Postganglionic sympathetic neurons, but not locus coeruleus optostimulation, activates neuromuscular transmission in the adult mouse in vivo.

Recent work demonstrated that sympathetic neurons innervate the skeletal muscle near the neuromuscular junction (NMJ), and muscle sympathectomy and sympathomimetic agents strongly influence motoneuron synaptic vesicle release ex vivo. Moreover, reports attest that the pontine nucleus locus coeruleus (LC) projects to preganglionic sympathetic neurons and regulates human mobility and skeletal muscle physiology. Thus, we hypothesized that peripheral and central sympathetic neurons projecting directly or indirectly to the skeletal muscle regulate NMJ transmission. The aim of this study was to define the specific neuronal groups in the peripheral and central nervous systems that account for such regulation in adult mice in vivo by using optogenetics and NMJ transmission recordings in 3-5-month-old, male and female ChR2(H134R/EYFP)/TH-Cre mice. After detecting ChR2(H134R)/EYFP fluorescence in the paravertebral ganglia and LC neurons, we tested whether optostimulating the plantar nerve near the lumbricalis muscle or LC neurons effectively modulates motor nerve terminal synaptic vesicle release in living mice. Nerve optostimulation increased motor synaptic vesicle release in vitro and in vivo, while the presynaptic adrenoceptor blockers propranolol (ß1/ß2) and atenolol (ß1) prevented this outcome. The effect is primarily presynaptic since miniature end-plate potential (MEPP) kinetics remained statistically unmodified after stimulation. In contrast, optostimulation of LC neurons did not regulate NMJ transmission. In summary, we conclude that postganglionic sympathetic neurons, but not LC neurons, increased NMJ transmission by acting on presynaptic ß1-adrenergic receptors in vivo.

Bidirectional Control of Alcohol-drinking Behaviors Through Locus Coeruleus Optoactivation.

The relationship between stress and alcohol-drinking behaviors has been intensively explored; however, neuronal substrates and neurotransmitter dynamics responsible for a causal link between these conditions are still unclear. Here, we optogenetically manipulated locus coeruleus (LC) norepinephrine (NE) activity by applying distinct stimulation protocols in order to explore how phasic and tonic NE release dynamics control alcohol-drinking behaviors. Our results clearly demonstrate contrasting behavioral consequences of LC-NE circuitry activation during low and high frequency stimulation. Specifically, applying tonic stimulation during a standard operant drinking session resulted in increased intake, while phasic stimulation decreased this measure. Furthermore, stimulation during extinction probe trials, when the lever press response was not reinforced, did not significantly alter alcohol-seeking behavior if a tonic pattern was applied. However, phasic stimulation substantially suppressed the number of lever presses, indicating decreased alcohol seeking under the same experimental condition. Given the well-established correlative link between stress and increased alcohol consumption, here we provide the first evidence that tonic LC-NE activity plays a causal role in stress-associated increases in drinking.

Opposite Consequences of Tonic and Phasic Increases in Accumbal Dopamine on Alcohol-Seeking Behavior.

Despite many years of work on dopaminergic mechanisms of alcohol addiction, much of the evidence remains mostly correlative in nature. Fortunately, recent technological advances have provided the opportunity to explore the causal role of alterations in neurotransmission within circuits involved in addictive behaviors. Here, we address this critical gap in our knowledge by integrating an optogenetic approach and an operant alcohol self-administration paradigm to assess directly how accumbal dopamine (DA) release dynamics influences the appetitive (seeking) component of alcohol-drinking behavior. We show that appetitive reward-seeking behavior in rats trained to self-administer alcohol can be shaped causally by ventral tegmental area-nucleus accumbens (VTA-NAc) DA neurotransmission. Our findings reveal that phasic patterns of DA release within this circuit enhance a discrete measure of alcohol seeking, whereas tonic patterns of stimulation inhibit this behavior. Moreover, we provide mechanistic evidence that tonic-phasic interplay within the VTA-NAc DA circuit underlies these seemingly paradoxical effects.

Real-Time Accumbal Dopamine Response to Negative Stimuli: Effects of Ethanol.

Activity in the mesolimbic dopamine (DA) pathway is known to have a role in reward processing and related behaviors. The mesolimbic DA response to reward has been well-examined, while the response to aversive or negative stimuli has been studied to a lesser extent and produced inconclusive results. However, a brief increase in the DA concentration in terminals during nociceptive activation has become an established but not well-characterized phenomenon. Consequently, the interpretation of the significance of this neurochemical response is still elusive. The present study was designed to further explore these increases in subsecond DA dynamics triggered by negative stimuli using voltammetry in anesthetized rats. Our experiments revealed that repeated exposure to a tail pinch resulted in more efficacious DA release in rat nucleus accumbens. This fact may suggest a protective nature of immediate DA efflux. Furthermore, a sensitized DA response to a neutral stimulus, such as a touch, was discovered following several noxious pinches, while a touch applied before these pinches did not trigger DA release. Finally, it was found that the pinch-evoked DA efflux was significantly decreased by ethanol acutely administrated at an analgesic dose. Taken together, these results support the hypothesis that subsecond DA release in the nucleus accumbens may serve as an endogenous antinociceptive signal.

In vivo voltammetric evidence that locus coeruleus activation predominantly releases norepinephrine in the infralimbic cortex: Effect of acute ethanol.

Using fast-scan cyclic voltammetry paired with pharmacology, the authors show that infralimbic catecholamine release following locus coeruleus stimulation is noradrenergic, but not dopaminergic, and not affected by acute ethanol. With previous work, these data suggest differential effects of ethanol on prefrontal norepinephrine and dopamine, a region important in addiction-related pathways.

Optogenetically-induced tonic dopamine release from VTA-nucleus accumbens projections inhibits reward consummatory behaviors.

Recent optogenetic studies demonstrated that phasic dopamine release in the nucleus accumbens may play a causal role in multiple aspects of natural and drug reward-related behaviors. The role of tonic dopamine release in reward consummatory behavior remains unclear. The current study used a combinatorial viral-mediated gene delivery approach to express ChR2 on mesolimbic dopamine neurons in rats. We used optical activation of this dopamine circuit to mimic tonic dopamine release in the nucleus accumbens and to explore the causal relationship between this form of dopamine signaling within the ventral tegmental area (VTA)-nucleus accumbens projection and consumption of a natural reward. Using a two bottle choice paradigm (sucrose vs. water), the experiments revealed that tonic optogenetic stimulation of mesolimbic dopamine transmission significantly decreased reward consummatory behaviors. Specifically, there was a significant decrease in the number of bouts, licks and amount of sucrose obtained during the drinking session. Notably, activation of VTA dopamine cell bodies or dopamine terminals in the nucleus accumbens resulted in identical behavioral consequences. No changes in water intake were evident under the same experimental conditions. Collectively, these data demonstrate that tonic optogenetic stimulation of VTA-nucleus accumbens dopamine release is sufficient to inhibit reward consummatory behavior, possibly by preventing this circuit from engaging in phasic activity that is thought to be essential for reward-based behaviors.

Real time adenosine fluctuations detected with fast-scan cyclic voltammetry in the rat striatum and motor cortex.

BACKGROUND: Adenosine serves many functions within the CNS, including inhibitory and excitatory control of neurotransmission. The understanding of adenosine dynamics in the brain is of fundamental importance. The goal of the present study was to explore subsecond adenosine fluctuations in the rat brain in vivo. METHOD: Long Evans rats were anesthetized and a carbon fiber electrode was positioned in the motor cortex or dorsal striatum. Real time electrochemical recordings were made at the carbon fiber electrodes every 100ms by applying a triangular waveform (-0.4 to +1.5V, 400V/s). Adenosine spikes were identified by the background-subtracted cyclic voltammogram. RESULTS: The frequency of detected adenosine spikes was relatively stable in both tested regions, and the time intervals between spikes were regular and lasted from 1 to 5s within an animal. Spike frequency ranged from 0.5 to 1.5Hz in both the motor cortex and the dorsal striatum. Average spike amplitudes were 85±11 and 66±7nM for the motor cortex and the dorsal striatum, respectively. COMPARISON WITH EXISTING METHODS: The current study established that adenosine signaling can operate on a fast time scale (within seconds) to modulate brain functions. CONCLUSIONS: This finding suggests that spontaneous adenosine release may play a fast, dynamic role in regulating an organism's response to external events. Therefore, adenosine transmission in the brain may have characteristics similar to those of classical neurotransmitters, such as dopamine and norepinephrine.

Understanding the role α7 nicotinic receptors play in dopamine efflux in nucleus accumbens.

Neuronal nicotinic acetylcholine receptors (NNRs) of the α7 subtype have been shown to contribute to the release of dopamine in the nucleus accumbens. The site of action and the underlying mechanism, however, are unclear. Here we applied a circuit modeling approach, supported by electrochemical in vivo recordings, to clarify this issue. Modeling revealed two potential mechanisms for the drop in accumbal dopamine efflux evoked by the selective α7 partial agonist TC-7020. TC-7020 could desensitize α7 NNRs located predominantly on dopamine neurons or glutamatergic afferents to them or, alternatively, activate α7 NNRs located on the glutamatergic afferents to GABAergic interneurons in the ventral tegmental area. Only the model based on desensitization, however, was able to explain the neutralizing effect of coapplied PNU-120596, a positive allosteric modulator. According to our results, the most likely sites of action are the preterminal α7 NNRs controlling glutamate release from cortical afferents to the nucleus accumbens. These findings offer a rationale for the further investigation of α7 NNR agonists as therapy for diseases associated with enhanced mesolimbic dopaminergic tone, such as schizophrenia and addiction.

Optogenetic stimulation of VTA dopamine neurons reveals that tonic but not phasic patterns of dopamine transmission reduce ethanol self-administration.

There is compelling evidence that acute ethanol exposure stimulates ventral tegmental area (VTA) dopamine cell activity and that VTA-dependent dopamine release in terminal fields within the nucleus accumbens plays an integral role in the regulation of ethanol drinking behaviors. Unfortunately, due to technical limitations, the specific temporal dynamics linking VTA dopamine cell activation and ethanol self-administration are not known. In fact, establishing a causal link between specific patterns of dopamine transmission and ethanol drinking behaviors has proven elusive. Here, we sought to address these gaps in our knowledge using a newly developed viral-mediated gene delivery strategy to selectively express Channelrhodopsin-2 (ChR2) on dopamine cells in the VTA of wild-type rats. We then used this approach to precisely control VTA dopamine transmission during voluntary ethanol drinking sessions. The results confirmed that ChR2 was selectively expressed on VTA dopamine cells and delivery of blue light pulses to the VTA induced dopamine release in accumbal terminal fields with very high temporal and spatial precision. Brief high frequency VTA stimulation induced phasic patterns of dopamine release in the nucleus accumbens. Lower frequency stimulation, applied for longer periods mimicked tonic increases in accumbal dopamine. Notably, using this optogenetic approach in rats engaged in an intermittent ethanol drinking procedure, we found that tonic, but not phasic, stimulation of VTA dopamine cells selectively attenuated ethanol drinking behaviors. Collectively, these data demonstrate the effectiveness of a novel viral targeting strategy that can be used to restrict opsin expression to dopamine cells in standard outbred animals and provide the first causal evidence demonstrating that tonic activation of VTA dopamine neurons selectively decreases ethanol self-administration behaviors.

Terminal effects of optogenetic stimulation on dopamine dynamics in rat striatum.

In this study, the first in-depth analysis of optically induced dopamine release using fast-scan cyclic voltammetry on striatal slices from rat brain was performed. An adeno-associated virus that expresses Channelrhodopsin-2 was injected in the substantia nigra. Tissue was collected and sectioned into 400µm-thick coronal slices 4 weeks later. Blue laser light (473nm) was delivered through a fiber optic inserted into slice tissue. Experiments revealed some difference between maximal amplitudes measured from optically and electrically evoked dopamine effluxes. Specifically, there was an increase in the amplitude of dopamine release induced by electrical stimulation in comparison with light stimulations. However, we found that dopamine release is more sensitive to changes in the pulse width in the case of optical stimulation. Light-stimulated dopamine was increased as the stimulation pulse widened. There was no difference with repeated stimulations at five minute intervals between stimulation sources and dopamine signal was stable during recording sessions, while one minute intervals resulted in a decline in the amplitude from both sources. Optical stimulation can also produce an artifact that is distinguishable from dopamine by the cyclic voltammogram. These results confirm that optical stimulation of dopamine is a sound approach for future pharmacological studies in slices.

Optogenetic control of striatal dopamine release in rats.

Optogenetic control over neuronal firing has become an increasingly elegant method to dissect the microcircuitry of mammalian brains. To date, examination of these manipulations on neurotransmitter release has been minimal. Here we present the first in-depth analysis of optogenetic stimulation on dopamine neurotransmission in the dorsal striatum of urethane-anesthetized rats. By combining the tight spatial and temporal resolution of both optogenetics and fast-scan cyclic voltammetry we have determined the parameters necessary to control phasic dopamine release in the dorsal striatum of rats in vivo. The kinetics of optically induced dopamine release mirror established models of electrically evoked release, indicating that potential artifacts of electrical stimulation on ion channels and the dopamine transporter are negligible. Furthermore a lack of change in extracellular pH indicates that optical stimulation does not alter blood flow. Optical control over dopamine release is highly reproducible and flexible. We are able to repeatedly evoke concentrations of dopamine release as small as a single dopamine transient (50 nM). An inverted U-shaped frequency response curve exists with maximal stimulation inducing dopamine effluxes exceeding 500 nM. Taken together, these results have obvious implications for understanding the neurobiological basis of dopaminergic-based disorders and provide the framework to effectively manipulate dopamine patterns.