Medial anterior prefrontal cortex stimulation downregulates implicit reactions to threats and prevents the return of fear.

Downregulating emotional overreactions toward threats is fundamental for developing treatments for anxiety and post-traumatic disorders. The prefrontal cortex (PFC) is critical for top-down modulatory processes, and despite previous studies adopting repetitive transcranial magnetic stimulation (rTMS) over this region provided encouraging results in enhancing extinction, no studies have hitherto explored the effects of stimulating the medial anterior PFC (aPFC, encompassing the Brodmann area 10) on threat memory and generalization. Here we showed that rTMS over the aPFC applied before threat memory retrieval immediately decreases implicit reactions to learned and novel stimuli in humans. These effects enduringly persisted 1 week later in the absence of rTMS. No effects were detected on explicit recognition. Critically, rTMS over the aPFC resulted in a more pronounced reduction of defensive responses compared to rTMS targeting the dorsolateral PFC. These findings reveal a previously unexplored prefrontal region, the modulation of which can efficiently and durably inhibit implicit reactions to learned threats. This represents a significant advancement toward the long-term deactivation of exaggerated responses to threats.

Hippocampus-to-amygdala pathway drives the separation of remote memories of related events.

The mammalian brain can store and retrieve memories of related events as distinct memories and remember common features of those experiences. How it computes this function remains elusive. Here, we show in rats that recent memories of two closely timed auditory fear events share overlapping neuronal ensembles in the basolateral amygdala (BLA) and are functionally linked. However, remote memories have reduced neuronal overlap and are functionally independent. The activity of parvalbumin (PV)-expressing neurons in the BLA plays a crucial role in forming separate remote memories. Chemogenetic blockade of PV preserves individual remote memories but prevents their segregation, resulting in reciprocal associations. The hippocampus drives this process through specific excitatory connections with BLA GABAergic interneurons. These findings provide insights into the neuronal mechanisms that minimize the overlap between distinct remote memories and enable the retrieval of related memories separately.

Stabilizing Immature Dendritic Spines in the Auditory Cortex: A Key Mechanism for mTORC1-Mediated Enhancement of Long-Term Fear Memories.

Mammalian target of rapamycin (mTOR) pathway has emerged as a key molecular mechanism underlying memory processes. Although mTOR inhibition is known to block memory processes, it remains elusive whether and how an enhancement of mTOR signaling may improve memory processes. Here we found in male mice that the administration of VO-OHpic, an inhibitor of the phosphatase and tensin homolog (PTEN) that negatively modulates AKT-mTOR pathway, enhanced auditory fear memory for days and weeks, while it left short-term memory unchanged. Memory enhancement was associated with a long-lasting increase in immature-type dendritic spines of pyramidal neurons into the auditory cortex. The persistence of spine remodeling over time arose by the interplay between PTEN inhibition and memory processes, as VO-OHpic induced only a transient immature spine growth in the somatosensory cortex, a region not involved in long-term auditory memory. Both the potentiation of fear memories and increase in immature spines were hampered by rapamycin, a selective inhibitor of mTORC1. These data revealed that memory can be potentiated over time by the administration of a selective PTEN inhibitor. In addition to disclosing new information on the cellular mechanisms underlying long-term memory maintenance, our study provides new insights on the molecular processes that aid enhancing memories over time.SIGNIFICANCE STATEMENT The neuronal mechanisms that may help improve the maintenance of long-term memories are still elusive. The inhibition of mammalian-target of rapamycin (mTOR) signaling shows that this pathway plays a crucial role in synaptic plasticity and memory formation. However, whether its activation may strengthen long-term memory storage is unclear. We assessed the consequences of positive modulation of AKT-mTOR pathway obtained by VO-OHpic administration, a phosphatase and tensin homolog inhibitor, on memory retention and underlying synaptic modifications. We found that mTOR activation greatly enhanced memory maintenance for weeks by producing a long-lasting increase of immature-type dendritic spines in pyramidal neurons of the auditory cortex. These results offer new insights on the cellular and molecular mechanisms that can aid enhancing memories over time.

Comparative Study of Different H2S Donors as Vasodilators and Attenuators of Superoxide-Induced Endothelial Damage.

In the last years, research proofs have confirmed that hydrogen sulfide (H2S) plays an important role in various physio-pathological processes, such as oxidation, inflammation, neurophysiology, and cardiovascular protection; in particular, the protective effects of H2S in cardiovascular diseases were demonstrated. The interest in H2S-donating molecules as tools for biological and pharmacological studies has grown, together with the understanding of H2S importance. Here we performed a comparative study of a series of H2S donor molecules with different chemical scaffolds and H2S release mechanisms. The compounds were tested in human serum for their stability and ability to generate H2S. Their vasorelaxant properties were studied on rat aorta strips, and the capacity of the selected compounds to protect NO-dependent endothelium reactivity in an acute oxidative stress model was tested. H2S donors showed different H2S-releasing kinetic and produced amounts and vasodilating profiles; in particular, compound 6 was able to attenuate the dysfunction of relaxation induced by pyrogallol exposure, showing endothelial protective effects. These results may represent a useful basis for the rational development of promising H2S-releasing agents also conjugated with other pharmacophores.

Prior fear learning enables the rapid assimilation of new fear memories directly into cortical networks.

Long-term memory formation involves the reorganization of brain circuits, termed system consolidation. Whether and how a prior fear experience influences system consolidation of new memories is poorly understood. In rats, we found that prior auditory fear learning allows the secondary auditory cortex to immediately encode new auditory memories, with these new memories purely requiring the activation of cellular mechanisms of synaptic consolidation within secondary auditory cortex. Similar results were obtained in the anterior cingulate cortex for contextual fear memories. Moreover, prior learning enabled connections from these cortices to the basolateral amygdala (BLA) to support recent memory retention. We propose that the reorganization of circuits that characterizes system consolidation occurs only in the first instance that an event is learned, subsequently allowing the immediate assimilation of new analogous events in final storage sites.

Expression of IGF-2 Receptor in the Auditory Cortex Improves the Precision of Recent Fear Memories and Maintains Detailed Remote Fear Memories Over Time.

Traumatic memories may become less precise over time and lead to the development of fear responses to novel stimuli, a process referred to as time-dependent fear generalization. The conditions that cause the growth of fear generalization over time are poorly understood. Here, we found that, in male rats, the level of discrimination at the early time point contributes to determining whether fear generalization will develop with the passage of time or not, suggesting a link between the precision of recent memory and the stability of remote engrams. We also found that the expression of insulin-like growth factor 2 receptor in layer 2/3 of the auditory cortex is linked to the precision of recent memories and to the stability of remote engrams and the development of fear generalization over time. These findings provide new insights on the neural mechanisms that underlie the time-dependent development of fear generalization that may occur over time after a traumatic event.

Implicit and explicit systems differently predict possible dangers.

One strategy to address new potential dangers is to generate defensive responses to stimuli that remind learned threats, a phenomenon called fear generalization. During a threatening experience, the brain encodes implicit and explicit memory traces. Nevertheless, there is a lack of studies comparing implicit and explicit response patterns to novel stimuli. Here, by adopting a discriminative threat conditioning paradigm and a two-alternative forced-choice recognition task, we found that the implicit reactions were selectively elicited by the learned threat and not by a novel similar but perceptually discriminable stimulus. Conversely, subjects explicitly misidentified the same novel stimulus as the learned threat. This generalization response was not due to stress-related interference with learning, but related to the embedded threatening value. Therefore, we suggest a dissociation between implicit and explicit threat recognition profiles and propose that the generalization of explicit responses stems from a flexible cognitive mechanism dedicated to the prediction of danger.

The auditory cortex and the emotional valence of sounds.

How and where sensory stimuli, such as tones or lights, are linked to valence is an important unresolved question in the field of neuroscience. The auditory cortex is essential to analyse the identity and the behavioural importance of tones paired with emotional events. On the contrary, whether the auditory cortex may also encode information on the emotional-motivational valence of sounds is much more controversial. Here, we reviewed recent studies showing that the activity of cortical neurons reflects information about the content of emotional stimuli paired with tones. Critically, the blockade of these neuronal processes prevents animals from recognising sounds as aversive or pleasant. Based on these findings, we proposed a conceptual model in which the auditory cortex may incorporate ascending information from subcortical nuclei about the valence of sounds in sound representations and may consequently drive the activity of subcortical structures towards emotionally laden tones. This hypothesis may also have important implications in the characterisation of neural circuits engaged by maladaptive affective disorders, such as phobias.

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.

Sox2 conditional mutation in mouse causes ataxic symptoms, cerebellar vermis hypoplasia, and postnatal defects of Bergmann glia.

Sox2 is a transcription factor active in the nervous system, within different cell types, ranging from radial glia neural stem cells to a few specific types of differentiated glia and neurons. Mutations in the human SOX2 transcription factor gene cause various central nervous system (CNS) abnormalities, involving hippocampus and eye defects, as well as ataxia. Conditional Sox2 mutation in mouse, with different Cre transgenes, previously recapitulated different essential features of the disease, such as hippocampus and eye defects. In the cerebellum, Sox2 is active from early embryogenesis in the neural progenitors of the cerebellar primordium; Sox2 expression is maintained, postnatally, within Bergmann glia (BG), a differentiated cell type essential for Purkinje neurons functionality and correct motor control. By performing Sox2 Cre-mediated ablation in the developing and postnatal mouse cerebellum, we reproduced ataxia features. Embryonic Sox2 deletion (with Wnt1Cre) leads to reduction of the cerebellar vermis, known to be commonly related to ataxia, preceded by deregulation of Otx2 and Gbx2, critical regulators of vermis development. Postnatally, BG is progressively disorganized, mislocalized, and reduced in mutants. Sox2 postnatal deletion, specifically induced in glia (with GLAST-CreERT2), reproduces the BG defect, and causes (milder) ataxic features. Our results define a role for Sox2 in cerebellar function and development, and identify a functional requirement for Sox2 within postnatal BG, of potential relevance for ataxia in mouse mutants, and in human patients.

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.

Acute administration of nicotine into the higher order auditory Te2 cortex specifically decreases the fear-related charge of remote emotional memories.

Nicotine elicits several behavioural effects on mood as well as on stress and anxiety processes. Recently, it was found that the higher order components of the sensory cortex, such as the secondary auditory cortex Te2, are essential for the long-term storage of remote fear memories. Therefore, in the present study, we examined the effects of acute nicotine injection into the higher order auditory cortex Te2, on the remote emotional memories of either threat or incentive experiences in rats. We found that intra-Te2 nicotine injection decreased the fear-evoked responses to a tone previously paired with footshock. This effect was cue- and dose-specific and was not due to any interference with auditory stimuli processing, innate anxiety and fear processes, or with motor responses. Nicotine acts acutely in the presence of threat stimuli but it did not determine the permanent degradation of the fear-memory trace, since memories tested one week after nicotine injection were unaffected. Remarkably, nicotine did not affect the memory of a similar tone that was paired to incentive stimuli. We conclude from our results that nicotine, when acting acutely in the auditory cortex, relieves the fear charge embedded by learned stimuli.