Multimodal Analysis of Cell Types in a Hypothalamic Node Controlling Social Behavior in Mice

The advent and recent advances of single-cell RNA sequencing (scRNA-seq) have yielded transformative insights into our understanding of cellular diversity in the central nervous system (CNS) with unprecedented detail. However, due to current experimental and computational limitations on defining transcriptomic cell types (T-types) and the multiple phenotypic features of cell types in the CNS, an integrative and multimodal approach should be required for the comprehensive classification of cell types. To this end, performing multimodal analysis of scRNA-seq in hypothalamus would be very beneficial in that hypothalamus, controlling homeostatic and innate survival behaviors which known to be highly conserved across a wide range of species and encoded in hard-wired brain circuits, is likely to display the more straightforward relationship between transcriptomic identity, axonal projections, and behavioral activation, respectively. In my dissertation, I have been focused on the cell type characterizations of a hypothalamic node controlling innate social behavior in mice, the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl). VMHvl only contains ~4,000 neurons per hemisphere in mice but due to its behavioral, anatomical, and molecular heterogeneity, which T-types in VMHvl are related to connectivity and behavioral function is largely unknown. In Chapter II, I described my main thesis work to perform scRNA-seq in VMHvl using two independent platforms: SMART-seq2 (~4,500 neurons sequenced) and 10x (~78,000 neurons sequenced). Specifically, 17 joint VMHvl T-types including several sexually dimorphic clusters were identified by canonical correlation analysis (CCA) in Seurat, and the majority of them were validated by multiplexed single-molecule FISH (seqFISH). Correspondence between transcriptomic identity, and axonal projections or behavioral activation, respectively, was also investigated. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity. In Chapter III, I will discuss about future directions for a deeper and better understanding of VMHvl cell types. Briefly, my previous data from whole-cell patch clamp recording in VMHvl slices suggested that there were at least 4 distinct electrophysiological cell types (E-types). Additionally, two distinct neuromodulatory effects on VMHvl were observed (persistently activated by vasopressin/oxytocin vs. silenced by nitric oxide) by monitoring populational activities using two-photon Ca2+ imaging in slices. Based on the results from the first part and combined with advanced molecular techniques (e.g. Patch-seq and CRISPR-Cas9), we can further dissect out the cellular diversity in VMHvl and their functional implications.</p

Computational models of attachment and self-attachment

We explore, using a variety of models grounded in computational neuroscience, the dynamics of attachment formation and change. In the first part of the thesis we consider the formation of the traditional organised forms of attachment (as defined by Mary Ainsworth) within the context of the free energy principle, showing how each type of attachment might arise in infant agents who minimise free energy over interoceptive states while interacting with caregivers with varying responsiveness. We show how exteroceptive cues (in the form of disrupted affective communication from the caregiver) can result in disorganised forms of attachment (as first uncovered by Mary Main) in infants of caregivers who consistently increase stress on approach, but can have an organising (towards ambivalence) effect in infants of inconsistent caregivers. The second part of the thesis concerns Self-Attachment: a new self-administrable attachment-based psychotherapy recently introduced by Abbas Edalat, which aims to induce neural plasticity in order to retrain an individual's suboptimal attachment schema. We begin with a model of the hypothesised neurobiological underpinnings of the Self-Attachment bonding protocols, which are concerned with the formation of an abstract, self-directed bond. Finally, using neuroscientific findings related to empathy and the self-other distinction within the context of pain, we propose a simple spiking neural model for how empathic states might serve to motivate application of the aforementioned bonding protocols.Open Acces

Functional Substrates of Social Odor Processing within the Corticomedial Amygdala: Implications for Reproductive Behavior in Male Syrian Hamsters

Adaptive reproductive behavior requires the ability to recognize and approach possible mating partners in the environment. Syrian hamsters (Mesocricetus auratus) provide a useful animal model by which to study the neural processing of sexual signals, as mate recognition in this species relies almost exclusively on the perception of social odors. In the laboratory, male hamsters prefer to investigate female odors compared to male odors, and this opposite-sex odor preference provides a sensitive measure of the underlying neural processing of sexual stimuli. In addition to chemosensory cues, reproductive behavior in hamsters also requires sufficient levels of circulating gonadal steroid hormones, which reflect the reproductive state of the animal. These chemosensory and hormone signals are processed within an interconnected network of ventral forebrain nuclei, and within this network, the posteromedial cortical amygdala (PMCo) and medial amygdala (MA) are the only nuclei that both receive substantial chemosensory input and are also highly sensitive to steroid hormones. Although a large body of evidence suggests that the MA is critical for generating attraction to sexual odors, the specific role of the PMCo in regulating odor-guided aspects of male reproductive behavior has never been directly tested. Furthermore, detailed analyses of the MA suggest that separate, but interconnected sub-regions within this nucleus process odors differently. Specifically, the anterior MA (MeA) receives the majority of chemosensory input and responds to a variety of social odors, whereas the posterodorsal MA (MePD) receives less chemosensory input but contains the vast majority of steroid receptors. In order to further elucidate how the PMCo and/or MA process sexual odors, this dissertation addressed the following research questions: (1) Is the PMCo required for the expression of either opposite-sex odor preferences or male copulatory behavior? (2) Are functional interactions between MeA and MePD required for the expression of opposite-sex odor preferences? (3) How do MeA and MePD regulate odor responses within the MePD and MeA, respectively? (4) Are odor and/or hormone cues conveyed directly between MeA and MePD? Together, these experiments provide a comprehensive analysis of the functional and neuroanatomical substrates by which the brain processes sexual odors and generates appropriate behavioral responses to these stimuli

Optimized Design of a Self-Biased Amplifier for Seizure Detection Supplied by Piezoelectric Nanogenerator: Metaheuristic Algorithms versus ANN-Assisted Goal Attainment Method

This work is dedicated to parameter optimization for a self-biased amplifier to be used in preamplifiers for the diagnosis of seizures in neuro-diseases such as epilepsy. For the sake of maximum compactness, which is obligatory for all implantable devices, power is to be supplied by a piezoelectric nanogenerator (PENG). Several meta-heuristic optimization algorithms and an ANN (artificial neural network)-assisted goal attainment method were applied to the circuit, aiming to provide us with the set of optimal design parameters which ensure the minimal overall area of the preamplifier. These parameters are the slew rate, load capacitor, gain–bandwidth product, maximal input voltage, minimal input voltage, input voltage, reference voltage, and dissipation power. The results are re-evaluated and compared in the Cadence 180 nm SCL environment. It has been observed that, among the metaheuristic algorithms, the whale optimization technique reached the best values at low computational cost, decreased complexity, and the highest convergence speed. However, all metaheuristic algorithms were outperformed by the ANN-assisted goal attainment method, which produced a roughly 50% smaller overall area of the preamplifier. All the techniques described here are applicable to the design and optimization of wearable or implantable circuits

AN INVESTIGATION OF THE ROLE OF AMYGDALAR CIRCUITS IN THE PRODUCTION OF SOCIAL BEHAVIOR

Adaptive social behaviors allow animals to survive, thrive, and successfully reproduce. These behaviors, including mating, parenting, affiliation, and aggression, can be stereotyped in response to specific stimuli but often display sex-specific, and interoceptive-dependent variations in their execution. A conserved set of brain regions collectively known as the social behavior network (SBN) interprets sensory information about social cues and generates an appropriate behavioral response. In this dissertation I present 5 chapters. Chapter 1 introduces historical research focusing on the neural circuits that drive social behavior and the potential impact of environmental factors on the activity of these circuits. Chapter 2 describes a new technique that uses magnetohydrodynamic-based tissue clearing to investigate intact neural circuits rapidly and efficiently. Chapter 3 uses this approach to interrogate the synaptic connections of a primary hub for social sensory integration, the medial amygdala (MeA). I focused on neurons in the MeA that express an enzyme that plays an important role in the development of sex-specific social behaviors: aromatase and identified the sources of synaptic input to this population. These inputs included regions involved in maintaining metabolic homeostasis, production of socio-sexual behaviors, fear/anxiety, parenting, and aggressive behaviors –suggesting an expanded view of social behavior production. I demonstrate that the brain regions involved in the production of social behavior have broad access to internal physiological and external environmental information. Chapter 4, demonstrates the impact of external environmental factors on the behaviors produced in response to a social stimulus, as well as, on the early sensory representation of these stimuli in the AOB. Predator presence influences an animals’ responses to conspecific stimuli even when not presented concurrently. This effect was observed in males and females and in response to male and female stimuli, demonstrating a generalizable impact of environmental conditions on the sensory representation of social stimuli. Chapter 5 summarizes these findings in a broader context, arguing for an expanded role for the SBN in integrating internal and external environmental information with sensory perceptions of social stimuli to produce appropriate behavioral response for not only a specific social stimulus, but a specific environmental context

Neuroendokrine Effekte der endokrinen Disruptoren Vinclozolin und Equol in der erwachsenen männlichen Ratte

Zahlreiche anthropogene Chemikalien und Pflanzeninhaltsstoffe können die Funktion der endogenen Sexualsteroide durch eine direkte Interaktion mit deren spezifischen nukleären Rezeptoren beeinflussen. Derartige Substanzen werden als „Endokrine Disruptoren (EDs)“ bezeichnet. Es mehren sich aber die Hinweise, dass zumindest einige EDs hinsichtlich der Interaktion mit Steroidrezeptoren eine geringe Selektivität aufweisen, i.e. diese promisken EDs binden an mehr als einen nukleären Rezeptor. Ein Beispiel für einen derartigen promisken endokrinen ED ist das Fungizid Vinclozolin (VZ), welches am Androgenrezeptor (AR) antagonistische, am Östrogenrezeptor (ER) hingegen agonistische Wirkungen ausüben kann. Ein zweites Beispiel ist Equol (EQ), ein Metabolit des Isoflavonoids Daidzein. Es ist vielfach gezeigt worden, dass Equol mit dem ER interagiert, aber neueste Daten weisen auf eine mögliche Androgene Wirkung von EQ hin. Trotz der eindeutigen negativen Wirkungen von EDs auf die Entwicklung und Funktion reproduktiver Organe gibt es keine Untersuchungen zur möglichen Wirkung von VZ und EQ auf die Funktion des ZNS, insbesondere die neuroendokrine Regulation der hypothalomo-hypophysio-gonadalen Achse (HPG-Achse). Daher ist die Zielsetzung der vorliegenden Studie die Untersuchung, ob und wie VZ und EQ die neuroendokrine Regulation der HPG-Achse in erwachsenen, männlichen Ratten nach einer fünftägigen oralen Behandlung beeinflussen. Als Zielparameter wurde die Genexpression in der medialen, präoptischen Region des vorderen Hypothalamus (MPOA/AH), des mediobasalen Hypothalamus/Eminentia mediana (MBH/ME) sowie im Striatum, Hippocampus, Amygdala sowie der Hypophyse untersucht. Als Referenzorgane für steroidregulierte Gewebe wurden Prostata, Samenblase und Nebenhoden gewählt. Im Fall des Equols wurde zusätzlich der Effekt diese EDs auf das Sexualverhalten der männlichen Ratten untersucht. Als Goldstandard für ein reine Antiandrogen wurde Flutamid (FLUT) verwendet. Nach Beendigung der Behandlung, bzw, des Verhaltenstests, wurden die Tiere tierschutzgerecht getötet, das Blut gesammelt und die oben genannten Organe präpariert. Von der Prostata, der Samenblase, dem Nebenhoden und der Leber wurde das Frischgewicht bestimmt. In der Blutprobe wurden die Konzentrationen der Hormone LH, Testosteron (T), Dihydrotestosteron (DHT), TSH, T4, T3 sowie die Konzentrationen der Testsubstanzen selbst durch Radioimmunoassay bzw. HPLC bestimmt. Die Genexpression von Steroidrezeptoren sowie von steroidrezeptor-regulierten Genen in den oben genannten Organen wurde per Taqman-PCR gemessen. Die VZ-Behandlung resultierte in einem signifikant geringeren Gewicht der Nebenhoden und erhöhten Serumspiegeln von LH und T. Im Hypothalamus beeinflusste VZ die Expression von GnRH und von ER"Α" und ER"Β". In den extrahypothalamischen Arealen verursachte VZ eine Veränderung der Expression sowohl des ARs als auch der ERs. Die hypophysäre Expression von LH, von TERP-1/-2 sowie des GnRH-Rezeptors wurde durch VZ hochreguliert. In der Prostata hingegen reduzierte VZ die Expression des ER"Β"und erhöhte die mRNA-Spiegel des AR. In der Samenblase bewirkte die Behandlung mit VZ eine erhöhte Expression des AR und des ER"Α". Im Nebenhoden erhöhte sich unter VZ-Behandlung die Expression des AR und ER"Β". Die reduzierten Serumwerte von TSH und T4 werden wahrscheinlich durch einen direkten Effekt von VZ auf die thyreoptropen Zellen der Hypophyse hervorgerufen. FLUT erzeugte das erwartete Muster von Effekten eines AR-Antagonisten, i.e. reduzierte Gewichte der Prostata, der Samenblase und des Nebenhodens und erhöhte Serumspiegel von LH, T und DHT ohne Effekte auf die Prolaktinspiegel. EQ hingegen induzierte gegensätzliche Effekte. EQ als auch FLUT veränderten im Hypothalamus die Expression von GnRH und des GnRH-Rezeptors während in den extrahypothlamischen Arealen beide Substanzen die Expression von ER"Α" und AR in identischer Weise veränderten. In der Hypophyse hingegen waren unter EQ bzw. FLUT-Behandlung gegenläufige Effekte auf die Expression von ER"Α", TERP-1/-2, des GnRH-Rezeptors, der LH"Α"und "Β"-Untereinheit zu messen. Die Expression des ER"Α" und des AR in der Prostata wurde sowohl durch EQ als auch durch FLUT beeinflusst. Die fünftägige Behandlung der männlichen Ratten mit drei verschiedenen Dosierungen von EQ führte in keiner Behandlungsgruppe zu einer Veränderung des Sexualverhaltens. FLUT hingegen unterdrückte das Sexualverhalten in allen getesteten Tieren komplett. Die Serum T4- und T3- Spiegel wurden durch EQ und FLUT signifikant reduziert, was auf eine direkte Wirkung der Testsubstanzen auf die thyreotrophen Zellen der Hypophyse hinweist. Möglicherweise trägt auch der erhöhte hepatische Metabolismus der Schilddrüsenhormone zu deren reduzierten Serumspiegeln bei. Diese Interpretation einer Enzyminduktion steht im Einklang mit den erhöhten Lebergewichten. Zusammenfassend können aus der vorliegenden Studie folgende Schlussfolgerungen gezogen werden: 1. In vivo ist VZ kein reines Antiandrogen, sondern besitzt sowohl AR-antagonistische als ER-agonistische Eigenschaften im Gehirn, der Hypophyse und akzessorischen Geschlechtsorganen. 2. Equol hat eine ausgeprägte ED-Aktivität auf die HPG-Achse, die ausschließlich auf eine östrogene Wirkung zurückzuführen ist. Weder im Gehirn, der Hypophyse noch in peripheren Organen wie der Prostata konnten eine signifikante antiandrogene Wirkung von EQ nachgewiesen werden. 3. Die Referenzsubstanz FLUT erwies sich als ein starkes Antiandrogen hinsichtlich der neuroendokrinen Regulation der HPG-Achse und des androgeninduzierten männlichen Sexualverhaltens

The role of endogenous opioids and brain neurotransmitters in the generation of the LH surge in the rat

It has been suggested that a multiplicity of neurotransmitter systems regulate the release of gonadotrophin-releasing hormone (GnRH) from their neurons in the hypothalamus which in turn control luteinising hormone (LH) secretion from the anterior pituitary. An example of such neurotransmitter systems is that secreting the endogenous opioid peptides which have a profound inhibitory influence on the LH secretion. Recently, it has been reported that a reduction in the activity of these peptides in the hypothalamus may be the initial neural stimulus for the generation of the pre-ovulatory LH surge which induces ovulation. Furthermore, this inhibitory opioidergic action may involve alterations in the activity of monoaminergic neurons which make synaptic contacts with the GnRH neurons in the hypothalamus. Naloxone-, the opioid antagonist, induced LH release can be prevented by prior administration of a-adrenergic blockers. The central noradrenergic system is believed to be an essential component of the GnRH pulse-generating mechanism. The present study was undertaken to determine (i) the modulating effects of specific mu-, kappa- and delta-opioid receptor agonists and antagonists on hypothalamic monoaminergic content and on LH release and (ii) the inter-relationship between the opioid peptidergic and aminergic systems in the control of GnRH activity. The results indicate that multiple opioid receptor subtypes are involved in the opioid suppression of the pre-ovulatory LH surge. It appears that opioid modulation of noradrenaline, dopamine and serotonin release and/or turnover within the specific areas of the hypothalamus via mu-, kappa- and delta-opioid receptors is inhibitory at the time of the LH surge. This study supports the concept that an increase in the hypothalamic noradrenergic activity is a critical event in triggering the surge release of GnRH and LH. Both the dopaminergic and serotonergic neurotransmitter systems may also facilitate the pre-ovulatory LH surge, although their effects are thought to be of minor importance in this process. In view of the unexpected interference by both urethane and ketamine anaesthesia on plasma LH levels, it is recommended that use of general anaesthetics in neuroendocrine studies should be avoided if at all possible. (Abstract shortened by ProQuest.)

Behavioral Sex Differences Caused by Distinct Vasopressin Sources

Dysfunction in social communication is a prominent aspect of many psychopathologies and social disorders including autism, schizophrenia, and social anxiety. Consequently, development of clinical treatment for these disorders requires an understanding of neural circuitry underlying social communication. Sex differences are a persistent feature of social disorders, where autism is more prevalent in males, while social anxiety occurs more frequently in females. A critical gap in knowledge exists in understanding the role of sex-differences in the control of social behavior and communication. A reasonable hypothesis is that differences in neural circuitry underlie sex-differentiated dysfunctions in social behavior and communication. A well-studied circuit in this regard is the sexually dimorphic expression of the neuropeptide arginine vasopressin (AVP). AVP in the nervous system originates from several distinct sources which are, in turn, regulated by different inputs and regulatory factors. Using modern molecular approaches, we can begin to define the specific role of AVP cell populations in social behavior. We demonstrate a behavioral function for the sexually dimorphic AVP neurons in the bed nucleus of the stria terminalis (BNST) and in the paraventricular nucleus of the hypothalamus (PVN). Collectively, our results indicate that AVP cell groups appear to play opposite roles in social investigation by males and females, as BNST-AVP cell ablations and BNST AVP knockdown reduced male social approach, while PVN-AVP cell ablations increased female social approach. We next utilized circuit level tracing techniques to map the inputs and outputs of BNST and medial amygdala (MeA) AVP cells, which are the major source of sexually dimorphic AVP expression. Finally, we tested the function of several sexually dimorphic BNST-AVP projection areas, such as, the lateral septum (LS), lateral habenula (LHb), and dorsal raphe (DR). In male mice, but not female mice, optogenetic stimulation of the BNST AVP terminals in the LS increased their social investigation and anxiety-like behavior in the elevated-zero maze. Antagonism of V1aR in the LS blocked optogenetic-mediated increases in male social investigation and anxiety-like behavior. Therefore, activation of a distinct BNST-LS AVP circuit modulates sex-specific social approach and anxiety-like behavior, which is mediated by V1aR within the LS. This work suggests that sex differences in the neurochemical underpinnings of social behavior may contribute to sex differences in disorders of social behavior and communication

Brain mechanisms of sexual disinhibition in the male rats by alcohol and amphetamine

The expression of sexual behavior requires a proper balance between inhibition and excitation at cognitive, hormonal and neurochemical levels. Inhibition of sexual behavior is considered to be important and even crucial to maintain proper social cohesion and adaptation (i.e. in response to threat, aversion, culture, fertility). In humans, spreading of sexually transmitted diseases is often due to inappropriate and risky sexual behavior (e.g. multiple partners, no use of condoms) or to a loosening of inhibitions under the influence of drugs. It is therefore important to understand where these drugs (recreational, or prescription) act to induce disinhibited sexual behavior. In this thesis, the mechanisms of action of two drugs associated famously with sexual disinhibition but possessing two largely opposite mechanisms of action, alcohol and d-amphetamine, are investigated. This is done using a conditioned partner avoidance paradigm in which male rats are trained to avoid copulation with females bearing a conditioned inhibitory olfactory cue associated with non reward. This paradigm has been used previously to reveal the disinhibitory properties of low doses of alcohol. In the first chapter, it is established that d-amphetamine disrupts conditioned partner avoidance as d-amphetamine-treated males still copulate with females bearing the conditioned inhibitory cue. Furthermore, similar to alcohol, exposure to the inhibitory cue alone under the influence of d-amphetamine activates brain regions associated with sexual behavior and sexual reward. As alcohol is known to induce disinhibition of behavior in general and of sexual behavior in particular, the focus of the second and third chapters is to examine the potential neurotransmitters involved in the disinhibitory effect of alcohol. Using microdialysis, it is found that levels of dopamine in the nucleus accumbens are increased when alcohol-treated male rats are exposed to the sexually conditioned inhibitory cue. This increase is due to activation of dopaminergic neurons from the ventral tegmental area. On the other hand, neither alcohol nor the conditioned cue has an effect on glutamate or GABA levels in the nucleus accumbens. Together these data demonstrate that dopamine release in the nucleus accumbens via activation of dopaminergic neurons in the ventral tegmental area plays a permissive role in the disinhibitory effects of alcohol (and likely amphetamine) on the sexual behavior of male rats

Socially driven changes in neural and behavioural plasticity in zebrafish

Tese de doutoramento, Biologia (Etologia), Universidade de Lisboa, Faculdade de Ciências, 2015Social competence, the ability of individuals to regulate the expression of their social behaviour in order to optimize their social relationships in a group, is especially benefic for individuals living in complex social environments, and implies the ability to perceive social cues and produce appropriate behavioural output responses (Social Plasticity). Numerous examples of social competence can be found in nature, where individuals extract social information from the environment, and change their behavioural response based on the collected information. At the neuronal level, two major plasticity mechanisms have been proposed to underlie social plasticity, structural reorganization and biochemical switching of the neuronal networks underlying behaviour. The neural substrate for behavioural plasticity has been identified as the social decision-making (SDM) network, such that the same neural circuitry may underlie the expression of different behaviours depending on social context. The goal of this work is to study the proximate mechanism underlying behavioural flexibility in the context of experience-dependent behavioural shifts, in an integrative framework. For this purpose we exposed male zebrafish to two types of social interactions: (1) real-opponent interactions, from which a Winner and Loser emerged; and (2) Mirror-elicited interactions, that produced individuals that did not experience a change in social status, despite expressing similar levels of aggressive behaviour to those participating in real-opponent fights. In a first set of experiments, we studied the influence of neuromodulators on social plasticity mechanisms, by characterizing the endocrine response to social challenges, as well as the social modulation of brain monoamines and nonapeptides. Next we tested the SDM network hypothesis by contrasting changes in functional localization vs. connectivity across this network. Finally we characterized changes in expression of key genes for different neuroplasticity mechanisms in response to changes in social status. Our research suggests different social plasticity mechanisms underlying Winners and Losers both at physiological and molecular levels, for Mirror-fighters, where the experience of winning or losing was decoupled for the fighting experience, few changes were detected. This, by itself suggests a pivotal role of social perception in triggering shifts between socially driven behavioural states