Early developmental emergence of human amygdala–prefrontal connectivity after maternal deprivation

Under typical conditions, medial prefrontal cortex (mPFC) connections with the amygdala are immature during childhood and become adult-like during adolescence. Rodent models show that maternal deprivation accelerates this development, prompting examination of human amygdala–mPFC phenotypes following maternal deprivation. Previously institutionalized youths, who experienced early maternal deprivation, exhibited atypical amygdala–mPFC connectivity. Specifically, unlike the immature connectivity (positive amygdala–mPFC coupling) of comparison children, children with a history of early adversity evidenced mature connectivity (negative amygdala–mPFC coupling) and thus, resembled the adolescent phenotype. This connectivity pattern was mediated by the hormone cortisol, suggesting that stress-induced modifications of the hypothalamic–pituitary–adrenal axis shape amygdala–mPFC circuitry. Despite being age-atypical, negative amygdala–mPFC coupling conferred some degree of reduced anxiety, although anxiety was still significantly higher in the previously institutionalized group. These findings suggest that accelerated amygdala–mPFC development is an ontogenetic adaptation in response to early adversity.Even brief exposure to stressful experiences early in life can have life-long impact on brain development and socioemotional functioning. Adverse or deprived caregiving is an example of a highly potent early life stressor in altricial species. Animal studies of maternal deprivation have demonstrated long-term effects on socioemotional and brain development (1–5), with particular influences on frontoamygdala circuitry. The timing of stress exposure and cellular properties of this circuitry may render it particularly vulnerable to early adversity (6). Consistently, rodent and nonhuman primate models show that the amygdala is highly susceptible to early environmental adversity due to its early structural development and readiness to respond to stressors (7–9). Human studies of early adverse caregiving have demonstrated structural volume abnormalities in the amygdala that were associated with increased trait anxiety and emotion dysregulation (10, 11) and increased amygdala reactivity to emotional stimuli (12).Abnormally rapid brain development following early adversity may be a response that reprioritizes developmental goals to match the demands of an adverse early environment. Early life caregiving adversity in rodents alters amygdala–medial prefrontal cortex (mPFC) circuits that in adulthood serve to regulate the activity of the amygdala (13, 14), perhaps through accelerating development of the circuitry. For example, maternal deprivation results in the early emergence of adult-like fear learning based in frontoamygdala circuitry (5) and earlier emergence of amygdala function (8, 15) and structural maturation (16). Maternal separation has also been associated with increased development of neurons in mPFC (17). Such changes can lead to adult-like fear extinction learning and mPFC-mediated down-regulation of the amygdala, even though as a group, these stressed animals can be more fearful and anxious (18). Maternal absence acts to accelerate amygdala–prefrontal functional development via premature elevations of glucocorticoids (8, 19), suggesting that maternal absence acts on amygdala-related circuitry through alterations of the hypothalamic–pituitary–adrenal (HPA) axis, consistent with the established close relationship between the amygdala and HPA axis (20). Whether a similar neurohormonal process explains affective development in humans following early maternal absence is currently unknown.Whereas the hypothesis of accelerated development has not been tested in humans, the conservation of frontoamygdala phenotypes across species would predict a common mechanism through which early life stress influences neuroaffective development. Such changes are important to understand given affective behaviors, including emotion regulation and fear learning, that rely on the amygdala and its connections with mPFC (21–27). In typical human development, childhood and adolescence is a period of large change in frontoamygdala phenotypes (28, 29), with amygdala–mPFC connectivity being markedly immature during childhood (28). The transition from childhood into adolescence has been characterized by a developmental shift in amygdala–mPFC functional connectivity. Specifically, adolescents and adults exhibit inverse (negative) correlations between amygdala and mPFC in response to emotional stimuli, a pattern of connectivity that has been characterized as indexing top-down inhibitory connections (24, 27, 28). Children exhibit a positive amygdala–mPFC coupling phenotype (28) that is associated with greater emotional reactivity, which is typically characteristic of young children. This shift from an immature (positive) amygdala–mPFC coupling phenotype to a mature (negative) coupling phenotype parallels age-related attenuation of amygdala signal and mediates maturation of emotional behavior (28). The goal of the current study was to examine childhood amygdala–mPFC phenotypes following early life adversity with the hypothesis that early life adversity would accelerate development of amygdala–mPFC circuitry. We examined the effects of early adversity among previously institutionalized (PI) children who have a history of early life maternal deprivation.     

Single dose of l-dopa makes extinction memories context-independent and prevents the return of fear

Traumatic events can engender persistent excessive fear responses to trauma reminders that may return even after successful treatment. Extinction, the laboratory analog of behavior therapy, does not erase conditioned fear memories but generates competing, fear-inhibitory “extinction memories” that, however, are tied to the context in which extinction occurred. Accordingly, a dominance of fear over extinction memory expression—and, thus, return of fear—is often observed if extinguished fear stimuli are encountered outside the extinction (therapy) context. We show that postextinction administration of the dopamine precursor l-dopa makes extinction memories context-independent, thus strongly reducing the return of fear in both mice and humans. Reduced fear is accompanied by decreased amygdala and enhanced ventromedial prefrontal cortex activation in both species. In humans, ventromedial prefrontal cortex activity is predicted by enhanced resting-state functional coupling of the area with the dopaminergic midbrain during the postextinction consolidation phase. Our data suggest that dopamine-dependent boosting of extinction memory consolidation is a promising avenue to improving anxiety therapy.In extinction, conditioned fear responses (CRs) are diminished by using repeated exposure to the conditioned fear stimulus (CS) in the absence of the aversive unconditioned stimulus (UCS) with which it had previously been paired (1). Most extinction protocols do not, or only partly, delete the original CS–UCS association (or fear memory) but result in the formation of an inhibitory CS–no-UCS association (extinction memory) (2, 3). At later CS exposures (test), the competition between both memory traces is thought to determine the level of conditioned responding. Dissimilarity between the test and the extinction context impairs the retrieval or expression of the extinction memory in favor of the retrieval/expression of the fear memory, which is thought to not require gating or occasion-setting by any particular context (2). In the laboratory, context dissimilarity is achieved by testing in a physically different context than extinction (renewal), by again administering UCSs shortly before testing (reinstatement) or by simply letting sufficient time pass between extinction and testing (spontaneous recovery) (2). The return of fear observed in these situations is considered a laboratory model of relapse after successful extinction-based psychotherapy of conditions such as posttraumatic stress disorder, panic disorder, or social phobia (2, 4, 5).To generate strong and long-lasting memories, new learning must initiate cascades of molecular events that ultimately result in changes in protein expression and/or posttranslational protein modification (6–8). Animal studies have shown that release of the neurotransmitter dopamine is critically important in many such consolidation processes and specifically promotes stable forms of long-term potentiation, a cellular correlate of long-term memory (9–13). In the domain of extinction, administration of dopamine receptor antagonists before or after extinction consistently impairs animals’ capacity to later express extinction, leading to enhanced CRs at test (i.e., return of fear) (14–16). Conversely, extinction expression is improved in animals that receive the combined dopamine and norepinephrine reuptake blocker methylphenidate directly after extinction (17), suggesting that dopamine contributes to extinction memory consolidation. These findings led us to ask whether administration of the dopamine precursor l-dopa directly after extinction (i.e., during memory consolidation) would enhance extinction memory formation and thereby prevent the return of fear.     

Altered fear learning across development in both mouse and human

The only evidence-based behavioral treatment for anxiety and stress-related disorders involves desensitization techniques that rely on principles of extinction learning. However, 40% of patients do not respond to this treatment. Efforts have focused on individual differences in treatment response, but have not examined when, during development, such treatments may be most effective. We examined fear-extinction learning across development in mice and humans. Parallel behavioral studies revealed attenuated extinction learning during adolescence. Probing neural circuitry in mice revealed altered synaptic plasticity of prefrontal cortical regions implicated in suppression of fear responses across development. The results suggest a lack of synaptic plasticity in the prefrontal regions, during adolescence, is associated with blunted regulation of fear extinction. These findings provide insight into optimizing treatment outcomes for when, during development, exposure therapies may be most effective.Fear learning is a highly adaptive, evolutionarily conserved process that allows one to respond appropriately to cues associated with danger. In the case of psychiatric disorders, however, fear may persist long after an environmental threat has passed. This unremitting and often debilitating form of fear is a core component of many anxiety disorders, including posttraumatic stress disorder (PTSD), and involves exaggerated and inappropriate fear responses. Existing treatments, such as exposure therapy, are based on principles of fear extinction, during which cues previously associated with threat are presented in the absence of the initial aversive event until cues are considered safe and fear responses are reduced. Extinction-based exposure therapies have the strongest empirical evidence for benefitting adult patients suffering from PTSD (1), yet a comparative lack of knowledge about the development of fear neural circuitry prohibits similarly successful treatment outcomes in children and adolescents (2). Adolescence, in particular, is a developmental stage when the incidence of anxiety disorders peaks in humans (3–6), and it is estimated that over 75% of adults with fear-related disorders met diagnostic criteria as children and adolescents (7, 8). Because of insufficient or inaccurate diagnoses and a dearth of pediatric and adolescent specialized treatments, fewer than one in five children or adolescents are expected to receive treatment for their anxiety disorders (9), leaving a vast number with inadequate or no treatment (2, 10). The increased frequency of anxiety disorders in pediatric and adolescent populations highlights the importance of understanding neural mechanisms of fear regulation from a developmental perspective, as existing therapies directly rely upon principles of fear-extinction learning. Converging evidence from human and rodent studies suggests that insufficient top-down regulation of subcortical structures (11–14), such as the amygdala, may coincide with diminished prototypical extinction learning (15), as well as ongoing fine-tuning of excitatory–inhibitory balance in the prefrontal cortex that may coincide with diminished prototypical extinction learning (16). Because top-down prefrontal regulation has been postulated to mediate extinction learning and may determine the efficacy of exposure therapy often used as part of cognitive behavioral therapy, it is important to discern how changes in the development of prefrontal circuitry influences fear extinction. Studying the development of fear learning and memory in humans, while examining, in parallel, the underlying neural mechanisms in rodent models, may offer insights into optimizing treatment strategies for developing populations by clarifying when, during development, a particular intervention or treatment may be more or less effective.     

Prefrontal neurons encode context-based response execution and inhibition in reward seeking and extinction

The prefrontal cortex (PFC) guides execution and inhibition of behavior based on contextual demands. In rodents, the dorsal/prelimbic (PL) medial PFC (mPFC) is frequently considered essential for execution of goal-directed behavior (“go”) whereas ventral/infralimbic (IL) mPFC is thought to control behavioral suppression (“stop”). This dichotomy is commonly seen for fear-related behaviors, and for some behaviors related to cocaine seeking. Overall, however, data for reward-directed behaviors are ambiguous, and few recordings of PL/IL activity have been performed to demonstrate single-neuron correlates. We recorded neuronal activity in PL and IL during discriminative stimulus driven sucrose seeking followed by multiple days of extinction of the reward-predicting stimulus. Contrary to a generalized PL-go/IL-stop hypothesis, we found cue-evoked activity in PL and IL during reward seeking and extinction. Upon analyzing this activity based on resultant behavior (lever press or withhold), we found that neurons in both areas encoded contextually appropriate behavioral initiation (during reward seeking) and withholding (during extinction), where context was dictated by response–outcome contingencies. Our results demonstrate that PL and IL signal contextual information for regulation of behavior, irrespective of whether that involves initiation or suppression of behavioral responses, rather than topographically encoding go vs. stop behaviors. The use of context to optimize behavior likely plays an important role in maximizing utility-promoting exertion of activity when behaviors are rewarded and conservation of energy when not.The prefrontal cortex (PFC) is involved in directing behavior and inhibiting inappropriate responses (1–4). Rodent medial PFC (mPFC) is functionally heterogeneous: prelimbic cortex (PL) is thought to be involved in behavioral execution, and infralimbic cortex (IL) in behavioral suppression, particularly during extinction (4). Fear conditioning studies support this dichotomy. PL stimulation elicits, and inactivation impairs, conditioned fear expression, and PL neurons fire during fear-related cues. Conversely, IL stimulation enhances, and inactivation blocks, extinction of fear conditioning, and IL neurons fire for extinguished cues that previously predicted an aversive stimulus (5, 6).A similar dichotomy is proposed for appetitive behaviors, although support for this is not unequivocal (7). PL inactivation during discriminative stimulus (DS)-driven reward-seeking reduces cue-driven behaviors (8). However, PL inactivation also increases nonspecific (8) and premature lever responding (9, 10), and PL is important in inhibiting responses during a stop-signal reaction time task (11). IL inactivation during DS task performance increases responses generally, including those triggered by a nonrewarded stimulus (NS) (8), increases premature responding on five-choice serial reaction time tasks (12), and increases spontaneous recovery and reinstatement (13). Cocaine seeking is also associated with activation of PL, and extinction with activity in IL (4). However, IL is also implicated in driving seeking of cocaine, heroin, and other drugs of abuse, and PL activation inhibits compulsive cocaine seeking (7). Thus, even though the PL/IL go/stop dichotomy has been demonstrated in a number of studies, there are also a significant number of studies calling it into question (7). The goal of the present study was to test whether the PL/IL functional dichotomy seen in fear conditioning was also present in an appetitively motivated task and its extinction. This view predicts that PL neurons would fire during reward seeking, and IL neurons during suppression of seeking, particularly after extinction. Instead, we found that both regions displayed rapid, stimulus-evoked activity changes closely linked to contextually appropriate behavior, largely independent of whether the behavior involved response execution or inhibition.     

Hippocampal molecular mechanisms involved in the enhancement of fear extinction caused by exposure to novelty

Exposure to a novel environment enhances the extinction of contextual fear. This has been explained by tagging of the hippocampal synapses used in extinction, followed by capture of proteins from the synapses that process novelty. The effect is blocked by the inhibition of hippocampal protein synthesis following the novelty or the extinction. Here, we show that it can also be blocked by the postextinction or postnovelty intrahippocampal infusion of the NMDA receptor antagonist 2-amino-5-phosphono pentanoic acid; the inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII), autocamtide-2–related inhibitory peptide; or the blocker of L-voltage–dependent calcium channels (L-VDCCs), nifedipine. Inhibition of proteasomal protein degradation by β-lactacystin has no effect of its own on extinction or on the influence of novelty thereon but blocks the inhibitory effects of all the other substances except that of rapamycin on extinction, suggesting that their action depends on concomitant synaptic protein turnover. Thus, the tagging-and-capture mechanism through which novelty enhances fear extinction involves more molecular processes than hitherto thought: NMDA receptors, L-VDCCs, CaMKII, and synaptic protein turnover.Frey and Morris (1, 2) and their collaborators (3–7) proposed a mechanism whereby relatively “weak” hippocampal long-term potentiation (LTP) or long-term depression (LTD) lasting only a few minutes can nevertheless “tag” the synapses involved with proteins synthesized ad hoc, so that other plasticity-related proteins (PRPs) produced at other sets of synapses by other LTPs or LTDs can be captured by the tagged synapses and strengthen their activity to “long” LTPs or LTDs lasting hours or days (8). LTDs and LTPs can “cross”-tag each other; that is, LTDs can enhance both LTDs and LTPs, and vice versa (6, 8). Because many learned behaviors rely on hippocampal LTP or LTD (7–9), among them the processing of novelty (9, 10) and the making of extinction (11–13), interactions between consecutive learnings can also be explained by the “tagging-and-capture” hypothesis (9, 10, 13), whose application to behavior became known as “behavioral tagging and capture” (5, 7, 9, 13). Typically, exposure to a novel environment [e.g., a nonanxiogenic 50 × 50 × 40-cm open field (OF) (5, 7, 9, 10, 14)] is interpolated before testing for another task, which becomes enhanced (4–10, 13). The usual reaction to novelty is orienting and exploration (14), followed by habituation of this response (14–16). Habituation is perhaps the simplest form of learning, and it consists of inhibition of the orienting/exploratory response (14, 16).We recently showed that the brief exposure of rats to a novel environment (the OF) within a limited time window enhances the extinction of contextual fear conditioning (CFC) through a mechanism of synaptic tagging and capture (13), which is a previously unidentified example of behavioral tagging of inhibitory learning. Fear extinction is most probably due to LTD in the hippocampus (11, 12), although the possibility that it may also involve LTP is not discarded (13). The enhancement of extinction by novelty probably relies on the habituation to the novel environment, which is also probably due to LTD (15, 16). The enhancement of extinction by the exposure to novelty depends on hippocampal gene expression and ribosomal protein synthesis following extinction training and on both ribosomal and nonribosomal protein synthesis caused by the novel experience (13). Nonribosomal protein synthesis that can be blocked by rapamycin is believed to be dendritic (13, 17), so it would be strategically located for tagging-and-capture processes, but it has not been studied in synaptic tagging to date (3–8) or in other forms of behavioral tagging (7–10). As occurs with the interactions between LTPs and/or LTDs (4), the enhancement of extinction by novelty relies on hippocampal but not amygdalar processes (13).Recent findings indicate that several hippocampal processes related to learning and memory, such as the reconsolidation of spatial learning, are highly dependent on NMDA glutamate receptors, calcium/calmodulin protein kinase II (CaMKII), and long-term voltage channel blockers (L-VDCCs), which, in turn, rely on the proteasomal degradation of proteins (18). Here, we study the effects of an NMDA blocker, 2-amino-5-phosphono pentanoic acid (AP5); the L-VDCC blocker nifedipine (Nife); a CaMKII inhibitor, the autocamtide-2–related inhibitory peptide (AIP); and the irreversible proteasome blocker β-lactacystin (12, 13) on the interaction between novelty and extinction (11). As will be seen, we found that both the setting up of tags by extinction and the presumable production of PRPs by the processing of novelty are dependent on NMDA receptors, CaMKII, and L-VDCCs. This endorses and expands the hypothesis that the novelty–extinction interaction relies on synaptic tagging and capture (13).     

From the Cover: Intergenerational transmission of emotional trauma through amygdala-dependent mother-to-infant transfer of specific fear

Emotional trauma is transmitted across generations. For example, children witnessing their parent expressing fear to specific sounds or images begin to express fear to those cues. Within normal range, this is adaptive, although pathological fear, such as occurs in posttraumatic stress disorder or specific phobias, is also socially transmitted to children and is thus of clinical concern. Here, using a rodent model, we report a mother-to-infant transfer of fear to a novel peppermint odor, which is dependent on the mother expressing fear to that smell in pups’ presence. Examination of pups’ neural activity using c-Fos early gene expression and ¹⁴C 2-deoxyglucose autoradiography during mother-to-infant fear transmission revealed lateral and basal amygdala nuclei activity, with a causal role highlighted by pharmacological inactivation of pups’ amygdala preventing the fear transmission. Maternal presence was not needed for fear transmission, because an elevation of pups’ corticosterone induced by the odor of the frightened mother along with a novel peppermint odor was sufficient to produce pups’ subsequent aversion to that odor. Disruption of axonal tracts from the Grueneberg ganglion, a structure implicated in alarm chemosignaling, or blockade of pups’ alarm odor-induced corticosterone increase prevented transfer of fear. These memories are acquired at younger ages compared with amygdala-dependent odor-shock conditioning and are more enduring following minimal conditioning. Our results provide clues to understanding transmission of specific fears across generations and its dependence upon maternal induction of pups’ stress response paired with the cue to induce amygdala-dependent learning plasticity. Results are discussed within the context of caregiver emotional responses and adaptive vs. pathological fears social transmission.Children, including infants, use their parents’ emotions to guide their behavior and learn about safety and danger (1–4). The infant’s ability to regulate behavior in novel situations using the caregiver’s emotional expression is known as social referencing and occurs in humans and nonhuman primates (1). Although parental physical presence itself or particular cues indicating parental presence, such as voice, touch, or smell typically signal safety for the child, infants are especially responsive to the caregiver’s communication during threats (3–5). This social learning is critical for enhancing survival through an adaptation to the environment but also provides transmission of pathological fears, such as occurs in posttraumatic stress disorder (PTSD) or in specific phobias (3–7).Despite existing evidence that children are sensitive to parental fear and anxiety, the neurobiological mechanisms for the transmission of parental specific fear to the offspring have remained elusive (2–7). Animal studies investigating the impact of parental stress on the offspring focused on the history of parental trauma, quality of maternal care, and resultant overall behavioral alterations in the offspring (7, 8). However, to develop efficient survival strategies, progenies must learn about specific environmental threats triggering parental fear (9).Most of what we know about fear learning comes from studies using fear conditioning (FC) (10). In FC, a neutral sensory cue [conditioned stimulus (CS)] is paired with a noxious event [unconditioned stimulus (US)]. Animal studies indicate that the amygdala’s lateral and basal nuclei (LBA) play an important role in FC (10). However, FC in infant rats is naturally attenuated until postnatal day (PND) 10 due to low levels of the stress hormone corticosterone (CORT) during the stress hyporesponsive period (11–15). This fear suppression continues in older pups (until PND 16) in the mother’s presence due to social buffering (attenuation) of the shock-induced CORT increase (15).To study the intergenerational transmission of fear to specific triggers, we developed a mother-to-infant social fear learning paradigm. In social fear learning, an organism learns fear through an exposure to a conspecific expressing fear to a discrete CS. Social fear learning may thus serve as a model explaining how defense responses to specific triggers are transmitted between individuals. Social fear learning has been demonstrated in primates, including humans and in rodents, and involves the amygdala (16–19).     

Extinction learning,which consists of the inhibition of retrieval,can be learned without retrieval

In the present study we test the hypothesis that extinction is not a consequence of retrieval in unreinforced conditioned stimulus (CS) presentation but the mere perception of the CS in the absence of a conditioned response. Animals with cannulae implanted in the CA1 region of hippocampus were subjected to extinction of contextual fear conditioning. Muscimol infused intra-CA1 before an extinction training session of contextual fear conditioning (CFC) blocks retrieval but not consolidation of extinction measured 24 h later. Additionally, this inhibition of retrieval does not affect early persistence of extinction when tested 7 d later or its spontaneous recovery after 2 wk. Furthermore, both anisomycin, an inhibitor of ribosomal protein synthesis, and rapamycin, an inhibitor of extraribosomal protein synthesis, given into the CA1, impair extinction of CFC regardless of whether its retrieval was blocked by muscimol. Therefore, retrieval performance in the first unreinforced session is not necessary for the installation, maintenance, or spontaneous recovery of extinction of CFC.In animal experiments, retrieval can be defined as the behavioral expression of recalled memories; the actual performance of retrieval is usually thought to destabilize memories and trigger two opposite protein synthesis-dependent processes: reconsolidation (1–7) and extinction (8–13). Reconsolidation is viewed as a consequence of the labilization of consolidated memories at the time of the unreinforced retrieval, which renders them open to strengthening and updating (3–7, 14–20), whereas extinction is viewed as a form of learning to inhibit retrieval of original memory (8–12). Pavlov observed more than a century ago (8) that extinguished responses can recover spontaneously with the passage of time, which indicates that extinction does not erase memories. This was corroborated and expanded by Konorski (9) and Rescorla (10, 11).In contextual fear conditioning (CFC), animals learn to associate a context used as a conditioned stimulus (CS) and relatively mild foot shocks used as an unconditioned stimulus (US). The conditioned response (CR), usually measured, is the increase of the time spent freezing in unreinforced sessions carried out later. Most accounts consider that extinction begins in the first unreinforced retrieval session because of labilization of the memory (3, 14, 15), or by the mismatch between what the animals expect and what really happens at the time of retrieval (19), or both. However, it is always possible that, alternatively, the CS itself in the absence of a CR may trigger memory labilization (3) and make it susceptible both to extinction (13) and to its counterpart, reconsolidation (14, 20).Bermudez-Rattoni and his group showed that the performance of unreinforced retrieval is not necessary to trigger reconsolidation of conditioned taste aversion (5, 21) or of object recognition learning (6, 16). The methodology used by this group is very straightforward: it consists of the pharmacological blockade of retrieval with microinfusions of ciano-nitro-quinoxaline-dione, an antagonist of AMPA receptors (16, 21), or of the GABA_A receptor agonist muscimol (Mus) (5) given into the basolateral amygdala (BLA) for conditioned taste aversion or into the perirhinal cortex for object recognition (6). In both tasks, the drugs selectively blocked retrieval but spared reconsolidation, suggesting that the two neural processes are independent from each other (16, 21). In contrast, a glutamate NMDA receptor antagonist blocked the reconsolidation of conditioned taste aversion when given into the BLA (21) and that of object recognition when infused into the perirhinal cortex (16).In the present study we test the hypothesis that extinction is not a consequence of retrieval in unreinforced CS presentations (13, 22) but to the mere perception of the CS in the absence of a CR. This is important because, depending on the answer, the origin of both postretrieval processes (extinction and perhaps reconsolidation) will have to be searched for in sensory or sensory-motor rather than in cognitive or behavioral events, and the unstabilization of the memory being studied may depend on the former.     

Metabotropic glutamate receptor 3 activation is required for long-term depression in medial prefrontal cortex and fear extinction

Clinical studies have revealed that genetic variations in metabotropic glutamate receptor 3 (mGlu₃) affect performance on cognitive tasks dependent upon the prefrontal cortex (PFC) and may be linked to psychiatric conditions such as schizophrenia, bipolar disorder, and addiction. We have performed a series of studies aimed at understanding how mGlu₃ influences PFC function and cognitive behaviors. In the present study, we found that activation of mGlu₃ can induce long-term depression in the mouse medial PFC (mPFC) in vitro. Furthermore, in vivo administration of a selective mGlu₃ negative allosteric modulator impaired learning in the mPFC-dependent fear extinction task. The results of these studies implicate mGlu₃ as a major regulator of PFC function and cognition. Additionally, potentiators of mGlu₃ may be useful in alleviating prefrontal impairments associated with several CNS disorders.Metabotropic glutamate receptor 3 (mGlu₃) has become of increasing clinical interest due to its genetic association with psychiatric conditions. For example, several studies have identified single-nucleotide polymorphisms (SNPs) in GRM3, the human gene encoding mGlu₃, that are associated with poor performance on cognitive tests that are dependent on function of the prefrontal cortex (PFC) and hippocampus (1, 2). Additionally, these SNPs have also been associated with variations in functional magnetic resonance imaging (fMRI) indexes of prefrontal cortical activity during working memory tasks (1, 3). Moreover, converging lines of evidence indicate that GRM3 represents a major locus associated with schizophrenia (1, 2, 4), bipolar disorder (5, 6), and substance abuse disorders (6–8). Because mGlu₃ is densely expressed in PFC (9), a brain region implicated as a site of pathology in these disorders (10–12), this genetic evidence has led to an increased interest in determining the role of mGlu₃ in regulating PFC function and behavior.Previous studies have revealed that pharmacological activation of group II mGlu receptors (mGlu₂ and mGlu₃) results in long-term depression (LTD) of excitatory transmission in layer V of the rat medial prefrontal cortex (mPFC) (13–16). Although it is not known whether induction of LTD in the mPFC is mediated by mGlu₂ or mGlu₃, previous studies suggest that presynaptically localized mGlu₂ is typically responsible for inhibition of synaptic transmission by group II mGlu receptor agonists at other synapses (17–23). However, evidence suggests that induction of LTD in the mPFC is dependent upon activation of a postsynaptic group II mGlu receptor (15, 16), suggesting that this response is mechanistically distinct from presynaptic effects of group II mGlu receptor agonists on transmission at other synapses. Unfortunately, a lack of pharmacological agents that can selectively antagonize mGlu₃ or mGlu₂ has impaired progress in this area. To allow us to begin studies aimed at understanding the role of mGlu₃ in regulation of mPFC function, we developed a series of negative allosteric modulators (NAMs) that are highly selective for mGlu₃ and are suitable for in vivo use (24). In addition, we now report characterization of a highly selective mGlu₂ NAM. We used these compounds, along with mGlu₂ and mGlu₃ knockout (KO) mice, to evaluate the respective roles of mGlu₂ and mGlu₃ in acute regulation of synaptic transmission and induction of LTD in mPFC. Interestingly, we found that mGlu₂ is involved in acute inhibition of synaptic transmission in the mPFC, but that induction of LTD at this synapse by group II mGlu receptor agonists is mediated exclusively by mGlu₃. Furthermore, we found that mGlu₃ NAMs impair extinction of conditioned fear, a behavioral task that is critically dependent upon the integrity of the mPFC. These data suggest that mGlu₃ plays an essential role in the regulation of a specific form of synaptic plasticity in the mPFC that could be important for forms of cognitive function that require depression of excitatory inputs to mPFC and are thought to be disrupted in patients suffering from a range of CNS disorders.     

Neural correlates of dueling affective reactions to win–win choices

Win–win choices cause anxiety, often more so than decisions lacking the opportunity for a highly desired outcome. These anxious feelings can paradoxically co-occur with positive feelings, raising important implications for individual decision styles and general well-being. Across three studies, people chose between products that varied in personal value. Participants reported feeling most positive and most anxious when choosing between similarly high-valued products. Behavioral and neural results suggested that this paradoxical experience resulted from parallel evaluations of the expected outcome (inducing positive affect) versus the cost of choosing a response (inducing anxiety). Positive feelings were reduced when there was no high-value option, and anxiety was reduced when only one option was highly valued. Dissociable regions within the striatum and the medial prefrontal cortex (mPFC) tracked these dueling affective reactions during choice. Ventral regions, associated with stimulus valuation, tracked positive feelings and the value of the best item. Dorsal regions, associated with response valuation, tracked anxiety. In addition to tracking anxiety, the dorsal mPFC was associated with conflict during the current choice, and activity levels across individual items predicted whether that choice would later be reversed during an unexpected reevaluation phase. By revealing how win–win decisions elicit responses in dissociable brain systems, these results help resolve the paradox of win–win choices. They also provide insight into behaviors that are associated with these two forms of affect, such as why we are pulled toward good options but may still decide to delay or avoid choosing among them.In a famous thought experiment, a hungry donkey is placed exactly between two equal bales of hay and, unable to decide which to approach, starves. Human decision makers face problems similar to the metaphorical donkey. Whether deciding between schools to attend or desserts to order, choices involving equally good outcomes (“win–win” choices) can generate anxiety along with the positive feelings one has about the rewarding prospects (1). Although the positive feelings may lead individuals to prefer having more good options, the anxiety can lead them to delay choosing, choose suboptimally, or make no choice at all (2–5). These seemingly contradictory preferences, particularly in situations where a “wrong” choice has negligible costs, represent a paradox for many decision scientists (6). The potential impact of negative choice experiences on important medical and financial decisions (7, 8) and on general well-being (6, 9, 10) gives the paradox far-reaching consequences. However, despite substantial research on the impact of choice conflict on behavior (2, 5, 7, 8) and postchoice feelings (1, 9, 11), little is known about the basis of the dueling affective reactions to the choice itself.One possibility is that positive and anxious feelings to win–win choices are tied to separate components of the neural circuitry for decision making. Brain regions that determine how good an item is and the costs of performing the response required to obtain it are supported by separate corticostriatal circuits (12–16). Ventral regions of the striatum and the medial prefrontal cortex (mPFC) associate stimuli and contexts with their expected outcomes, whether or not those outcomes are directly relevant to one’s response (17–20). Dorsal regions of the striatum and mPFC associate possible actions (including “internal actions”: i.e., control signals) with their expected outcomes and modify these actions according to current demands (13, 14, 16, 21–24). One of the most well-studied demands encoded by the dorsal mPFC is response conflict (21, 25), including instances of choice conflict similar to those described at the outset (26–30). Whether dorsal mPFC activity correlates with the anxiety elicited by choice conflict and/or predicts future adjustments to prior choices remains an open question.Here, we explored the neural systems underlying the dueling affective states evoked by win–win decisions. In two functional MRI (fMRI) experiments and one behavioral follow-up, participants made a series of decisions between real products that they cared about. Choices between similarly high-value options were rated as the most positive and anxiety-inducing. Activity in dissociable regions correlated with these competing experiences. Ventral mPFC and striatum correlated with the positive experience of the choice offers whereas dorsal mPFC and striatum correlated with the anxiety associated with making the choice. Activity within the dorsal mPFC also predicted postscan choice adjustment (i.e., changes of mind). These findings suggest that win–win choices give rise to separate assessments of the value of options versus the cost of choosing among them, leading to a paradoxical experience that is as anxiety-provoking as it is positive. Our results may have broad implications for understanding the behavioral correlates of these affective experiences, such as indecisiveness and decision avoidance.     

From the Cover: Optogenetic stimulation of infralimbic PFC reproduces ketamine’s rapid and sustained antidepressant actions

Ketamine produces rapid and sustained antidepressant actions in depressed patients, but the precise cellular mechanisms underlying these effects have not been identified. Here we determined if modulation of neuronal activity in the infralimbic prefrontal cortex (IL-PFC) underlies the antidepressant and anxiolytic actions of ketamine. We found that neuronal inactivation of the IL-PFC completely blocked the antidepressant and anxiolytic effects of systemic ketamine in rodent models and that ketamine microinfusion into IL-PFC reproduced these behavioral actions of systemic ketamine. We also found that optogenetic stimulation of the IL-PFC produced rapid and long-lasting antidepressant and anxiolytic effects and that these effects are associated with increased number and function of spine synapses of layer V pyramidal neurons. The results demonstrate that ketamine infusions or optogenetic stimulation of IL-PFC are sufficient to produce long-lasting antidepressant behavioral and synaptic responses similar to the effects of systemic ketamine administration.The NMDA receptor antagonist ketamine produces rapid and robust therapeutic responses in treatment-resistant (1, 2) as well as bipolar depressed patients (3). Preclinical studies report that ketamine also rapidly increases the number and function of spine synapses in the medial prefrontal cortex (mPFC) and that these effects are associated with rapid antidepressant behavioral responses in rodent models (4). These findings represent a major advance for the treatment of depression, although the widespread use of ketamine is limited by side effects (e.g., psychotomimetic and dissociative symptoms) and abuse potential. Further studies of the mechanisms underlying the actions of ketamine could lead to novel rapid antidepressant treatments with fewer side effects.Neuroimaging studies in humans demonstrate that ketamine increases the activity of PFC (5–7), consistent with evidence of rapid increases of glutamate transmission in rodent PFC (8, 9). In addition, depressed patients are reported to have reduced activity in the PFC (10) that is normalized with treatment (11). Rodent studies also demonstrate that long-term stress causes neuronal atrophy of mPFC neurons (12, 13) that is rapidly reversed by ketamine (14). Subregions of the mPFC, including infralimbic (IL) and prelimbic (PrL), have been implicated in diverse cognitive and emotional processes, including fear learning, extinction, and anxiety (15–18). However, the role of PFC activity in the behavioral responses to ketamine has not been examined.Here we examined the antidepressant behavioral effects of neuronal inactivation or direct infusions of ketamine into the IL-PFC and compared these effects with PrL-PFC. Using optogenetics, we also examined the antidepressant and anxiolytic effects of neuronal activation in IL-PFC and determined the impact on pyramidal cell spine number and function to assess long-term neuroplasticity.     

PACAP receptor gene polymorphism impacts fear responses in the amygdala and hippocampus

We have recently found higher circulating levels of pituitary adenylate cyclase-activating polypeptide (PACAP) associated with posttraumatic stress disorder (PTSD) symptoms in a highly traumatized cohort of women but not men. Furthermore, a single nucleotide polymorphism in the PACAP receptor gene ADCYAP1R1, adenylate cyclase activating polypeptide 1 receptor type 1, was associated with individual differences in PTSD symptoms and psychophysiological markers of fear and anxiety. The current study outlines an investigation of individual differences in brain function associated with ADCYAP1R1 genotype. Forty-nine women who had experienced moderate to high levels of lifetime trauma participated in a functional MRI task involving passive viewing of threatening and neutral face stimuli. Analyses focused on the amygdala and hippocampus, regions that play central roles in the pathophysiology of PTSD and are known to have high densities of PACAP receptors. The risk genotype was associated with increased reactivity of the amygdala and hippocampus to threat stimuli and decreased functional connectivity between the amygdala and hippocampus. The findings indicate that the PACAP system modulates medial temporal lobe function in humans. Individual differences in ADCYAP1R1 genotype may contribute to dysregulated fear circuitry known to play a central role in PTSD and other anxiety disorders.Posttraumatic stress disorder (PTSD) is an anxiety disorder estimated to affect 7% of the population (1), with symptoms that are highly debilitating and associated with a range of major physical health conditions (2, 3). PTSD disproportionally affects women over men (1, 4), and mechanisms for this sex difference have not yet been defined. We recently identified a single nucleotide polymorphism (SNP) in the gene coding for the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (ADCYAP1R1, adenylate cyclase activating polypeptide 1 receptor type 1) that predicts PTSD in women and not men (5–9). This SNP, rs2267735, is located on a canonical estrogen response element, indicating that estrogen levels may influence expression of the receptor. The ADCYAP1R1 polymorphism has been shown to predict exaggerated arousal responses characteristic of PTSD in autonomic psychophysiology (5, 10), but no study has yet examined the extent to which this polymorphism influences fear responses in the human brain. The current study tests the hypothesis that ADCYAP1R1 polymorphism influences brain regions that underlie emotional arousal, using functional MRI (fMRI) in a sample of women who have experienced civilian trauma.PTSD symptom clusters include hyperarousal, reexperiencing, avoidance, and numbing (11). Recently, evidence has accumulated to support the idea that hyperarousal is predictive of PTSD after trauma, whereas other symptoms are products of the disorder (12). In particular, pretrauma reactivity of the amygdala—a brain region responsible for coordinating and maintaining multiple components of emotional arousal (13)—appears to be a predisposing risk factor for the maintenance of PTSD symptoms (14–16). Additional brain regions implicated in the pathophysiology of PTSD include the ventromedial and dorsolateral prefrontal cortex, the anterior cingulate cortex, the hippocampus, and the insula (17, 18), each of which plays a role in regulating aspects of the emotional response.Genetic profiles appear to be the initiating predictor of vulnerability to psychiatric disorders. In the case of PTSD, however, environmental factors play a similarly critical role, with a major example being the specific traumatic experience that is a necessary component of the disorder. The combined influences of genetics and the environment are also apparent in twin studies indicating that genetic factors account for 30–70% of PTSD risk, with higher estimates for women than men (19, 20), and the remaining variance is attributable to experience. Such interacting genetic and environmental influences create complex symptom profiles that can vary greatly from individual to individual (19, 21), introducing wide error margins in predicting initial vulnerability, progress of symptoms over time, or effective therapeutic courses for the individual.Relative to genetic predictors or specific experiences, neurobiological phenotypes may provide greater power to predict psychopathology, as brain structure and function reflect an aggregate of genetic and environmental factors. Identifying such intermediate phenotypes, whose action falls between risk factors and psychiatric outcomes, will be critical to our ability to predict and understand disorders such as PTSD, helping to link multiple levels of research from proteins and cellular mechanisms to patient outcomes (21). Here we investigated the effects of an ADCYAP1R1 polymorphism on amygdala and hippocampal function, in a sample of adult women who have experienced moderate to high levels of civilian trauma. To disentangle genetic effects from the complex effects of PTSD and other psychiatric symptoms, genotype groups were matched for childhood and adult trauma levels and PTSD and depression symptom severity.Our primary hypotheses focused on the amygdala because of its role in regulating emotional arousal and as a possible predisposing factor in PTSD. We also examined the hippocampus, another brain region that plays a central role in PTSD (22), where PACAP binding sites are particularly dense (23) and where PACAP has been shown to act in a neuroprotective or neurotrophic manner (24–26) and may facilitate synaptic plasticity (25, 27). We predicted that individuals with the risk polymorphism would show exaggerated amygdala responses to threat stimuli and decreased functional connectivity between the amygdala and the hippocampus and medial prefrontal cortex, regions that regulate amygdala reactivity (28, 29).     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Extinction reverses olfactory fear-conditioned increases in neuron number and glomerular size

Although much work has investigated the contribution of brain regions such as the amygdala, hippocampus, and prefrontal cortex to the processing of fear learning and memory, fewer studies have examined the role of sensory systems, in particular the olfactory system, in the detection and perception of cues involved in learning and memory. The primary sensory receptive field maps of the olfactory system are exquisitely organized and respond dynamically to cues in the environment, remaining plastic from development through adulthood. We have previously demonstrated that olfactory fear conditioning leads to increased odorant-specific receptor representation in the main olfactory epithelium and in glomeruli within the olfactory bulb. We now demonstrate that olfactory extinction training specific to the conditioned odor stimulus reverses the conditioning-associated freezing behavior and odor learning-induced structural changes in the olfactory epithelium and olfactory bulb in an odorant ligand-specific manner. These data suggest that learning-induced freezing behavior, structural alterations, and enhanced neural sensory representation can be reversed in adult mice following extinction training.Increasing evidence suggests that the cellular, neuroanatomical, and receptive field organizations of vertebrate sensory systems are continually reshaped throughout adulthood by cues from the external environment. Activity-dependent changes are known to occur both during critical periods of development and also in the adult brain, allowing the animal to optimally perform behaviors based on the demands of the surrounding environment. Postmitotic organizational changes, along with activity-dependent plasticity, have been largely implicated in shaping sensory circuits from development through adulthood (1–4). In particular, the olfactory sensory system of adult mice exhibits functional and neuroanatomical learning-dependent changes following olfactory fear conditioning in adulthood (5–7). The M71-LacZ transgenic mouse line expresses LacZ under the M71 odorant receptor (OR) promoter (encoded by the olfactory receptor 151 gene, Olfr151) (8) in the M71 OR-expressing, acetophenone-responsive population of olfactory sensory neurons (OSNs). Using this line, we previously demonstrated an increased number of M71-expressing OSNs in the main olfactory epithelium (MOE) of adult mice following olfactory fear conditioning to acetophenone (5, 7), an odorant that activates the M71/M72 ORs (9, 10). This increase in receptor-specific OSNs within the MOE was directly correlated with an increase in the area of M71+ axons innervating the M71 glomeruli within the olfactory bulbs (OBs). Behaviorally, these olfactory fear-conditioned mice also exhibited enhanced fear-potentiated startle (FPS) and freezing specific to the conditioned odor stimulus. Notably such changes were never seen with equivalent odorant exposure alone but only when the odorant was paired with an aversive or appetitive cue (5, 7), suggesting the critical importance of behavioral learning facilitating these structural and functional alterations.Reversing the behavioral and neuroanatomical effects of such emotional learning is important for our understanding of disorders such as posttraumatic stress disorder (PTSD), in which exposure-based psychotherapy is widely used for treatment. Notably, extinction training in rodent fear-conditioning models closely parallels many aspects of exposure-based psychotherapy in humans where exposure to nonreinforced presentations of the previously acquired conditioned stimulus (CS) reduces acquired fear responses such as freezing to the CS (11, 12). In the current study, we demonstrate that previously acquired structural changes within the primary olfactory system are reversed with olfactory fear extinction specific to the conditioned odorant cue.     

Endogenous cannabinoid release within prefrontal-limbic pathways affects memory consolidation of emotional training

Previous studies have provided extensive evidence that administration of cannabinoid drugs after training modulates the consolidation of memory for an aversive experience. The present experiments investigated whether the memory consolidation is regulated by endogenously released cannabinoids. The experiments first examined whether the endocannabinoids anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) are released by aversive training. Inhibitory avoidance training with higher footshock intensity produced increased levels of AEA in the amygdala, hippocampus, and medial prefrontal cortex (mPFC) shortly after training in comparison with levels assessed in rats trained with lower footshock intensity or unshocked controls exposed only to the training apparatus. In contrast, 2-AG levels were not significantly elevated. The additional finding that posttraining infusions of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which selectively increases AEA levels at active synapses, administered into the basolateral complex of the amygdala (BLA), hippocampus, or mPFC enhanced memory strongly suggests that the endogenously released AEA modulates memory consolidation. Moreover, in support of the view that this emotional training-associated increase in endocannabinoid neurotransmission, and its effects on memory enhancement, depends on the integrity of functional interactions between these different brain regions, we found that disruption of BLA activity blocked the training-induced increases in AEA levels as well as the memory enhancement produced by URB597 administered into the hippocampus or mPFC. Thus, the findings provide evidence that emotionally arousing training increases AEA levels within prefrontal-limbic circuits and strongly suggest that this cannabinoid activation regulates emotional arousal effects on memory consolidation.It is well-established that stressful or emotionally arousing experiences are well-remembered (1). In addition, there is extensive evidence that the basolateral complex of the amygdala (BLA), hippocampus, and medial prefrontal cortex (mPFC) are all crucially involved in mediating emotional arousal effects on memory consolidation (2, 3). The consolidation of memories of arousing experiences requires an orchestration of neural activity in these brain systems, and the cannabinoid system has emerged as a key modulator of such function (1, 4–6). Endogenous cannabinoid ligands [termed endocannabinoids, mainly N-arachidonoyl ethanolamine (anandamide; AEA) and 2-arachidonoyl glycerol (2-AG)] are released from postsynaptic membranes and feedback in a retrograde manner onto either excitatory or inhibitory presynaptic terminals, thus suppressing both excitatory and inhibitory signaling within specific neuronal circuits (7).Extensive evidence indicates that exogenous cannabinoids administered into this neural circuitry modulate memory processing of emotionally arousing training (8–12). We previously showed that the synthetic cannabinoid agonist WIN55,212-2, administered into the BLA immediately after inhibitory avoidance training, enhances the consolidation of long-term memory (8). Conversely, inhibition of endogenous cannabinoid signaling within the BLA with posttraining infusions of the cannabinoid type 1 (CB1) receptor antagonist AM251 impairs inhibitory avoidance memory (8). We recently reported that the level of emotional arousal at the time of training is an important factor in determining cannabinoid effects on memory (13): WIN55,212-2 administration enhanced long-term object recognition memory when rats were trained under a high arousal condition but was ineffective with low-arousing training (13). Although the findings of our prior studies, as well as those of other investigators (6, 8, 11–13), indicate that administration of cannabinoids can modulate memory consolidation, studies have not yet investigated whether AEA or 2-AG are released physiologically in response to emotionally arousing training and normally play a role in creating strong memories for these experiences. The present experiments investigated this issue. Rats were trained on an inhibitory avoidance task under different arousal conditions and were killed after training for determination of AEA and 2-AG levels in the amygdala, hippocampus, and mPFC. To investigate whether training-induced endocannabinoid release contributes to memory consolidation, we pharmacologically blocked AEA degradation in the BLA, hippocampus, or mPFC by locally infusing the fatty acid amide hydrolase (FAAH) inhibitor URB597 immediately after inhibitory avoidance training. In contrast to direct cannabinoid receptor agonists, URB597 selectively augments AEA signaling at active synapses, thus mimicking the physiological condition normally occurring after an emotionally arousing training experience.The experiments also investigated whether training-associated endocannabinoid neurotransmission within this neurocircuitry depends on functional interactions between the BLA, hippocampus, and mPFC. Extensive evidence indicates that the BLA contributes to enhancement of memory for emotional events primarily by integrating neuromodulatory influences and modulating neural activity and synaptic plasticity in other brain regions (1). The BLA is known to project both directly and indirectly to the hippocampus (14, 15) and mPFC (16, 17), and it has been shown that a disruption of BLA activity blocks the memory-enhancing effects of drugs administered directly into either the hippocampus or mPFC (18, 19). Therefore, in this last series of experiments, we investigated whether an intact BLA activity is required to allow for a normal endocannabinoid response in the hippocampus and mPFC after an aversive training experience.     

Extinction during reconsolidation of threat memory diminishes prefrontal cortex involvement

Controlling learned defensive responses through extinction does not alter the threat memory itself, but rather regulates its expression via inhibitory influence of the prefrontal cortex (PFC) over amygdala. Individual differences in amygdala–PFC circuitry function have been linked to trait anxiety and posttraumatic stress disorder. This finding suggests that exposure-based techniques may actually be least effective in those who suffer from anxiety disorders. A theoretical advantage of techniques influencing reconsolidation of threat memories is that the threat representation is altered, potentially diminishing reliance on this PFC circuitry, resulting in a more persistent reduction of defensive reactions. We hypothesized that timing extinction to coincide with threat memory reconsolidation would prevent the return of defensive reactions and diminish PFC involvement. Two conditioned stimuli (CS) were paired with shock and the third was not. A day later, one stimulus (reminded CS+) but not the other (nonreminded CS+) was presented 10 min before extinction to reactivate the threat memory, followed by extinction training for all CSs. The recovery of the threat memory was tested 24 h later. Extinction of the nonreminded CS+ (i.e., standard extinction) engaged the PFC, as previously shown, but extinction of the reminded CS+ (i.e., extinction during reconsolidation) did not. Moreover, only the nonreminded CS+ memory recovered on day 3. These results suggest that extinction during reconsolidation prevents the return of defensive reactions and diminishes PFC involvement. Reducing the necessity of the PFC–amygdala circuitry to control defensive reactions may help overcome a primary obstacle in the long-term efficacy of current treatments for anxiety disorders.Efforts to control maladaptive defensive reactions through extinction or exposure therapy are sometimes short-lived because these techniques do not significantly alter the threat memory itself, but rather regulate its expression via the prefrontal cortex’s (PFC) inhibition of the amygdala (1, 2). Individual variation in the integrity of this amygdala–prefrontal circuitry has been linked to trait anxiety and posttraumatic stress disorder, suggesting that exposure-based techniques may be least effective in those who suffer from anxiety disorders (3–9).Recently, it has been shown in mice (10, 11), rats (12), and humans (13–16) that precisely timing behavioral extinction to coincide with memory reconsolidation can persistently inhibit the return of defensive reactions (but see refs. 17–19 for a discussion of boundary conditions). Reconsolidation is the state to which memories enter upon retrieval, which makes them prone to interference (20–22). Behavioral interference of reconsolidation using extinction has been linked to alterations in glutamate receptor function in the amygdala, which plays a critical role in memory plasticity (10, 12). These findings are consistent with the suggestion that, in contrast to standard extinction training, extinction during reconsolidation may lead to long-lasting changes in the original threat memory (13, 16, 23).To date, the impact of extinction occurring during threat memory reconsolidation on PFC involvement is unknown in humans and other species. Animal studies of standard extinction training (i.e., repeated presentations of a conditioned stimulus without the aversive outcome) show that extinction learning and recall are mediated via the infralimbic (IL) region of the medial PFC and its connections with the amygdala; IL projections activate inhibitory cells within the amygdala that block the generation of the defense response (24, 25). Functional MRI (fMRI) studies of extinction in humans typically show a decrease in blood-oxygenation level-dependent (BOLD) signal in the ventral medial PFC (vmPFC; the human homolog of IL) in acquisition and early extinction, and a gradual increase in BOLD activity with the progression of extinction training (26, 27). If extinction occurs during reconsolidation, how might the vmPFC’s role change? One possibility is that processes occurring during reconsolidation alter the extinction circuitry, diminishing vmPFC involvement. To test this hypothesis, we used fMRI to examine the vmPFC during behavioral interference of reconsolidation in humans.BOLD responses were assessed during a 3-d protocol previously shown to interfere with reconsolidation (16). On day 1, two conditioned stimuli (CS+) were paired with a mild wrist shock (US, unconditioned stimulus); the third was not (CS−). On day 2, one CS+ (reminded CS+) but not the other (nonreminded CS+) was presented 10 min before extinction to reactivate the threat memory, followed by extinction training for all CSs. In this protocol, the reminded CS+ is presumably undergoing extinction during reconsolidation and the nonreminded CS+ is undergoing standard extinction training. On day 3, the threat memory was reinstated using four unsignaled shocks, followed by a test of recovery of the threat memory and another extinction session.We found that timing extinction training to coincide with threat memory reconsolidation prevents the return of defensive reactions, and indeed significantly diminishes PFC involvement. During extinction, only the nonreminded CS+ engaged the vmPFC, but not the reminded CS+ or the CS−. The vmPFC, moreover, showed enhanced functional connectivity with the amygdala only during extinction of the nonreminded, but not the reminded CS+. This altered connectivity during extinction of the reminded CS+ may play a role in enabling extinction learning training to more persistently modify the original threat-memory trace within the amygdala, thus preventing the return of defensive reactions on subsequent recovery tests. Reducing the necessity of the prefrontal–amygdala circuitry to control learned defensive reactions may help overcome a primary obstacle in the long-term efficacy of current treatments for anxiety disorders.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.