Spatiotemporal Distributions of Acoustic Propagation in Skull During Ultrasound Neuromodulation.

There is widespread interest and concern about the evidence and hypothesis that the auditory system is involved in ultrasound neuromodulation. We have addressed this problem by performing acoustic shear wave simulations in mouse skull and behavioral experiments in deaf mice. The simulation results showed that shear waves propagating along the skull did not reach sufficient acoustic pressure in the auditory cortex to modulate neurons. Behavioral experiments were subsequently performed to awaken anesthetized mice with ultrasound targeting the motor cortex or ventral tegmental area (VTA). The experimental results showed that ultrasound stimulation (US) of the target areas significantly increased arousal scores even in deaf mice, whereas the loss of ultrasound gel abolished the effect. Immunofluorescence staining also showed that ultrasound can modulate neurons in the target area, whereas neurons in the auditory cortex required the involvement of the normal auditory system for activation. In summary, the shear waves propagating along the skull cannot reach the auditory cortex and induce neuronal activation. Ultrasound neuromodulation-induced arousal behavior needs direct action on functionally relevant stimulation targets in the absence of auditory system participation.

Precise Ultrasound Neuromodulation in a Deep Brain Region Using Nano Gas Vesicles as Actuators.

Ultrasound is a promising new modality for non-invasive neuromodulation. Applied transcranially, it can be focused down to the millimeter or centimeter range. The ability to improve the treatment's spatial resolution to a targeted brain region could help to improve its effectiveness, depending upon the application. The present paper details a neurostimulation scheme using gas-filled nanostructures, gas vesicles (GVs), as actuators for improving the efficacy and precision of ultrasound stimuli. Sonicated primary neurons display dose-dependent, repeatable Ca2+ responses, closely synced to stimuli, and increased nuclear expression of the activation marker c-Fos in the presence of GVs. GV-mediated ultrasound triggered rapid and reversible Ca2+ responses in vivo and could selectively evoke neuronal activation in a deep-seated brain region. Further investigation indicate that mechanosensitive ion channels are important mediators of this effect. GVs themselves and the treatment scheme are also found not to induce significant cytotoxicity, apoptosis, or membrane poration in treated cells. Altogether, this study demonstrates a simple and effective method to achieve enhanced and better-targeted neurostimulation with non-invasive low-intensity ultrasound.

Ventral tegmental area GABA neurons mediate stress-induced blunted reward-seeking in mice.

Decreased pleasure-seeking (anhedonia) forms a core symptom of depression. Stressful experiences precipitate depression and disrupt reward-seeking, but it remains unclear how stress causes anhedonia. We recorded simultaneous neural activity across limbic brain areas as mice underwent stress and discovered a stress-induced 4 Hz oscillation in the nucleus accumbens (NAc) that predicts the degree of subsequent blunted reward-seeking. Surprisingly, while previous studies on blunted reward-seeking focused on dopamine (DA) transmission from the ventral tegmental area (VTA) to the NAc, we found that VTA GABA, but not DA, neurons mediate stress-induced blunted reward-seeking. Inhibiting VTA GABA neurons disrupts stress-induced NAc oscillations and rescues reward-seeking. By contrast, mimicking this signature of stress by stimulating NAc-projecting VTA GABA neurons at 4 Hz reproduces both oscillations and blunted reward-seeking. Finally, we find that stress disrupts VTA GABA, but not DA, neural encoding of reward anticipation. Thus, stress elicits VTA-NAc GABAergic activity that induces VTA GABA mediated blunted reward-seeking.

Deep brain optogenetics without intracranial surgery.

Achieving temporally precise, noninvasive control over specific neural cell types in the deep brain would advance the study of nervous system function. Here we use the potent channelrhodopsin ChRmine to achieve transcranial photoactivation of defined neural circuits, including midbrain and brainstem structures, at unprecedented depths of up to 7 mm with millisecond precision. Using systemic viral delivery of ChRmine, we demonstrate behavioral modulation without surgery, enabling implant-free deep brain optogenetics.

Cue-Evoked Dopamine Neuron Activity Helps Maintain but Does Not Encode Expected Value.

Cue-evoked midbrain dopamine (DA) neuron activity reflects expected value, but its influence on reward assessment is unclear. In mice performing a trial-based operant task, we test if bidirectional manipulations of cue or operant-associated DA neuron activity drive learning as a result of under- or overexpectation of reward value. We target optogenetic manipulations to different components of forced trials, when only one lever is presented, and assess lever biases on choice trials in the absence of photomanipulation. Although lever biases are demonstrated to be flexible and sensitive to changes in expected value, augmentation of cue or operant-associated DA signaling does not significantly alter choice behavior, and blunting DA signaling during any component of the forced trials reduces choice trial responses on the associated lever. These data suggest cue-evoked DA helps maintain cue-value associations but does not encode expected value as to set the benchmark against which received reward is judged.

A Midbrain Circuit that Mediates Headache Aversiveness in Rats.

Migraines are a major health burden, but treatment is limited because of inadequate understanding of neural mechanisms underlying headache. Imaging studies of migraine patients demonstrate changes in both pain-modulatory circuits and reward-processing regions, but whether these changes contribute to the experience of headache is unknown. Here, we demonstrate a direct connection between the ventrolateral periaqueductal gray (vlPAG) and the ventral tegmental area (VTA) that contributes to headache aversiveness in rats. Many VTA neurons receive monosynaptic input from the vlPAG, and cranial nociceptive input increases Fos expression in VTA-projecting vlPAG neurons. Activation of PAG inputs to the VTA induces avoidance behavior, while inactivation of these projections induces a place preference only in animals with headache. This work identifies a distinct pathway that mediates cranial nociceptive aversiveness.

Manipulating midbrain dopamine neurons and reward-related behaviors with light-controllable nicotinic acetylcholine receptors.

Dopamine (DA) neurons of the ventral tegmental area (VTA) integrate cholinergic inputs to regulate key functions such as motivation and goal-directed behaviors. Yet the temporal dynamic range and mechanism of action of acetylcholine (ACh) on the modulation of VTA circuits and reward-related behaviors are not known. Here, we used a chemical-genetic approach for rapid and precise optical manipulation of nicotinic neurotransmission in VTA neurons in living mice. We provide direct evidence that the ACh tone fine-tunes the firing properties of VTA DA neurons through ß2-containing (ß2*) nicotinic ACh receptors (nAChRs). Furthermore, locally photo-antagonizing these receptors in the VTA was sufficient to reversibly switch nicotine reinforcement on and off. By enabling control of nicotinic transmission in targeted brain circuits, this technology will help unravel the various physiological functions of nAChRs and may assist in the design of novel therapies relevant to neuropsychiatric disorders.

Pontomesencephalic Tegmental Afferents to VTA Non-dopamine Neurons Are Necessary for Appetitive Pavlovian Learning.

The ventral tegmental area (VTA) receives phenotypically distinct innervations from the pedunculopontine tegmental nucleus (PPTg). While PPTg-to-VTA inputs are thought to play a critical role in stimulus-reward learning, direct evidence linking PPTg-to-VTA phenotypically distinct inputs in the learning process remains lacking. Here, we used optogenetic approaches to investigate the functional contribution of PPTg excitatory and inhibitory inputs to the VTA in appetitive Pavlovian conditioning. We show that photoinhibition of PPTg-to-VTA cholinergic or glutamatergic inputs during cue presentation dampens the development of anticipatory approach responding to the food receptacle during the cue. Furthermore, we employed in vivo optetrode recordings to show that photoinhibition of PPTg cholinergic or glutamatergic inputs significantly decreases VTA non-dopamine (non-DA) neural activity. Consistently, photoinhibition of VTA non-DA neurons disrupts the development of cue-elicited anticipatory approach responding. Taken together, our study reveals a crucial regulatory mechanism by PPTg excitatory inputs onto VTA non-DA neurons during appetitive Pavlovian conditioning.

Optogenetic Modulation of Locomotor Activity on Free-Behaving Rats.

The technology of optogenetics provides a new method to modulate neural activity with spatial specificity and millisecond-temporal scale. This nonelectrical modulation method also gives chance for simultaneous electrophysiological recording during stimulations. Here, we describe our locomotor activity modulation on free-behaving rats using optogenetic techniques. The target sites of the rat brain were dorsal periaqueductal gray (dPAG) and ventral tegmental area (VTA) for the modulation of defensive and reward behaviors, respectively.

Leptin regulates the reward value of nutrient.

We developed an assay for quantifying the reward value of nutrient and used it to analyze the effects of metabolic state and leptin. In this assay, mice chose between two sippers, one of which dispensed water and was coupled to optogenetic activation of dopaminergic (DA) neurons and the other of which dispensed natural or artificial sweeteners. This assay measured the reward value of sweeteners relative to lick-induced optogenetic activation of DA neurons. Mice preferred optogenetic stimulation of DA neurons to sucralose, but not to sucrose. However, the mice preferred sucralose plus optogenetic stimulation versus sucrose. We found that food restriction increased the value of sucrose relative to sucralose plus optogenetic stimulation, and that leptin decreased it. Our data suggest that leptin suppresses the ability of sucrose to drive taste-independent DA neuronal activation and provide new insights into the mechanism of leptin's effects on food intake.

Short-acting cocaine and long-acting GBR-12909 both elicit rapid dopamine uptake inhibition following intravenous delivery.

The rewarding effects of cocaine have been reported to occur within seconds of administration. Extensive evidence suggests that these actions involve the ability of cocaine to inhibit the dopamine (DA) transporter. We recently showed that 1.5 mg/kg i.v. cocaine inhibits DA uptake within 5 s. Despite this evidence, there remains a lack of consensus regarding how quickly i.v. cocaine and other DA uptake inhibitors elicit DA uptake inhibition. The current studies sought to better characterize the onset of cocaine-induced DA uptake inhibition and to compare these effects to those obtained with the high-affinity, long-acting DA transporter inhibitor, GBR-12909 (1-(2-bis(4-fluorphenyl)-methoxy)-ethyl)-4-(3-phenyl-propyl)piperazine). Using in vivo fast scan cyclic voltammetry, we showed that i.v. cocaine (0.75, 1.5, and 3.0 mg/kg) significantly inhibited DA uptake in the nucleus accumbens of anesthetized rats within 5 s. DA uptake inhibition peaked at 30 s and returned to baseline levels in approximately 1 h. The effects of cocaine were dose-dependent, with the 3.0 mg/kg dose producing greater uptake inhibition at the early time points and exhibiting a longer latency to return to baseline. Further, the blood-brain barrier impermeant cocaine-methiodide had no effect on DA uptake or peak height, indicating that the generalized peripheral effects of cocaine do not contribute to the CNS alterations measured here. Finally, we show that GBR-12909 (0.75, 1.5, and 3.0 mg/kg) also significantly inhibited DA uptake within 5 s post-injection, although the peak effect and return to baseline were markedly delayed compared with cocaine, particularly at the highest dose. Combined, these observations indicate that the central effects of dopamine uptake inhibitors occur extremely rapidly following i.v. drug delivery.

High-frequency afferent stimulation induces long-term potentiation of field potentials in the ventral tegmental area.

Excitatory synapses on dopamine neurons in the VTA can undergo both long-term potentiation and depression. Additionally, drug-induced plasticity has been found at VTA synapses, and is proposed to play a role in reward-related learning and addiction by modifying dopamine cell firing. LTP at these synapses is difficult to generate experimentally in that it requires an undisturbed intracellular milieu and is often small in magnitude. Here, we demonstrate the induction of LTP as a property of evoked field potentials within the VTA. Excitatory field potentials were recorded extracellularly from VTA neurons in acute horizontal midbrain slices. Using extracellular and intracellular recording techniques, we found that evoked field potentials originate within the VTA itself and are largely composed of AMPA receptor-mediated EPSPs and action potentials triggered by activation of glutamatergic synapses on both dopamine and GABA neurons. High-frequency afferent stimulation (HFS) induced LTP of the field potential. The induction of this LTP was blocked by application of the NMDAR antagonist, d-APV, prior to HFS. As reported previously, glutamatergic synapses on GABA neurons did not express LTP while those on dopamine neurons did. We conclude that the potentiation of glutamatergic synapses on dopamine neurons is a major contributor to NMDA receptor-dependent LTP of the field potential. Field potential recordings may provide a convenient approach to explore the basic electrophysiological properties of VTA neurons and the development of addiction-related processes in this brain region.

Neurobiology of 50-kHz ultrasonic vocalizations in rats: electrode mapping, lesion, and pharmacology studies.

Fifty-kHz ultrasonic vocalizations have been proposed to reflect a positive appetitive affective state in rats, being consistently linked to the positive appetitive behavior. In the first study, we examined the brain substrates of 50-kHz ultrasonic vocalizations (USVs) by using localized electrical stimulation of the brain (ESB) at various sites that are known to mediate reward. We found that the brain areas that produced ESB-induced 50-kHz calls are the areas that have previously been shown to support the most vigorous self-stimulation behavior (prefrontal cortex, nucleus accumbens, ventral pallidum, lateral preoptic area, lateral hypothalamus, ventral tegmental area, and raphe). Importantly, all animals that showed repeatable ESB-induced 50-kHz USVs demonstrated self-stimulation behavior. In the second study, conditioned place preference was assessed following microinjection of the mu-opiate agonist Tyr-D-Ala-Gly-N-methyl-Phe-Gly-ol (DAMGO) directly into the ventral tegmental area (VTA) at a dose previously found to be rewarding. Animals that showed more 50-kHz USVs in response to drug injections compared to vehicle injections showed significant place preferences, whereas animals that did not show elevated vocalization to DAMGO did not show place preference. In experiment 3, we examined the effect of VTA electrolytic lesions, 6-OHDA lesions, and the effect of the D1/D2 dopamine antagonist flupenthixol (0 and 0.8 mg/kg, i.p.) on 50-kHz ultrasonic vocalizations. We found that these manipulations all selectively reduced 50-kHz ultrasonic vocalizations, and that these effects could be disassociated from any side effects. These data are consistent with the proposition that 50-kHz calls are tightly linked to reward in rats and that the neural circuit of 50-kHz calls closely overlaps that of ESB self-stimulation reward, drug reward, and the mesolimbic dopamine system.

Hyperactivity of the facial nucleus produced by chronic electrical stimulation in rats.

OBJECTIVE: There are two hypotheses for the pathogenesis of hemifacial spasm (HFS): abnormal cross-transmission between the facial nerve fibers at a site of vascular compression, and hyperactivity of the facial nucleus. To further elucidate the mechanism of HFS, we established an animal model. We applied chronic electrical stimulation (CES) to the facial nucleus in rats, and clarified functional and morphological changes in the nucleus. METHOD: Under anesthesia, a novel intracranial electrode was stereotactically implanted in the facial nucleus of six rats. CES of the facial nucleus via the implanted electrode was applied for 5 min daily for three weeks (CES animals). Facial electromyograms (EMGs) were recorded at rest and during electrical stimulation to study the excitability of the facial nucleus at 1, 2, and 4 weeks after initiating CES. As control animals, six rats were implanted with intracranial electrodes, but did not undergo CES. Electrophysiological studies of the control animals were performed using the same protocol as in the CES animals. RESULT: Spontaneous abnormal movement of the facial muscle mimicking HFS did not occur. Four weeks after starting CES, one of the six CES animals developed an abnormal EMG response with a latency of 10 ms. No control animals developed such a response. CONCLUSIONS: CES of the facial nucleus can produce an abnormal EMG response very similar to the abnormal muscle response (AMR) characteristic of HFS patients. Kindling-like hyperactivity of the facial nucleus induced by CES is the cause of the AMR, suggesting a pathogenesis of HFS.

Long-term neuroadaptations produced by withdrawal from repeated cocaine treatment: role of dopaminergic receptors in modulating cortical excitability.

Dopamine (DA) modulates neuronal activity in the prefrontal cortex (PFC) and is necessary for optimal cognitive function. Dopamine transmission in the PFC is also important for the behavioral adaptations produced by repeated exposure to cocaine. Therefore, we investigated the effects of repeated cocaine treatment followed by withdrawal (2-4 weeks) on the responsivity of cortical cells to electrical stimulation of the ventral tegmental area (VTA) and to systemic administration of DA D1 or D2 receptor antagonists. Cortical cells in cocaine- and saline-treated animals exhibited a similar decrease in excitability after the administration of D1 receptor antagonists. In contrast, cortical neurons from cocaine-treated rats exhibited a lack of D2-mediated regulation relative to saline rats. Furthermore, in contrast to saline-treated animals, VTA stimulation did not increase cortical excitability in the cocaine group. These data suggest that withdrawal from repeated cocaine administration elicits some long-term neuroadaptations in the PFC, including (1) reduced D2-mediated regulation of cortical excitability, (2) reduced responsivity of cortical cells to phasic increases in DA, and (3) a trend toward an overall decrease in excitability of PFC neurons.

Altered prefrontal cortical metabolic response to mesocortical activation in adult animals with a neonatal ventral hippocampal lesion.

BACKGROUND: Adult animals with a neonatal ventral hippocampal lesion (NVHL) exhibit deficits in working memory and sensorimotor gating similar to those observed in schizophrenia. As cognitive deficits in this disorder are typically associated with changes in cortical metabolic levels, we investigated here whether an NVHL affects metabolic responses to ventral tegmental area (VTA) activation, a procedure that elicits abnormal cell firing in the prefrontal cortex (PFC) of NVHL animals. METHODS: Prefrontal cortex metabolic activity was determined by measuring cytochrome oxidase I (CO-I) staining. Cytochrome oxidase I levels were quantified by densitometry in pre- and postpubertal sham-operated and lesioned rats that received one or three series of fifteen 20-Hz trains of VTA stimuli every 20 seconds. RESULTS: Ventral tegmental area stimulation yielded higher levels of PFC CO-I in NVHL animals when compared with the sham-operated group, an effect that appeared only after puberty. Increasing the series of burst stimulations further elevated CO-I in sham-operated, but not in NVHL animals. CONCLUSIONS: Increased PFC CO-I activity after VTA burst stimulation in NVHL rats highlights the enhanced energy demand that could be linked to the exaggerated response to stress observed in these animals. The inability to further increase the response with higher mesocortical activity, as observed in sham-operated animals, could be expression of a reduced PFC functional capacity in lesioned animals. Thus, a hyperexcitable PFC with a reduced ability to further increase activity could be a plausible pathophysiological scenario for schizophrenia. Human functional studies could be interpreted in the light of this conceptual framework.

Microinjection of procaine and electrolytic lesion in the ventral tegmental area suppresses hippocampal theta rhythm in urethane-anesthetized rats.

The midbrain ventral tegmental area (VTA), a key structure of the mesocorticolimbic system is anatomically connected with the hippocampal formation. In addition mesocortical dopamine was found to influence hippocampus-related memory and hippocampal synaptic plasticity, both being linked to the theta rhythm. Therefore, the aim of the present study was to evaluate the possible role of the VTA in the regulation of the hippocampal theta activity. The study was performed on urethane-anesthetized male Wistar rats in which theta rhythm was evoked by tail pinch. It was found that unilateral, temporal inactivation of the VTA by means of direct procaine injection resulted in bilateral suppression of the hippocampal theta which manifested as a loss of synchronization of hippocampal EEG and respective reduction of the power and also the frequency of the 3-6 Hz theta band. Depression of the power of the 3-6 Hz component of the EEG signal was also seen in spontaneous hippocampal EEG after procaine. The permanent destruction of the VTA by means of unilateral electrocoagulation evoked a long-lasting, mainly ipsilateral depression of the power of the theta with some influence on its frequency. Simultaneously, there was a substantial increase of the power in higher frequency bands indicating decrease of a synchrony of the hippocampal EEG activity. On the basis of these results indicating impairment of synchronization of the hippocampal activity the VTA may be considered as another part of the brainstem theta synchroning system.

Slower extinction of responses maintained by intra-cranial self-stimulation (ICSS) in an animal model of attention-deficit/hyperactivity disorder (ADHD).

Children with attention-deficit/hyperactivity disorder (ADHD) show performance deficits and excessive motor activity during extinction and in situations where no reinforcer can be identified, suggesting an extinction deficit in ADHD possibly linked to dopamine dysfunction. The present study examined extinction of responding previously maintained by intra-cranial self-stimulation (ICSS) in spontaneously hypertensive rats (SHR), an animal model of ADHD using three different extinction procedures. Delivery of electrical pulses were terminated altogether or presented independently of responding using two different current intensities. The results showed that more responses were retained in the SHR, especially during the initial transition from ICSS-maintained responding to response-independent delivery of electrical pulses with current reduced relative to that given during reinforcement. Slower extinction of previously reinforced behavior is suggested as an alternative explanation for the frequently observed increased behavioral output that has previously been interpreted as "disinhibition" of behavior in ADHD.

Simultaneous AMPA/kainate receptor blockade and dopamine D(2/3) receptor stimulation in the nucleus accumbens decreases brain stimulation reward in rats.

Interactions between dopamine (DA) and glutamate (GLU) in the mesocorticolimbic pathway of the brain may influence motivation and reward. Previous work from this laboratory has demonstrated that alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor blockade may potentiate decreases in exploratory motor activity induced by the DA D(2/3) receptor agonist 7-OH-DPAT in the nucleus accumbens septi (NAS). This study investigated the interaction of AMPA/kainate receptor antagonists CNQX or NBQX with 7-OH-DPAT on ventral tegmental area (VTA) brain stimulation reward (BSR). Effects of these compounds, alone and combined, were measured in male Sprague-Dawley rats stereotaxically implanted with a unilateral VTA electrode and bilateral guide cannulae in the NAS core or shell subregions. Rate-frequency analysis was used to assess BSR frequency thresholds and maximum response rates of rats trained to lever-press for reinforcing electrical stimulation. When given alone, CNQX (0.5 microg), NBQX (0.5 microg), or 7-OH-DPAT (5.0 microg) did not affect BSR frequency thresholds. Co-administration of CNQX or NBQX with 7-OH-DPAT synergistically increased BSR frequency thresholds, indicative of decreased reward. These data indicate that simultaneous AMPA/kainate receptor blockade and DA D(2/3) receptor stimulation in the NAS may act synergistically to inhibit motivated behaviours such as electrical brain self-stimulation.

Short- and long-term effects of interleukin-2 on weight, food intake, and hedonic mechanisms in the rat.

In the present work, we investigated the short- and long-term effects of a single systemic injection of rat recombinant interleukin-2 on weight, food intake, and brain stimulation reward thresholds elicited from the ventral tegmental area. An inverted U-shaped dose-function was obtained with 0.5 microg producing the greatest increases in the threshold for rewarding brain stimulation which were sustained during the month long tests. No differences between groups in terms of maximum response rates, a measure of performance, were observed. Although all injected groups showed a minor decline in the rate of weight gain over time, percent efficiency of food utilization (percent weight gain/food intake) was the same across groups, suggesting that metabolic function was not affected by the cytokine. In animals with bilateral ventral tegmental area implants, there was no consistent correspondence between the threshold change obtained from ipsilateral stimulation and that associated with the contralateral site; side-to-side differences ranged from 0 to 100%, suggesting a specific interaction between cytokine activity and the locus of rewarding brain stimulation. These data suggest that peripheral IL-2 significantly modifies hedonic processes arising from medial forebrain bundle stimulation in a long-term manner. We further suggest that since this modulation appears to be notably site-specific, IL-2 receptors or its metabolites may not be evenly distributed within the medial forebrain bundle.