DREADD-mediated modulation of locus coeruleus inputs to mPFC improves strategy set-shifting.

Appropriate modification of behavior in response to our dynamic environment is essential for adaptation and survival. This adaptability allows organisms to maximize the utility of behavior-related energy expenditure. Modern theories of locus coeruleus (LC) function implicate a pivotal role for the noradrenergic nucleus in mediating switches between focused behavior during periods of high utility (exploit) versus disengagement of behavior and exploration of other, more rewarding opportunities. Two modes of activity in LC neurons have been characterized as elements in an Adaptive Gain Theory (AGT) of LC function. In this theory, during periods of accurate and focused behavior, LC neurons exhibit task-related phasic bursts. However, as behavioral utility wanes, phasic activity is suppressed and baseline (tonic) impulse activity increases to facilitate exploration. Our experiments sought to exogenously induce an elevated pattern of activity in LC neurons and their medial prefrontal cortical (mPFC) targets to test the tenets of the AGT. This theory posits that tonic activation immediately following a rule change should increase exploration and thereby improve performance on a set-shifting task. Indeed, DREADD mediated stimulation of LC terminals within mPFC decreased trials to reach criterion. However, this effect resulted from improved application of the new rule once the original rule is jettisoned rather than earlier disengagement from the old, ineffective strategy. Such improvements were not seen with global manipulation of LC, consistent with the view that LC-mediated exploration involves specific sub-circuits targeting mPFC. These findings extend our understanding of the role of LC in PFC and flexible behavior.

Histologic validation of locus coeruleus MRI contrast in post-mortem tissue.

The locus coeruleus (LC) noradrenergic system regulates arousal and modulates attention through its extensive projections across the brain. LC dysfunction has been implicated in a broad range of neurodevelopmental, neurodegenerative and psychiatric disorders, as well as in the cognitive changes observed during normal aging. Magnetic resonance imaging (MRI) has been used to characterize the human LC (elevated contrast relative to surrounding structures), but there is limited understanding of the factors underlying putative LC contrast that are critical to successful biomarker development and confidence in localizing nucleus LC. We used ultra-high-field 7 T magnetic resonance imaging (MRI) to acquire T1-weighted microscopy resolution images (78 µm in-plane resolution) of the LC from post-mortem tissue samples. Histological analyses were performed to characterize the distribution of tyrosine hydroxylase (TH) and neuromelanin in the scanned tissue, which allowed for direct comparison with MR microscopy images. Our results indicate that LC-MRI contrast corresponds to the location of neuromelanin cells in LC; these also correspond to norepinephrine neurons. Thus, neuromelanin appears to serve as a natural contrast agent for nucleus LC that can be used to localize nucleus LC and may have the potential to characterize neurodegenerative disease.

Fos activation of selective afferents to ventral tegmental area during cue-induced reinstatement of cocaine seeking in rats.

Ventral tegmental area (VTA) dopamine neurons are crucial for appetitive responses to Pavlovian cues, including cue-induced reinstatement of drug seeking. However, it is unknown which VTA inputs help activate these neurons, transducing stimuli into salient cues that drive drug-seeking behavior. Here we examined 56 VTA afferents from forebrain and midbrain that are Fos activated during cue-induced reinstatement. We injected the retrograde tracer cholera toxin ß subunit (CTb) unilaterally into rostral or caudal VTA of male rats. All animals were trained to self-administer cocaine, then extinguished of this behavior. On a final test day, animals were exposed to response-contingent cocaine-associated cues, extinction conditions, a non-cocaine-predictive CS-, or a novel environment, and brains were processed to visualize CTb and Fos immunoreactivity to identify VTA afferents activated in relation to behaviors. VTA-projecting neurons in subregions of medial accumbens shell, ventral pallidum, elements of extended amygdala, and lateral septum (but not prefrontal cortex) were activated specifically during cue-induced cocaine seeking, and some of these were also activated proportionately to the degree of cocaine seeking. Surprisingly, though efferents from the lateral hypothalamic orexin field were also Fos activated during reinstatement, these were largely non-orexinergic. Also, VTA afferents from the rostromedial tegmental nucleus and lateral habenula were specifically activated during extinction and CS- tests, when cocaine was not expected. These findings point to a select set of subcortical nuclei which provide reinstatement-related inputs to VTA, translating conditioned stimuli into cocaine-seeking behavior.

A septal-hypothalamic pathway drives orexin neurons, which is necessary for conditioned cocaine preference.

Orexins (also called hypocretins) have been shown to be importantly involved in reward and addiction, but little is known about the circuitry that regulates orexin neuronal activity during drug-seeking behaviors. Here, we examined inputs to the lateral hypothalamus (LH) orexin cell field from the lateral septum (LS) using tract-tracing and Fos immunohistochemistry after cocaine (10 mg/kg) conditioned place preference (CPP) in Sprague Dawley rats. We found that neurons in rostral LS (LSr) that project to LH are Fos-activated in proportion to cocaine CPP, and that inhibition of LSr neurons with local baclofen and muscimol microinjection (0.3/0.03 nmol) blocks expression of Fos in LH orexin cells and cocaine preference. In addition, using local inactivation in LS and orexin antisense morpholinos in LH, we found that LSr influences on LH orexin neurons are critical for the expression of cocaine preference. These results indicate that LSr activates LH orexin neurons during cocaine place preference, and that this circuit is essential for expression of cocaine place preference.

Novel neurons in ventral tegmental area fire selectively during the active phase of the diurnal cycle.

The ventral tegmental area (VTA) contains dopamine (DA) and gamma-aminobutyric acid (GABA) neurons involved in motivation and behavioral state. These phenomena are also influenced by circadian factors. The goal of our studies was to examine the impulse activity of neurochemically identified VTA neurons during dark (active) vs light (rest) phases of the circadian cycle. Using extracellular single-unit recordings with juxtacellular labeling in anesthetized rats, we found multiple neuronal subpopulations including 'novel neurons' that selectively fired during the dark phase. These novel neurons were electrophysiologically categorized into two groups, 'novel wide-spike' and 'novel thin-spike' neurons. Characterization of novel wide-spike neurons found they were consistently non-dopaminergic and non-GABAergic [tyrosine hydroxylase (TH)(-), glutamic acid decarboxylase (GAD)(-)]. However, they were inhibited by the D2 agonist quinpirole, an effect that could be reversed by the D2 antagonist eticlopride. Physiologically, they were fast firing (mean = 18.9 +/- 1.2 spikes/s), low bursting neurons (median = 6.2 +/- 3.0% of spikes in bursts) with spike durations > or = 2.0 ms, but slightly shorter than TH(+) neurons. They were also consistently non-responsive to footpad stimulation. The novel thin-spike neurons were neurochemically heterogeneous, and were located more ventrally than thin-spike neurons found during the light phase. These findings reveal previously unknown populations of VTA neurons whose activities are sensitive to diurnal phase, and whose functions may be in the temporal regulation of arousal and motivational processes.

Dopamine terminals from the ventral tegmental area gate intrinsic inhibition in the prefrontal cortex.

Spike frequency adaptation (SFA or accommodation) and calcium-activated potassium channels that underlie after-hyperpolarization potentials (AHP) regulate repetitive firing of neurons. Precisely how neuromodulators such as dopamine from the ventral tegmental area (VTA) regulate SFA and AHP (together referred to as intrinsic inhibition) in the prefrontal cortex (PFC) remains unclear. Using whole cell electrophysiology, we measured intrinsic inhibition in prelimbic (PL) layer 5 pyramidal cells of male adult rats. Results demonstrate that bath application of dopamine reduced intrinsic inhibition (EC50: 25.0 µmol/L). This dopamine action was facilitated by coapplication of cocaine (1 µmol/L), a blocker of dopamine reuptake. To evaluate VTA dopamine terminals in PFC slices, we transfected VTA dopamine cells of TH::Cre rats in vivo with Cre-dependent AAVs to express channelrhodopsin-2 (ChR2) or designer receptors exclusively activated by designer drugs (DREADDS). In PFC slices from these animals, stimulation of VTA terminals with either blue light to activate ChR2 or bath application of clozapine-N-oxide (CNO) to activate Gq-DREADDs produced a similar reduction in intrinsic inhibition in PL neurons. Electrophysiological recordings from cells expressing retrograde fluorescent tracers showed that this plasticity occurs in PL neurons projecting to the accumbens core. Collectively, these data highlight an ability of VTA terminals to gate intrinsic inhibition in the PFC, and under appropriate circumstances, enhance PL neuronal firing. These cellular actions of dopamine may be important for dopamine-dependent behaviors involving cocaine and cue-reward associations within cortical-striatal circuits.