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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.
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Miedo , Corteza Prefrontal , Estimulación Magnética Transcraneal , Humanos , Miedo/fisiología , Corteza Prefrontal/fisiología , Masculino , Adulto Joven , Femenino , Adulto , Extinción Psicológica/fisiologíaRESUMEN
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.
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Amígdala del Cerebelo , Hipocampo , Parvalbúminas , Animales , Hipocampo/fisiología , Hipocampo/metabolismo , Ratas , Masculino , Amígdala del Cerebelo/fisiología , Parvalbúminas/metabolismo , Complejo Nuclear Basolateral/fisiología , Complejo Nuclear Basolateral/metabolismo , Interneuronas/fisiología , Interneuronas/metabolismo , Memoria/fisiología , Miedo/fisiología , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/fisiología , Neuronas/fisiología , Neuronas/metabolismo , Vías Nerviosas/fisiologíaRESUMEN
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.
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Corteza Auditiva , Proteínas Proto-Oncogénicas c-akt , Masculino , Ratones , Animales , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Corteza Auditiva/metabolismo , Espinas Dendríticas/metabolismo , Tensinas/metabolismo , Memoria a Largo Plazo/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Memoria a Corto Plazo/fisiología , Sirolimus/farmacología , Miedo/fisiología , Monoéster Fosfórico Hidrolasas/metabolismo , MamíferosRESUMEN
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.
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Corteza Auditiva , Complejo Nuclear Basolateral , Animales , Corteza Auditiva/fisiología , Complejo Nuclear Basolateral/fisiología , Miedo/fisiología , Aprendizaje/fisiología , Memoria/fisiología , RatasRESUMEN
Past aversive experiences shape our ability to deal with future dangers, through the encoding of implicit and explicit memory traces and through the ability to generalize defensive reactions to new stimuli resembling learned threats. Numerous evidence demonstrate that sleep is important for the consolidation of memories related to threatening events. However, there is a lack of studies examining the effects of sleep deprivation on the retrieval of consolidated threat memories, and previous studies on the role of sleep in threat generalization have produced mixed results. To address these issues, here we adopted a differential threat conditioning and a delayed (second half of the night) sleep deprivation during the first or the seventh night after learning. We found no effects of sleep deprivation on either implicit or explicit threat memories, regardless of its occurrence timing. Conversely, implicit but not explicit responses to novel cues similar to a learned threat displayed a widened generalization pattern, but only if sleep deprivation took place during the first night after conditioning and not if it occurred during the seventh night after conditioning. Therefore, we propose that sleeping after exposure to danger may support optimal implicit discrimination processes to evaluate new signals in the future and that even a brief period of sleeplessness may widen threat generalization to new stimuli, which is a hallmark of several threat-related disorders.
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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.
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Corteza Auditiva , Animales , Corteza Auditiva/fisiología , Miedo/fisiología , Masculino , Memoria/fisiología , Memoria a Largo Plazo , Ratas , Receptor IGF Tipo 2RESUMEN
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.
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Condicionamiento Clásico/fisiología , Miedo/fisiología , Memoria/fisiología , Encéfalo/fisiología , Condicionamiento Operante/fisiología , Miedo/psicología , Femenino , Respuesta Galvánica de la Piel/fisiología , Generalización Psicológica/fisiología , Humanos , Aprendizaje/fisiología , Masculino , Reconocimiento en Psicología/fisiología , Adulto JovenRESUMEN
Clonal development (clonogenicity) is an inherent property of a subset of postnatal bone marrow (BM) adherent stromal mesenchymal stem cells (MSCs) from which a multipotent progeny develops in culture. Our data suggest that clonogenicity and BM-MSC expansion are two distinct biological events. This hypothesis is based on the following observations: (1) the beginning of clonal growth is a property strictly dependent on serum and independent of the social context, (2) the expansion of individual clone is influenced by events deriving from a social context during initial growth, (3) clonogenic cells grown in a social context in presence of serum can emancipate themselves to generate a secondary different progeny, and (4) the ability of socially generated clones to develop an inherent potential for further growth suggests that quorum sensing may operate in BM-MSC cultures and determine the potential growth of clonal strains.
RESUMEN
: Subendothelial cells (pericytes) are the clonogenic, multipotent and self-renewing skeletal stem cells (SSCs) found in bone marrow (BM) stroma. They express genes maintaining hematopoietic stem cell (HMC) niche identity and, transplanted in immunocompromised mice, organize the hematopoietic microenvironment (HME) generating humanized bone/BM ossicles. To create a mouse model of hematogenous metastasis of human prostate cancer (PC) cells to human bone/BM, we injected PC cells in the blood circulatory system of Severe Combined Immunodeficiency (SCID)/beige mice bearing heterotopic ossicles. Results indicate that PC cells could efficiently home to mice-implanted extraskeletal BM ossicles, but were not able to colonize mice skeletal segments. In humanized bone/BM ossicles, early foci of PC cells occupied a perisinusoidal position, in close contact with perivascular stromal cells. These findings demonstrate the importance of the SSC compartment in recreating a suitable environment to metastatic PC cells. Our data support the hypothesis that BM SSCs committed to a pericyte fate can specify for homing niches of PC cells, suggesting an involvement of specific interactions with subendothelial stromal cells in extravasation of circulating metastatic PC cells to BM.
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Artificial receptors designed for adoptive immune therapies need to absolve dual functions: antigen recognition and abilities to trigger the lytic machinery of reprogrammed effector T lymphocytes. In this way, CAR-T cells deliver their cytotoxic hit to cancer cells expressing targeted tumor antigens, bypassing the limitation of HLA-restricted antigen recognition. Expanding technologies have proposed a wide repertoire of soluble and cellular "immunological weapons" to kill tumor cells; they include monoclonal antibodies recognizing tumor associated antigens on tumor cells and immune cell checkpoint inhibition receptors expressed on tumor specific T cells. Moreover, a wide range of formidable chimeric antigen receptors diversely conceived to sustain quality, strength and duration of signals delivered by engineered T cells have been designed to specifically target tumor cells while minimize off-target toxicities. The latter immunological weapons have shown distinct efficacy and outstanding palmarès in curing leukemia, but limited and durable effects for solid tumors. General experience with checkpoint inhibitors and CAR-T cell immunotherapy has identified a series of variables, weaknesses and strengths, influencing the clinical outcome of the oncologic illness. These aspects will be shortly outlined with the intent of identifying the still "missing strategy" to combat epithelial cancers.
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Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Receptores Quiméricos de Antígenos/uso terapéutico , Animales , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/inmunología , Antineoplásicos Inmunológicos/uso terapéutico , Humanos , Inmunidad Celular , Inmunidad Humoral , Neoplasias/inmunología , Neoplasias/patología , Medicina de Precisión/métodos , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Escape del TumorRESUMEN
[This corrects the article DOI: 10.3389/fphys.2014.00203.].
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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.
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Corteza Auditiva/fisiología , Percepción Auditiva/fisiología , Emociones/fisiología , Miedo/fisiología , Motivación/fisiología , Estimulación Acústica , Animales , HumanosRESUMEN
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.
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Corteza Auditiva/fisiología , Percepción Auditiva/fisiología , Discriminación en Psicología/fisiología , Miedo/fisiología , Ritmo Gamma , Corteza Prefrontal/fisiología , Estimulación Acústica , Animales , Complejo Nuclear Basolateral/fisiología , Condicionamiento Clásico , Sincronización Cortical , Generalización Psicológica , Masculino , Vías Nerviosas/fisiología , Neuronas/fisiología , Optogenética , Ratas WistarRESUMEN
Bone, cartilage, and marrow adipocytes are generated by skeletal progenitors, but the relationships between lineages and mechanisms controlling their differentiation are poorly understood. We established mouse clonal skeletal progenitors with distinct differentiation properties and analyzed their transcriptome. Unipotent osteogenic and adipogenic cells expressed specific transcriptional programs, whereas bipotent clones combined expression of those genes and did not show a unique signature. We tested potential regulators of lineage commitment and found that in the presence of interferon-γ (IFNγ) adipogenic clones can be induced to osteogenesis and that their adipogenic capacity is inhibited. Analysis of IFNγ-regulated genes showed that lineage signatures and fate commitment of skeletal progenitors were controlled by EGR1 and EGR2. Knockdown experiments revealed that EGR1 is a positive regulator of the adipogenic transcriptional program and differentiation capacity, whereas EGR2 inhibits the osteogenic program and potency. Therefore, our work revealed transcriptional signatures of osteogenic and adipogenic lineages and mechanism triggering cell fate.
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Adipogénesis/genética , Diferenciación Celular/genética , Evolución Clonal/genética , Osteogénesis/genética , Células Madre/citología , Células Madre/metabolismo , Transcripción Genética , Animales , Biomarcadores , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Perfilación de la Expresión Génica , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Reproducibilidad de los Resultados , Transducción de Señal , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
In the presence of new stimuli, it is crucial for survival to react with defensive responses in the presence of stimuli that resemble threats but also to not react with defensive behavior in response to new harmless stimuli. Here, we show that in the presence of new uncertain stimuli with sensory features that produce an ambiguous interpretation, discriminative processes engage a subset of excitatory and inhibitory neurons within the lateral amygdala (LA) that are partially different from those engaged by fear processes. Inducing the pharmacogenetic deletion of this neuronal ensemble caused fear generalization but left anxiety-like response, fear memory and extinction processes intact. These data reveal that two opposite neuronal processes account for fear discrimination and generalization within the LA and suggest a potential pathophysiological mechanism for the impaired discrimination that characterizes fear-related disorders.
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Amígdala del Cerebelo/fisiología , Conducta Animal , Miedo , Neuronas/fisiología , Animales , Animales Modificados Genéticamente , Ansiedad , Complejo Nuclear Basolateral/fisiología , Condicionamiento Psicológico , Extinción Psicológica , Hibridación Fluorescente in Situ , Masculino , Aprendizaje por Laberinto , Memoria , Modelos Neurológicos , Ratas , Ratas Sprague-DawleyRESUMEN
Adoptive cell therapy of solid tumors with reprogrammed T cells can be considered the "next generation" of cancer hallmarks. CAR-T cells fail to be as effective as in liquid tumors for the inability to reach and survive in the microenvironment surrounding the neoplastic foci. The intricate net of cross-interactions occurring between tumor components, stromal and immune cells leads to an ineffective anergic status favoring the evasion from the host's defenses. Our goal is hereby to trace the road imposed by solid tumors to CAR-T cells, highlighting pitfalls and strategies to be developed and refined to possibly overcome these hurdles.
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Terapia Genética/métodos , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Receptores Quiméricos de Antígenos/inmunología , Animales , Edición Génica , Técnicas de Transferencia de Gen , Humanos , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Receptores Quiméricos de Antígenos/genética , Microambiente TumoralRESUMEN
Memories of fearful events can be maintained throughout the lifetime of animals. Here we showed that lesions of the lateral nucleus (LA) performed shortly after training impaired the retention of long-term memories, assessed by the concomitant measurement of two dissociable defensive responses, freezing and avoidance in rats. Strikingly, when LA lesions were performed four weeks after training, rats did not show freezing to a learned threat stimulus, but they were able to direct their responses away from it. Similar results were found when the central nucleus (CeA) was lesioned four weeks after training, whereas lesions of the basal nucleus (BA) suppressed avoidance without affecting freezing. LA and BA receive parallel inputs from the auditory cortex, and optogenetic inhibition of these terminals hampered both freezing and avoidance. We therefore propose that, at variance with the traditional serial flow of information model, long-term fearful memories recruit two parallel circuits in the amygdala, one relying on the LA-to-CeA pathway and the other relying solely on BA, which operate independently and mediate distinct defensive responses.
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Complejo Nuclear Basolateral/patología , Núcleo Amigdalino Central/patología , Miedo/fisiología , Memoria/fisiología , Estimulación Acústica , Animales , Reacción de Prevención/fisiología , Complejo Nuclear Basolateral/metabolismo , Conducta Animal , Núcleo Amigdalino Central/metabolismo , Halorrodopsinas/genética , Halorrodopsinas/metabolismo , Masculino , Microscopía Confocal , Ratas , Ratas WistarRESUMEN
MiR-204 and 211 enforced expression in murine mesenchymal stromal cells (MSCs) has been shown to induce adipogenesis and impair osteogenesis, through RUNX2 down-modulation. This mechanism has been suggested to play a role in osteoporosis associated with obesity. However, two further fundamental MSC functions, chondrogenesis and hematopoietic supporting activity, have not yet been explored. To this end, we transduced, by a lenti-viral vector, miR-204 and 211 in a model primary human MSC line, opportunely chosen among our MSC collection for displaying all properties of canonical bone marrow MSCs, except adipogenesis. Enforced expression of miR-204&211 in these cells, rescued adipogenesis, and inhibited osteogenesis, as previously reported in murine MSCs, but, surprisingly, also damaged cartilage formation and hematopoietic supporting activity, which were never explored before. RUNX2 has been previously indicated as the target of miR-204&211, whose down modulation is responsible for the switch from osteogenesis to adipogenesis. However, the additional disruption of chondrogenesis and hematopoietic supporting activity, which we report here, might depend on diverse miR-204&211 targets. To investigate this hypothesis, permanent RUNX2 knock-down was performed. Sh-RUNX2 fully reproduced the phenotypes induced by miR-204&211, confirming that RUNX2 down modulation is the major event leading to the reported functional modification on our MSCs. It seems thus apparent that RUNX2, a recognized master gene for osteogenesis, might rule all four MSC commitment and differentiation processes. Hence, the formerly reported role of miR204&211 and RUNX2 in osteoporosis and obesity, coupled with our novel observation showing inhibition of cartilage differentiation and hematopoietic support, strikingly resemble the clinical traits of metabolic syndrome, where osteoarthritis, osteoporosis, anaemia and obesity occur together. Our observations, corroborating and extending previous observations, suggest that miR-204&211-RUNX2 axis in human MSCs is possibly involved in the pathogenesis of this rapidly growing disease in industrialized countries, for possible therapeutic intervention to regenerate former homeostasis.
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Recent observation identifies subendothelial (mural) cells expressing MCAM, a specific system of clonogenic, self-renewing, osteoprogenitors (a.k.a, "mesenchymal stem cells") in the microvascular compartment of post-natal human bone marrow (BM). In this study, we used MCAM/CD146, as a marker to localize, isolate and assay subendothelial clonogenic cells from the microvasculature of postnatal human skeletal muscle. We show here that these cells share with their BM counterpart, anatomic position (subendothelial/adventitial) and ex vivo clonogenicity (CFU-Fs). When assayed under the stringent conditions, these cells display a high spontaneous myogenic potential (independent of co-culture with myoblasts or of in vivo fusion with local myoblasts), which is otherwise only attained in cultures of satellite cells. These muscle-derived mural cells activated a myogenic program in culture. Cultured CD146+ cells expressed the myogenic factors (Pax7, Pax3 and Myf5), NCAM/CD56, desmin as well as proteins characteristic of more advanced myogenic differentiation, such as myosin heavy chain. In vivo, these cells spontaneously generate myotubes and myofibrils. These data identify the anatomy and phenotype of a novel class of committed myogenic progenitor in human post-natal skeletal muscle of subendothelial cells associated with the abluminal surface of microvascular compartment distinct from satellite cells.