufenamic acid. These data suggest that nigral plateau potentials are mediated by a calcium-activated nonselective cation conductance (I(CAN)) that is activated by calcium entry predominantly through L-type calcium channels. In many nigral neurons, I(CAN) is masked by tetraethylammonium-sensitive potassium conductances, but plateaus can be evoked after increasing calcium conductances. The I(CAN)-mediated plateau potential in nigral GABAergic neurons likely affects the way these neurons integrate input and may represent a mechanism contributing to the rhythmic firing of these neurons seen in pathological conditions such as Parkinson's disease.

Decoding cortical brain states from widefield calcium imaging data using visibility graph.

Widefield optical imaging of neuronal populations over large portions of the cerebral cortex in awake behaving animals provides a unique opportunity for investigating the relationship between brain function and behavior. In this paper, we demonstrate that the temporal characteristics of calcium dynamics obtained through widefield imaging can be utilized to infer the corresponding behavior. Cortical activity in transgenic calcium reporter mice (n=6) expressing GCaMP6f in neocortical pyramidal neurons is recorded during active whisking (AW) and no whisking (NW). To extract features related to the temporal characteristics of calcium recordings, a method based on visibility graph (VG) is introduced. An extensive study considering different choices of features and classifiers is conducted to find the best model capable of predicting AW and NW from calcium recordings. Our experimental results show that temporal characteristics of calcium recordings identified by the proposed method carry discriminatory information that are powerful enough for decoding behavior.

Morphological and physiological properties of parvalbumin- and calretinin-containing gamma-aminobutyric acidergic neurons in the substantia nigra.

Evidence for the existence of different populations of gamma-aminobutyric acid (GABA)-ergic neurons in the substantia nigra comes partially from anatomical studies, which have shown there to be little if any overlap between the calcium-binding proteins parvalbumin and calretinin in individual neurons, suggesting that these may represent neuronal subtypes with distinct electrophysiological and/or anatomical properties. We obtained whole-cell recordings from neurons in the substantia nigra pars reticulata in rat brain slices and labeled them with biocytin, followed by immunocytochemical staining for parvalbumin and calretinin. In other cases, neurons were retrogradely labeled from the thalamus or tectum and immunocytochemically identified to determine their projection sites. Intracellularly stained neurons were found to have a variety of somatic sizes and shapes. Reconstructions revealed that all parvalbumin- and calretinin-positive neurons issued at least one axon collateral, which ramified within the substantia nigra pars reticulata and/or pars compacta. Local collaterals were of medium caliber and branched modestly, expressing many long, smooth segments that then issued numerous en passant or terminal boutons, consistent with previous in vivo studies. There were no clear differences in the electrophysiological or morphological properties of neurons expressing parvalbumin or calretinin. Retrograde tracing experiments revealed that both parvalbumin- and calretinin-containing neurons project nonpreferentially to the thalamus or tectum. In sum, the parvalbumin- and calretinin-containing GABAergic neurons of the substantia nigra pars reticulata cannot be differentiated on the basis of their electrophysiological properties, morphological properties, or target nuclei, and both parvalbumin- and calretinin-containing projection neurons issue local axon collaterals that arborize within the substantia nigra.

GABAergic control of substantia nigra dopaminergic neurons.

At least 70% of the afferents to substantia nigra dopaminergic neurons are GABAergic. The vast majority of these arise from the neostriatum, the external globus pallidus and the substantia nigra pars reticulata. Nigral dopaminergic neurons express both GABA(A) and GABA(B) receptors, and are inhibited by local application of GABA(A) or GABA(B) agonists in vivo and in vitro. However, in vivo, synaptic responses elicited by stimulation of neostriatal or pallidal afferents, or antidromic activation of nigral pars reticulata GABAergic projection neurons are mediated predominantly or exclusively by GABA(A) receptors. The clearest and most consistent role for the nigral GABA(B) receptor in vivo is as an inhibitory autoreceptor that presynaptically modulates GABA(A) synaptic responses that originate from all three principal GABAergic inputs. The firing pattern of dopaminergic neurons is also effectively modulated by GABAergic inputs in vivo. Local blockade of nigral GABA(A) receptors causes dopaminergic neurons to shift to a burst firing pattern regardless of the original firing pattern. This is accompanied by a modest increase in spontaneous firing rate. The GABAergic inputs from the axon collaterals of the pars reticulata projection neurons seem to be a particularly important source of a GABA(A) tone to the dopaminergic neurons, inhibition of which leads to burst firing. The globus pallidus exerts powerful control over the pars reticulata input, and through the latter, disynaptically over the dopaminergic neurons. Inhibition of pallidal output leads to a slight decrease in firing of the dopaminergic neurons due to disinhibition of the pars reticulata neurons whereas increased firing of pallidal neurons leads to burst firing in dopaminergic neurons that is associated with a modest increase in spontaneous firing rate and a significant increase in extracellular levels of dopamine in the neostriatum. The pallidal disynaptic disinhibitory control of the dopaminergic neurons dominates the monosynaptic inhibitory influence because of a differential sensitivity to GABA of the two nigral neuron types. Nigral GABAergic neurons are more sensitive to GABA(A)-mediated inhibition than dopaminergic neurons, in part due to a more hyperpolarized GABA(A) reversal potential. The more depolarized GABA(A) reversal potential in the dopaminergic neurons is due to the absence of KCC2, the chloride transporter responsible for setting up a hyperpolarizing Cl(-) gradient in most mature CNS neurons. The data reviewed in this chapter have made it increasingly clear that in addition to the effects that nigral GABAergic output neurons have on their target nuclei outside of the basal ganglia, local interactions between GABAergic projection neurons and dopaminergic neurons are crucially important to the functioning of the nigral dopaminergic neurons.

Aberrant Cortical Activity in Multiple GCaMP6-Expressing Transgenic Mouse Lines.

Transgenic mouse lines are invaluable tools for neuroscience but, as with any technique, care must be taken to ensure that the tool itself does not unduly affect the system under study. Here we report aberrant electrical activity, similar to interictal spikes, and accompanying fluorescence events in some genotypes of transgenic mice expressing GCaMP6 genetically encoded calcium sensors. These epileptiform events have been observed particularly, but not exclusively, in mice with Emx1-Cre and Ai93 transgenes, of either sex, across multiple laboratories. The events occur at >0.1 Hz, are very large in amplitude (>1.0 mV local field potentials, >10% df/f widefield imaging signals), and typically cover large regions of cortex. Many properties of neuronal responses and behavior seem normal despite these events, although rare subjects exhibit overt generalized seizures. The underlying mechanisms of this phenomenon remain unclear, but we speculate about possible causes on the basis of diverse observations. We encourage researchers to be aware of these activity patterns while interpreting neuronal recordings from affected mouse lines and when considering which lines to study.

Pupil Dynamics Reflect Behavioral Choice and Learning in a Go/NoGo Tactile Decision-Making Task in Mice.

The eye's pupil undergoes dynamic changes in diameter associated with cognitive effort, motor activity and emotional state, and can be used to index brain state across mammalian species. Recent studies in head-fixed mice have linked arousal-related pupil dynamics with global neural activity as well as the activity of specific neuronal populations. However, it has remained unclear how pupil dynamics in mice report trial-by-trial performance of behavioral tasks, and change on a longer time scale with learning. We measured pupil dynamics longitudinally as mice learned to perform a Go/NoGo tactile decision-making task. Mice learned to discriminate between two textures presented to the whiskers by licking in response to the Go texture (Hit trial) or withholding licking in response to the NoGo texture (Correct Reject trial, CR). Characteristic pupil dynamics were associated with behavioral choices: large-amplitude pupil dilation prior to and during licking accompanied Hit and False Alarm (FA) responses, while smaller amplitude dilation followed by constriction accompanied CR responses. With learning, the choice-dependent pupil dynamics became more pronounced, including larger amplitude dilations in both Hit and FA trials and earlier onset dilations in Hit and CR trials. A more pronounced constriction was also present in CR trials. Furthermore, pupil dynamics predicted behavioral choice increasingly with learning to greater than 80% accuracy. Our results indicate that pupil dynamics reflect behavioral choice and learning in head-fixed mice, and have implications for understanding decision- and learning-related neuronal activity in pupil-linked neural circuits.

Peripheral optogenetic stimulation induces whisker movement and sensory perception in head-fixed mice.

We discovered that optical stimulation of the mystacial pad in Emx1-Cre;Ai27D transgenic mice induces whisker movements due to activation of ChR2 expressed in muscles controlling retraction and protraction. Using high-speed videography in anesthetized mice, we characterize the amplitude of whisker protractions evoked by varying the intensity, duration, and frequency of optogenetic stimulation. Recordings from primary somatosensory cortex (S1) in anesthetized mice indicated that optogenetic whisker pad stimulation evokes robust yet longer latency responses than mechanical whisker stimulation. In head-fixed mice trained to report optogenetic whisker pad stimulation, psychometric curves showed similar dependence on stimulus duration as evoked whisker movements and S1 activity. Furthermore, optogenetic stimulation of S1 in expert mice was sufficient to substitute for peripheral stimulation. We conclude that whisker protractions evoked by optogenetic activation of whisker pad muscles results in cortical activity and sensory perception, consistent with the coding of evoked whisker movements by reafferent sensory input.

Cell type-specific differences in chloride-regulatory mechanisms and GABA(A) receptor-mediated inhibition in rat substantia nigra.

The regulation of intracellular chloride has important roles in neuronal function, especially by setting the magnitude and direction of the Cl- flux gated by GABA(A) receptors. Previous studies have shown that GABA(A)-mediated inhibition is less effective in dopaminergic than in GABAergic neurons in substantia nigra. We studied whether this phenomenon may be related to a difference in Cl-regulatory mechanisms. Light-microscopic immunocytochemistry revealed that the potassium-chloride cotransporter 2 (KCC2) was localized only in the dendrites of nondopaminergic (primarily GABAergic) neurons in the substantia nigra, whereas the voltage-sensitive chloride channel 2 (ClC-2) was observed only in the dopaminergic neurons of the pars compacta. Electron-microscopic immunogold labeling confirmed that KCC2 is localized in the dendritic plasma membrane of GABAergic neurons close to inhibitory synapses. Confocal microscopy showed that ClC-2 was selectively expressed in the somatic and dendritic cell membranes of the dopaminergic neurons. Gramicidin-perforated-patch recordings revealed that the GABA(A) IPSP reversal potential was significantly less negative and had a much smaller hyperpolarizing driving force in dopaminergic than in GABAergic neurons. The GABA(A) reversal potential was significantly less negative in bicarbonate-free buffer in dopaminergic but not in GABAergic neurons. The present study suggests that KCC2 is responsible for maintaining the low intracellular Cl- concentration in nigral GABAergic neurons, whereas a sodium-dependent anion (Cl--HCO3-) exchanger and ClC-2 are likely to serve this role in dopaminergic neurons. The relatively low efficacy of GABAA-mediated inhibition in nigral dopaminergic neurons compared with nigral GABAergic neurons may be related to their lack of KCC2.

Insulin enhances striatal dopamine release by activating cholinergic interneurons and thereby signals reward.

Insulin activates insulin receptors (InsRs) in the hypothalamus to signal satiety after a meal. However, the rising incidence of obesity, which results in chronically elevated insulin levels, implies that insulin may also act in brain centres that regulate motivation and reward. We report here that insulin can amplify action potential-dependent dopamine (DA) release in the nucleus accumbens (NAc) and caudate-putamen through an indirect mechanism that involves striatal cholinergic interneurons that express InsRs. Furthermore, two different chronic diet manipulations in rats, food restriction (FR) and an obesogenic (OB) diet, oppositely alter the sensitivity of striatal DA release to insulin, with enhanced responsiveness in FR, but loss of responsiveness in OB. Behavioural studies show that intact insulin levels in the NAc shell are necessary for acquisition of preference for the flavour of a paired glucose solution. Together, these data imply that striatal insulin signalling enhances DA release to influence food choices.

Regulation of Substantia Nigra Pars Reticulata GABAergic Neuron Activity by H2O2 via Flufenamic Acid-Sensitive Channels and KATP Channels.

Substantia nigra pars reticulata (SNr) GABAergic neurons are key output neurons of the basal ganglia. Given the role of these neurons in motor control, it is important to understand factors that regulate their firing rate and pattern. One potential regulator is hydrogen peroxide (H2O2), a reactive oxygen species that is increasingly recognized as a neuromodulator. We used whole-cell current clamp recordings of SNr GABAergic neurons in guinea-pig midbrain slices to determine how H2O2 affects the activity of these neurons and to explore the classes of ion channels underlying those effects. Elevation of H2O2 levels caused an increase in the spontaneous firing rate of SNr GABAergic neurons, whether by application of exogenous H2O2 or amplification of endogenous H2O2 through inhibition of glutathione peroxidase with mercaptosuccinate. This effect was reversed by flufenamic acid (FFA), implicating transient receptor potential (TRP) channels. Conversely, depletion of endogenous H2O2 by catalase, a peroxidase enzyme, decreased spontaneous firing rate and firing precision of SNr neurons, demonstrating tonic control of firing rate by H2O2. Elevation of H2O2 in the presence of FFA revealed an inhibition of tonic firing that was prevented by blockade of ATP-sensitive K(+) (K(ATP)) channels with glibenclamide. In contrast to guinea-pig SNr neurons, the dominant effect of H2O2 elevation in mouse SNr GABAergic neurons was hyperpolarization, indicating a species difference in H2O2-dependent regulation. Thus, H2O2 is an endogenous modulator of SNr GABAergic neurons, acting primarily through presumed TRP channels in guinea-pig SNr, with additional modulation via K(ATP) channels to regulate SNr output.