Subcortico-amygdala pathway processes innate and learned threats.

Behavioral flexibility and timely reactions to salient stimuli are essential for survival. The subcortical thalamic-basolateral amygdala (BLA) pathway serves as a shortcut for salient stimuli ensuring rapid processing. Here, we show that BLA neuronal and thalamic axonal activity in mice mirror the defensive behavior evoked by an innate visual threat as well as an auditory learned threat. Importantly, perturbing this pathway compromises defensive responses to both forms of threats, in that animals fail to switch from exploratory to defensive behavior. Despite the shared pathway between the two forms of threat processing, we observed noticeable differences. Blocking ß-adrenergic receptors impairs the defensive response to the innate but not the learned threats. This reduced defensive response, surprisingly, is reflected in the suppression of the activity exclusively in the BLA as the thalamic input response remains intact. Our side-by-side examination highlights the similarities and differences between innate and learned threat-processing, thus providing new fundamental insights.

Thalamus sends information about arousal but not valence to the amygdala.

RATIONALE: The basolateral amygdala (BLA) and medial geniculate nucleus of the thalamus (MGN) have both been shown to be necessary for the formation of associative learning. While the role that the BLA plays in this process has long been emphasized, the MGN has been less well-studied and surrounded by debate regarding whether the relay of sensory information is active or passive. OBJECTIVES: We seek to understand the role the MGN has within the thalamoamgydala circuit in the formation of associative learning. METHODS: Here, we use optogenetics and in vivo electrophysiological recordings to dissect the MGN-BLA circuit and explore the specific subpopulations for evidence of learning and synthesis of information that could impact downstream BLA encoding. We employ various machine learning techniques to investigate function within neural subpopulations. We introduce a novel method to investigate tonic changes across trial-by-trial structure, which offers an alternative approach to traditional trial-averaging techniques. RESULTS: We find that the MGN appears to encode arousal but not valence, unlike the BLA which encodes for both. We find that the MGN and the BLA appear to react differently to expected and unexpected outcomes; the BLA biased responses toward reward prediction error and the MGN focused on anticipated punishment. We uncover evidence of tonic changes by visualizing changes across trials during inter-trial intervals (baseline epochs) for a subset of cells. CONCLUSION: We conclude that the MGN-BLA projector population acts as both filter and transferer of information by relaying information about the salience of cues to the amygdala, but these signals are not valence-specified.

Dissociated Role of Thalamic and Cortical Input to the Lateral Amygdala for Consolidation of Long-Term Fear Memory.

Post-encoding coordinated reactivation of memory traces distributed throughout interconnected brain regions is thought to be critical for consolidation of memories. However, little is known about the role of neural circuit pathways during post-learning periods for consolidation of memories. To investigate this question, we optogenetically silenced the inputs from both auditory cortex and thalamus in the lateral amygdala (LA) for 15 min immediately following auditory fear conditioning (FC) and examined its effect on fear memory formation in mice of both sexes. Optogenetic inhibition of both inputs disrupted long-term fear memory formation tested 24 h after FC. This effect was specific such that the same inhibition did not affect short-term memory and context-dependent memory. Moreover, long-term memory was intact if the inputs were inhibited at much later time points after FC (3 h or 1 d after FC), indicating that optical inhibition for 15 min itself does not produce any nonspecific deleterious effect on fear memory retrieval. Selective inhibition of thalamic input was sufficient to impair consolidation of auditory fear memory. In contrast, selective inhibition of cortical input disrupted remote fear memory without affecting recent memory. These results reveal a dissociated role of thalamic and cortical input to the LA during early post-learning periods for consolidation of long-term fear memory.SIGNIFICANCE STATEMENT Coordinated communications between brain regions are thought to be essential during post-learning periods for consolidation of memories. However, the role of specific neural circuit pathways in this process has been scarcely explored. Using a precise optogenetic inhibition of auditory input pathways, either thalamic or cortical or both, to the LA during post-training periods, we here show that thalamic input is required for consolidation of both recent and remote fear memory, whereas cortical input is crucial for consolidation of remote fear memory. These results reveal a dissociated role of auditory input pathways to the LA for consolidation of long-term fear memory.

Associative and plastic thalamic signaling to the lateral amygdala controls fear behavior.

Decades of research support the idea that associations between a conditioned stimulus (CS) and an unconditioned stimulus (US) are encoded in the lateral amygdala (LA) during fear learning. However, direct proof for the sources of CS and US information is lacking. Definitive evidence of the LA as the primary site for cue association is also missing. Here, we show that calretinin (Calr)-expressing neurons of the lateral thalamus (Calr+LT neurons) convey the association of fast CS (tone) and US (foot shock) signals upstream from the LA in mice. Calr+LT input shapes a short-latency sensory-evoked activation pattern of the amygdala via both feedforward excitation and inhibition. Optogenetic silencing of Calr+LT input to the LA prevents auditory fear conditioning. Notably, fear conditioning drives plasticity in Calr+LT neurons, which is required for appropriate cue and contextual fear memory retrieval. Collectively, our results demonstrate that Calr+LT neurons provide integrated CS-US representations to the LA that support the formation of aversive memories.

Orexin as a modulator of fear-related behavior: Hypothalamic control of noradrenaline circuit.

Fear is an important physiological function for survival. It appears when animals or humans are confronted with an environmental threat. The amygdala has been shown to play a highly important role in emergence of fear. Hypothalamic orexin neurons are activated by fearful stimuli to evoke a 'defense reaction' with an increase in arousal level and sympathetic outflow to deal with the imminent danger. However, how this system contributes to the emergence of fear-related behavior is not well understood. Orexin neurons in the hypothalamus send excitatory innervations to noradrenergic neurons in the locus coeruleus (NALC) which express orexin receptor 1 (OX1R) and send projections to the lateral amygdala (LA). Inhibition of this di-synaptic orexin → NALC → LA pathway by pharmacological or opto/chemogenetic methods reduces cue-induced fear expression. Excitatory manipulation of this pathway induces freezing, a fear-related behavior that only occurs when the environment contains some elements suggestive of danger. Although, fear memory helps animals respond to a context or cue previously paired with an aversive stimulus, fear-related behavior is sometimes evoked even in a distinct context containing some similar elements, which is known as fear generalization. Our recent observation suggests that the orexin → NALC → LA pathway might contribute to this response. This review focuses on recent advances regarding the role of hypothalamic orexin neurons in behavioral fear expression. We also discuss the potential effectiveness of orexin receptor antagonists for treating excessive fear response or overgeneralization seen in anxiety disorder and post-traumatic stress disorder (PTSD).

Basolateral amygdala - nucleus accumbens circuitry regulates optimal cue-guided risk/reward decision making.

Maladaptive decision making is a characteristic feature of substance use disorder and pathological gambling. Studies in humans and animals have implicated neural circuits that include the basolateral amygdala (BLA) and nucleus accumbens (NAc) in facilitating risk/reward decision making. However, the preclinical literature has focussed primarily on situations where animals use internally-generated information to adapt to changes in reward likelihood, whereas many real-life situations require the use of external stimuli to facilitate context-appropriate behavior. We recently developed the "Blackjack" task, to measure cued risk/reward decision making requiring rats to chose between Small/Certain and Large/Risky rewards, with auditory cues at the start of each trial explicitly informing that the probability of obtaining a large reward was either good (50%) or poor (12.5%). Here we investigated the contribution of the BLA and its interaction with the NAc in guiding these types of decisions. In well-trained male rats, bilateral inactivation of the BLA induced suboptimal decision making, primarily by reducing risky choice on good-odds trials. In comparison, pharmacological disconnection of the BLA and NAc-shell also induced suboptimal decision making, diverting choice from more preferred option by reducing or increasing risky choice on good vs. poor odds trials respectively. Together, these results suggest that the BLA-NAc circuitry plays a crucial role in integrating information provided by discriminative stimuli. Furthermore, this circuitry may aid in guiding action selection of advantageous options in situations to maximize rewards. Finally, they suggest that perturbations in optimal decision making observed in substance abuse and gambling disorders may be driven in part by dysfunction within this circuitry.

Interhemispheric Connectivity Potentiates the Basolateral Amygdalae and Regulates Social Interaction and Memory.

Impaired interhemispheric connectivity is commonly found in various psychiatric disorders, although how interhemispheric connectivity regulates brain function remains elusive. Here, we use the mouse amygdala, a brain region that is critical for social interaction and fear memory, as a model to demonstrate that contralateral connectivity intensifies the synaptic response of basolateral amygdalae (BLA) and regulates amygdala-dependent behaviors. Retrograde tracing and c-FOS expression indicate that contralateral afferents widely innervate BLA non-randomly and that some BLA neurons innervate both contralateral BLA and the ipsilateral central amygdala (CeA). Our optogenetic and electrophysiological studies further suggest that contralateral BLA input results in the synaptic facilitation of BLA neurons, thereby intensifying the responses to cortical and thalamic stimulations. Finally, pharmacological inhibition and chemogenetic disconnection demonstrate that BLA contralateral facilitation is required for social interaction and memory. Our study suggests that interhemispheric connectivity potentiates the synaptic dynamics of BLA neurons and is critical for the full activation and functionality of amygdalae.

Novel objects elicit greater activation in the basolateral complex of the amygdala of wild rats compared with laboratory rats.

Wild animals tend to avoid novel objects that do not elicit clear avoidance behaviors in domesticated animals. We previously found that the basolateral complex of the amygdala (BLA) and dorsal bed nucleus of the stria terminalis (dBNST) were larger in trapped wild rats compared with laboratory rats. Based on these findings, we hypothesized that the BLA and/or dBNST would be differentially activated when wild and laboratory rats showed different avoidance behaviors towards novel objects. In this study, we placed novel objects at one end of the home cage. We measured the time spent in that half of the cage and expressed the data as a percentage of the time spent in that region with no object placement. We found that this percentage was lower in the wild rats compared with the laboratory rats. These behavioral differences were accompanied by increased Fos expression in the BLA, but not in the dBNST, of the wild rats. These results suggest that wild rats show greater BLA activation compared with laboratory rats in response to novel objects. We also found increased Fos expression in the paraventricular nucleus of the hypothalamus, ventral BNST, and ventromedial hypothalamus, but not in the central amygdala of wild rats. Taken together, our data represent new information regarding differences in behavioral and neural responses towards novel objects in wild vs. laboratory rats.

Neural circuits underlying a psychotherapeutic regimen for fear disorders.

A psychotherapeutic regimen that uses alternating bilateral sensory stimulation (ABS) has been used to treat post-traumatic stress disorder. However, the neural basis that underlies the long-lasting effect of this treatment-described as eye movement desensitization and reprocessing-has not been identified. Here we describe a neuronal pathway driven by the superior colliculus (SC) that mediates persistent attenuation of fear. We successfully induced a lasting reduction in fear in mice by pairing visual ABS with conditioned stimuli during fear extinction. Among the types of visual stimulation tested, ABS provided the strongest fear-reducing effect and yielded sustained increases in the activities of the SC and mediodorsal thalamus (MD). Optogenetic manipulation revealed that the SC-MD circuit was necessary and sufficient to prevent the return of fear. ABS suppressed the activity of fear-encoding cells and stabilized inhibitory neurotransmission in the basolateral amygdala through a feedforward inhibitory circuit from the MD. Together, these results reveal the neural circuit that underlies an effective strategy for sustainably attenuating traumatic memories.

Coherent Activity between the Prelimbic and Auditory Cortex in the Slow-Gamma Band Underlies Fear Discrimination.

The medial prefrontal cortex and the basolateral amygdala (BLA) are essential for discriminating between harmful and safe stimuli. The primary auditory cortex (Te1) sends projections to both sites, but whether and how it interacts with these areas during fear discrimination are poorly understood. Here we show that in male rats that can differentiate between a new tone and a threatening one, the selective optogenetic inhibition of Te1 axon terminals into the prelimbic (PL) cortex shifted discrimination to fear generalization. Meanwhile, no effects were detected when Te1 terminals were inhibited in the BLA. Using a combination of local field potential and multiunit recordings, we show that in animals that discriminate successfully between a new tone and a harmful one, the activity of the Te1 and the PL cortex becomes immediately and tightly synchronized in the slow-gamma range (40-70 Hz) at the onset of the new tone. This enhanced synchronization was not present in other frequency ranges, such as the theta range. Critically, the level of gamma synchrony predicted the behavioral choice (i.e., no freezing or freezing) of the animals. Moreover, in the same rats, gamma synchrony was absent before the fear-learning trial and when animals should discriminate between an olfactory stimulus and the auditory harmful one. Thus, our findings reveal that the Te1 and the PL cortex dynamically establish a functional connection during auditory fear-discrimination processes, and that this corticocortical oscillatory mechanism drives the behavioral choice of the animals.SIGNIFICANCE STATEMENT Identifying neural networks that infer safety versus danger is of great interest in the scientific field. Fear generalization reduces the chances of an animal's survival and leads to psychiatric diseases, such as post-traumatic stress disorders and phobias in humans. Here we demonstrate that animals able to differentiate a new tone from a previous threating tone showed synchronization between the prefrontal and primary auditory cortices. Critically, this connectivity precedes and predicts the behavioral outcome of the animal. Optogenetic inhibition of this functional connectivity leads to fear generalization. To the best of our knowledge, this study is the first to demonstrate that a corticocortical dialogue occurring between sensory and prefrontal areas is a key node for fear-discrimination processes.

Serotonin gating of cortical and thalamic glutamate inputs onto principal neurons of the basolateral amygdala.

The basolateral amygdala (BLA) is a key site for crossmodal association of sensory stimuli and an important relay in the neural circuitry of emotion. Indeed, the BLA receives substantial glutamatergic inputs from multiple brain regions including the prefrontal cortex and thalamic nuclei. Modulation of glutamatergic transmission in the BLA regulates stress- and anxiety-related behaviors. Serotonin (5-HT) also plays an important role in regulating stress-related behavior through activation of both pre- and postsynaptic 5-HT receptors. Multiple 5-HT receptors are expressed in the BLA, where 5-HT has been reported to modulate glutamatergic transmission. However, the 5-HT receptor subtype mediating this effect is not yet clear. The aim of this study was to use patch-clamp recordings from BLA neurons in an ex vivo slice preparation to examine 1) the effect of 5-HT on extrinsic sensory inputs, and 2) to determine if any pathway specificity exists in 5-HT regulation of glutamatergic transmission. Two independent input pathways into the BLA were stimulated: the external capsule to mimic cortical input, and the internal capsule to mimic thalamic input. Bath application of 5-HT reversibly reduced the amplitude of evoked excitatory postsynaptic currents (eEPSCs) induced by stimulation of both pathways. The decrease was associated with an increase in the paired-pulse ratio and coefficient of variation of eEPSC amplitude, suggesting 5-HT acts presynaptically. Moreover, the effect of 5-HT in both pathways was mimicked by the selective 5-HT1B receptor agonist CP93129, but not by the 5-HT1A receptor agonist 8-OH DPAT. Similarly the effect of exogenous 5-HT was blocked by the 5-HT1B receptor antagonist GR55562, but not affected by the 5-HT1A receptor antagonist WAY 100635 or the 5-HT2 receptor antagonists pirenperone and MDL 100907. Together these data suggest 5-HT gates cortical and thalamic glutamatergic inputs into the BLA by activating presynaptic 5-HT1B receptors.

Synaptic Plasticity, Engrams, and Network Oscillations in Amygdala Circuits for Storage and Retrieval of Emotional Memories.

The neuronal circuits of the basolateral amygdala (BLA) are crucial for acquisition, consolidation, retrieval, and extinction of associative emotional memories. Synaptic plasticity in BLA neurons is essential for associative emotional learning and is a candidate mechanism through which subsets of BLA neurons (commonly termed "engram") are recruited during learning and reactivated during memory retrieval. In parallel, synchronous oscillations in the theta and gamma bands between the BLA and interconnected structures have been shown to occur during consolidation and retrieval of emotional memories. Understanding how these cellular and network phenomena interact is vital to decipher the roles of emotional memory formation and storage in the healthy and pathological brain. Here, we review data on synaptic plasticity, engrams, and network oscillations in the rodent BLA. We explore mechanisms through which synaptic plasticity, engrams, and long-range synchrony might be interconnected.

Distinct recruitment of basolateral amygdala-medial prefrontal cortex pathways across Pavlovian appetitive conditioning.

Associative learning can enable environmental cues to signal food and stimulate feeding, independent of physiological hunger. Two forebrain regions necessary in cue driven feeding, the basolateral area of the amygdala and the medial prefrontal cortex, communicate via extensive, topographically organized connections. The basolateral nucleus (BLA) sends extensive projections to the prelimbic cortex (PL), and our aim here was to determine if this pathway was selectively recruited during cue-food associative learning. The anterior and posterior basolateral nuclei are recruited during different phases of cue-food learning, and thus we examined whether distinct pathways that originate in these nuclei and project to the PL are differently recruited during early and late stages of learning. To accomplish this we used neuroanatomical tract tracing combined with the detection of Fos induction. To identify projecting neurons within the BLA, prior to training, rats received a retrograde tracer, Fluoro-Gold (FG) into the PL. Rats were given either one or ten sessions of tone-food presentations (Paired group) or tone-only presentations (Control group). The Paired group learned the tone-food association quickly and robustly and had greater Fos induction within the anterior and posterior BLA during early and late learning compared to the Control group. Notably, the Paired group had more double-labeled neurons (FG + Fos) during late training compared to the Control group, specifically in the anterior BLA. This demonstrates selective recruitment of the anterior BLA-PL pathway by late cue-food learning. These findings indicate plasticity and specificity in the BLA-PL pathways across cue-food associative learning.

Auditory Tones and Foot-Shock Recapitulate Spontaneous Sub-Threshold Activity in Basolateral Amygdala Principal Neurons and Interneurons.

In quiescent states such as anesthesia and slow wave sleep, cortical networks show slow rhythmic synchronized activity. In sensory cortices this rhythmic activity shows a stereotypical pattern that is recapitulated by stimulation of the appropriate sensory modality. The amygdala receives sensory input from a variety of sources, and in anesthetized animals, neurons in the basolateral amygdala (BLA) show slow rhythmic synchronized activity. Extracellular field potential recordings show that these oscillations are synchronized with sensory cortex and the thalamus, with both the thalamus and cortex leading the BLA. Using whole-cell recording in vivo we show that the membrane potential of principal neurons spontaneously oscillates between up- and down-states. Footshock and auditory stimulation delivered during down-states evokes an up-state that fully recapitulates those occurring spontaneously. These results suggest that neurons in the BLA receive convergent input from networks of cortical neurons with slow oscillatory activity and that somatosensory and auditory stimulation can trigger activity in these same networks.

Higher-Order Sensory Cortex Drives Basolateral Amygdala Activity during the Recall of Remote, but Not Recently Learned Fearful Memories.

Negative experiences are quickly learned and long remembered. Key unresolved issues in the field of emotional memory include identifying the loci and dynamics of memory storage and retrieval. The present study examined neural activity in the higher-order auditory cortex Te2 and basolateral amygdala (BLA) and their crosstalk during the recall of recent and remote fear memories. To this end, we obtained local field potentials and multiunit activity recordings in Te2 and BLA of rats that underwent recall at 24 h and 30 d after the association of an acoustic conditioned (CS, tone) and an aversive unconditioned stimulus (US, electric shock). Here we show that, during the recall of remote auditory threat memories in rats, the activity of the Te2 and BLA is highly synchronized in the theta frequency range. This functional connectivity stems from memory consolidation processes because it is present during remote, but not recent, memory retrieval. Moreover, the observed increase in synchrony is cue and region specific. A preponderant Te2-to-BLA directionality characterizes this dialogue, and the percentage of time Te2 theta leads the BLA during remote memory recall correlates with a faster latency to freeze to the auditory conditioned stimulus. The blockade of this information transfer via Te2 inhibition with muscimol prevents any retrieval-evoked neuronal activity in the BLA and animals are unable to retrieve remote memories. We conclude that memories stored in higher-order sensory cortices drive BLA activity when distinguishing between learned threatening and neutral stimuli. SIGNIFICANCE STATEMENT: How and where in the brain do we store the affective/motivational significance of sensory stimuli acquired through life experiences? Scientists have long investigated how "limbic" structures, such as the amygdala, process affective stimuli. Here we show that retrieval of well-established threat memories requires the functional interplay between higher-order components of the auditory cortex and the amygdala via synchrony in the theta range. This functional connectivity is a result of memory consolidation processes and is characterized by a predominant cortical to amygdala direction of information transfer. This connectivity is predictive of the animals' ability to recognize auditory stimuli as aversive. In the absence of this necessary cortical activity, the amygdala is unable to distinguish between frightening and neutral stimuli.

Synaptic competition in the lateral amygdala and the stimulus specificity of conditioned fear: a biophysical modeling study.

Competitive synaptic interactions between principal neurons (PNs) with differing intrinsic excitability were recently shown to determine which dorsal lateral amygdala (LAd) neurons are recruited into a fear memory trace. Here, we explored the contribution of these competitive interactions in determining the stimulus specificity of conditioned fear associations. To this end, we used a realistic biophysical computational model of LAd that included multi-compartment conductance-based models of 800 PNs and 200 interneurons. To reproduce the continuum of spike frequency adaptation displayed by PNs, the model included three subtypes of PNs with high, intermediate, and low spike frequency adaptation. In addition, the model network integrated spatially differentiated patterns of excitatory and inhibitory connections within LA, dopaminergic and noradrenergic inputs, extrinsic thalamic and cortical tone afferents to simulate conditioned stimuli as well as shock inputs for the unconditioned stimulus. Last, glutamatergic synapses in the model could undergo activity-dependent plasticity. Our results suggest that plasticity at both excitatory (PN-PN) and di-synaptic inhibitory (PN-ITN and, particularly, ITN-PN) connections are major determinants of the synaptic competition governing the assignment of PNs to the memory trace. The model also revealed that training-induced potentiation of PN-PN synapses promotes, whereas that of ITN-PN synapses opposes, stimulus generalization. Indeed, suppressing plasticity of PN-PN synapses increased, whereas preventing plasticity of interneuronal synapses decreased the CS specificity of PN recruitment. Overall, our results indicate that the plasticity configuration imprinted in the network by synaptic competition ensures memory specificity. Given that anxiety disorders are characterized by tendency to generalize learned fear to safe stimuli or situations, understanding how plasticity of intrinsic LAd synapses regulates the specificity of learned fear is an important challenge for future experimental studies.

[Analyzing A- and 0-rhythms of Conditioned Avoidance Reflex in the Rat Brain With Granger Causality].

It has been shown by the method of frequency decomposition of conditional Granger causality that under the execution of conditioned avoidance reflex θ-rhythm from the ventral hippocampus concurrently influences the ventral tegmental area and the series-connected basolateral amygdala and medial prefrontal cortex. Under the expectation of conditioned signal δ-rhythm from the prefrontal cortex influ- ences the ventral tegmental area and the amygdala.

Neural activation patterns underlying basolateral amygdala influence on intra-accumbens opioid-driven consummatory versus appetitive high-fat feeding behaviors in the rat.

The present study explored the role of the amygdala in mediating a unique pattern of feeding behavior driven by intra-accumbens (intra-Acb) opioid activation in the rat. Temporary inactivation of the basolateral amygdala (BLA), via GABAA agonist muscimol administration prevents increased consumption following intra-Acb opioid administration of the selective µ-opioid agonist D-Ala2, NMe-Phe4, Glyol5-enkephalin (DAMGO), yet leaves food approach behaviors intact, particularly after consumption has ended. One interpretation is that inactivation of the BLA selectively blocks neural activity underlying DAMGO-driven consummatory (consumption) but not appetitive (approach) behaviors. The present experiments take advantage of this temporal dissociation of consumption and approach behaviors to investigate their associated neural activity. Following either intra-Acb saline or DAMGO administration, with or without BLA muscimol administration, rats were given 2-hr access to a limited amount of high-fat diet. Immediately following the feeding session, rats were sacrificed and brains assayed for neural activity patterns across critical brain regions known to regulate both appetitive and consummatory feeding behaviors. The results show that intra-Acb DAMGO administration increased c-Fos activation in orexin neurons within the perifornical area of the hypothalamus and that this increase in activation is blocked by BLA muscimol inactivation. Intra-Acb DAMGO administration significantly increased c-Fos activation within dopaminergic neurons of the ventral tegmental area, compared to saline controls, and BLA inactivation had no effect on this increase. Overall, these data provide underlying circuitry that may mediate the selective influence of the BLA on driving consummatory, but not appetitive, feeding behaviors in a model of hedonically driven feeding behavior.