A distinct cortical code for socially learned threat.

Animals can learn about sources of danger while minimizing their own risk by observing how others respond to threats. However, the distinct neural mechanisms by which threats are learned through social observation (known as observational fear learning1-4 (OFL)) to generate behavioural responses specific to such threats remain poorly understood. The dorsomedial prefrontal cortex (dmPFC) performs several key functions that may underlie OFL, including processing of social information and disambiguation of threat cues5-11. Here we show that dmPFC is recruited and required for OFL in mice. Using cellular-resolution microendoscopic calcium imaging, we demonstrate that dmPFC neurons code for observational fear and do so in a manner that is distinct from direct experience. We find that dmPFC neuronal activity predicts upcoming switches between freezing and moving state elicited by threat. By combining neuronal circuit mapping, calcium imaging, electrophysiological recordings and optogenetics, we show that dmPFC projections to the midbrain periaqueductal grey (PAG) constrain observer freezing, and that amygdalar and hippocampal inputs to dmPFC opposingly modulate observer freezing. Together our findings reveal that dmPFC neurons compute a distinct code for observational fear and coordinate long-range neural circuits to select behavioural responses.

A cortico-amygdala neural substrate for endocannabinoid modulation of fear extinction.

Preclinical and clinical studies implicate endocannabinoids (eCBs) in fear extinction, but the underlying neural circuit basis of these actions is unclear. Here, we employed in vivo optogenetics, eCB biosensor imaging, ex vivo electrophysiology, and CRISPR-Cas9 gene editing in mice to examine whether basolateral amygdala (BLA)-projecting medial prefrontal cortex (mPFC) neurons represent a neural substrate for the effects of eCBs on extinction. We found that photoexcitation of mPFC axons in BLA during extinction mobilizes BLA eCBs. eCB biosensor imaging showed that eCBs exhibit a dynamic stimulus-specific pattern of activity at mPFC→BLA neurons that tracks extinction learning. Furthermore, using CRISPR-Cas9-mediated gene editing, we demonstrated that extinction memory formation involves eCB activity at cannabinoid CB1 receptors expressed at vmPFC→BLA synapses. Our findings reveal the temporal characteristics and a neural circuit basis of eCBs' effects on fear extinction and inform efforts to target the eCB system as a therapeutic approach in extinction-deficient neuropsychiatric disorders.

Intercalated amygdala clusters orchestrate a switch in fear state.

Adaptive behaviour necessitates the formation of memories for fearful events, but also that these memories can be extinguished. Effective extinction prevents excessive and persistent reactions to perceived threat, as can occur in anxiety and 'trauma- and stressor-related' disorders1. However, although there is evidence that fear learning and extinction are mediated by distinct neural circuits, the nature of the interaction between these circuits remains poorly understood2-6. Here, through a combination of in vivo calcium imaging, functional manipulations, and slice physiology, we show that distinct inhibitory clusters of intercalated neurons (ITCs) in the mouse amygdala exert diametrically opposed roles during the acquisition and retrieval of fear extinction memory. Furthermore, we find that the ITC clusters antagonize one another through mutual synaptic inhibition and differentially access functionally distinct cortical- and midbrain-projecting amygdala output pathways. Our findings show that the balance of activity between ITC clusters represents a unique regulatory motif that orchestrates a distributed neural circuitry, which in turn regulates the switch between high- and low-fear states. These findings suggest that the ITCs have a broader role in a range of amygdala functions and associated brain states that underpins the capacity to adapt to salient environmental demands.

Behavioral and Myelin-Related Abnormalities after Blast-Induced Mild Traumatic Brain Injury in Mice.

In civilian and military settings, mild traumatic brain injury (mTBI) is a common consequence of impacts to the head, sudden blows to the body, and exposure to high-energy atmospheric shockwaves from blast. In some cases, mTBI from blast exposure results in long-term emotional and cognitive deficits and an elevated risk for certain neuropsychiatric diseases. Here, we tested the effects of mTBI on various forms of auditory-cued fear learning and other measures of cognition in male C57BL/6J mice after single or repeated blast exposure (blast TBI; bTBI). bTBI produced an abnormality in the temporal organization of cue-induced freezing behavior in a conditioned trace fear test. Spatial working memory, evaluated by the Y-maze task performance, was also deleteriously affected by bTBI. Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis for glial markers indicated an alteration in the expression of myelin-related genes in the hippocampus and corpus callosum 1-8 weeks after bTBI. Immunohistochemical and ultrastructural analyses detected bTBI-related myelin and axonal damage in the hippocampus and corpus callosum. Together, these data suggest a possible link between blast-induced mTBI, myelin/axonal injury, and cognitive dysfunction.

Effects of optogenetic photoexcitation of infralimbic cortex inputs to the basolateral amygdala on conditioned fear and extinction.

Deficiencies in the ability to extinguish fear is a hallmark of Trauma- and stressor-related disorders, Anxiety disorders, and certain other neuropsychiatric conditions. Hence, a greater understanding of the brain mechanisms involved in the inhibition of fear is of significant translational relevance. Previous studies in rodents have shown that glutamatergic projections from the infralimbic prefrontal cortex (IL) to basolateral amygdala (BLA) play a crucial instructional role in the formation of extinction memories, and also indicate that variation in the strength of this input correlates with extinction efficacy. To further examine the relationship between the IL→BLA pathway and extinction we expressed three different titers of the excitatory opsin, channelrhodopsin (ChR2), in IL neurons and photostimulated their projections in the BLA during partial extinction training. The behavioral effects of photoexcitation differed across the titer groups: the low titer had no effect, the medium titer selectively facilitated extinction memory formation, and the high titer produced both an acute suppression of fear and a decrease in fear during (light-free) extinction retrieval. We discuss various possible explanations for these titer-specific effects, including the possibility of IL-mediated inhibition of BLA fear-encoding neurons under conditions of sufficiently strong photoexcitation. These findings further support the role of IL→BLA pathway in regulating fear and highlight the importance of methodological factors in optogenetic studies of neural circuits underling behavior.

Excitation of Diverse Classes of Cholecystokinin Interneurons in the Basal Amygdala Facilitates Fear Extinction.

There is growing evidence that interneurons (INs) orchestrate neural activity and plasticity in corticoamygdala circuits to regulate fear behaviors. However, defining the precise role of cholecystokinin-expressing INs (CCK INs) remains elusive due to the technical challenge of parsing this population from CCK-expressing principal neurons (CCK PNs). Here, we used an intersectional genetic strategy in CCK-Cre;Dlx5/6-Flpe double-transgenic mice to study the anatomical, molecular and electrophysiological properties of CCK INs in the basal amygdala (BA) and optogenetically manipulate these cells during fear extinction. Electrophysiological recordings confirmed that this strategy targeted GABAergic cells and that a significant proportion expressed functional cannabinoid CB1 receptors; a defining characteristic of CCK-expressing basket cells. However, immunostaining showed that subsets of the genetically-targeted cells expressed either neuropeptide Y (NPY; 29%) or parvalbumin (PV; 17%), but not somatostatin (SOM) or Ca2+/calmodulin-dependent protein kinase II (CaMKII)-α. Further morphological and electrophysiological analyses showed that four IN types could be identified among the EYFP-expressing cells: CCK/cannabinoid receptor type 1 (CB1R)-expressing basket cells, neurogliaform cells, PV+ basket cells, and PV+ axo-axonic cells. At the behavioral level, in vivo optogenetic photostimulation of the targeted population during extinction acquisition led to reduced freezing on a light-free extinction retrieval test, indicating extinction memory facilitation; whereas photosilencing was without effect. Conversely, non-selective (i.e., inclusive of INs and PNs) photostimulation or photosilencing of CCK-targeted cells, using CCK-Cre single-transgenic mice, impaired extinction. These data reveal an unexpectedly high degree of phenotypic complexity in a unique population of extinction-modulating BA INs.

Identification of a novel gene regulating amygdala-mediated fear extinction.

Recent years have seen advances in our understanding of the neural circuits associated with trauma-related disorders, and the development of relevant assays for these behaviors in rodents. Although inherited factors are known to influence individual differences in risk for these disorders, it has been difficult to identify specific genes that moderate circuit functions to affect trauma-related behaviors. Here, we exploited robust inbred mouse strain differences in Pavlovian fear extinction to uncover quantitative trait loci (QTL) associated with this trait. We found these strain differences to be resistant to developmental cross-fostering and associated with anatomical variation in basolateral amygdala (BLA) perineuronal nets, which are developmentally implicated in extinction. Next, by profiling extinction-driven BLA expression of QTL-linked genes, we nominated Ppid (peptidylprolyl isomerase D, a member of the tetratricopeptide repeat (TPR) protein family) as an extinction-related candidate gene. We then showed that Ppid was enriched in excitatory and inhibitory BLA neuronal populations, but at lower levels in the extinction-impaired mouse strain. Using a virus-based approach to directly regulate Ppid function, we demonstrated that downregulating BLA-Ppid impaired extinction, while upregulating BLA-Ppid facilitated extinction and altered in vivo neuronal extinction encoding. Next, we showed that Ppid colocalized with the glucocorticoid receptor (GR) in BLA neurons and found that the extinction-facilitating effects of Ppid upregulation were blocked by a GR antagonist. Collectively, our results identify Ppid as a novel gene involved in regulating extinction via functional actions in the BLA, with possible implications for understanding genetic and pathophysiological mechanisms underlying risk for trauma-related disorders.

Regulation of myelin structure and conduction velocity by perinodal astrocytes.

The speed of impulse transmission is critical for optimal neural circuit function, but it is unclear how the appropriate conduction velocity is established in individual axons. The velocity of impulse transmission is influenced by the thickness of the myelin sheath and the morphology of electrogenic nodes of Ranvier along axons. Here we show that myelin thickness and nodal gap length are reversibly altered by astrocytes, glial cells that contact nodes of Ranvier. Thrombin-dependent proteolysis of a cell adhesion molecule that attaches myelin to the axon (neurofascin 155) is inhibited by vesicular release of thrombin protease inhibitors from perinodal astrocytes. Transgenic mice expressing a dominant-negative fragment of VAMP2 in astrocytes, to reduce exocytosis by 50%, exhibited detachment of adjacent paranodal loops of myelin from the axon, increased nodal gap length, and thinning of the myelin sheath in the optic nerve. These morphological changes alter the passive cable properties of axons to reduce conduction velocity and spike-time arrival in the CNS in parallel with a decrease in visual acuity. All effects were reversed by the thrombin inhibitor Fondaparinux. Similar results were obtained by viral transfection of tetanus toxin into astrocytes of rat corpus callosum. Previously, it was unknown how the myelin sheath could be thinned and the functions of perinodal astrocytes were not well understood. These findings describe a form of nervous system plasticity in which myelin structure and conduction velocity are adjusted by astrocytes. The thrombin-dependent cleavage of neurofascin 155 may also have relevance to myelin disruption and repair.

Chronic stress dysregulates amygdalar output to the prefrontal cortex.

Chronic stress contributes to the neuropathology of mental health disorders, including those associated with anxiety. The basolateral amygdala (BLA) coordinates emotional behavioral responses through glutamatergic outputs to downstream regions such as the prefrontal cortex (PFC), nucleus accumbens core (NAcc) and bed nucleus of the stria terminalis (BNST). We explored the effects of chronic stress on BLA outputs to the PFC, NAcc and BNST using slice electrophysiology combined with optogenetics in two inbred mouse strains with distinct stress-induced anxiety responses. We found that ten consecutive days of chronic restraint stress enhanced pre-synaptic glutamate release at BLA-to-PFC synapses in C57BL/6J mice, but reduced pre-synaptic glutamate release at these synapses in DBA/2J mice. To assess the behavioral relevance of enhanced glutamate output at BLA-to-PFC synapses, we approximated the effects of chronic stress on the BLA-PFC circuit using optogenetics. We found that photostimulation of the BLA-PFC circuit in unstressed C57BL/6J mice produced persistent (i.e., post-stimulation) increased anxiety-like behavior and hyperactivity in the elevated plus-maze - a profile consistent with prototypical behavioral responses of stressed C57BL/6J mice. These data demonstrate that chronic stress dysregulates the BLA-PFC circuit by altering pre-synaptic glutamate release from BLA outputs, and provide a mechanism by which chronic stress can lead to increased anxiety.

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.

Everything in Its Right Place: A Prefrontal-Midbrain Circuit for Contextual Fear Discrimination.

In this issue of Neuron, Rozeske et al. (2018) use an ingenuous behavioral paradigm to change pertinent sensory stimuli defining a given context to interrogate how the dorsomedial prefrontal cortex (dmPFC) and periaqueductal gray (PAG) interact during contextual fear discrimination.

BDNF mRNA abundance regulated by antidromic action potentials and AP-LTD in hippocampus.

Action-potential-induced LTD (AP-LTD) is a form of synaptic plasticity that reduces synaptic strength in CA1 hippocampal neurons firing antidromically during sharp-wave ripples. This firing occurs during slow-wave sleep and quiet moments of wakefulness, which are periods of offline replay of neural sequences learned during encoding sensory information. Here we report that rapid and persistent down-regulation of different mRNA transcripts of the BDNF gene accompanies AP-LTD, and that AP-LTD is abolished in mice with the BDNF gene knocked out in CA1 hippocampal neurons. These findings increase understanding of the mechanism of AP-LTD and the cellular mechanisms of memory consolidation.

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.

Synaptic plasticity by antidromic firing during hippocampal network oscillations.

Learning and other cognitive tasks require integrating new experiences into context. In contrast to sensory-evoked synaptic plasticity, comparatively little is known of how synaptic plasticity may be regulated by intrinsic activity in the brain, much of which can involve nonclassical modes of neuronal firing and integration. Coherent high-frequency oscillations of electrical activity in CA1 hippocampal neurons [sharp-wave ripple complexes (SPW-Rs)] functionally couple neurons into transient ensembles. These oscillations occur during slow-wave sleep or at rest. Neurons that participate in SPW-Rs are distinguished from adjacent nonparticipating neurons by firing action potentials that are initiated ectopically in the distal region of axons and propagate antidromically to the cell body. This activity is facilitated by GABA(A)-mediated depolarization of axons and electrotonic coupling. The possible effects of antidromic firing on synaptic strength are unknown. We find that facilitation of spontaneous SPW-Rs in hippocampal slices by increasing gap-junction coupling or by GABA(A)-mediated axon depolarization resulted in a reduction of synaptic strength, and electrical stimulation of axons evoked a widespread, long-lasting synaptic depression. Unlike other forms of synaptic plasticity, this synaptic depression is not dependent upon synaptic input or glutamate receptor activation, but rather requires L-type calcium channel activation and functional gap junctions. Synaptic stimulation delivered after antidromic firing, which was otherwise too weak to induce synaptic potentiation, triggered a long-lasting increase in synaptic strength. Rescaling synaptic weights in subsets of neurons firing antidromically during SPW-Rs might contribute to memory consolidation by sharpening specificity of subsequent synaptic input and promoting incorporation of novel information.

Restoration of synaptic plasticity and learning in young and aged NCAM-deficient mice by enhancing neurotransmission mediated by GluN2A-containing NMDA receptors.

Neural cell adhesion molecule (NCAM) is the predominant carrier of the unusual glycan polysialic acid (PSA). Deficits in PSA and/or NCAM expression cause impairments in hippocampal long-term potentiation and depression (LTP and LTD) and are associated with schizophrenia and aging. In this study, we show that impaired LTP in adult NCAM-deficient (NCAM(-/-)) mice is restored by increasing the activity of the NMDA subtype of glutamate receptor (GluN) through either reducing the extracellular Mg2+ concentration or applying d-cycloserine (DCS), a partial agonist of the GluN glycine binding site. Pharmacological inhibition of the GluN2A subtype reduced LTP to the same level in NCAM(-/-) and wild-type (NCAM(+/+)) littermate mice and abolished the rescue by DCS in NCAM(-/-) mice, suggesting that the effects of DCS are mainly mediated by GluN2A. The insufficient contribution of GluN to LTD in NCAM(-/-) mice was also compensated for by DCS. Furthermore, impaired contextual and cued fear conditioning levels were restored in NCAM(-/-) mice by administration of DCS before conditioning. In 12-month-old NCAM(-/-), but not NCAM(+/+) mice, there was a decline in LTP compared with 3-month-old mice that could be rescued by DCS. In 24-month-old mice of both genotypes, there was a reduction in LTP that could be fully restored by DCS in NCAM(+/+) mice but only partially restored in NCAM(-/-) mice. Thus, several deficiencies of NCAM(-/-) mice can be ameliorated by enhancing GluN2A-mediated neurotransmission with DCS.

Synaptic cell adhesion molecules.

During development of the nervous system following axon pathfinding, synaptic connections are established between neurons. Specific cell adhesion molecules (CAMs) accumulate at pre- and postsynaptic sites and trigger synaptic differentiation through interactions with intra- and extracellular scaffolds. These interactions are important to align pre- and postsynaptic transduction machineries and to couple the sites of cell-to-cell adhesion to the cytoskeleton and signaling complexes necessary to accumulate and recycle presynaptic vesicles, components of exo- and endocytic zones, and postsynaptic receptors. In mature brains, CAMs contribute to regulation of synaptic efficacy and plasticity, partially via direct interactions with postsynaptic neurotransmitter receptors and presynaptic voltage-gated ion channels. This chapter is to highlight the major classes of synaptic CAMs, their multiple functions, and the multistage concerted interactions between different CAMs and other components of synapses.

The extracellular matrix molecule hyaluronic acid regulates hippocampal synaptic plasticity by modulating postsynaptic L-type Ca(2+) channels.

Although the extracellular matrix plays an important role in regulating use-dependent synaptic plasticity, the underlying molecular mechanisms are poorly understood. Here we examined the synaptic function of hyaluronic acid (HA), a major component of the extracellular matrix. Enzymatic removal of HA with hyaluronidase reduced nifedipine-sensitive whole-cell Ca(2+) currents, decreased Ca(2+) transients mediated by L-type voltage-dependent Ca(2+) channels (L-VDCCs) in postsynaptic dendritic shafts and spines, and abolished an L-VDCC-dependent component of long-term potentiation (LTP) at the CA3-CA1 synapses in the hippocampus. Adding exogenous HA, either by bath perfusion or via local delivery near recorded synapses, completely rescued this LTP component. In a heterologous expression system, exogenous HA rapidly increased currents mediated by Ca(v)1.2, but not Ca(v)1.3, subunit-containing L-VDCCs, whereas intrahippocampal injection of hyaluronidase impaired contextual fear conditioning. Our observations unveil a previously unrecognized mechanism by which the perisynaptic extracellular matrix influences use-dependent synaptic plasticity through regulation of dendritic Ca(2+) channels.

Improved reversal learning and working memory and enhanced reactivity to novelty in mice with enhanced GABAergic innervation in the dentate gyrus.

The balance between excitation and inhibition controls fundamental aspects of the hippocampal function. Here, we report an increase in the ratio of inhibitory to excitatory neurons in the dentate gyrus, accompanied by γ-aminobutyric acid(A) (GABA(A)) receptor-dependent impairment of synaptic plasticity and enhancement of activity-dependent changes in excitability in anesthetized adult mice deficient for the extracellular matrix glycoprotein tenascin-R (TNR). TNR-deficient mice showed faster reversal learning, improved working memory, and enhanced reactivity to novelty than wild-type littermates. Remarkably, in wild-type and TNR-deficient mice, faster reversal learning rates correlated at the individual animal level with ratios of parvalbumin-positive interneurons to granule cells and densities of parvalbumin-positive terminals on somata of granule cells. Our data demonstrate that modification of the extracellular matrix by ablation of TNR leads to a new structural and functional design of the dentate gyrus, with enhanced GABAergic innervation, that is, enhanced ratio of inhibitory to excitatory cells, and altered plasticity, promoting working memory and reversal learning. In wild-type mice, the enhanced ratio of inhibitory to excitatory cells in the dentate gyrus also positively correlated with reversal learning, indicating that level of inhibition regulates specific aspects of learning independent of the TNR gene.