Monitoring norepinephrine release in vivo using next-generation GRABNE sensors.

Norepinephrine (NE) is an essential biogenic monoamine neurotransmitter. The first-generation NE sensor makes in vivo, real-time, cell-type-specific and region-specific NE detection possible, but its low NE sensitivity limits its utility. Here, we developed the second-generation GPCR-activation-based NE sensors (GRABNE2m and GRABNE2h) with a superior response and high sensitivity and selectivity to NE both in vitro and in vivo. Notably, these sensors can detect NE release triggered by either optogenetic or behavioral stimuli in freely moving mice, producing robust signals in the locus coeruleus and hypothalamus. With the development of a novel transgenic mouse line, we recorded both NE release and calcium dynamics with dual-color fiber photometry throughout the sleep-wake cycle; moreover, dual-color mesoscopic imaging revealed cell-type-specific spatiotemporal dynamics of NE and calcium during sensory processing and locomotion. Thus, these new GRABNE sensors are valuable tools for monitoring the precise spatiotemporal release of NE in vivo, providing new insights into the physiological and pathophysiological roles of NE.

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.

Neural dynamics in the limbic system during male social behaviors.

Sexual and aggressive behaviors are vital for species survival and individual reproductive success. Although many limbic regions have been found relevant to these behaviors, how social cues are represented across regions and how the network activity generates each behavior remains elusive. To answer these questions, we utilize multi-fiber photometry (MFP) to simultaneously record Ca2+ signals of estrogen receptor alpha (Esr1)-expressing cells from 13 limbic regions in male mice during mating and fighting. We find that conspecific sensory information and social action signals are widely distributed in the limbic system and can be decoded from the network activity. Cross-region correlation analysis reveals striking increases in the network functional connectivity during the social action initiation phase, whereas late copulation is accompanied by a "dissociated" network state. Based on the response patterns, we propose a mating-biased network (MBN) and an aggression-biased network (ABN) for mediating male sexual and aggressive behaviors, respectively.

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.

VMHvllCckar cells dynamically control female sexual behaviors over the reproductive cycle.

Sexual behavior is fundamental for the survival of mammalian species and thus supported by dedicated neural substrates. The ventrolateral part of ventromedial hypothalamus (VMHvl) is an essential locus for controlling female sexual behaviors, but recent studies revealed the molecular complexity and functional heterogeneity of VMHvl cells. Here, we identify the cholecystokinin A receptor (Cckar)-expressing cells in the lateral VMHvl (VMHvllCckar) as the key controllers of female sexual behaviors. The inactivation of VMHvllCckar cells in female mice diminishes their interest in males and sexual receptivity, whereas activating these cells has the opposite effects. Female sexual behaviors vary drastically over the reproductive cycle. In vivo recordings reveal reproductive-state-dependent changes in VMHvllCckar cell spontaneous activity and responsivity, with the highest activity occurring during estrus. These in vivo response changes coincide with robust alternation in VMHvllCckar cell excitability and synaptic inputs. Altogether, VMHvllCckar cells represent a key neural population dynamically controlling female sexual behaviors over the reproductive cycle.

Neural mechanisms of aggression across species.

Aggression is a social behavior essential for securing resources and defending oneself and family. Thanks to its indispensable function in competition and thus survival, aggression exists widely across animal species, including humans. Classical works from Tinbergen and Lorenz concluded that instinctive behaviors including aggression are mediated by hardwired brain circuitries that specialize in processing certain sensory inputs to trigger stereotyped motor outputs. They further suggest that instinctive behaviors are influenced by an animal's internal state and past experiences. Following this conceptual framework, here we review our current understanding regarding the neural substrates underlying aggression generation, highlighting an evolutionarily conserved 'core aggression circuit' composed of four subcortical regions. We further discuss the neural mechanisms that support changes in aggression based on the animal's internal state. We aim to provide an overview of features of aggression and the relevant neural substrates across species, highlighting findings in rodents, primates and songbirds.

Looming Danger: Unraveling the Circuitry for Predator Threats.

Threat avoidance, particularly from predators, is key for survival. Through the use of optogenetics, viral tracing, and electrophysiological recordings, Zhou and colleagues identified a superior colliculus to ventral tegmental area pathway in detecting alarming visual cues and mediating defensive behaviors in mice. These findings provide novel insight into the neural circuit underlying innate predator defense.

A Genetically Encoded Fluorescent Sensor for Rapid and Specific In Vivo Detection of Norepinephrine.

Norepinephrine (NE) is a key biogenic monoamine neurotransmitter involved in a wide range of physiological processes. However, its precise dynamics and regulation remain poorly characterized, in part due to limitations of available techniques for measuring NE in vivo. Here, we developed a family of GPCR activation-based NE (GRABNE) sensors with a 230% peak ΔF/F0 response to NE, good photostability, nanomolar-to-micromolar sensitivities, sub-second kinetics, and high specificity. Viral- or transgenic-mediated expression of GRABNE sensors was able to detect electrical-stimulation-evoked NE release in the locus coeruleus (LC) of mouse brain slices, looming-evoked NE release in the midbrain of live zebrafish, as well as optogenetically and behaviorally triggered NE release in the LC and hypothalamus of freely moving mice. Thus, GRABNE sensors are robust tools for rapid and specific monitoring of in vivo NE transmission in both physiological and pathological processes.

The Neural Mechanisms of Sexually Dimorphic Aggressive Behaviors.

Aggression is a fundamental social behavior that is essential for competing for resources and protecting oneself and families in both males and females. As a result of natural selection, aggression is often displayed differentially between the sexes, typically at a higher level in males than females. Here, we highlight the behavioral differences between male and female aggression in rodents. We further outline the aggression circuits in males and females, and compare their differences at each circuit node. Lastly, we summarize our current understanding regarding the generation of sexually dimorphic aggression circuits during development and their maintenance during adulthood. In both cases, gonadal steroid hormones appear to play crucial roles in differentiating the circuits by impacting on the survival, morphology, and intrinsic properties of relevant cells. Many other factors, such as environment and experience, may also contribute to sex differences in aggression and remain to be investigated in future studies.

Embryonic transcription factor expression in mice predicts medial amygdala neuronal identity and sex-specific responses to innate behavioral cues.

The medial subnucleus of the amygdala (MeA) plays a central role in processing sensory cues required for innate behaviors. However, whether there is a link between developmental programs and the emergence of inborn behaviors remains unknown. Our previous studies revealed that the telencephalic preoptic area (POA) embryonic niche is a novel source of MeA destined progenitors. Here, we show that the POA is comprised of distinct progenitor pools complementarily marked by the transcription factors Dbx1 and Foxp2. As determined by molecular and electrophysiological criteria this embryonic parcellation predicts postnatal MeA inhibitory neuronal subtype identity. We further find that Dbx1-derived and Foxp2+ cells in the MeA are differentially activated in response to innate behavioral cues in a sex-specific manner. Thus, developmental transcription factor expression is predictive of MeA neuronal identity and sex-specific neuronal responses, providing a potential developmental logic for how innate behaviors could be processed by different MeA neuronal subtypes.

Molecular and behavioral profiling of Dbx1-derived neurons in the arcuate, lateral and ventromedial hypothalamic nuclei.

BACKGROUND: Neurons in the hypothalamus function to regulate the state of the animal during both learned and innate behaviors, and alterations in hypothalamic development may contribute to pathological conditions such as anxiety, depression or obesity. Despite many studies of hypothalamic development and function, the link between embryonic development and innate behaviors remains unexplored. Here, focusing on the embryonically expressed homeodomain-containing gene Developing Brain Homeobox 1 (Dbx1), we explored the relationship between embryonic lineage, post-natal neuronal identity and lineage-specific responses to innate cues. We found that Dbx1 is widely expressed across multiple developing hypothalamic subdomains. Using standard and inducible fate-mapping to trace the Dbx1-derived neurons, we identified their contribution to specific neuronal subtypes across hypothalamic nuclei and further mapped their activation patterns in response to a series of well-defined innate behaviors. RESULTS: Dbx1-derived neurons occupy multiple postnatal hypothalamic nuclei including the lateral hypothalamus (LH), arcuate nucleus (Arc) and the ventral medial hypothalamus (VMH). Within these nuclei, Dbx1 (+) progenitors generate a large proportion of the Pmch-, Nesfatin-, Cart-, Hcrt-, Agrp- and ERα-expressing neuronal populations, and to a lesser extent the Pomc-, TH- and Aromatase-expressing populations. Inducible fate-mapping reveals distinct temporal windows for development of the Dbx1-derived LH and Arc populations, with Agrp(+) and Cart(+) populations in the Arc arising early (E7.5-E9.5), while Pmch(+) and Hcrt(+) populations in the LH derived from progenitors expressing Dbx1 later (E9.5-E11.5). Moreover, as revealed by c-Fos labeling, Dbx1-derived cells in male and female LH, Arc and VMH are responsive during mating and aggression. In contrast, Dbx1-lineage cells in the Arc and LH have a broader behavioral tuning, which includes responding to fasting and predator odor cues. CONCLUSION: We define a novel fate map of the hypothalamus with respect to Dbx1 expression in hypothalamic progenitor zones. We demonstrate that in a temporally regulated manner, Dbx1-derived neurons contribute to molecularly distinct neuronal populations in the LH, Arc and VMH that have been implicated in a variety of hypothalamic-driven behaviors. Consistent with this, Dbx1-derived neurons in the LH, Arc and VMH are activated during stress and other innate behavioral responses, implicating their involvement in these diverse behaviors.

Preliminary Findings Show Maternal Hypothyroidism May Contribute to Abnormal Cortical Morphology in Offspring.

In rodents, insufficient thyroid hormone (TH) gestationally has adverse effects on cerebral cortex development. Comparable studies of humans examining how TH insufficiency affects cortical morphology are limited to children with congenital hypothyroidism or offspring of hypothyroxinemic women; effects on cortex of children born to women with clinically diagnosed hypothyroidism are not known. We studied archived MRI scans from 22 children aged 10-12 years born to women treated for preexisting or de novo hypothyroidism in pregnancy (HYPO) and 24 similar age and sex controls from euthyroid women. FreeSurfer Image Analysis Suite software was used to measure cortical thickness (CT) and a vertex-based approach served to compare HYPO versus control groups and Severe versus Mild HYPO subgroups as well as to perform regression analyses examining effects of trimester-specific maternal TSH on CT. Results showed that relative to controls, HYPO had multiple regions of both cortical thinning and thickening, which differed for left and right hemispheres. In HYPO, thinning was confined to medial and mid-lateral regions of each hemisphere and thickening to superior regions (primarily frontal) of the left hemisphere and inferior regions (particularly occipital and temporal) of the right. The Severe HYPO subgroup showed more thinning than Mild in frontal and temporal regions and more thickening in bilateral posterior and frontal regions. Maternal TSH values predicted degree of thinning and thickening within multiple brain regions, with the pattern and direction of correlations differing by trimester. Notably, some correlations remained when cases born to women with severe hypothyroidism were removed from the analyses, suggesting that mild variations of maternal TH may permanently affect offspring cortex. We conclude that maternal hypothyroidism during pregnancy has long-lasting manifestations on the cortical morphology of their offspring with specific effects reflecting both severity and timing of maternal TH insufficiency.

Do children with congenital hypothyroidism exhibit abnormal cortical morphology?

BACKGROUND: Given thyroid hormone (TH)'s essential role in multiple aspects of early brain development, children with congenital hypothyroidism (CH) detected and treated early may still display subtle cognitive and behavioral impairments as well as brain abnormalities. However, effects on their cortical development are not yet known. We used an automated neuroimaging technique to determine if these children differ in cortical thickness (CT) from typically developing controls (TDC) and if the regions showing CT differences reflect severity of initial hypothyroidism and predict later neuropsychological functioning. METHODS: FreeSurfer Image Analysis Suite was used on archived MRI scans from 41 CH and 42 TDC children aged 9-16 y. Vertex-based procedures were used to compare groups and perform correlations between CT and indices of disease severity and neuropsychological outcome. RESULTS: The CH group showed multiple regions of cortical thinning or cortical thickening within right and left hemispheres relative to TDC. CT values were significantly correlated with early T4 and thyroid-stimulating hormone (TSH) levels and current neuropsychological test indices. CONCLUSION: The developing cortex is sensitive to early TH loss in CH. Different patterns of cortical thinning or cortical thickening among brain regions may reflect timing of TH deficiency relative to timing of cortical development.