Vagal sensory neurons mediate the Bezold-Jarisch reflex and induce syncope.

Visceral sensory pathways mediate homeostatic reflexes, the dysfunction of which leads to many neurological disorders1. The Bezold-Jarisch reflex (BJR), first described2,3 in 1867, is a cardioinhibitory reflex that is speculated to be mediated by vagal sensory neurons (VSNs) that also triggers syncope. However, the molecular identity, anatomical organization, physiological characteristics and behavioural influence of cardiac VSNs remain mostly unknown. Here we leveraged single-cell RNA-sequencing data and HYBRiD tissue clearing4 to show that VSNs that express neuropeptide Y receptor Y2 (NPY2R) predominately connect the heart ventricular wall to the area postrema. Optogenetic activation of NPY2R VSNs elicits the classic triad of BJR responses-hypotension, bradycardia and suppressed respiration-and causes an animal to faint. Photostimulation during high-resolution echocardiography and laser Doppler flowmetry with behavioural observation revealed a range of phenotypes reflected in clinical syncope, including reduced cardiac output, cerebral hypoperfusion, pupil dilation and eye-roll. Large-scale Neuropixels brain recordings and machine-learning-based modelling showed that this manipulation causes the suppression of activity across a large distributed neuronal population that is not explained by changes in spontaneous behavioural movements. Additionally, bidirectional manipulation of the periventricular zone had a push-pull effect, with inhibition leading to longer syncope periods and activation inducing arousal. Finally, ablating NPY2R VSNs specifically abolished the BJR. Combined, these results demonstrate a genetically defined cardiac reflex that recapitulates characteristics of human syncope at physiological, behavioural and neural network levels.

Pathogenic SYNGAP1 mutations impair cognitive development by disrupting maturation of dendritic spine synapses.

Mutations that cause intellectual disability (ID) and autism spectrum disorder (ASD) are commonly found in genes that encode for synaptic proteins. However, it remains unclear how mutations that disrupt synapse function impact intellectual ability. In the SYNGAP1 mouse model of ID/ASD, we found that dendritic spine synapses develop prematurely during the early postnatal period. Premature spine maturation dramatically enhanced excitability in the developing hippocampus, which corresponded with the emergence of behavioral abnormalities. Inducing SYNGAP1 mutations after critical developmental windows closed had minimal impact on spine synapse function, whereas repairing these pathogenic mutations in adulthood did not improve behavior and cognition. These data demonstrate that SynGAP protein acts as a critical developmental repressor of neural excitability that promotes the development of life-long cognitive abilities. We propose that the pace of dendritic spine synapse maturation in early life is a critical determinant of normal intellectual development.

Syngap1 regulates experience-dependent cortical ensemble plasticity by promoting in vivo excitatory synapse strengthening.

A significant proportion of autism risk genes regulate synapse function, including plasticity, which is believed to contribute to behavioral abnormalities. However, it remains unclear how impaired synapse plasticity contributes to network-level processes linked to adaptive behaviors, such as experience-dependent ensemble plasticity. We found that Syngap1, a major autism risk gene, promoted measures of experience-dependent excitatory synapse strengthening in the mouse cortex, including spike-timing-dependent glutamatergic synaptic potentiation and presynaptic bouton formation. Synaptic depression and bouton elimination were normal in Syngap1 mice. Within cortical networks, Syngap1 promoted experience-dependent increases in somatic neural activity in weakly active neurons. In contrast, plastic changes to highly active neurons from the same ensemble that paradoxically weaken with experience were unaffected. Thus, experience-dependent excitatory synapse strengthening mediated by Syngap1 shapes neuron-specific plasticity within cortical ensembles. We propose that other genes regulate neuron-specific weakening within ensembles, and together, these processes function to redistribute activity within cortical networks during experience.

Basolateral amygdala corticotropin releasing factor receptor 2 interacts with nonmuscle myosin II to destabilize memory in males.

Preclinical studies show that inhibiting the actin motor ATPase nonmuscle myosin II (NMII) with blebbistatin (Blebb) in the basolateral amgydala (BLA) depolymerizes actin, resulting in an immediate, retrieval-independent disruption of methamphetamine (METH)-associated memory in male and female adult and adolescent rodents. The effect is highly selective, as NMII inhibition has no effect in other relevant brain regions (e.g., dorsal hippocampus [dPHC], nucleus accumbens [NAc]), nor does it interfere with associations for other aversive or appetitive stimuli, including cocaine (COC). To understand the mechanisms responsible for drug specific selectivity we began by investigating, in male mice, the pharmacokinetic differences in METH and COC brain exposure . Replicating METH's longer half-life with COC did not render the COC association susceptible to disruption by NMII inhibition. Therefore, we next assessed transcriptional differences. Comparative RNA-seq profiling in the BLA, dHPC and NAc following METH or COC conditioning identified crhr2, which encodes the corticotropin releasing factor receptor 2 (CRF2), as uniquely upregulated by METH in the BLA. CRF2 antagonism with Astressin-2B (AS2B) had no effect on METH-associated memory after consolidation, allowing for determination of CRF2 influences on NMII-based susceptibility. Pretreatment with AS2B prevented the ability of Blebb to disrupt an established METH-associated memory. Alternatively, combining CRF2 overexpression and agonist treatment, urocortin 3 (UCN3), in the BLA during conditioning rendered COC-associated memory susceptible to disruption by NMII inhibition, mimicking the Blebb-induced, retrieval-independent memory disruption seen with METH. These results suggest that BLA CRF2 receptor activation during memory formation in male mice can prevent stabilization of the actin-myosin cytoskeleton supporting the memory, rendering it vulnerable to disruption by NMII inhibition. CRF2 represents an interesting target for BLA-dependent memory destabilization via downstream effects on NMII.

Methamphetamine Learning Induces Persistent and Selective Nonmuscle Myosin II-Dependent Spine Motility in the Basolateral Amygdala.

Nonmuscle myosin II inhibition (NMIIi) in the basolateral amygdala (BLA), but not dorsal hippocampus (CA1), selectively disrupts memories associated with methamphetamine (METH) days after learning, without retrieval. However, the molecular mechanisms underlying this selective vulnerability remain poorly understood. A known function of NMII is to transiently activate synaptic actin dynamics with learning. Therefore, we hypothesized that METH-associated learning perpetuates NMII-driven actin dynamics in synapses, leading to an extended window of vulnerability for memory disruption. We used time-lapse two-photon imaging of dendritic spine motility in acutely prepared brain slices from female and male mice following METH-associated learning as a readout of actin-myosin dynamics. Spine motility was persistently increased in the BLA, but not in CA1. Consistent with the memory disrupting effect of intra-BLA NMII inhibition, METH-induced changes to BLA spine dynamics were reversed by a single systemic injection of an NMII inhibitor. Intra-CA1 NMII inhibition, on the other hand, did not disrupt METH-associated memory. Thus, we report identification of a previously unknown ability for spine actin dynamics to persist days after stimulation and that this is under the control of NMII. Further, these perpetual NMII-driven spine actin dynamics in BLA neurons may contribute to the unique susceptibility of METH-associated memories.SIGNIFICANCE STATEMENT There are no Food and Drug Administration-approved pharmacotherapies to prevent relapse to the use of stimulants, such as methamphetamine (METH). Environmental cues become associated with drug use, such that the memories can elicit strong motivation to seek the drug during abstinence. We previously reported that the storage of METH-associated memories is uniquely vulnerable to immediate, retrieval-independent, and lasting disruption by direct actin depolymerization or by inhibiting the actin driver nonmuscle myosin II (NMII) in the BLA or systemically. Here we report a potential structural mechanism responsible for the unique vulnerability of METH-associated memories and METH-seeking behavior to NMII inhibition within the BLA.

SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons.

SYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. De novo loss-of-function variants in this gene cause a neurodevelopmental disorder defined by cognitive impairment, social-communication disorder, and early-onset seizures. Cell biological studies in mouse and rat neurons have shown that Syngap1 regulates developing excitatory synapse structure and function, with loss-of-function variants driving formation of larger dendritic spines and stronger glutamatergic transmission. However, studies to date have been limited to mouse and rat neurons. Therefore, it remains unknown how SYNGAP1 loss of function impacts the development and function of human neurons. To address this, we used CRISPR/Cas9 technology to ablate SYNGAP1 protein expression in neurons derived from a commercially available induced pluripotent stem cell line (hiPSC) obtained from a human female donor. Reducing SynGAP protein expression in developing hiPSC-derived neurons enhanced dendritic morphogenesis, leading to larger neurons compared with those derived from isogenic controls. Consistent with larger dendritic fields, we also observed a greater number of morphologically defined excitatory synapses in cultures containing these neurons. Moreover, neurons with reduced SynGAP protein had stronger excitatory synapses and expressed synaptic activity earlier in development. Finally, distributed network spiking activity appeared earlier, was substantially elevated, and exhibited greater bursting behavior in SYNGAP1 null neurons. We conclude that SYNGAP1 regulates the postmitotic maturation of human neurons made from hiPSCs, which influences how activity develops within nascent neural networks. Alterations to this fundamental neurodevelopmental process may contribute to the etiology of SYNGAP1-related disorders.SIGNIFICANCE STATEMENTSYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum disorder, and epileptic encephalopathy. While this gene is well studied in rodent neurons, its function in human neurons remains unknown. We used CRISPR/Cas9 technology to disrupt SYNGAP1 protein expression in neurons derived from an induced pluripotent stem cell line. We found that induced neurons lacking SynGAP expression exhibited accelerated dendritic morphogenesis, increased accumulation of postsynaptic markers, early expression of synapse activity, enhanced excitatory synaptic strength, and early onset of neural network activity. We conclude that SYNGAP1 regulates the postmitotic differentiation rate of developing human neurons and disrupting this process impacts the function of nascent neural networks. These altered developmental processes may contribute to the etiology of SYNGAP1 disorders.

SynGAP is expressed in the murine suprachiasmatic nucleus and regulates circadian-gated locomotor activity and light-entrainment capacity.

The suprachiasmatic nucleus (SCN) of the hypothalamus functions as the master circadian clock. The phasing of the SCN oscillator is locked to the daily solar cycle, and an intracellular signaling cassette from the small GTPase Ras to the p44/42 mitogen-activated protein kinase (ERK/MAPK) pathway is central to this entrainment process. Here, we analyzed the expression and function of SynGAP-a GTPase-activating protein that serves as a negative regulator of Ras signaling-within the murine SCN. Using a combination of immunohistochemical and Western blotting approaches, we show that SynGAP is broadly expressed throughout the SCN. In addition, temporal profiling assays revealed that SynGAP expression is regulated over the circadian cycle, with peak expression occurring during the circadian night. Further, time-of-day-gated expression of SynGAP was not observed in clock arrhythmic BMAL1 null mice, indicating that the daily oscillation in SynGAP is driven by the inherent circadian timing mechanism. We also show that SynGAP phosphorylation at serine 1138-an event that has been found to modulate its functional efficacy-is regulated by clock time and is responsive to photic input. Finally, circadian phenotypic analysis of Syngap1 heterozygous mice revealed enhanced locomotor activity, increased sensitivity to light-evoked clock entrainment, and elevated levels of light-evoked MAPK activity, which is consistent with the role of SynGAP as a negative regulator of MAPK signaling. These findings reveal that SynGAP functions as a modulator of SCN clock entrainment, an effect that may contribute to sleep and circadian abnormalities observed in patients with SYNGAP1 gene mutations.

Correction: MicroRNA regulation of persistent stress-enhanced memory.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

MicroRNA regulation of persistent stress-enhanced memory.

Disruption of persistent, stress-associated memories is relevant for treating posttraumatic stress disorder (PTSD) and related syndromes, which develop in a subset of individuals following a traumatic event. We previously developed a stress-enhanced fear learning (SEFL) paradigm in inbred mice that produces PTSD-like characteristics in a subset of mice, including persistently enhanced memory and heightened cFos in the basolateral amygdala complex (BLC) with retrieval of the remote (30-day-old) stress memory. Here, the contribution of BLC microRNAs (miRNAs) to stress-enhanced memory was investigated because of the molecular complexity they achieve through their ability to regulate multiple targets simultaneously. We performed small-RNA sequencing (smRNA-Seq) and quantitative proteomics on BLC tissue collected from mice 1 month after SEFL and identified persistently changed microRNAs, including mir-135b-5p, and proteins associated with PTSD-like heightened fear expression. Viral-mediated overexpression of mir-135b-5p in the BLC of stress-resilient animals enhanced remote fear memory expression and promoted spontaneous renewal 14 days after extinction. Conversely, inhibition of BLC mir-135b-5p in stress-susceptible animals had the opposite effect, promoting a resilient-like phenotype. mir-135b-5p is highly conserved across mammals and was detected in post mortem human amygdala, as well as human serum samples. The mir-135b passenger strand, mir-135b-3p, was significantly elevated in serum from PTSD military veterans, relative to combat-exposed control subjects. Thus, miR-135b-5p may be an important therapeutic target for dampening persistent, stress-enhanced memory and its passenger strand a potential biomarker for responsivity to a mir-135-based therapeutic.

Design, Optimization, and Study of Small Molecules That Target Tau Pre-mRNA and Affect Splicing.

Approximately 95% of human genes are alternatively spliced, and aberrant splicing events can cause disease. One pre-mRNA that is alternatively spliced and linked to neurodegenerative diseases is tau (microtubule-associated protein tau), which can cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and can contribute to Alzheimer's disease. Here, we describe the design of structure-specific lead small molecules that directly target tau pre-mRNA from sequence. This was followed by hit expansion and analogue synthesis to further improve upon these initial lead molecules. The emergent compounds were assessed for functional activity in a battery of assays, including binding assays and an assay that mimics molecular recognition of tau pre-mRNA by a U1 small nuclear ribonucleoprotein (snRNP) splicing factor. Compounds that emerged from these studies had enhanced potency and selectivity for the target RNA relative to the initial hits, while also having significantly improved drug-like properties. The compounds are shown to directly target tau pre-mRNA in cells, via chemical cross-linking and isolation by pull-down target profiling, and to rescue disease-relevant splicing of tau pre-mRNA in a variety of cellular systems, including primary neurons. More broadly, this study shows that lead, structure-specific compounds can be designed from sequence and then further optimized for their physicochemical properties while at the same time enhancing their activity.

SynGAP splice variants display heterogeneous spatio-temporal expression and subcellular distribution in the developing mammalian brain.

The SynGAP protein is a major regulator of synapse biology and neural circuit function. Genetic variants linked to epilepsy and intellectual disability disrupt synaptic function and neural excitability. SynGAP has been involved in multiple signaling pathways and can regulate small GTPases with very different roles. Yet, the molecular bases behind this pleiotropy are poorly understood. We hypothesize that different SynGAP isoforms will mediate different sets of functions and that deciphering their spatio-temporal expression and subcellular localization will accelerate understanding their multiple functions. Using isoform-specific antibodies recognizing SynGAP in mouse and human samples we found distinctive developmental expression patterns for all SynGAP isoforms in five mouse brain areas. Particularly noticeable was the delayed expression of SynGAP-α1 isoforms, which directly bind to postsynaptic density-95, in cortex and hippocampus during the first 2 weeks of postnatal development. Suggesting that during this period other isoforms would have a more prominent role. Furthermore, we observed subcellular localization differences between isoforms, particularly throughout postnatal development. Consistent with previous reports, SynGAP was enriched in the postsynaptic density in the mature forebrain. However, SynGAP was predominantly found in non-synaptic locations in a period of early postnatal development highly sensitive to SynGAP levels. While, α1 isoforms were always found enriched in the postsynaptic density, α2 isoforms changed from a non-synaptic to a mostly postsynaptic density localization with age and ß isoforms were always found enriched in non-synaptic locations. The differential expression and subcellular distribution of SynGAP isoforms may contribute to isoform-specific regulation of small GTPases, explaining SynGAP pleiotropy.

The sixth international RASopathies symposium: Precision medicine-From promise to practice.

The RASopathies are a group of genetic disorders that result from germline pathogenic variants affecting RAS-mitogen activated protein kinase (MAPK) pathway genes. RASopathies share RAS/MAPK pathway dysregulation and share phenotypic manifestations affecting numerous organ systems, causing lifelong and at times life-limiting medical complications. RASopathies may benefit from precision medicine approaches. For this reason, the Sixth International RASopathies Symposium focused on exploring precision medicine. This meeting brought together basic science researchers, clinicians, clinician scientists, patient advocates, and representatives from pharmaceutical companies and the National Institutes of Health. Novel RASopathy genes, variants, and animal models were discussed in the context of medication trials and drug development. Attempts to define and measure meaningful endpoints for treatment trials were discussed, as was drug availability to patients after trial completion.

microRNA mir-598-3p mediates susceptibility to stress enhancement of remote fear memory.

microRNAs (miRNAs) have emerged as potent regulators of learning, recent memory, and extinction. However, our understanding of miRNAs directly involved in regulating complex psychiatric conditions perpetuated by aberrant memory, such as in posttraumatic stress disorder (PTSD), remains limited. To begin to address the role of miRNAs in persistent memories, we performed small-RNA sequencing on basolateral amygdala (BLA) tissue and identified miRNAs altered by auditory fear conditioning (FC) one month after training. mir-598-3p, a highly conserved miRNA previously unstudied in the brain, was down-regulated in the BLA. Further decreasing BLA mir-598-3p levels did not increase strength of the remote fear memory. Given that stress is a critical component in PTSD, we next assessed the impact of stress and stress-enhanced fear learning (SEFL) on mir-598-3p levels, finding the miRNA is elevated in the BLA of male, but not female, mice susceptible to the effects of stress in SEFL. Accordingly, intra-BLA inhibition of mir-598-3p interfered with expression and extinction of the remote fear memory in male, but not female, mice. This effect could not be attributed to an anxiolytic effect of miRNA inhibition. Finally, bioinformatic analysis following quantitative proteomics on BLA tissue collected 30 d post-SEFL training identified putative mir-598-3p targets and related pathways mediating the differential susceptibility, with evidence for regulation of the actin cytoskeleton, the core mediator of structural plasticity. Taken together, the results suggest BLA mir-598-3p may be recruited by stress to mediate a critical switch from a salient remote fear memory to one that is enhanced and extinction-resistant.

Atypical Endocannabinoid Signaling Initiates a New Form of Memory-Related Plasticity at a Cortical Input to Hippocampus.

Endocannabinoids (ECBs) depress transmitter release at sites throughout the brain. Here, we describe another form of ECB signaling that triggers a novel form of long-term potentiation (LTP) localized to the lateral perforant path (LPP) which conveys semantic information from cortex to hippocampus. Two cannabinoid CB1 receptor (CB1R) signaling cascades were identified in hippocampus. The first is pregnenolone sensitive, targets vesicular protein Munc18-1 and depresses transmitter release; this cascade is engaged by CB1Rs in Schaffer-Commissural afferents to CA1 but not in the LPP, and it does not contribute to LTP. The second cascade is pregnenolone insensitive and LPP specific; it entails co-operative CB1R/ß1-integrin signaling to effect synaptic potentiation via stable enhancement of transmitter release. The latter cascade is engaged during LPP-dependent learning. These results link atypical ECB signaling to the encoding of a fundamental component of episodic memory and suggest a novel route whereby endogenous and exogenous cannabinoids affect cognition.

Social stress-potentiated methamphetamine seeking.

Human studies of substance use disorder show that psychological stress and drug availability interact following rehabilitation, contributing to the high relapse potential. Social stressors trigger particularly strong motivation for drug, but how this affects neuronal function to increase relapse is unknown. Animal models, which allow for the dissection of neural mechanisms, primarily utilize physical stressors to trigger relapse. To recapitulate psychosocial post-rehabilitation challenges in animals, we developed a model of social stress-potentiated methamphetamine (METH) seeking. Rats receive a single social defeat (SD) session after completion of self-administration and extinction of lever pressing. While a reminder of the SD was insufficient to reinstate METH seeking on its own, rats that received a reminder of SD followed by a METH-priming injection displayed potentiated reinstatement over METH-priming alone. Examination of neuronal activation patterns of the METH-primed reinstatement session identified c-Fos-immunoreactivity in the basolateral amygdala (BLA) as correlated with SD score, a measure of defeat latency. Rapidly defeated rats showed potentiated METH-primed reinstatement and elevated BLA c-Fos compared with controls. Conversely, rats that were undefeated during the social stress did not show potentiated METH-primed reinstatement or elevated BLA c-Fos. Interestingly, inactivation of the BLA with baclofen/muscimol prior to the stress reminder and METH-priming generated a potentiation of METH seeking in the undefeated rats, suggesting the BLA may mediate resilience to the stressor. This model provides a tool for the further dissection of neural mechanisms mediating social stress-potentiated relapse and for the development of relapse-reducing therapeutics.

Species-conserved SYNGAP1 phenotypes associated with neurodevelopmental disorders.

SYNGAP1 loss-of-function variants are causally associated with intellectual disability, severe epilepsy, autism spectrum disorder and schizophrenia. While there are hundreds of genetic risk factors for neurodevelopmental disorders (NDDs), this gene is somewhat unique because of the frequency and penetrance of loss-of-function variants found in patients combined with the range of brain disorders associated with SYNGAP1 pathogenicity. These clinical findings indicate that SYNGAP1 regulates fundamental neurodevelopmental processes that are necessary for brain development. Here, we describe four phenotypic domains that are controlled by Syngap1 expression across vertebrate species. Two domains, the maturation of cognitive functions and maintenance of excitatory-inhibitory balance, are defined exclusively through a review of the current literature. Two additional domains are defined by integrating the current literature with new data indicating that SYNGAP1/Syngap1 regulates innate survival behaviors and brain structure. These four phenotypic domains are commonly disrupted in NDDs, suggesting that a deeper understanding of developmental Syngap1 functions will be generalizable to other NDDs of known or unknown etiology. Therefore, we discuss the known molecular and cellular functions of Syngap1 and consider how these functions may contribute to the emergence of disease-relevant phenotypes. Finally, we identify major unexplored areas of Syngap1 neurobiology and discuss how a deeper understanding of this gene may uncover general principles of NDD pathobiology.

The role of nonmuscle myosin II in polydrug memories and memory reconsolidation.

Using pharmacologic and genetic approaches targeting actin or the actin-driving molecular motor, nonmuscle myosin II (NMII), we previously discovered an immediate, retrieval-independent, and long-lasting disruption of methamphetamine- (METH-) and amphetamine-associated memories. A single intrabasolateral amygdala complex infusion or systemic administration of the NMII inhibitor Blebbistatin (Blebb) is sufficient to produce this disruption, which is selective, having no retrieval-independent effect on memories for fear, food reward, cocaine, or morphine. However, it was unclear if Blebb treatment would disrupt memories of other stimulants and amphetamine class drugs, such as nicotine (NIC) or mephedrone (MEPH; bath salts). Moreover, many individuals abuse multiple drugs, but it was unknown if Blebb could disrupt polydrug memories, or if the inclusion of another substance would render Blebb no longer able to disrupt METH-associated memories. Therefore, the present study had two primary goals: (1) to determine the ability of Blebb to disrupt NIC- or MEPH-associated memories, and (2) to determine the ability of METH to modify other unconditioned stimulus (US) associations' susceptibility to Blebb. To this end, using the conditional place preference model, mice were conditioned to NIC and MEPH alone or METH in combination with NIC, morphine, or foot shock. We report that, unlike METH, there was no retrieval-independent effect of Blebb on NIC- or MEPH-associated memories. However, similar to cocaine, reconsolidation of the memory for both drugs was disrupted. Further, when combined with METH administration, NIC- and morphine-, but not fear-, associated memories were rendered susceptible to disruption by Blebb. Given the high rate of polydrug use and the resurgence of METH use, these results have important implications for the treatment of substance use disorder.

Memory disrupting effects of nonmuscle myosin II inhibition depend on the class of abused drug and brain region.

Depolymerizing actin in the amygdala through nonmuscle myosin II inhibition (NMIIi) produces a selective, lasting, and retrieval-independent disruption of the storage of methamphetamine-associated memories. Here we report a similar disruption of memories associated with amphetamine, but not cocaine or morphine, by NMIIi. Reconsolidation appeared to be disrupted with cocaine. Unlike in the amygdala, methamphetamine-associated memory storage was not disrupted by NMIIi in the hippocampus, nucleus accumbens, or orbitofrontal cortex. NMIIi in the hippocampus did appear to disrupt reconsolidation. Identification of the unique mechanisms responsible for NMII-mediated, amygdala-dependent disruption of memory storage associated with the amphetamine class may enable induction of retrieval-independent vulnerability to other pathological memories.

Nonmuscle myosin II inhibition disrupts methamphetamine-associated memory in females and adolescents.

Memories associated with drug use can trigger strong motivation for the drug, which increases relapse vulnerability in substance use disorder (SUD). Currently there are no treatments for relapse to abuse of psychostimulants, such as methamphetamine (METH). We previously reported that storage of memories associated with METH, but not those for fear or food reward, and the concomitant spine density increase are disrupted in a retrieval-independent manner by depolymerizing actin in the basolateral amygdala complex (BLC) of adult male rats and mice. Similar results are achieved in males through intra-BLC or systemic inhibition of nonmuscle myosin II (NMII), a molecular motor that directly drives actin polymerization. Given the substantial differences in physiology between genders, we sought to determine if this immediate and selective disruption of METH-associated memory extends to adult females. A single intra-BLC infusion of the NMII inhibitor Blebbistatin (Blebb) produced a long-lasting disruption of context-induced drug seeking for at least 30days in female rats that mirrored our prior results in males. Furthermore, a single systemic injection of Blebb prior to testing disrupted METH-associated memory and the concomitant increase in BLC spine density in females. Importantly, as in males, the same manipulation had no effect on an auditory fear memory or associated BLC spine density. In addition, we established that the NMII-based disruption of METH-associated memory extends to both male and female adolescents. These findings provide further support that small molecular inhibitors of NMII have strong therapeutic potential for the prevention of relapse to METH abuse triggered by associative memories.

Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment.

The mechanisms of neuronal death in protein misfolding neurodegenerative diseases such as Alzheimer's, Parkinson's and prion diseases are poorly understood. We used a highly toxic misfolded prion protein (TPrP) model to understand neurotoxicity induced by prion protein misfolding. We show that abnormal autophagy activation and neuronal demise is due to severe, neuron-specific, nicotinamide adenine dinucleotide (NAD(+)) depletion. Toxic prion protein-exposed neuronal cells exhibit dramatic reductions of intracellular NAD(+) followed by decreased ATP production, and are completely rescued by treatment with NAD(+) or its precursor nicotinamide because of restoration of physiological NAD(+) levels. Toxic prion protein-induced NAD(+) depletion results from PARP1-independent excessive protein ADP-ribosylations. In vivo, toxic prion protein-induced degeneration of hippocampal neurons is prevented dose-dependently by intracerebral injection of NAD(+). Intranasal NAD(+) treatment of prion-infected sick mice significantly improves activity and delays motor impairment. Our study reveals NAD(+) starvation as a novel mechanism of autophagy activation and neurodegeneration induced by a misfolded amyloidogenic protein. We propose the development of NAD(+) replenishment strategies for neuroprotection in prion diseases and possibly other protein misfolding neurodegenerative diseases.