A temporal shift in the circuits mediating retrieval of fear memory.

Fear memories allow animals to avoid danger, thereby increasing their chances of survival. Fear memories can be retrieved long after learning, but little is known about how retrieval circuits change with time. Here we show that the dorsal midline thalamus of rats is required for the retrieval of auditory conditioned fear at late (24 hours, 7 days, 28 days), but not early (0.5 hours, 6 hours) time points after learning. Consistent with this, the paraventricular nucleus of the thalamus (PVT), a subregion of the dorsal midline thalamus, showed increased c-Fos expression only at late time points, indicating that the PVT is gradually recruited for fear retrieval. Accordingly, the conditioned tone responses of PVT neurons increased with time after training. The prelimbic (PL) prefrontal cortex, which is necessary for fear retrieval, sends dense projections to the PVT. Retrieval at late time points activated PL neurons projecting to the PVT, and optogenetic silencing of these projections impaired retrieval at late, but not early, time points. In contrast, silencing of PL inputs to the basolateral amygdala impaired retrieval at early, but not late, time points, indicating a time-dependent shift in retrieval circuits. Retrieval at late time points also activated PVT neurons projecting to the central nucleus of the amygdala, and silencing these projections at late, but not early, time points induced a persistent attenuation of fear. Thus, the PVT may act as a crucial thalamic node recruited into cortico-amygdalar networks for retrieval and maintenance of long-term fear memories.

A NeuroD1 AAV-Based Gene Therapy for Functional Brain Repair after Ischemic Injury through In Vivo Astrocyte-to-Neuron Conversion.

Adult mammalian brains have largely lost neuroregeneration capability except for a few niches. Previous studies have converted glial cells into neurons, but the total number of neurons generated is limited and the therapeutic potential is unclear. Here, we demonstrate that NeuroD1-mediated in situ astrocyte-to-neuron conversion can regenerate a large number of functional new neurons after ischemic injury. Specifically, using NeuroD1 adeno-associated virus (AAV)-based gene therapy, we were able to regenerate one third of the total lost neurons caused by ischemic injury and simultaneously protect another one third of injured neurons, leading to a significant neuronal recovery. RNA sequencing and immunostaining confirmed neuronal recovery after cell conversion at both the mRNA level and protein level. Brain slice recordings found that the astrocyte-converted neurons showed robust action potentials and synaptic responses at 2 months after NeuroD1 expression. Anterograde and retrograde tracing revealed long-range axonal projections from astrocyte-converted neurons to their target regions in a time-dependent manner. Behavioral analyses showed a significant improvement of both motor and cognitive functions after cell conversion. Together, these results demonstrate that in vivo cell conversion technology through NeuroD1-based gene therapy can regenerate a large number of functional new neurons to restore lost neuronal functions after injury.

Neuroscience Research and Mentoring in Puerto Rico: What Succeeds in This Environment?

Twenty years ago, I arrived in Puerto Rico from New York City to establish a neuroscience laboratory and research program on extinction of conditioned fear. The lab's first research paper appeared in the Journal of Neuroscience (Quirk et al., 2000) and has been cited >900 times. The success of this project in Puerto Rico far surpassed my original expectations. Therefore, I thought it might be useful to identify the factors responsible for this success, with the hope of facilitating the development of laboratories in diverse settings. A description of our lab practices is interspersed with personal statements from trainees hailing from Puerto Rico and other parts of Latin America. Creating an effective research and training environment depends less on the director's personality and more on the proper practice of activities that foster intellectual growth, such as journal clubs, lab meetings, and philosophy of science retreats. On a personal level, this project has been enormously gratifying. The unique environment in Puerto Rico fostered my best work, and I am very happy to have established my laboratory here.

The Storytelling Brain: How Neuroscience Stories Help Bridge the Gap between Research and Society.

Active communication between researchers and society is necessary for the scientific community's involvement in developing science-based policies. This need is recognized by governmental and funding agencies that compel scientists to increase their public engagement and disseminate research findings in an accessible fashion. Storytelling techniques can help convey science by engaging people's imagination and emotions. Yet, many researchers are uncertain about how to approach scientific storytelling, or feel they lack the tools to undertake it. Here we explore some of the techniques intrinsic to crafting scientific narratives, as well as the reasons why scientific storytelling may be an optimal way of communicating research to nonspecialists. We also point out current communication gaps between science and society, particularly in the context of neurodiverse audiences and those that include neurological and psychiatric patients. Present shortcomings may turn into areas of synergy with the potential to link neuroscience education, research, and advocacy.

Less fear, more diversity.

Fear is an instinctual response that's adaptive and critical for survival when it is short-lived but can lead to anxiety disorders when chronic. Studying how the brain controls our fears helps us understand the mechanisms required to recover from traumatic experiences and what goes wrong when we don't. Research in rodents has identified neural circuits and molecular mechanisms regulating fear expression. Rodent work has been amenable to translation to humans and has led to improvements in clinical therapies for anxiety disorders. The societal benefit of this type of research is magnified when performed in minority-serving institutions, offering high-caliber training opportunities to increase ethnic diversity in science.

Revisiting the role of infralimbic cortex in fear extinction with optogenetics.

Previous rodent studies have implicated the infralimbic (IL) subregion of the medial prefrontal cortex in extinction of auditory fear conditioning. However, these studies used pharmacological inactivation or electrical stimulation techniques, which lack temporal precision and neuronal specificity. Here, we used an optogenetic approach to either activate (with channelrhodopsin) or silence (with halorhodopsin) glutamatergic IL neurons during conditioned tones delivered in one of two phases: extinction training or extinction retrieval. Activating IL neurons during extinction training reduced fear expression and strengthened extinction memory the following day. Silencing IL neurons during extinction training had no effect on within-session extinction, but impaired the retrieval of extinction the following day, indicating that IL activity during extinction tones is necessary for the formation of extinction memory. Surprisingly, however, silencing IL neurons optogenetically or pharmacologically during the retrieval of extinction 1 day or 1 week following extinction training had no effect. Our findings suggest that IL activity during extinction training likely facilitates storage of extinction in target structures, but contrary to current models, IL activity does not appear to be necessary for retrieval of extinction memory.

Neural structures mediating expression and extinction of platform-mediated avoidance.

Individuals use both passive and active defensive responses to environmental threats. Much is known about the neural circuits of passive defensive responses (e.g., freezing), but less is known about the substrates of active defensive responses (e.g., avoidance). We developed an active avoidance task in which rats learn to avoid a tone-signaled footshock by stepping onto a nearby platform. An advantage of this task is that freezing, which can interfere with avoidance, is reduced, thereby facilitating comparison of the effects of manipulations on avoidance versus freezing. After 10 d of avoidance training, rats were infused with muscimol to pharmacologically inactivate the prelimbic cortex (PL), infralimbic cortex (IL), ventral striatum (VS), or basolateral amygdala (BLA). Inactivating PL, VS, or BLA all impaired avoidance expression, but these areas differed with respect to freezing. Inactivating BLA decreased freezing consistent with loss of the tone-shock association, whereas inactivation of VS increased freezing consistent with loss of avoidance memory. Inactivation of PL had no effect on freezing. Inactivation of IL did not impair avoidance expression but did impair avoidance extinction. Our findings suggest that active avoidance is mediated by prefrontal-striatal circuits, which may be overactive in individuals suffering from trauma-related disorders.

Deep brain stimulation of the ventral striatum enhances extinction of conditioned fear.

Deep brain stimulation (DBS) of the ventral capsule/ventral striatum (VC/VS) reduces symptoms of intractable obsessive-compulsive disorder (OCD), but the mechanism of action is unknown. OCD is characterized by avoidance behaviors that fail to extinguish, and DBS could act, in part, by facilitating extinction of fear. We investigated this possibility by using auditory fear conditioning in rats, for which the circuits of fear extinction are well characterized. We found that DBS of the VS (the VC/VS homolog in rats) during extinction training reduced fear expression and strengthened extinction memory. Facilitation of extinction was observed for a specific zone of dorsomedial VS, just above the anterior commissure; stimulation of more ventrolateral sites in VS impaired extinction. DBS effects could not be obtained with pharmacological inactivation of either dorsomedial VS or ventrolateral VS, suggesting an extrastriatal mechanism. Accordingly, DBS of dorsomedial VS (but not ventrolateral VS) increased expression of a plasticity marker in the prelimbic and infralimbic prefrontal cortices, the orbitofrontal cortex, the amygdala central nucleus (lateral division), and intercalated cells, areas known to learn and express extinction. Facilitation of fear extinction suggests that, in accord with clinical observations, DBS could augment the effectiveness of cognitive behavioral therapies for OCD.

Specific Patterns of Endogenous Functional Connectivity Are Associated With Harm Avoidance in Obsessive-Compulsive Disorder.

BACKGROUND: Individuals with obsessive-compulsive disorder (OCD) show persistent avoidance behaviors, often in the absence of actual threat. Quality-of-life costs and heterogeneity support the need for novel brain-behavior intervention targets. Informed by mechanistic and anatomical studies of persistent avoidance in rodents and nonhuman primates, our goal was to test whether connections within a hypothesized persistent avoidance-related network predicted OCD-related harm avoidance (HA), a trait measure of persistent avoidance. We hypothesized that 1) HA, not an OCD diagnosis, would be associated with altered endogenous connectivity in at least one connection in the network; 2) HA-specific findings would be robust to comorbid symptoms; and 3) reliable findings would replicate in a holdout testing subsample. METHODS: Using resting-state functional connectivity magnetic resonance imaging, cross-validated elastic net for feature selection, and Poisson generalized linear models, we tested which connections significantly predicted HA in our training subsample (n = 73; 71.8% female; healthy control group n = 36, OCD group n = 37); robustness to comorbidities; and replicability in a testing subsample (n = 30; 56.7% female; healthy control group n = 15, OCD group n = 15). RESULTS: Stronger inverse connectivity between the right dorsal anterior cingulate cortex and right basolateral amygdala and stronger positive connectivity between the right ventral anterior insula and left ventral striatum were associated with greater HA across groups. Network connections did not discriminate OCD diagnostic status or predict HA-correlated traits, suggesting sensitivity to trait HA. The dorsal anterior cingulate cortex-basolateral amygdala relationship was robust to controlling for comorbidities and medication in individuals with OCD and was also predictive of HA in our testing subsample. CONCLUSIONS: Stronger inverse dorsal anterior cingulate cortex-basolateral amygdala connectivity was robustly and reliably associated with HA across groups and in OCD. Results support the relevance of a cross-species persistent avoidance-related network to OCD, with implications for precision-based approaches and treatment.

Fear signaling in the prelimbic-amygdala circuit: a computational modeling and recording study.

The acquisition and expression of conditioned fear depends on prefrontal-amygdala circuits. Auditory fear conditioning increases the tone responses of lateral amygdala neurons, but the increase is transient, lasting only a few hundred milliseconds after tone onset. It was recently reported that that the prelimbic (PL) prefrontal cortex transforms transient lateral amygdala input into a sustained PL output, which could drive fear responses via projections to the lateral division of basal amygdala (BL). To explore the possible mechanisms involved in this transformation, we developed a large-scale biophysical model of the BL-PL network, consisting of 850 conductance-based Hodgkin-Huxley-type cells, calcium-based learning, and neuromodulator effects. The model predicts that sustained firing in PL can be derived from BL-induced release of dopamine and norepinephrine that is maintained by PL-BL interconnections. These predictions were confirmed with physiological recordings from PL neurons during fear conditioning with the selective ß-blocker propranolol and by inactivation of BL with muscimol. Our model suggests that PL has a higher bandwidth than BL, due to PL's decreased internal inhibition and lower spiking thresholds. It also suggests that variations in specific microcircuits in the PL-BL interconnection can have a significant impact on the expression of fear, possibly explaining individual variability in fear responses. The human homolog of PL could thus be an effective target for anxiety disorders.

Fear extinction as a model for translational neuroscience: ten years of progress.

The psychology of extinction has been studied for decades. Approximately 10 years ago, however, there began a concerted effort to understand the neural circuits of extinction of fear conditioning, in both animals and humans. Progress during this period has been facilitated by a high degree of coordination between rodent and human researchers examining fear extinction. Here we review the major advances and highlight new approaches to understanding and exploiting fear extinction. Research in fear extinction could serve as a model for translational research in other areas of behavioral neuroscience.

A Novel Insular/Orbital-Prelimbic Circuit That Prevents Persistent Avoidance in a Rodent Model of Compulsive Behavior.

BACKGROUND: A common symptom of obsessive-compulsive disorder is the persistent avoidance of cues incorrectly associated with negative outcomes. This maladaptation becomes increasingly evident as subjects fail to respond to extinction-based treatments such as exposure-with-response prevention therapy. While previous studies have highlighted the role of the insular-orbital cortex in fine-tuning avoidance-based decisions, little is known about the projections from this area that might modulate compulsive-like avoidance. METHODS: Here, we used anatomical tract-tracing, single-unit recording, and optogenetics to characterize the projections from the insular-orbital cortex. To model exposure-with-response prevention and persistent avoidance in rats, we used the platform-mediated avoidance task followed by extinction-with-response prevention training. RESULTS: Using tract-tracing and unit recording, we found that projections from the agranular insular/lateral orbital (AI/LO) cortex to the prefrontal cortex predominantly target the rostral portion of the prelimbic (rPL) cortex and excite rPL neurons. Photoinhibiting this projection induced persistent avoidance after extinction-with-response prevention training, an effect that was still present 1 week later. Consistent with this, photoexcitation of this projection prevented persistent avoidance in overtrained rats. This projection to rPL appears to be key for AI/LO's effects, considering that there was no effect of photoinhibiting AI/LO projections to the ventral striatum or basolateral amygdala. CONCLUSIONS: Our findings suggest that projections from the AI/LO to the rPL decreases the likelihood of avoidance behavior following extinction. In humans, this connectivity may share some homology of projections from lateral prefrontal cortices (i.e., ventrolateral prefrontal cortex, orbitofrontal cortex, and insula) to other prefrontal areas and the anterior cingulate cortex, suggesting that reduced activity in these pathways may contribute to obsessive-compulsive disorder.

Memory for fear extinction requires mGluR5-mediated activation of infralimbic neurons.

Consolidation of fear extinction involves enhancement of N-methyl D aspartate (NMDA) receptor-dependent bursting in the infralimbic region (IL) of the medial prefrontal cortex (mPFC). Previous studies have shown that systemic blockade of metabotropic glutamate receptor type 5 (mGluR5) reduces bursting in the mPFC and mGluR5 agonists enhance NMDA receptor currents in vitro, suggesting that mGluR5 activation in IL may contribute to fear extinction. In the current study, rats injected with the mGluR5 antagonist 2-methyl-6-(phenylethyl)-pyridine (MPEP) systemically, or intra-IL, prior to extinction exhibited normal within-session extinction, but were impaired in their ability to recall extinction the following day. To directly determine whether mGluR5 stimulation enhances the burst firing of IL neurons, we used patch-clamp electrophysiology in prefrontal slices. The mGluR5 agonist, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), increased intrinsic bursting in IL neurons. Increased bursting was correlated with a reduction in the slow after hyperpolarizing potential and was prevented by coapplication of MPEP. CHPG did not increase NMDA currents, suggesting that an NMDA receptor-independent enhancement of IL bursting via stimulation of mGluR5 receptors contributes to fear extinction. Therefore, the mGluR5 receptor could be a suitable target for pharmacological adjuncts to extinction-based therapies for anxiety disorders.

Consolidation of fear extinction requires NMDA receptor-dependent bursting in the ventromedial prefrontal cortex.

Extinction of conditioned fear is an active learning process requiring N-methyl-D-aspartate receptors (NMDARs), but the timing, location, and neural mechanisms of NMDAR-mediated processing in extinction are a matter of debate. Here we show that infusion of the NMDAR antagonist CPP into the ventromedial prefrontal cortex (vmPFC) prior to, or immediately after, extinction training impaired 24 hr recall of extinction. These findings indicate that consolidation of extinction requires posttraining activation of NMDARs within the vmPFC. Using multichannel unit recording, we observed that CPP selectively reduced burst firing in vmPFC neurons, suggesting that bursting in vmPFC is necessary for consolidation of extinction. In support of this, we found that the degree of bursting in infralimbic vmPFC neurons shortly after extinction predicted subsequent recall of extinction. We suggest that NMDAR-dependent bursting in the infralimbic vmPFC initiates calcium-dependent molecular cascades that stabilize extinction memory, thereby allowing for successful recall of extinction.

Erasing fear memories with extinction training.

Decades of behavioral studies have confirmed that extinction does not erase classically conditioned fear memories. For this reason, research efforts have focused on the mechanisms underlying the development of extinction-induced inhibition within fear circuits. However, recent studies in rodents have uncovered mechanisms that stabilize and destabilize fear memories, opening the possibility that extinction might be used to erase fear memories. This symposium focuses on several of these new developments, which involve the timing of extinction training. Extinction-induced erasure of fear occurs in very young rats, but is lost with the development of perineuronal nets in the amygdala that render fear memories impervious to extinction. Moreover, extinction administered during the reconsolidation phase, when fear memory is destabilized, updates the fear association as safe, thereby preventing the return of fear, in both rats and humans. The use of modified extinction protocols to eliminate fear memories complements existing pharmacological strategies for strengthening extinction.

Time-Dependent Recruitment of Prelimbic Prefrontal Circuits for Retrieval of Fear Memory.

The long-lasting nature of fear memories is essential for survival, but the neural circuitry for retrieval of these associations changes with the passage of time. We previously reported a time-dependent shift from prefrontal-amygdalar circuits to prefrontal-thalamic circuits for the retrieval of auditory fear conditioning. However, little is known about the time-dependent changes in the originating site, the prefrontal cortex. Here we monitored the responses of prelimbic (PL) prefrontal neurons to conditioned tones at early (2 h) vs. late (4 days) timepoints following training. Using c-Fos, we find that PL neurons projecting to the amygdala are activated early after learning, but not later, whereas PL neurons projecting to the paraventricular thalamus (PVT) show the opposite pattern. Using unit recording, we find that PL neurons in layer V (the origin of projections to amygdala) showed cue-induced excitation at earlier but not later timepoints, whereas PL neurons in Layer VI (the origin of projections to PVT) showed cue-induced inhibition at later, but not earlier, timepoints, along with an increase in spontaneous firing rate. Thus, soon after conditioning, there are conditioned excitatory responses in PL layer V which influence the amygdala. With the passage of time, however, retrieval of fear memories shifts to inhibitory responses in PL layer VI which influence the midline thalamus.

Characterizing Different Strategies for Resolving Approach-Avoidance Conflict.

The ability of animals to maximize benefits and minimize costs during approach-avoidance conflicts is an important evolutionary tool, but little is known about the emergence of specific strategies for conflict resolution. Accordingly, we developed a simple approach-avoidance conflict task in rats that pits the motivation to press a lever for sucrose against the motivation to step onto a distant platform to avoid a footshock delivered at the end of a 30 s tone (sucrose is available only during the tone). Rats received conflict training for 16 days to give them a chance to optimize their strategy by learning to properly time the expression of both behaviors across the tone. Rats unexpectedly separated into three distinct subgroups: those pressing early in the tone and avoiding later (Timers, 49%); those avoiding throughout the tone (Avoidance-preferring, 32%); and those pressing throughout the tone (Approach-preferring, 19%). The immediate early gene cFos revealed that Timers showed increased activity in the ventral striatum and midline thalamus relative to the other two subgroups, Avoidance-preferring rats showed increased activity in the amygdala, and Approach-preferring rats showed decreased activity in the prefrontal cortex. This pattern is consistent with low fear and high behavioral flexibility in Timers, suggesting the potential of this task to reveal the neural mechanisms of conflict resolution.

Building a Diverse Workforce and Thinkforce to Reduce Health Disparities.

The Research Centers in Minority Institutions (RCMI) Program was congressionally mandated in 1985 to build research capacity at institutions that currently and historically recruit, train, and award doctorate degrees in the health professions and health-related sciences, primarily to individuals from underrepresented and minority populations. RCMI grantees share similar infrastructure needs and institutional goals. Of particular importance is the professional development of multidisciplinary teams of academic and community scholars (the "workforce") and the harnessing of the heterogeneity of thought (the "thinkforce") to reduce health disparities. The purpose of this report is to summarize the presentations and discussion at the RCMI Investigator Development Core (IDC) Workshop, held in conjunction with the RCMI Program National Conference in Bethesda, Maryland, in December 2019. The RCMI IDC Directors provided information about their professional development activities and Pilot Projects Programs and discussed barriers identified by new and early-stage investigators that limit effective career development, as well as potential solutions to overcome such obstacles. This report also proposes potential alignments of professional development activities, targeted goals and common metrics to track productivity and success.

Sustained conditioned responses in prelimbic prefrontal neurons are correlated with fear expression and extinction failure.

During auditory fear conditioning, it is well established that lateral amygdala (LA) neurons potentiate their response to the tone conditioned stimulus, and that this potentiation is required for conditioned fear behavior. Conditioned tone responses in LA, however, last only a few hundred milliseconds and cannot be responsible for sustained fear responses to a tone lasting tens of seconds. Recent evidence from inactivation and stimulation studies suggests that the prelimbic (PL) prefrontal cortex is necessary for expression of learned fears, but the timing of PL tone responses and correlations with fear behavior have not been studied. Using multichannel unit recording techniques in behaving rats, we observed sustained conditioned tone responses in PL that were correlated with freezing behavior on a second-to-second basis during the presentation of a 30 s tone. PL tone responses were also correlated with conditioned freezing across different experimental phases (habituation, conditioning, extinction). Moreover, the persistence of PL responses after extinction training was associated with failure to express extinction memory. Together with previous inactivation findings, the present results suggest that PL transforms transient amygdala inputs to a sustained output that drives conditioned fear responses and gates the expression of extinction. Given the relatively long latency of conditioned responses we observed in PL (approximately 100 ms after tone onset), we propose that PL integrates inputs from the amygdala, hippocampus, and other cortical sources to regulate the expression of fear memories.