Neuronal Subtypes and Connectivity of the Adult Mouse Paralaminar Amygdala.

The paralaminar nucleus of the amygdala (PL) comprises neurons that exhibit delayed maturation. PL neurons are born during gestation but mature during adolescent ages, differentiating into excitatory neurons. These late-maturing PL neurons contribute to the increase in size and cell number of the amygdala between birth and adulthood. However, the function of the PL upon maturation is unknown, as the region has only recently begun to be characterized in detail. In this study, we investigated key defining features of the adult mouse PL; the intrinsic morpho-electric properties of its neurons, and its input and output circuit connectivity. We identify two subtypes of excitatory neurons in the PL based on unsupervised clustering of electrophysiological properties. These subtypes are defined by differential action potential firing properties and dendritic architecture, suggesting divergent functional roles. We further uncover major axonal inputs to the adult PL from the main olfactory network and basolateral amygdala. We also find that axonal outputs from the PL project reciprocally to these inputs and to diverse targets including the amygdala, frontal cortex, hippocampus, hypothalamus, and brainstem. Thus, the adult mouse PL is centrally placed to play a major role in the integration of olfactory sensory information, to coordinate affective and autonomic behavioral responses to salient odor stimuli.

Neuronal subtypes and connectivity of the adult mouse paralaminar amygdala.

The paralaminar nucleus of the amygdala (PL) is comprised of neurons which exhibit delayed maturation. PL neurons are born during gestation but mature during adolescent ages, differentiating into excitatory neurons. The PL is prominent in the adult amygdala, contributing to its increased neuron number and relative size compared to childhood. However, the function of the PL is unknown, as the region has only recently begun to be characterized in detail. In this study, we investigated key defining features of the adult PL; the intrinsic morpho-electric properties of its neurons, and its input and output connectivity. We identify two subtypes of excitatory neurons in the PL based on unsupervised clustering of electrophysiological properties. These subtypes are defined by differential action potential firing properties and dendritic architecture, suggesting divergent functional roles. We further uncover major axonal inputs to the adult PL from the main olfactory network and basolateral amygdala. We also find that axonal outputs from the PL project reciprocally to major inputs, and to diverse targets including the amygdala, frontal cortex, hippocampus, hypothalamus, and brainstem. Thus, the adult PL is centrally placed to play a major role in the integration of olfactory sensory information, likely coordinating affective and autonomic behavioral responses to salient odor stimuli. Significance Statement: Mammalian amygdala development includes a growth period from childhood to adulthood, believed to support emotional and social learning. This amygdala growth is partly due to the maturation of neurons during adolescence in the paralaminar amygdala. However, the functional properties of these neurons are unknown. In our recent studies, we characterized the paralaminar amygdala in the mouse. Here, we investigate the properties of the adult PL in the mouse, revealing the existence of two neuronal subtypes that may play distinct functional roles in the adult brain. We further reveal the brain-wide input and output connectivity of the PL, indicating that the PL combines olfactory cues for emotional processing and delivers information to regions associated with reward and autonomic states.

Delayed maturation and migration of excitatory neurons in the juvenile mouse paralaminar amygdala.

The human amygdala paralaminar nucleus (PL) contains many immature excitatory neurons that undergo prolonged maturation from birth to adulthood. We describe a previously unidentified homologous PL region in mice that contains immature excitatory neurons and has previously been considered part of the amygdala intercalated cell clusters or ventral endopiriform cortex. Mouse PL neurons are born embryonically, not from postnatal neurogenesis, despite a subset retaining immature molecular and morphological features in adults. During juvenile-adolescent ages (P21-P35), the majority of PL neurons undergo molecular, structural, and physiological maturation, and a subset of excitatory PL neurons migrate into the adjacent endopiriform cortex. Alongside these changes, PL neurons develop responses to aversive and appetitive olfactory stimuli. The presence of this homologous region in both humans and mice points to the significance of this conserved mechanism of neuronal maturation and migration during adolescence, a key time period for amygdala circuit maturation and related behavioral changes.

Connectivity and molecular profiles of Foxp2- and Dbx1-lineage neurons in the accessory olfactory bulb and medial amygdala.

In terrestrial vertebrates, the olfactory system is divided into main (MOS) and accessory (AOS) components that process both volatile and nonvolatile cues to generate appropriate behavioral responses. While much is known regarding the molecular diversity of neurons that comprise the MOS, less is known about the AOS. Here, focusing on the vomeronasal organ (VNO), the accessory olfactory bulb (AOB), and the medial amygdala (MeA), we reveal that populations of neurons in the AOS can be molecularly subdivided based on their ongoing or prior expression of the transcription factors Foxp2 or Dbx1, which delineate separate populations of GABAergic output neurons in the MeA. We show that a majority of AOB neurons that project directly to the MeA are of the Foxp2 lineage. Using single-neuron patch-clamp electrophysiology, we further reveal that in addition to sex-specific differences across lineage, the frequency of excitatory input to MeA Dbx1- and Foxp2-lineage neurons differs between sexes. Together, this work uncovers a novel molecular diversity of AOS neurons, and lineage and sex differences in patterns of connectivity.

Transcriptionally defined amygdala subpopulations play distinct roles in innate social behaviors.

Social behaviors are innate and supported by dedicated neural circuits, but the molecular identities of these circuits and how they are established developmentally and shaped by experience remain unclear. Here we show that medial amygdala (MeA) cells originating from two embryonically parcellated developmental lineages have distinct response patterns and functions in social behavior in male mice. MeA cells expressing the transcription factor Foxp2 (MeAFoxp2) are specialized for processing male conspecific cues and are essential for adult inter-male aggression. By contrast, MeA cells derived from the Dbx1 lineage (MeADbx1) respond broadly to social cues, respond strongly during ejaculation and are not essential for male aggression. Furthermore, MeAFoxp2 and MeADbx1 cells show differential anatomical and functional connectivity. Altogether, our results suggest a developmentally hardwired aggression circuit at the MeA level and a lineage-based circuit organization by which a cell's embryonic transcription factor profile determines its social information representation and behavioral relevance during adulthood.

An unbiased seed-based RNAi selection screen identifies small RNAs that inhibit androgen signaling and prostate cancer cell growth.

Blocking androgen receptor signaling is the mainstay of therapy for advanced prostate cancer (PCa). However, acquired resistance to single agents targeting this pathway results in the development of lethal castration-resistant PCa. Combination therapy approaches represent a promising strategy for the treatment of advanced disease. Here, we explore a therapeutic strategy for PCa based on the ability of shRNAs/siRNAs to function essentially as miRNAs and, via seed sequence complementarity, induce RNA interference of numerous targets simultaneously. We developed a library that contained shRNAs with all possible seed sequence combinations to identify those ones that most potently reduce cell growth and viability when expressed in PCa cells. Validation of some of these RNAi sequences indicated that the toxic effect is associated with seed sequence complementarity to the 3' UTR of AR coregulatory and essential genes. In fact, expression of siRNAs containing the identified toxic seed sequences led to global inhibition of AR-mediated gene expression and reduced expression of cell-cycle genes. When tested in mice, the toxic shRNAs also inhibited castration-resistant PCa and exhibited therapeutic efficacy in pre-established tumors. Our findings highlight RNAi of androgen signaling networks as a promising therapeutic strategy for PCa.

Onco-condensates: formation, multi-component organization, and biological functions.

Numerous cellular processes occur in the context of condensates, a type of large, membrane-less biomolecular assembly generated through phase separation. These condensates function as a hub of diversified cellular events by concentrating the required components. Cancer frequently coopts biomolecular condensation mechanisms to promote survival and/or proliferation. Onco-condensates, which refer to those that have causal roles or are critically involved in tumorigenicity, operate to abnormally elevate biological output of a proliferative process, or to suppress a tumor-suppressive pathway, thereby promoting oncogenesis. Here, we summarize advances regarding how multi-component onco-condensates are established and organized to promote oncogenesis, with those related to chromatin and transcription deregulation used as showcases. A better understanding should enable development of new means of targeting onco-condensates as potential therapeutics.

Hardwired to attack: Transcriptionally defined amygdala subpopulations play distinct roles in innate social behaviors.

Social behaviors are innate and supported by dedicated neural circuits, but it remains unclear whether these circuits are developmentally hardwired or established through social experience. Here, we revealed distinct response patterns and functions in social behavior of medial amygdala (MeA) cells originating from two embryonically parcellated developmental lineages. MeA cells in male mice that express the transcription factor Foxp2 (MeAFoxp2) are specialized for processing male conspecific cues even before puberty and are essential for adult inter-male aggression. In contrast, MeA cells derived from the Dbx1-lineage (MeADbx1) respond broadly to social cues and are non-essential for male aggression. Furthermore, MeAFoxp2 and MeADbx1 cells show differential anatomical and functional connectivity. Altogether, our results support a developmentally hardwired aggression circuit at the level of the MeA and we propose a lineage-based circuit organization by which a cell's embryonic transcription factor profile determines its social information representation and behavior relevance during adulthood.

Sex-Specific Social Behavior and Amygdala Proteomic Deficits in Foxp2 +/- Mutant Mice.

In humans, mutations in the transcription factor encoding gene, FOXP2, are associated with language and Autism Spectrum Disorders (ASD), the latter characterized by deficits in social interactions. However, little is known regarding the function of Foxp2 in male or female social behavior. Our previous studies in mice revealed high expression of Foxp2 within the medial subnucleus of the amygdala (MeA), a limbic brain region highly implicated in innate social behaviors such as mating, aggression, and parental care. Here, using a comprehensive panel of behavioral tests in male and female Foxp2 +/- heterozygous mice, we investigated the role Foxp2 plays in MeA-linked innate social behaviors. We reveal significant deficits in olfactory processing, social interaction, mating, aggressive, and parental behaviors. Interestingly, some of these deficits are displayed in a sex-specific manner. To examine the consequences of Foxp2 loss of function specifically in the MeA, we conducted a proteomic analysis of microdissected MeA tissue. This analyses revealed putative sex differences expression of a host of proteins implicated in neuronal communication, connectivity, and dopamine signaling. Consistent with this, we discovered that MeA Foxp2-lineage cells were responsive to dopamine with differences between males and females. Thus, our findings reveal a central and sex-specific role for Foxp2 in social behavior and MeA function.

Seed-mediated RNA interference of androgen signaling and survival networks induces cell death in prostate cancer cells.

Resistance to anti-androgen therapy in prostate cancer (PCa) is often driven by genetic and epigenetic aberrations in the androgen receptor (AR) and coregulators that maintain androgen signaling activity. We show that specific small RNAs downregulate expression of multiple essential and androgen receptor-coregulatory genes, leading to potent androgen signaling inhibition and PCa cell death. Expression of different short hairpin/small interfering RNAs (sh-/siRNAs) designed to target TMEFF2 preferentially reduce viability of PCa but not benign cells, and growth of murine xenografts. Surprisingly, this effect is independent of TMEFF2 expression. Transcriptomic and sh/siRNA seed sequence studies indicate that expression of these toxic shRNAs lead to downregulation of androgen receptor-coregulatory and essential genes through mRNA 3' UTR sequence complementarity to the seed sequence of the toxic shRNAs. These findings reveal a form of the "death induced by survival gene elimination" mechanism in PCa cells that mainly targets AR signaling, and that we have termed androgen network death induced by survival gene elimination (AN-DISE). Our data suggest that AN-DISE may be a novel therapeutic strategy for PCa.

Sex Differences in Biophysical Signatures across Molecularly Defined Medial Amygdala Neuronal Subpopulations.

The medial amygdala (MeA) is essential for processing innate social and non-social behaviors, such as territorial aggression and mating, which display in a sex-specific manner. While sex differences in cell numbers and neuronal morphology in the MeA are well established, if and how these differences extend to the biophysical level remain unknown. Our previous studies revealed that expression of the transcription factors, Dbx1 and Foxp2, during embryogenesis defines separate progenitor pools destined to generate different subclasses of MEA inhibitory output neurons. We have also previously shown that Dbx1-lineage and Foxp2-lineage neurons display different responses to innate olfactory cues and in a sex-specific manner. To examine whether these neurons also possess sex-specific biophysical signatures, we conducted a multidimensional analysis of the intrinsic electrophysiological profiles of these transcription factor defined neurons in the male and female MeA. We observed striking differences in the action potential (AP) spiking patterns across lineages, and across sex within each lineage, properties known to be modified by different voltage-gated ion channels. To identify the potential mechanism underlying the observed lineage-specific and sex-specific differences in spiking adaptation, we conducted a phase plot analysis to narrow down putative ion channel candidates. Of these candidates, we found a subset expressed in a lineage-biased and/or sex-biased manner. Thus, our results uncover neuronal subpopulation and sex differences in the biophysical signatures of developmentally defined MeA output neurons, providing a potential physiological substrate for how the male and female MeA may process social and non-social cues that trigger innate behavioral responses.

Identification of amygdala-expressed genes associated with autism spectrum disorder.

BACKGROUND: Studies of individuals with autism spectrum disorder (ASD) have revealed a strong multigenic basis with the identification of hundreds of ASD susceptibility genes. ASD is characterized by social deficits and a range of other phenotypes, implicating complex genetics and involvement of a variety of brain regions. However, how mutations and mis-expression of select gene sets are associated with the behavioral components of ASD remains unknown. We reasoned that for genes to be associated with ASD core behaviors they must be: (1) expressed in brain regions relevant to ASD social behaviors and (2) expressed during the ASD susceptible window of brain development. METHODS: Focusing on the amygdala, a brain region whose dysfunction has been highly implicated in the social component of ASD, we mined publicly available gene expression databases to identify ASD-susceptibility genes expressed during human and mouse amygdala development. We found that a large cohort of known ASD susceptibility genes is expressed in the developing human and mouse amygdala. We further performed analysis of single-nucleus RNA-seq (snRNA-seq) data from microdissected amygdala tissue from five ASD and five control human postmortem brains ranging in age from 4 to 20 years to elucidate cell type specificity of amygdala-expressed genes and their dysregulation in ASD. RESULTS: Our analyses revealed that of the high-ranking ASD susceptibility genes, 80 are expressed in both human and mouse amygdala during fetal to early postnatal stages of development. Our human snRNA-seq analyses revealed cohorts of genes with altered expression in the ASD amygdala postnatally, especially within excitatory neurons, with dysregulated expression of seven genes predicted from our datamining pipeline. LIMITATIONS: We were limited by the ages for which we were able to obtain human tissue; therefore, the results from our datamining pipeline approach will require validation, to the extent possible, in human tissue from earlier developmental stages. CONCLUSIONS: Our pipeline narrows down the number of amygdala-expressed genes possibly involved in the social pathophysiology of ASD. Our human single-nucleus gene expression analyses revealed that ASD is characterized by changes in gene expression in specific cell types in the early postnatal amygdala.

Biomimetic 2-Imino-Nazarov Cyclizations via Eneallene Aziridination.

Amidoallyl cations are appealing three-carbon synthons for the preparation of complex amine-containing carbocycles; however, methods to generate and utilize these reactive species are limited and underexplored compared to those for oxallyl cations. Here we disclose a bioinspired strain-driven ring opening of bicyclic methyleneaziridines to 2-amidopentadienyl cation intermediates that readily engage in Nazarov cyclizations. Advantages of this strategy include ease of generation and improved reactivity compared to 3-pentadienyl cations, control over the ultimate position of the alkene, the potential for high dr between vicinal stereocenters, and the ability to further elaborate the products to fully substituted aminocyclopentanes. Experimental and computational studies support a dual role for the Rh2Ln complex as both a nitrene transfer catalyst and a Lewis acid promoter, insight that provides a framework for the future development of asymmetric 2-imino-Nazarov cyclizations.

Kcnn2 blockade reverses learning deficits in a mouse model of fetal alcohol spectrum disorders.

Learning disabilities are hallmarks of congenital conditions caused by prenatal exposure to harmful agents. These include fetal alcohol spectrum disorders (FASDs) with a wide range of cognitive deficiencies, including impaired motor skill development. Although these effects have been well characterized, the molecular effects that bring about these behavioral consequences remain to be determined. We previously found that the acute molecular responses to alcohol in the embryonic brain are stochastic, varying among neural progenitor cells. However, the pathophysiological consequences stemming from these heterogeneous responses remain unknown. Here we show that acute responses to alcohol in progenitor cells altered gene expression in their descendant neurons. Among the altered genes, an increase of the calcium-activated potassium channel Kcnn2 in the motor cortex correlated with motor learning deficits in a mouse model of FASD. Pharmacologic blockade of Kcnn2 improves these learning deficits, suggesting Kcnn2 blockers as a new intervention for learning disabilities in FASD.

HuR Reduces Radiation-Induced DNA Damage by Enhancing Expression of ARID1A.

Tumor suppressor ARID1A, a subunit of the chromatin remodeling complex SWI/SNF, regulates cell cycle progression, interacts with the tumor suppressor TP53, and prevents genomic instability. In addition, ARID1A has been shown to foster resistance to cancer therapy. By promoting non-homologous end joining (NHEJ), ARID1A enhances DNA repair. Consequently, ARID1A has been proposed as a promising therapeutic target to sensitize cancer cells to chemotherapy and radiation. Here, we report that ARID1A is regulated by human antigen R (HuR), an RNA-binding protein that is highly expressed in a wide range of cancers and enables resistance to chemotherapy and radiation. Our results indicate that HuR binds ARID1A mRNA, thereby increasing its stability in breast cancer cells. We further find that ARID1A expression suppresses the accumulation of DNA double-strand breaks (DSBs) caused by radiation and can rescue the loss of radioresistance triggered by HuR inhibition, suggesting that ARID1A plays an important role in HuR-driven resistance to radiation. Taken together, our work shows that HuR and ARID1A form an important regulatory axis in radiation resistance that can be targeted to improve radiotherapy in breast cancer patients.

A TMEFF2-regulated cell cycle derived gene signature is prognostic of recurrence risk in prostate cancer.

BACKGROUND: The clinical behavior of prostate cancer (PCa) is variable, and while the majority of cases remain indolent, 10% of patients progress to deadly forms of the disease. Current clinical predictors used at the time of diagnosis have limitations to accurately establish progression risk. Here we describe the development of a tumor suppressor regulated, cell-cycle gene expression based prognostic signature for PCa, and validate its independent contribution to risk stratification in several radical prostatectomy (RP) patient cohorts. METHODS: We used RNA interference experiments in PCa cell lines to identify a gene expression based gene signature associated with Tmeff2, an androgen regulated, tumor suppressor gene whose expression shows remarkable heterogeneity in PCa. Gene expression was confirmed by qRT-PCR. Correlation of the signature with disease outcome (time to recurrence) was retrospectively evaluated in four geographically different cohorts of patients that underwent RP (834 samples), using multivariate logistical regression analysis. Multivariate analyses were adjusted for standard clinicopathological variables. Performance of the signature was compared to previously described gene expression based signatures using the SigCheck software. RESULTS: Low levels of TMEFF2 mRNA significantly (p < 0.0001) correlated with reduced disease-free survival (DFS) in patients from the Memorial Sloan Kettering Cancer Center (MSKCC) dataset. We identified a panel of 11 TMEFF2 regulated cell cycle related genes (TMCC11), with strong prognostic value. TMCC11 expression was significantly associated with time to recurrence after prostatectomy in four geographically different patient cohorts (2.9 ≤ HR ≥ 4.1; p ≤ 0.002), served as an independent indicator of poor prognosis in the four RP cohorts (1.96 ≤ HR ≥ 4.28; p ≤ 0.032) and improved the prognostic value of standard clinicopathological markers. The prognostic ability of TMCC11 panel exceeded previously published oncogenic gene signatures (p = 0.00017). CONCLUSIONS: This study provides evidence that the TMCC11 gene signature is a robust independent prognostic marker for PCa, reveals the value of using highly heterogeneously expressed genes, like Tmeff2, as guides to discover prognostic indicators, and suggests the possibility that low Tmeff2 expression marks a distinct subclass of PCa.

Rigor and reproducibility in rodent behavioral research.

Behavioral neuroscience research incorporates the identical high level of meticulous methodologies and exacting attention to detail as all other scientific disciplines. To achieve maximal rigor and reproducibility of findings, well-trained investigators employ a variety of established best practices. Here we explicate some of the requirements for rigorous experimental design and accurate data analysis in conducting mouse and rat behavioral tests. Novel object recognition is used as an example of a cognitive assay which has been conducted successfully with a range of methods, all based on common principles of appropriate procedures, controls, and statistics. Directors of Rodent Core facilities within Intellectual and Developmental Disabilities Research Centers contribute key aspects of their own novel object recognition protocols, offering insights into essential similarities and less-critical differences. Literature cited in this review article will lead the interested reader to source papers that provide step-by-step protocols which illustrate optimized methods for many standard rodent behavioral assays. Adhering to best practices in behavioral neuroscience will enhance the value of animal models for the multiple goals of understanding biological mechanisms, evaluating consequences of genetic mutations, and discovering efficacious therapeutics.

PAC1R Genotype to Phenotype Correlations in Autism Spectrum Disorder.

Amygdala dysfunction has been implicated in numerous neurodevelopmental disorders, including autism spectrum disorder (ASD). Previous studies in mice and humans, respectively, have linked Pac1r/PAC1R function to social behavior and PTSD-susceptibility. Based on this connection to social and emotional processing and the central role played by the amygdala in ASD, we examined a putative role for PAC1R in social deficits in ASD and determined the pattern of gene expression in the developing mouse and human amygdala. We reveal that Pac1r/PAC1R is expressed in both mouse and human amygdala from mid-neurogenesis through early postnatal stages, critical time points when altered brain trajectories are hypothesized to unfold in ASD. We further find that parents of autistic children carrying a previously identified PTSD-risk genotype (CC) report greater reciprocal social deficits compared to those carrying the non-risk GC genotype. Additionally, by exploring resting-state functional connectivity differences in a subsample of the larger behavioral sample, we find higher functional connectivity between the amygdala and right middle temporal gyrus in individuals with the CC risk genotype. Thus, using multimodal approaches, our data reveal that the amygdala-expressed PAC1R gene may be linked to severity of ASD social phenotype and possible alterations in brain connectivity, therefore potentially acting as a modifier of amygdala-related phenotypes. Autism Res 2019, 12: 200-211 © 2018 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: In this multimodal study across mouse and human, we examined expression patterns of Pac1r/PAC1R, a gene implicated in social behavior, and further explored whether a previously identified human PTSD-linked mutation in PAC1R can predict brain connectivity and social deficits in ASD. We find that PAC1R is highly expressed in the both the mouse and human amygdala. Furthermore, our human data suggest that PAC1R genotype is linked to severity of social deficits and functional amygdala connectivity in ASD.

Amygdala Corticofugal Input Shapes Mitral Cell Responses in the Accessory Olfactory Bulb.

Interconnections between the olfactory bulb and the amygdala are a major pathway for triggering strong behavioral responses to a variety of odorants. However, while this broad mapping has been established, the patterns of amygdala feedback connectivity and the influence on olfactory circuitry remain unknown. Here, using a combination of neuronal tracing approaches, we dissect the connectivity of a cortical amygdala [posteromedial cortical nucleus (PmCo)] feedback circuit innervating the mouse accessory olfactory bulb. Optogenetic activation of PmCo feedback mainly results in feedforward mitral cell (MC) inhibition through direct excitation of GABAergic granule cells. In addition, LED-driven activity of corticofugal afferents increases the gain of MC responses to olfactory nerve stimulation. Thus, through corticofugal pathways, the PmCo likely regulates primary olfactory and social odor processing.

Method for Small-Scale Production of Deuterochloroform.

Deuterochloroform (CDCl3) is a common deuterated solvent for nuclear magnetic resonance (NMR) analyses. The synthesis of significant amounts of CDCl3 for both research use and large undergraduate organic laboratories in a safe and inexpensive manner is appealing. Herein, we describe a convenient laboratory scale preparation of CDCl3 that employs a reduction and decarboxylation of hexachloro-2-propanone (HCP) catalyzed by various pyridines. A PVP catalyst gives cleaner reaction and greater catalyst stability through multiple rounds of recycling, justifying its higher cost compared to pyridine.