Auditory Conditioned Stimulus Research Articles

Pharmacological stimulation of infralimbic cortex after fear conditioning facilitates subsequent fear extinction

The infralimbic (IL) division of the medial prefrontal cortex (mPFC) is a crucial site for the extinction of conditioned fear memories in rodents. Recent work suggests that neuronal plasticity in the IL that occurs during (or soon after) fear conditioning enables subsequent IL-dependent extinction learning. We therefore hypothesized that pharmacological activation of the IL after fear conditioning would promote the extinction of conditioned fear. To test this hypothesis, we characterized the effects of post-conditioning infusions of the GABAA receptor antagonist, picrotoxin, into the IL on the extinction of auditory conditioned freezing in male and female rats. In four experiments, we found that picrotoxin injections performed immediately, 24 h, or 13 days after fear conditioning reduced conditioned freezing to the auditory conditioned stimulus (CS) during both extinction training and extinction retrieval; this effect was observed up to two weeks after picrotoxin infusions. Interestingly, inhibiting protein synthesis inhibition in the IL immediately after fear conditioning prevented the inhibition of freezing by picrotoxin injected 24 h later. Our data suggest that the IL encodes an inhibitory memory during the consolidation of fear conditioning that is necessary for future fear suppression.

Adrenalectomy exacerbates stress-induced impairment in fear discrimination: A causal role for kynurenic acid?

Impaired activity of the hypothalamic-pituitary axis and reduced blood levels of glucocorticoids (GCs) are signature features of stress-related maladies. Recent evidence suggests a possible role of the tryptophan metabolite kynurenic acid (KYNA) in this context. Here we investigated possible causal relationships in adult male rats, using stress-induced fear discrimination as a translationally relevant behavioral outcome measure. One week following adrenalectomy (ADX) or sham surgery, animals were for 2 h either physically restrained or exposed to a predator odor, which caused a much milder stress response. Extracellular KYNA levels were determined before, during and after stress by in vivo microdialysis in the prefrontal cortex. Separate cohorts underwent a fear discrimination procedure starting immediately after stress termination. Different auditory conditioned stimuli (CS) were either paired with a foot shock (CS+) or non-reinforced (CS−). One week later, fear was assessed by re-exposing the animals to each CS. Separate groups of rats were treated with the KYNA synthesis inhibitor BFF-816 prior to stress initiation to test a causal role of KYNA in fear discrimination. Restraint stress raised extracellular KYNA levels by ∼85 % in ADX rats for several hours, and these animals were unable to discriminate between CS+ and CS−. Both effects were prevented by BFF-816 and were not observed after exposure to predator odor or in sham-operated rats. These findings suggest that a causal connection exists between adrenal function, stress-induced KYNA increases, and behavioral deficits. Pharmacological inhibition of KYNA synthesis may therefore be an attractive, novel option for the treatment of stress-related disorders.

Augmenting glutamatergic, but not dopaminergic, activity in the nucleus accumbens shell disrupts responding to a discrete alcohol cue in an alcohol context.

Discrete alcohol cues and contexts are relapse triggers for people with alcohol use disorder exerting particularly powerful control over behaviour when they co-occur. Here, we investigated the neural substrates subserving the capacity for alcohol-associated contexts to elevate responding to an alcohol-predictive conditioned stimulus (CS). Specifically, rats were trained in a distinct 'alcohol context' to respond by entering a fluid port during a discrete auditory CS that predicted the delivery of alcohol and were familiarized with a 'neutral context' wherein alcohol was never available. When conditioned CS responding was tested by presenting the CS without alcohol, we found that augmenting glutamatergic activity in the nucleus accumbens (NAc) shell by microinfusing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) reduced responding to an alcohol CS in an alcohol, but not neutral, context. Further, AMPA microinfusion robustly affected behaviour, attenuating the number, duration and latency of CS responses selectively in the alcohol context. Although dopaminergic inputs to the NAc shell were previously shown to be necessary for CS responding in an alcohol context, here, chemogenetic excitation of ventral tegmental area (VTA) dopamine neurons and their inputs to the NAc shell did not affect CS responding. Critically, chemogenetic excitation of VTA dopamine neurons affected feeding behaviour and elevated c-fos immunoreactivity in the VTA and NAc shell, validating the chemogenetic approach. These findings enrich our understanding of the substrates underlying Pavlovian responding for alcohol and reveal that the capacity for contexts to modulate responding to discrete alcohol cues is delicately underpinned by the NAc shell.

Sex-biased effects of outcome devaluation by sensory-specific satiety on Pavlovian-conditioned behavior.

Goal-directed behavior relies on accurate mental representations of the value of expected outcomes. Disruptions to this process are a central feature of several neuropsychiatric disorders, including addiction. Goal-directed behavior is most frequently studied using instrumental paradigms paired with outcome devaluation, but cue-evoked behaviors in Pavlovian settings can also be goal-directed and therefore sensitive to changes in outcome value. Emerging literature suggests that male and female rats may differ in the degree to which their Pavlovian-conditioned responses are goal-directed, but interpretation of these findings is complicated by the tendency of female and male rats to engage in distinct types of Pavlovian responses when trained with localizable cues. Here, we used outcome devaluation via sensory-specific satiety to assess the behavioral responses in male and female Long Evans rats trained to respond to an auditory CS (conditioned stimulus) in a Pavlovian-conditioning paradigm. We found that satiety-induced devaluation led to a decrease in behavioral responding to the reward-predictive CS, with males showing an effect on both port entry latency and probability and females showing an effect only on port entry probability. Overall, our results suggest that outcome devaluation affects Pavlovian-conditioned responses in both male and female rats, but that females may be less sensitive to outcome devaluation.

EVs-mediated delivery of CB2 receptor agonist for Alzheimer's disease therapy

Alzheimer's disease (AD) is a typical neurodegenerative disease that leads to irreversible neuronal degeneration, and effective treatment remains elusive due to the unclear mechanism. We utilized biocompatible mesenchymal stem cell-derived extracellular vesicles as carriers loaded with the CB2 target medicine AM1241 (EVs-AM1241) to protect against neurodegenerative progression and neuronal function in AD model mice. According to the results, EVs-AM1241 were successfully constructed and exhibited better bioavailability and therapeutic effects than bare AM1241. The Morris water maze (MWM) and fear conditioning tests revealed that the learning and memory of EVs-AM1241-treated model mice were significantly improved. In vivo electrophysiological recording of CA1 neurons indicated enhanced response to an auditory conditioned stimulus following fear learning. Immunostaining and Western blot analysis showed that amyloid plaque deposition and amyloid β (Aβ)-induced neuronal apoptosis were significantly suppressed by EVs-AM1241. Moreover, EVs-AM1241 increased the number of neurons and restored the neuronal cytoskeleton, indicating that they enhanced neuronal regeneration. RNA sequencing revealed that EVs-AM1241 facilitated Aβ phagocytosis, promoted neurogenesis and ultimately improved learning and memory through the calcium-Erk signaling pathway. Our study showed that EVs-AM1241 efficiently reversed neurodegenerative pathology and enhanced neurogenesis in model mice, indicating that they are very promising particles for treating AD.

A novel automated software and video analysis system for quantifying physiological responses of conditioned fear in mice

BACKGROUND: Conditioned fear is an aversive learning process that is highly sensitive to sensory conditions such as environmental context, threat intensity and circadian rhythms. To better understand these external variables that impact conditioned fear learning, we developed a novel software application that allows for programmable remote conditioned stimulus (CS, ie., auditory tone) delivery and recording of group-housed mice in a homecage, or familiar environment. The goal of our study was therefore to leverage this technology to examine conditioned fear in the homecage across light-dark (LD) circadian cycles. METHODS: Male C57bl/6J mice underwent Pavlovian auditory fear conditioning (20 CS/US pairings), with a CS-only group as a control (n=4/group). Mice were then returned to the homecage, equipped with speaker and camera. Using a customized open-source software to remotely evoke an enhanced physiological state of fear in the homecage, mice received a select number of conditioned auditory stimuli (30sec. intermittent tones) throughout the LD cycle over 14 days. The software was configured to deliver six tones (6kHz) over a 24hr period (3 light, 3 dark). The first CS during each LD cycle was recorded and percent freezing behavior was manually scored. On day 15, to evaluate whether the HC-specific evoked fear response transfers to a new environment, mice were placed into a novel context and CS-dependent freezing was measured. RESULTS: Over 14 days, compared to controls, fear-conditioned pair-housed mice exhibited strong CS-evoked freezing in the homecage ( total freezing: FC 70.46%, CS-only 3.69%, F(2.29, 11.14)=10.78, p<0.01). Interestingly, when we evaluated CS-specific circadian responses, fear-conditioned mice exhibited enhanced freezing in the light versus the dark phase ( total freezing: light 84.56%, dark 56.36%, F(2.15, 6.46)=11.04, p<0.01). In fear-conditioned mice, there was a 36% reduction in total CS-dependent homecage freezing over 14 days. However, this was a context-dependent effect, as the reduction in freezing on day 14 was not observed in the novel context on day 15 ( t(3)=3.16, p=0.05). CONCLUSIONS: Here we demonstrate for the first time, using an open-source software developed in our laboratory, environment and circadian-dependent conditioned freezing responses in the homecage of group-housed mice. This study provides a proof-of-principle innovative approach for examining physiological outcomes of conditioned fear across ethologically relevant environments, which may help further the understanding for heightened physiological responses in neuropsychiatric diseases such as posttraumatic stress disorder (PTSD). To further elucidate impaired physiological mechanisms of disordered fear, future studies will integrate multimodal behavioral (DeepLabCut TM ) and cardiovascular telemetry-based approaches. Congressionally Directed Medical Research Programs (CDMRP) PR210574 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Glucocorticoid- β-adrenoceptors interactions in the infralimbic cortex in acquisition and consolidation of auditory fear memory extinction in rats

This study investigated the interactive effect of glucocorticoid and β-adrenoceptors in the infralimbic (IL) cortex on the acquisition and consolidation of fear extinction in rats' auditory fear conditioning (AFC) task. On day 1, rats underwent habituation for 9 min (12 tones, 10 s, 4 kHz, 80 dB, without footshock). On day 2 (conditioning), rats received 3 mild electrical footshocks (US; 2 s, 0.5 mA) paired with the auditory-conditioned stimulus (CS; tone: 30 s, 4 kHz, 80 dB). On days 3–5 (Ext 1–3), rats received 15 tones with no footshock in the test box. Intra-IL injection of corticosterone (CORT, 20 ng/0.5 μl per side) before Ext 1 and after Ext 1–2, respectively, facilitated the acquisition and consolidation of fear memory extinction. Intra-IL injection of the β2-adrenoceptor agonist clenbuterol (CLEN, 50 ng/0.5 μl per side) inhibited, but the β-adrenoceptor antagonist propranolol (PROP, 500 ng/0.5 μl per side) enhanced the facilitatory effects of CORT on fear memory extinction. CORT injection before the acquisition of fear extinction increased p-ERK levels in the IL. Co-injection of CORT with CLEN increased, but PROP decreased p-ERK activities. CORT injection after the consolidation of fear extinction increased p-CREB in the IL. Co-injection of CORT with CLEN increased, but PROP reduced p-CREB activities. Our findings show that corticosterone facilitates the acquisition and consolidation of fear memory extinction. GRs and β-adrenoceptors in the IL jointly regulate fear memory extinction via ERK and CREB signaling pathways. This pre-clinical animal study may highlight the effect of GRs and β-adrenoceptors of the IL cortex in regulating fear memory processes in fear-related disorders such as PTSD.

Sensory gating deficits and childhood trauma in the onset of first-episode schizophrenia

Studies have shown sensory gating deficits and severe childhood trauma in patients with schizophrenia; however, their relationship with this condition remains unclear. Here, we hypothesized that sensory gating deficits mediate the effects of childhood trauma on schizophrenia onset. We recruited 79 patients with first-episode schizophrenia (PFES) and 76 health controls (HC). The auditory conditioning (S1) and testing (S2) stimulus paradigm was used to detect P50 sensory gating. The Childhood Trauma Questionnaire (CTQ) was used to assess childhood trauma experiences. Compared with HC, the PFES group had more severe childhood trauma experiences together with sensory gating deficits. In a partial correlation analysis, sexual abuse was negatively correlated with the P50 S2 latency, physical neglect was negatively correlated with the S1 latency, while emotional neglect was positively correlated with the S2/S1 ratio and negatively correlated with the S1-S2 difference in the PFES group. However, there was no correlation between the CTQ total and each sub-scores and P50 indicators in the HC. The S1-S2 difference was the mediator between emotional neglect and the onset of schizophrenia. Childhood trauma might be associated with schizophrenia by influencing sensory gating deficits. Early intervention targeting childhood trauma might reduce the incidence of sensory gating deficits and thus schizophrenia.

Anxiolytic reversal of classically conditioned / chronic stress-induced gene expression and learning in the Stress Alternatives Model

Social decision-making is critically influenced by neurocircuitries that regulate stress responsiveness. Adaptive choices, therefore, are altered by stress-related neuromodulatory peptide systems, such as corticotropin releasing factor (CRF). Experimental designs that take advantage of ecologically salient fear-inducing stimuli allow for revelation of neural mechanisms that regulate the balance between pro- and anti-stress responsiveness. To accomplish this, we developed a social stress and conditioning protocol, the Stress Alternatives Model (SAM), that utilizes a simple dichotomous choice, and produces distinctive behavioral phenotypes (Escape or Stay). The experiments involve repeated social aggression, a potent unconditioned stimulus (US), from a novel larger conspecific (a 3X larger Rainbow trout). Prior to the social interaction, the smaller test fish is presented with an auditory conditioning stimulus (water off = CS). During the social aggression, an escape route is available, but is only large enough for the smaller test animal. Surprisingly, although the new aggressor provides vigorous attacks each day, only 50% of the test fish choose Escape. Stay fish, treated with the CRF1 antagonist antalarmin, a potent anxiolytic drug, on day 4, promotes Escape behavior for the last 4 days of the SAM protocol. The results suggest that the decision to Escape, required a reduction in stress reactivity. The Stay fish that chose Escape following anxiolytic treatment, learned how to use the escape route prior to stress reduction, as the Escape latency in these fish was significantly faster than first time escapers. In Escape fish, the use of the escape route is learned over several days, reducing the Escape latency over time in the SAM. Fear conditioning (water off + aggression) resulted in elevated hippocampal (DL) Bdnf mRNA levels, with coincident reduction in the AMPA receptor subunit Glua1 expression, a result that is reversed following a one-time treatment (during SAM aggression on day 4) with the anxiolytic CRF1 receptor antagonist antalarmin.

Ventral hippocampal shock encoding modulates the expression of trace cued fear

The hippocampus is crucial for associative fear learning when the anticipation of threat requires temporal or contextual binding of predictive stimuli as in trace and contextual fear conditioning. Compared with the dorsal hippocampus, far less is known about the contribution of the ventral hippocampus to fear learning. The ventral hippocampus, which is highly interconnected with defensive and emotional networks, has a prominent role in both innate and learned affective behaviors including anxiety, fear, and reward. Lesions or temporary inactivation of the ventral hippocampus impair both cued and contextual fear learning, but whether the ventral hippocampal role in learning is driven by affective processing, associative encoding, or both is not clear. Here, we used trace fear conditioning in mixed sex cohorts to assess the contribution of shock-encoding to the acquisition of cued and contextual fear memories. Trace conditioning requires subjects to associate an auditory conditional stimulus (CS) with a shock unconditional stimulus (UCS) that are separated in time by a 20-s trace interval. We first recorded neuronal activity in the ventral hippocampus during trace fear conditioning and found that ventral CA1 predominantly encoded the shock reinforcer. Potentiated firing to the CS was evident at testing, but no encoding of the trace interval was observed. We then tested the necessity of shock encoding for conditional fear acquisition by optogenetically silencing ventral hippocampal activity during the UCS on each trial of training. Contrary to our predictions, preventing hippocampal shock-evoked firing did not impair associative fear. Instead, it led to a more prolonged expression of CS freezing across test trials, an effect observed in males, but not females. Contextual fear learning was largely intact, although a subset of animals in each sex were differentially affected by shock-silencing. Taken together, the results show that shock encoding in the ventral hippocampus modulates the expression of learned fear in a sex-specific manner.

Corticostriatal Suppression of Appetitive Pavlovian Conditioned Responding.

The capacity to suppress learned responses is essential for animals to adapt in dynamic environments. Extinction is a process by which animals learn to suppress conditioned responding when an expected outcome is omitted. The infralimbic (IL) cortex to nucleus accumbens shell (NAcS) neural circuit is implicated in suppressing conditioned responding after extinction, especially in the context of operant cocaine-seeking behavior. However, the role of the IL-to-NAcS neural circuit in the extinction of responding to appetitive Pavlovian cues is unknown, and the psychological mechanisms involved in response suppression following extinction are unclear. We trained male Long Evans rats to associate a 10 s auditory conditioned stimulus (CS; 14 trials per session) with a sucrose unconditioned stimulus (US; 0.2 ml per CS) in a specific context, and then following extinction in a different context, precipitated a renewal of CS responding by presenting the CS alone in the original Pavlovian conditioning context. Unilateral, optogenetic stimulation of the IL-to-NAcS circuit selectively during CS trials suppressed renewal. In a separate experiment, IL-to-NAcS stimulation suppressed CS responding regardless of prior extinction and impaired extinction retrieval. Finally, IL-to-NAcS stimulation during the CS did not suppress the acquisition of Pavlovian conditioning but was required for the subsequent expression of CS responding. These results are consistent with multiple studies showing that the IL-to-NAcS neural circuit is involved in the suppression of operant cocaine-seeking, extending these findings to appetitive Pavlovian cues. The suppression of appetitive Pavlovian responding following IL-to-NAcS circuit stimulation, however, does not appear to be an extinction-dependent process.SIGNIFICANCE STATEMENT Extinction is a form of inhibitory learning through which animals learn to suppress conditioned responding in the face of nonreinforcement. We investigated the role of the IL cortex inputs to the NAcS in the extinction of responding to appetitive Pavlovian cues and the psychological mechanisms involved in response suppression following extinction. Using in vivo optogenetics, we found that stimulating the IL-to-NAcS neural circuit suppressed context-induced renewal of conditioned responding after extinction. In a separate experiment, stimulating the IL-to-NAcS circuit suppressed conditioned responding in an extinction-independent manner. These findings can be used by future research aimed at understanding how corticostriatal circuits contribute to behavioral flexibility and mental disorders that involve the suppression of learned behaviors.

The role of context conditioning in the reinstatement of responding to an alcohol-predictive conditioned stimulus

Re-exposure to an unconditioned stimulus (US) can reinstate extinguished conditioned responding elicited by a conditioned stimulus (CS). We tested the hypothesis that the reinstatement of responding to an appetitive CS is driven by an excitatory association formed between the US and the context that the US was ingested in during US re-exposure. Male, Long-Evans rats were acclimated to drinking alcohol (15%, v/v) in the home-cage, then trained to associate an auditory CS with an alcohol-US that was delivered into a fluid port for oral intake. During subsequent extinction sessions, the CS was presented as before, but without alcohol. After extinction, rats were re-exposed to alcohol as in training, but without the CS (alcohol re-exposure). 24 h later at test, the CS was presented as in training, but without alcohol. First, we tested the effect of extinguishing the context-alcohol association, formed during alcohol re-exposure, on reinstatement. Conducting four context extinction sessions across four days (spaced extinction) after the alcohol re-exposure session did not impact reinstatement. However, four context extinction sessions conducted across two days (massed extinction) prevented reinstatement. Next, we conducted alcohol re-exposure in a context that either differed from, or was the same as, the test context. One alcohol re-exposure session in a different context did not affect reinstatement, however, three alcohol re-exposure sessions in a different context significantly reduced reinstatement during the first CS trial. These results partially support the view that a context-US association formed during US re-exposure drives the reinstatement of responding to an appetitive, alcohol-predictive CS.

Contributions of postrhinal and perirhinal cortex to contextual information processing.

The role of the postrhinal cortex (POR) and the perirhinal cortex (PER) in processing relational or contextual information was examined with Pavlovian fear conditioning. Rats with electrolytic or neurotoxic lesions of the POR or PER were tested in 2 contextual fear conditioning paradigms. In Experiment 1, electrolytic lesions of the POR or PER produced impairments in contextual fear conditioning but not in conditioning to a phasic auditory conditioned stimulus. Neurotoxic lesions of the POR or PER likewise resulted in anterograde (Experiment 2) and retrograde (Experiment 3) deficits in fear conditioning to the training context in an unsignaled shock paradigm. The results suggest that operations performed on sensory information by the POR and PER are necessary to support contextual learning. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

A Signaled Locomotor Avoidance Action Is Fully Represented in the Neural Activity of the Midbrain Tegmentum

Animals, including humans, readily learn to avoid harmful and threatening situations by moving in response to cues that predict the threat (e.g., fire alarm, traffic light). During a negatively reinforced sensory-guided locomotor action, known as signaled active avoidance, animals learn to avoid a harmful unconditioned stimulus (US) by moving away when signaled by a harmless conditioned stimulus (CS) that predicts the threat. CaMKII-expressing neurons in the pedunculopontine tegmentum area (PPT) of the midbrain locomotor region have been shown to play a critical role in the expression of this learned behavior, but the activity of these neurons during learned behavior is unknown. Using calcium imaging fiber photometry in freely behaving mice, we show that PPT neurons sharply activate during presentation of the auditory CS that predicts the threat before onset of avoidance movement. PPT neurons activate further during the succeeding CS-driven avoidance movement, or during the faster US-driven escape movement. PPT neuron activation was weak during slow spontaneous movements but correlated sharply with movement speed and, therefore, with the urgency of the behavior. Moreover, using optogenetics, we found that these neurons must discharge during the signaled avoidance interval for naive mice to effectively learn the active avoidance behavior. As an essential hub for signaled active avoidance, neurons in the midbrain tegmentum process the conditioned cue that predicts the threat and discharge sharply relative to the speed or apparent urgency of the avoidance (learned) and escape (innate) responses.SIGNIFICANCE STATEMENTDuring signaled active avoidance behavior, subjects move away to avoid a threat when directed by an innocuous sensory stimulus. Using imaging methods in freely behaving mice, we found that the activity of neurons in a part of the midbrain, known as the pedunculopontime tegmentum, increases during the presentation of the innocuous sensory stimulus that predicts the threat and also during the expression of the learned behavior as mice move away to avoid the threat. In addition, inhibiting these neurons abolishes the ability of mice to learn the behavior. Thus, neurons in this part of the midbrain code and are essential for signaled active avoidance behavior.

Saccadic scanpath length: an index for human threat conditioning

Threat-conditioned cues are thought to capture overt attention in a bottom-up process. Quantification of this phenomenon typically relies on cue competition paradigms. Here, we sought to exploit gaze patterns during exclusive presentation of a visual conditioned stimulus, in order to quantify human threat conditioning. To this end, we capitalized on a summary statistic of visual search during CS presentation, scanpath length. During a simple delayed threat conditioning paradigm with full-screen monochrome conditioned stimuli (CS), we observed shorter scanpath length during CS+ compared to CS- presentation. Retrodictive validity, i.e., effect size to distinguish CS+ and CS-, was maximized by considering a 2-s time window before US onset. Taking into account the shape of the scan speed response resulted in similar retrodictive validity. The mechanism underlying shorter scanpath length appeared to be longer fixation duration and more fixation on the screen center during CS+ relative to CS- presentation. These findings were replicated in a second experiment with similar setup, and further confirmed in a third experiment using full-screen patterns as CS. This experiment included an extinction session during which scanpath differences appeared to extinguish. In a fourth experiment with auditory CS and instruction to fixate screen center, no scanpath length differences were observed. In conclusion, our study suggests scanpath length as a visual search summary statistic, which may be used as complementary measure to quantify threat conditioning with retrodictive validity similar to that of skin conductance responses.

Cholinergic neurotransmission in the basolateral amygdala during cued fear extinction

Cholinergic neuromodulation plays an important role in numerous cognitive functions including regulating arousal and attention, as well as associative learning and extinction processes. Further, studies demonstrate that cholinergic inputs from the basal forebrain cholinergic system influence physiological responses in the basolateral amygdala (BLA) as well as fear extinction processes. Since rodent models display individual differences in conditioned fear and extinction responses, this study investigated if cholinergic transmission in the BLA during fear extinction could contribute to differences between extinction resistant and extinction competent phenotypes in outbred Long-Evans male rats. Experiment 1 used in vivo microdialysis to test the hypothesis that acetylcholine (ACH) efflux in the BLA would increase with presentation of an auditory conditioned stimulus (CS+) during extinction learning. Acetylcholine efflux was compared in rats exposed to the CS+, a CS- (the tone never paired with a footshock), or to a context shift alone (without CS+ tone presentation). Consistent with acetylcholine's role in attention and arousal, ACH efflux in the BLA was increased in all three groups (CS+, CS-, Shift Alone) by the initial context shift into the extinction learning chamber, but returned more rapidly to baseline levels in the Shift Alone group (no CS+). In contrast, in the group exposed to the CS+, ACH efflux in the BLA remained elevated during continued presentation of conditioned cues and returned to baseline more slowly, leading to an overall increase in ACH efflux compared with the Shift Alone group. Based on the very dense staining in the BLA for acetylcholinesterase (ACHE), Experiment 2 examined if individual differences in fear extinction were associated with differences in cholinesterase enzyme activity (CHE) in the BLA and/or plasma with a separate cohort of animals. Cholinesterase activity (post-testing) in both the BLA and plasma was higher in extinction competent rats versus rats resistant to extinction learning. There was also a significant negative correlation between BLA CHE activity and freezing during extinction learning. Taken together, our results support a role for ACH efflux in the BLA during cued fear extinction that may be modulated by individual differences in ACHE activity, and are associated with behavioral responses during fear extinction. These findings implicate individual differences in cholinergic regulation in the susceptibility to disorders with dysregulation of extinction learning, such post-traumatic stress disorder (PTSD) in humans.

Human cerebral organoids establish subcortical projections in the mouse brain after transplantation

Numerous studies have used human pluripotent stem cell-derived cerebral organoids to elucidate the mystery of human brain development and model neurological diseases in vitro, but the potential for grafted organoid-based therapy in vivo remains unknown. Here, we optimized a culturing protocol capable of efficiently generating small human cerebral organoids. After transplantation into the mouse medial prefrontal cortex, the grafted human cerebral organoids survived and extended projections over 4.5 mm in length to basal brain regions within 1 month. The transplanted cerebral organoids generated human glutamatergic neurons that acquired electrophysiological maturity in the mouse brain. Importantly, the grafted human cerebral organoids functionally integrated into pre-existing neural circuits by forming bidirectional synaptic connections with the mouse host neurons. Furthermore, compared to control mice, the mice transplanted with cerebral organoids showed an increase in freezing time in response to auditory conditioned stimuli, suggesting the potentiation of the startle fear response. Our study showed that subcortical projections can be established by microtransplantation and may provide crucial insights into the therapeutic potential of human cerebral organoids for neurological diseases.

REM sleep promotes experience-dependent dendritic spine elimination in the mouse cortex

In many parts of the nervous system, experience-dependent refinement of neuronal circuits predominantly involves synapse elimination. The role of sleep in this process remains unknown. We investigated the role of sleep in experience-dependent dendritic spine elimination of layer 5 pyramidal neurons in the visual (V1) and frontal association cortex (FrA) of 1-month-old mice. We found that monocular deprivation (MD) or auditory-cued fear conditioning (FC) caused rapid spine elimination in V1 or FrA, respectively. MD- or FC-induced spine elimination was significantly reduced after total sleep or REM sleep deprivation. Total sleep or REM sleep deprivation also prevented MD- and FC-induced reduction of neuronal activity in response to visual or conditioned auditory stimuli. Furthermore, dendritic calcium spikes increased substantially during REM sleep, and the blockade of these calcium spikes prevented MD- and FC-induced spine elimination. These findings reveal an important role of REM sleep in experience-dependent synapse elimination and neuronal activity reduction.

NMDA receptors in the CeA and BNST differentially regulate fear conditioning to predictable and unpredictable threats

Considerable work demonstrates that Pavlovian fear conditioning depends on N-methyl-D-aspartate (NMDA) receptor-dependent plasticity within the amygdala. In addition, the bed nucleus of the stria terminalis (BNST) has also been implicated in fear conditioning, particularly in the expression of fear to poor predictors of threat. We recently found that the expression of backward (BW) fear conditioning, in which an auditory conditioned stimulus (CS) follows a footshock unconditioned stimulus (US), requires the BNST; the expression of forward (FW) fear conditioning was not disrupted by BNST inactivation. However, whether NMDA receptors within the BNST contribute to the acquisition of fear conditioning is unknown. Moreover, the central nucleus of the amygdala (CeA), which has extensive connections with the BNST, is critically involved in FW conditioning, however whether it participates in BW conditioning has not been explored. Here we test the specific hypothesis that the CeA and the BNST mediate the acquisition of FW and BW fear conditioning, respectively. Adult female and male rats were randomly assigned to receive bilateral infusions of the NMDA receptor antagonist, D,L-2-amino-5-phosphonovalerate (APV), into the CeA or BNST prior to FW or BW fear conditioning. We found that intra-CeA APV impaired the acquisition of both FW and BW conditioning, whereas intra-BNST APV produced selective deficits in BW conditioning. Moreover, APV in the BNST significantly reduced contextual freezing, whereas CeA NMDA receptor antagonism impeded early but not long-lasting contextual fear. Collectively, these data reveal that CeA and BNST NMDA receptors have unique roles in fear conditioning.

Auditory Fear Conditioning Alters Sensitivity of the Medial Prefrontal Cortex but this is not based on Frequency-dependent Integration

Although many studies have shown that the prelimbic (PL) cortex of the mPFC is involved in the formation of conditioned freezing behavior, few have considered the acoustic response characteristics of PL cortex. Importantly, the change in auditory response characteristics of the PL cortex after conditional fear learning is largely unknown. Here we used in vivo cell-attached recordings targeting the mPFC during the waking state. We confirmed that the mPFC of adult C57 mice have neurons that respond to noise and tone in the waking state, especially in the PL cortex. Interestingly, the data also confirmed that these neurons responded well to the intensity of sound but did not have frequency topological distribution characteristics. Furthermore, we found that the number of c-fos positive neurons in the PL cortex increased significantly after auditory fear conditioning. The auditory-induced local field potential recordings and in vivo cell-attached recordings demonstrated that the PL cortex was more sensitive to the auditory conditioned stimulus after the acquisition of conditioned fear. The proportion of neurons responding to noise was significantly increased, and the signal to noise ratio of the spikes were also increased. These data reveal that PL neurons themselves responded to the main information (sound intensity), while the secondary information (frequency) response was almost negligible after auditory fear conditioning. This phenomenon may be the functional basis for handling this type of emotional memory, and this response characteristic is thought to be emotional sensitization but does not change the nature of this response.