Chronic Ethanol During Adolescence Impacts Corticolimbic Dendritic Spines and Behavior.

BACKGROUND: Risk for alcohol use disorders (AUDs) in adulthood is linked to alcohol drinking during adolescence, but understanding of the neural and behavioral consequences of alcohol exposure during adolescence remains incomplete. Here, we examined the neurobehavioral impact of adolescent chronic intermittent EtOH (CIE) vapor exposure in mice. METHODS: C57BL/6J-background Thy1-EGFP mice were CIE-exposed during adolescence or adulthood and examined, as adults, for alterations in the density and morphology of dendritic spines in infralimbic (IL) cortex, prelimbic (PL) cortex, and basolateral amygdala (BLA). In parallel, adolescent- and adult-exposed C57BL/6J mice were tested as adults for 2-bottle EtOH drinking, sensitivity to EtOH intoxication (loss of righting reflex [LORR]), blood EtOH clearance, and measures of operant responding for food reward. RESULTS: CIE during adolescence decreased IL neuronal spine density and increased the head width of relatively wide-head IL and BLA spines, whereas CIE decreased head width of relatively narrow-head BLA spines. Adolescents had higher EtOH consumption prior to CIE than adults, while CIE during adulthood, but not adolescence, increased EtOH consumption relative to pre-CIE baseline. CIE produced a tolerance-like decrease in LORR sensitivity to EtOH challenge, irrespective of the age at which mice received CIE exposure. Mice exposed to CIE during adolescence, but not adulthood, required more sessions than AIR controls to reliably respond for food reward on a fixed-ratio (FR) 1, but not subsequent FR3, reinforcement schedule. On a progressive ratio reinforcement schedule, break point responding was higher in the adolescent- than the adult-exposed mice, regardless of CIE. Finally, footshock punishment markedly suppressed responding for reward in all groups. CONCLUSIONS: Exposure to CIE during adolescence altered dendritic spine density and morphology in IL and BLA neurons, in parallel with a limited set of behavioral alterations. Together, these data add to growing evidence that key corticolimbic circuits are vulnerable to the effects of alcohol during adolescence, with lasting, potentially detrimental, consequences for behavior.

Prefrontal single-unit firing associated with deficient extinction in mice.

The neural circuitry mediating fear extinction has been increasingly well studied and delineated. The rodent infralimbic subregion (IL) of the ventromedial prefrontal cortex (vmPFC) has been found to promote extinction, whereas the prelimbic cortex (PL) demonstrates an opposing, pro-fear, function. Studies employing in vivo electrophysiological recordings have observed that while increased IL single-unit firing and bursting predicts robust extinction retrieval, increased PL firing can correlate with sustained fear and poor extinction. These relationships between single-unit firing and extinction do not hold under all experimental conditions, however. In the current study, we further investigated the relationship between vmPFC and PL single-unit firing and extinction using inbred mouse models of intact (C57BL/6J, B6) and deficient (129S1/SvImJ, S1) extinction strains. Simultaneous single-unit recordings were made in the PL and vmPFC (encompassing IL) as B6 and S1 mice performed extinction training and retrieval. Impaired extinction retrieval in S1 mice was associated with elevated PL single-unit firing, as compared to firing in extinguishing B6 mice, consistent with the hypothesized pro-fear contribution of PL. Analysis of local field potentials also revealed significantly higher gamma power in the PL of S1 than B6 mice during extinction training and retrieval. In the vmPFC, impaired extinction in S1 mice was also associated with exaggerated single-unit firing, relative to B6 mice. This is in apparent contradiction to evidence that IL activity promotes extinction, but could reflect a (failed) compensatory effort by the vmPFC to mitigate fear-promoting activity in other regions, such as the PL or amygdala. In support of this hypothesis, augmenting IL activity via direct infusion of the GABAA receptor antagonist picrotoxin rescued impaired extinction retrieval in S1 mice. Chronic fluoxetine treatment produced modest reductions in fear during extinction retrieval and increased the number of Zif268-labeled cells in layer II of IL, but failed to increase vmPFC single-unit firing. Collectively, these findings further support the important contribution these cortical regions play in determining the balance between robust extinction on the one hand, and sustained fear on the other. Elucidating the precise nature of these roles could help inform understanding of the pathophysiology of fear-related anxiety disorders.

Dorsolateral Striatum Engagement Interferes with Early Discrimination Learning.

In current models, learning the relationship between environmental stimuli and the outcomes of actions involves both stimulus-driven and goal-directed systems, mediated in part by the DLS and DMS, respectively. However, though these models emphasize the importance of the DLS in governing actions after extensive experience has accumulated, there is growing evidence of DLS engagement from the onset of training. Here, we used in vivo photosilencing to reveal that DLS recruitment interferes with early touchscreen discrimination learning. We also show that the direct output pathway of the DLS is preferentially recruited and causally involved in early learning and find that silencing the normal contribution of the DLS produces plasticity-related alterations in a PL-DMS circuit. These data provide further evidence suggesting that the DLS is recruited in the construction of stimulus-elicited actions that ultimately automate behavior and liberate cognitive resources for other demands, but with a cost to performance at the outset of learning.

Neuronal localization of Ca(v)1.2 L-type calcium channels in the rat basolateral amygdala.

L-type high-voltage-activated calcium channels are involved in the conduction and integration of electrical activity and associative long-term potentiation in the basolateral amygdalar complex (BLC). However, little is known about the neuronal localization of these channels in this brain region. We used immunohistochemical techniques to determine which cell types in the BLC express the Ca(v)1.2 subtype of L-type calcium channels. Immunofluorescence experiments demonstrated that Ca(v)1.2 calcium channels were mainly found in somata and dendrites of pyramidal neurons that exhibited immunoreactivity for calcium/calmodulin-dependent protein kinase II (CaMK). Only a few parvalbumin-positive and calretinin-positive interneurons exhibited Ca(v)1.2 immunoreactivity. The presence of high levels of Ca(v)1.2 immunoreactivity in BLC pyramidal cells is consistent with physiological findings showing that calcium entry through L-type calcium channels in pyramidal cell dendrites in the lateral amygdala is required for associative LTP and the conversion of synaptic events into long-term emotional memory.

Prefrontal inputs to the amygdala instruct fear extinction memory formation.

Persistent anxiety after a psychological trauma is a hallmark of many anxiety disorders. However, the neural circuits mediating the extinction of traumatic fear memories remain incompletely understood. We show that selective, in vivo stimulation of the ventromedial prefrontal cortex (vmPFC)-amygdala pathway facilitated extinction memory formation, but not retrieval. Conversely, silencing the vmPFC-amygdala pathway impaired extinction formation and reduced extinction-induced amygdala activity. Our data demonstrate a critical instructional role for the vmPFC-amygdala circuit in the formation of extinction memories. These findings advance our understanding of the neural basis of persistent fear, with implications for posttraumatic stress disorder and other anxiety disorders.

Mechanisms to medicines: elucidating neural and molecular substrates of fear extinction to identify novel treatments for anxiety disorders.

The burden of anxiety disorders is growing, but the efficacy of available anxiolytic treatments remains inadequate. Cognitive behavioural therapy for anxiety disorders focuses on identifying and modifying maladaptive patterns of thinking and behaving, and has a testable analogue in rodents in the form of fear extinction. A large preclinical literature has amassed in recent years describing the neural and molecular basis of fear extinction in rodents. In this review, we discuss how this work is being harnessed to foster translational research on anxiety disorders and facilitate the search for new anxiolytic treatments. We begin by summarizing the anatomical and functional connectivity of a medial prefrontal cortex (mPFC)-amygdala circuit that subserves fear extinction, including new insights from optogenetics. We then cover some of the approaches that have been taken to model impaired fear extinction and associated impairments with mPFC-amygdala dysfunction. The principal goal of the review is to evaluate evidence that various neurotransmitter and neuromodulator systems mediate fear extinction by modulating the mPFC-amygdala circuitry. To that end, we describe studies that have tested how fear extinction is impaired or facilitated by pharmacological manipulations of dopamine, noradrenaline, 5-HT, GABA, glutamate, neuropeptides, endocannabinoids and various other systems, which either directly target the mPFC-amygdala circuit, or produce behavioural effects that are coincident with functional changes in the circuit. We conclude that there are good grounds to be optimistic that the progress in defining the molecular substrates of mPFC-amygdala circuit function can be effectively leveraged to identify plausible candidates for extinction-promoting therapies for anxiety disorders.

Limited convergence of rhinal cortical and dopaminergic inputs in the rat basolateral amygdala: an ultrastructural analysis.

The basolateral nuclear complex of the amygdala (BLC) receives robust sensory inputs from the rhinal cortices (RCx) that are important for the generation of emotional behavior. The BLC is also one of the main targets of the mesolimbic dopamine (DA) system. DA potentiates cortical sensory inputs to the BLC, which leads to an increase in the excitability of BLC pyramidal cells. These findings suggest that there may be convergence of RCx and DA inputs onto the dendrites of pyramidal cells in the BLC. In the present study we used dual-labeling immunohistochemistry and anterograde tract-tracing at the ultrastructural level to test this hypothesis in the rat brain. RCx axons were labeled by Phaseolus vulgaris leucoagglutinin (PHA-L) injections, whereas tyrosine hydroxylase (TH) was used as a marker for DA axons. The extent of convergence of these axons was analyzed in the posterior subdivision of the basolateral nucleus (BLp), which is densely innervated by both inputs. RCx synapses were asymmetrical and mainly contacted dendritic spines (86.4%) and dendritic shafts (12.1%). TH-positive (TH+) terminals also mainly formed synapses (symmetrical) and appositions with spines and shafts of dendrites. However, ultrastructural analysis found a very low percentage of RCx terminals converging with DA terminals onto unlabeled dendrites (9.4%) and axons (7.5 %), or exhibiting direct contacts with TH+ terminals (3.8%). These findings suggest that the association of specific behaviorally salient sensory stimuli with dopamine release in the BLC is not dependent on a point-to-point spatial relationship of cortical and DA inputs.