Disynaptic specificity of serial information flow for conditioned fear.

Memory encoding and retrieval rely on specific interactions across multiple brain areas. Although connections between individual brain areas have been extensively studied, the anatomical and functional specificity of neuronal circuit organization underlying information transfer across multiple brain areas remains unclear. Here, we combine transsynaptic viral tracing, optogenetic manipulations, and calcium dynamics recordings to dissect the multisynaptic functional connectivity of the amygdala. We identify a distinct basolateral amygdala (BLA) subpopulation that connects disynaptically to the periaqueductal gray (PAG) via the central amygdala (CeA). This disynaptic pathway serves as a core circuit element necessary for the learning and expression of conditioned fear and exhibits learning-related plasticity. Together, our findings demonstrate the utility of multisynaptic approaches for functional circuit analysis and indicate that disynaptic specificity may be a general feature of neuronal circuit organization.

NMDA-receptor-dependent plasticity in the bed nucleus of the stria terminalis triggers long-term anxiolysis.

Anxiety is controlled by multiple neuronal circuits that share robust and reciprocal connections with the bed nucleus of the stria terminalis (BNST), a key structure controlling negative emotional states. However, it remains unknown how the BNST integrates diverse inputs to modulate anxiety. In this study, we evaluated the contribution of infralimbic cortex (ILCx) and ventral subiculum/CA1 (vSUB/CA1) inputs in regulating BNST activity at the single-cell level. Using trans-synaptic tracing from single-electroporated neurons and in vivo recordings, we show that vSUB/CA1 stimulation promotes opposite forms of in vivo plasticity at the single-cell level in the anteromedial part of the BNST (amBNST). We find that an NMDA-receptor-dependent homosynaptic long-term potentiation is instrumental for anxiolysis. These findings suggest that the vSUB/CA1-driven LTP in the amBNST is involved in eliciting an appropriate response to anxiogenic context and dysfunction of this compensatory mechanism may underlie pathologic anxiety states.

A competitive inhibitory circuit for selection of active and passive fear responses.

When faced with threat, the survival of an organism is contingent upon the selection of appropriate active or passive behavioural responses. Freezing is an evolutionarily conserved passive fear response that has been used extensively to study the neuronal mechanisms of fear and fear conditioning in rodents. However, rodents also exhibit active responses such as flight under natural conditions. The central amygdala (CEA) is a forebrain structure vital for the acquisition and expression of conditioned fear responses, and the role of specific neuronal sub-populations of the CEA in freezing behaviour is well-established. Whether the CEA is also involved in flight behaviour, and how neuronal circuits for active and passive fear behaviour interact within the CEA, are not yet understood. Here, using in vivo optogenetics and extracellular recordings of identified cell types in a behavioural model in which mice switch between conditioned freezing and flight, we show that active and passive fear responses are mediated by distinct and mutually inhibitory CEA neurons. Cells expressing corticotropin-releasing factor (CRF+) mediate conditioned flight, and activation of somatostatin-positive (SOM+) neurons initiates passive freezing behaviour. Moreover, we find that the balance between conditioned flight and freezing behaviour is regulated by means of local inhibitory connections between CRF+ and SOM+ neurons, indicating that the selection of appropriate behavioural responses to threat is based on competitive interactions between two defined populations of inhibitory neurons, a circuit motif allowing for rapid and flexible action selection.

Temporal dynamics of parvalbumin-expressing axo-axonic and basket cells in the rat medial prefrontal cortex in vivo.

Axo-axonic interneurons, innervating exclusively axon initial segments, and parvalbumin-expressing basket interneurons, targeting somata, dendrites, and spines of pyramidal cells, have been proposed to control neuronal activity in prefrontal circuits. We recorded the spike-timing of identified neurons in the prelimbic cortex of anesthetized rats, and show that axo-axonic cells increase their firing during tail pinch-induced brain state-activation. In addition, axo-axonic cells differ from other GABAergic parvalbumin-expressing cells in their spike timing during DOWN- to UP-state transitions of slow oscillations and in their coupling to gamma and spindle oscillations. The distinct firing dynamics and synaptic targets of axo-axonic and other parvalbumin-expressing cells provide differential contributions to the temporal organization of prefrontal networks.

Lateral hypothalamic orexin/hypocretin neurons: A role in reward-seeking and addiction.

Orexins (synonymous with hypocretins) are recently discovered neuropeptides made exclusively in hypothalamus. Behavioral, anatomical, and neurophysiological studies show that a subset of these cells, specifically those in lateral hypothalamus (LH), are involved in reward processing and addictive behaviors. Fos expression in LH orexin neurons varied in proportion to conditioned place preference (CPP) for morphine, cocaine, or food. This relationship occurred both in drug-naïve rats and in animals during protracted morphine withdrawal, when drug preference was elevated but food preference was decreased. Inputs to the LH orexin cell field from lateral septum and bed nucleus of the stria terminalis were Fos-activated during cocaine CPP in proportion to the preference expressed in each animal. This implies that these inputs may be involved in driving the conditioned responses in LH orexin neurons. Related studies showed that LH orexin neurons that project to ventral tegmental area (VTA) had greater Fos induction in association with elevated morphine preference during protracted withdrawal than non-VTA-projecting orexin neurons, indicating that the VTA is an important site of action for orexin's role in reward processing. In addition, stimulation of LH orexin neurons, or microinjection of orexin into VTA, reinstated an extinguished morphine preference. In self-administration studies, the orexin 1 receptor antagonist SB-334867 (SB) blocked cocaine-seeking induced by discrete or contextual cues previously associated with cocaine, but not by a priming injection of cocaine. There was no effect of SB on cocaine self-administration itself, indicating that it did not interfere with the drug's reinforcing properties. Neurophysiological studies revealed that locally applied orexin often augmented responses of VTA dopamine (DA) neurons to activation of the medial prefrontal cortex (mPFC), consistent with the view that orexin facilitates activation of VTA DA neurons by stimulus-reward associations. This LH-to-VTA orexin pathway was found to be necessary for learning a morphine place preference. These findings are consistent with results showing that orexin facilitates glutamate-mediated responses, and is necessary for glutamate-dependent long-term potentiation in VTA DA neurons. We surmise from these studies that LH orexin neurons play an important role in reward processing and addiction and that LH orexin cells are an important input to VTA for behavioral effects associated with reward-paired stimuli.

Cannabinoid receptors in the bed nucleus of the stria terminalis control cortical excitation of midbrain dopamine cells in vivo.

The endocannabinoid system is involved in multiple physiological functions including reward. Cannabinoids potently control the activity of midbrain dopamine cells, but the contribution of cortical projections in this phenomenon is unclear. We show that the bed nucleus of the stria terminalis (BNST) efficient relays cortical excitation to dopamine neurons of the ventral tegmental area (VTA). Anatomical and in vivo electrophysiological evidence demonstrate that excitatory projections arising exclusively from the infralimbic cortex converge on BNST neurons, which in turn project to and excite >80% VTA dopamine cells. At the ultrastructural level, cannabinoid type 1 receptors are detected within the BNST on axon terminals arising from the infralimbic cortex. We found that intra-BNST infusion of a cannabinoid agonist inhibits the firing of dopamine cells evoked by stimulation of the infralimbic cortex. Our data identify a new neuronal substrate for the actions of cannabinoids in the reward pathway.