Transformations of neural representations in a social behaviour network.

Mating and aggression are innate social behaviours that are controlled by subcortical circuits in the extended amygdala and hypothalamus1-4. The bed nucleus of the stria terminalis (BNSTpr) is a node that receives input encoding sex-specific olfactory cues from the medial amygdala5,6, and which in turn projects to hypothalamic nuclei that control mating7-9 (medial preoptic area (MPOA)) and aggression9-14 (ventromedial hypothalamus, ventrolateral subdivision (VMHvl)), respectively15. Previous studies have demonstrated that male aromatase-positive BNSTpr neurons are required for mounting and attack, and may identify conspecific sex according to their overall level of activity16. However, neural representations in BNSTpr, their function and their transformations in the hypothalamus have not been characterized. Here we performed calcium imaging17,18 of male BNSTprEsr1 neurons during social behaviours. We identify distinct populations of female- versus male-tuned neurons in BNSTpr, with the former outnumbering the latter by around two to one, similar to the medial amygdala and MPOA but opposite to VMHvl, in which male-tuned neurons predominate6,9,19. Chemogenetic silencing of BNSTprEsr1 neurons while imaging MPOAEsr1 or VMHvlEsr1 neurons in behaving animals showed, unexpectedly, that the male-dominant sex-tuning bias in VMHvl was inverted to female-dominant whereas a switch from sniff- to mount-selective neurons during mating was attenuated in MPOA. Our data also indicate that BNSTprEsr1 neurons are not essential for conspecific sex identification. Rather, they control the transition from appetitive to consummatory phases of male social behaviours by shaping sex- and behaviour-specific neural representations in the hypothalamus.

Sexually Dimorphic Neurosteroid Synthesis Regulates Neuronal Activity in the Murine Brain.

Sex steroid hormones act on hypothalamic kisspeptin neurons to regulate reproductive neural circuits in the brain. Kisspeptin neurons start to express estrogen receptors in utero, suggesting steroid hormone action on these cells early during development. Whether neurosteroids are locally produced in the embryonic brain and impinge onto kisspeptin/reproductive neural circuitry is not known. To address this question, we analyzed aromatase expression, a key enzyme in estrogen synthesis, in male and female mouse embryos. We identified an aromatase neuronal network comprising ∼6000 neurons in the hypothalamus and amygdala. By birth, this network has become sexually dimorphic in a cluster of aromatase neurons in the arcuate nucleus adjacent to kisspeptin neurons. We demonstrate that male arcuate aromatase neurons convert testosterone to estrogen to regulate kisspeptin neuron activity. We provide spatiotemporal information on aromatase neuronal network development and highlight a novel mechanism whereby aromatase neurons regulate the activity of distinct neuronal populations expressing estrogen receptors.SIGNIFICANCE STATEMENT Sex steroid hormones, such as estradiol, are important regulators of neural circuits controlling reproductive physiology in the brain. Embryonic kisspeptin neurons in the hypothalamus express steroid hormone receptors, suggesting hormone action on these cells in utero Whether neurosteroids are locally produced in the brain and impinge onto reproductive neural circuitry is insufficiently understood. To address this question, we analyzed aromatase expression, a key enzyme in estradiol synthesis, in mouse embryos and identified a network comprising ∼6000 neurons in the brain. By birth, this network has become sexually dimorphic in a cluster of aromatase neurons in the arcuate nucleus adjacent to kisspeptin neurons. We demonstrate that male aromatase neurons convert testosterone to estradiol to regulate kisspeptin neuron activity.

Single cell plasticity and population coding stability in auditory thalamus upon associative learning.

Cortical and limbic brain areas are regarded as centres for learning. However, how thalamic sensory relays participate in plasticity upon associative learning, yet support stable long-term sensory coding remains unknown. Using a miniature microscope imaging approach, we monitor the activity of populations of auditory thalamus (medial geniculate body) neurons in freely moving mice upon fear conditioning. We find that single cells exhibit mixed selectivity and heterogeneous plasticity patterns to auditory and aversive stimuli upon learning, which is conserved in amygdala-projecting medial geniculate body neurons. Activity in auditory thalamus to amygdala-projecting neurons stabilizes single cell plasticity in the total medial geniculate body population and is necessary for fear memory consolidation. In contrast to individual cells, population level encoding of auditory stimuli remained stable across days. Our data identifies auditory thalamus as a site for complex neuronal plasticity in fear learning upstream of the amygdala that is in an ideal position to drive plasticity in cortical and limbic brain areas. These findings suggest that medial geniculate body's role goes beyond a sole relay function by balancing experience-dependent, diverse single cell plasticity with consistent ensemble level representations of the sensory environment to support stable auditory perception with minimal affective bias.

Posterior amygdala regulates sexual and aggressive behaviors in male mice.

Sexual and aggressive behaviors are fundamental to animal survival and reproduction. The medial preoptic nucleus (MPN) and ventrolateral part of the ventromedial hypothalamus (VMHvl) are essential regions for male sexual and aggressive behaviors, respectively. While key inhibitory inputs to the VMHvl and MPN have been identified, the extrahypothalamic excitatory inputs essential for social behaviors remain elusive. Here we identify estrogen receptor alpha (Esr1)-expressing cells in the posterior amygdala (PA) as a main source of excitatory inputs to the hypothalamus and key mediators for mating and fighting in male mice. We find two largely distinct PA subpopulations that differ in connectivity, gene expression, in vivo responses and social behavior relevance. MPN-projecting PAEsr1+ cells are activated during mating and are necessary and sufficient for male sexual behaviors, while VMHvl-projecting PAEsr1+ cells are excited during intermale aggression and promote attacks. These findings place the PA as a key node in both male aggression and reproduction circuits.

Nucleus Accumbens Cell Type- and Input-Specific Suppression of Unproductive Reward Seeking.

The nucleus accumbens (NAc) contributes to behavioral inhibition and compulsions, but circuit mechanisms are unclear. Recent evidence suggests that amygdala and thalamic inputs exert opposing control over behavior, much like direct and indirect pathway output neurons. Accordingly, opponent processes between these NAc inputs or cell types may underlie efficient reward seeking. We assess the contributions of these circuit elements to mouse operant behavior during recurring conditions when reward is and is not available. Although direct pathway stimulation is rewarding and indirect pathway stimulation aversive, the activity of both cell types is elevated during periods of behavioral suppression, and the inhibition of either cell-type selectively increases unproductive reward seeking. Amygdala and thalamic inputs are also necessary for behavioral suppression, even though they both support self-stimulation and innervate different NAc subregions. These data suggest that efficient reward seeking relies on complementary activity across NAc cell types and inputs rather than opponent processes between them.

Organization of primate amygdalar-thalamic pathways for emotions.

Studies on the thalamus have mostly focused on sensory relay nuclei, but the organization of pathways associated with emotions is not well understood. We addressed this issue by testing the hypothesis that the primate amygdala acts, in part, like a sensory structure for the affective import of stimuli and conveys this information to the mediodorsal thalamic nucleus, magnocellular part (MDmc). We found that primate sensory cortices innervate amygdalar sites that project to the MDmc, which projects to the orbitofrontal cortex. As in sensory thalamic systems, large amygdalar terminals innervated excitatory relay and inhibitory neurons in the MDmc that facilitate faithful transmission to the cortex. The amygdala, however, uniquely innervated a few MDmc neurons by surrounding and isolating large segments of their proximal dendrites, as revealed by three-dimensional high-resolution reconstruction. Physiologic studies have shown that large axon terminals are found in pathways issued from motor systems that innervate other brain centers to help distinguish self-initiated from other movements. By analogy, the amygdalar pathway to the MDmc may convey signals forwarded to the orbitofrontal cortex to monitor and update the status of the environment in processes deranged in schizophrenia, resulting in attribution of thoughts and actions to external sources.

Perineuronal net expression in the brain of a hibernating mammal.

During hibernation, mammals like the 13-lined ground squirrel cycle between physiological extremes. Most of the hibernation season is spent in bouts of torpor, where body temperature, heart rate, and cerebral blood flow are all very low. However, the ground squirrels periodically enter into interbout arousals (IBAs), where physiological parameters return to non-hibernating levels. During torpor, neurons in many brain regions shrink and become electrically quiescent, but reconnect and regain activity during IBA. Previous work showed evidence of extracellular matrix (ECM) changes occurring in the hypothalamus during hibernation that could be associated with this plasticity. Here, we examined expression of a specialized ECM structure, the perineuronal net (PNN), in the forebrain of ground squirrels in torpor, IBA, and summer (non-hibernating). PNNs are known to restrict plasticity, and could be important for retaining essential connections in the brain during hibernation. We found PNNs in three regions of the hypothalamus: ventrolateral hypothalamus, paraventricular nucleus (PVN), and anterior hypothalamic area. We also found PNNs throughout the cerebral cortex, amygdala, and lateral septum. The total area covered by PNNs within the PVN was significantly higher during IBA compared to non-hibernating and torpor (P < 0.01). Additionally, the amount of PNN coverage area per Nissl-stained neuron in the PVN was significantly higher in hibernation compared to non-hibernating (P < 0.05). No other significant differences were found across seasons. The PVN is involved in food intake and homeostasis, and PNNs found here could be essential for retaining vital life functions during hibernation.

µ-opioid receptor-mediated downregulation of midline thalamic pathways to basal and central amygdala.

Brain µ-opioid receptors (MOR) mediate reward and help coping with pain, social rejection, anxiety and depression. The dorsal midline thalamus (dMT) integrates visceral/emotional signals and biases behavior towards aversive or defensive states through projections to the amygdala. While a dense MOR expression in the dMT has been described, the exact cellular and synaptic mechanisms of µ-opioidergic modulation in the dMT-amygdala circuitry remain unresolved. Here, we hypothesized that MORs are important negative modulators of dMT-amygdala excitatory networks. Using retrograde tracers and targeted channelrhodopsin expression in combination with patch-clamp electrophysiology, we found that projections of dMT neurons onto both basal amygdala principal neurons (BA PN) and central amygdala (CeL) neurons are attenuated by stimulation of somatic or synaptic MORs. Importantly, dMT efferents to the amygdala drive feedforward excitation of centromedial amygdala neurons (CeM), which is dampened by MOR activation. This downregulation of excitatory activity in dMT-amygdala networks puts the µ-opioid system in a position to ameliorate aversive or defensive behavioral states associated with stress, withdrawal, physical pain or social rejection.

Stereotactic system for accurately targeting deep brain structures in awake head-fixed mice.

Deep brain nuclei, such as the amygdala, nucleus basalis, and locus coeruleus, play a crucial role in cognition and behavior. Nonetheless, acutely recording electrical activity from these structures in head-fixed awake rodents has been very challenging due to the fact that head-fixed preparations are not designed for stereotactic accuracy. We overcome this issue by designing the DeepTarget, a system for stereotactic head fixation and recording, which allows for accurately directing recording electrodes or other probes into any desired location in the brain. We then validated it by performing intracellular recordings from optogenetically tagged amygdalar neurons followed by histological reconstruction, which revealed that it is accurate and precise to within ~100 µm. Moreover, in another group of mice we were able to target both the mammillothalamic tract and subthalamic nucleus. This approach can be adapted to any type of extracellular electrode, fiber optic, or other probe in cases where high accuracy is needed in awake, head-fixed rodents.NEW & NOTEWORTHY Accurate targeting of recording electrodes in awake head-restrained rodents is currently beyond our reach. We developed a device for stereotactic implantation of a custom head bar and a recording system that together allow the accurate and precise targeting of any brain structure, including deep and small nuclei. We demonstrated this by performing histology and intracellular recordings in the amygdala of awake mice. The system enables the targeting of any probe to any location in the awake brain.

Sleep Regulation by Neurotensinergic Neurons in a Thalamo-Amygdala Circuit.

A crucial step in understanding the sleep-control mechanism is to identify sleep neurons. Through systematic anatomical screening followed by functional testing, we identified two sleep-promoting neuronal populations along a thalamo-amygdala pathway, both expressing neurotensin (NTS). Rabies-mediated monosynaptic retrograde tracing identified the central nucleus of amygdala (CeA) as a major source of GABAergic inputs to multiple wake-promoting populations; gene profiling revealed NTS as a prominent marker for these CeA neurons. Optogenetic activation and inactivation of NTS-expressing CeA neurons promoted and suppressed non-REM (NREM) sleep, respectively, and optrode recording showed they are sleep active. Further tracing showed that CeA GABAergic NTS neurons are innervated by glutamatergic NTS neurons in a posterior thalamic region, which also promote NREM sleep. CRISPR/Cas9-mediated NTS knockdown in either the thalamic or CeA neurons greatly reduced their sleep-promoting effect. These results reveal a novel thalamo-amygdala circuit for sleep generation in which NTS signaling is essential for both the upstream glutamatergic and downstream GABAergic neurons.

Long-term environmental enrichment affects microglial morphology in middle age mice.

Aging is associated with increased central nervous system inflammation, in large part due to dysfunctional microglia. Environmental enrichment (EE) provides a model for studying the dynamics of lifestyle factors in the development of age-related neuroinflammation and microglial dysfunction. EE results in improvements in learning and memory, metabolism, and mental health in a variety of animal models. We recently reported that implementing EE in middle age promotes healthy aging. In the present study, we investigated whether EE influences microglial morphology, and whether EE is associated with changes in expression of microglial and neuroinflammatory markers. Inflammatory cytokines and MHC-II were reduced following 12-month EE in 10-month-old mice. Long-term EE for 7.5 months resulted in broad increases in Iba1 expression in hippocampus, hypothalamus, and amygdala detected by immunohistochemistry. Quantification of microglial morphology reveal both hypertrophy and ramification in these three brain regions, without increases in microglial cell density. These data indicate that long-term EE implemented in middle age results in a microglial state distinct from that of normal aging in standard laboratory housing, in specific brain regions, associated with reduced neuroinflammatory markers and improvement of systemic metabolism.

Effect of electroacupuncture on the activity of corticotrophin-releasing hormone neurons in the hypothalamus and amygdala in rats exposed to restraint water-immersion stress.

OBJECTIVE: To investigate the effects of electroacupuncture (EA) treatment on gastric mucosal lesions and the activity of corticotrophin-releasing hormone (CRH) neurons in the paraventricular nucleus (PVN) of the hypothalamus and the central nucleus of the amygdala (CNA) in a rat model of restraint water-immersion stress (RWIS). METHODS: 24 male Wistar rats were randomly divided into three groups: normal, RWIS, and RWIS+EA (n=8 per group). Rats in the RWIS group and RWIS+EA group received RWIS for 3 hours. For rats in the RWIS+EA group, EA was applied at ST36 in the bilateral hind legs for 30 min before RWIS. Rats in the normal group did not receive stressors or EA treatment. The gastric mucosal lesions of each rat were evaluated by the erosion index (EI) according to the methods of Guth. The activity of CRH neurons in the PVN and CNA was measured by a dual immunohistochemical test for Fos and CRH in the brain sections. RESULTS: RWIS induced serious gastric mucosal lesions. The mean gastric EI was significantly decreased in the RWIS+EA group versus the RWIS group (P=0.005). Stress induced significant activation of CRH neurons in the PVN and CNA compared with the normal group (P<0.001 for both). The mean number of Fos+CRH immunoreactive neurons in the PVN and CNA were both decreased inRWIS+EA versusRWIS groups (P<0.001 and P=0.001). CONCLUSIONS: EA at ST36 can ameliorate RWIS-induced gastric mucosal lesions and suppress the Fos expression of CRH neurons in the PVN and CNA, suggesting a potentially therapeutic role for EA in stress-related gastric disorders.

Sensory overamplification in layer 5 auditory corticofugal projection neurons following cochlear nerve synaptic damage.

Layer 5 (L5) cortical projection neurons innervate far-ranging brain areas to coordinate integrative sensory processing and adaptive behaviors. Here, we characterize a plasticity in L5 auditory cortex (ACtx) neurons that innervate the inferior colliculus (IC), thalamus, lateral amygdala and striatum. We track daily changes in sound processing using chronic widefield calcium imaging of L5 axon terminals on the dorsal cap of the IC in awake, adult mice. Sound level growth functions at the level of the auditory nerve and corticocollicular axon terminals are both strongly depressed hours after noise-induced damage of cochlear afferent synapses. Corticocollicular response gain rebounded above baseline levels by the following day and remained elevated for several weeks despite a persistent reduction in auditory nerve input. Sustained potentiation of excitatory ACtx projection neurons that innervate multiple limbic and subcortical auditory centers may underlie hyperexcitability and aberrant functional coupling of distributed brain networks in tinnitus and hyperacusis.

Neuropeptide Y (NPY) or cocaine- and amphetamine-regulated transcript (CART) fiber innervation on central and medial amygdaloid neurons that project to the locus coeruleus and dorsal raphe in the rat.

The amygdaloid nuclear complex has been linked to the regulation of emotional behavior and energy regulation in that emotional stress might cause either reduction or enhancement of eating. We examined hypothalamic neuronal origin of feeding/arousal-related peptidergic fibers containing cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY) located in the rat amygdala along with its efferent projections to the brainstem monoaminergic nuclei. First, central (CeA) as well as medial (MeA) amygdala, among several amygdaloid subdivisions, exhibited the most prominent NPY or CART immunostaining which consisted of a substantial number of somata as well as labeled fibers. When we examined hypothalamic neuronal origin of NPY or CART fibers projecting to the CeA and MeA, medial and lateral arcuate nuclei were neuronal origins of NPY and CART fibers, respectively. However, the majority (>70%) of amygdala-projecting CART neurons which co-contained melanin-concentrating hormone (MCH) originated from the lateral hypothalamus (LH), zona incerta (ZI), and dorsal hypothalamic area (DA). This observation implied that the CeA as well as the MeA might receive potent second-order (and downstream) feeding-related CART input from the lateral hypothalamic regions in addition to first-order CART or NPY input from the Arc. Second, a large number of CeA neurons projected to the locus coeruleus (LC), whereas only a small number of MeA cells projected to the dorsal raphe (DR); none of the CeA or MeA cells provided dual projections to the LC and DR. Finally, a portion of MCH cells in the LH, ZI, and DA sent divergent axon collaterals to the CeA and LC. Considering that the CeA sends substantial GABAergic input to the LC, the present observation might serve as an anatomical substrate to support the potent hypnogenic role of MCH neurons in the LH regions during cataplexy and REM sleep.

NPY Induces Stress Resilience via Downregulation of Ih in Principal Neurons of Rat Basolateral Amygdala.

Neuropeptide Y (NPY) expression is tightly linked with the development of stress resilience in rodents and humans. Local NPY injections targeting the basolateral amygdala (BLA) produce long-term behavioral stress resilience in male rats via an unknown mechanism. Previously, we showed that activation of NPY Y1 receptors hyperpolarizes BLA principal neurons (PNs) through inhibition of the hyperpolarization-activated, depolarizing H-current, Ih The present studies tested whether NPY treatment induces stress resilience by modulating Ih NPY (10 pmol) was delivered daily for 5 d bilaterally into the BLA to induce resilience; thereafter, the electrophysiological properties of PNs and the expression of Ih in the BLA were characterized. As reported previously, increases in social interaction (SI) times persisted weeks after completion of NPY administration. In vitro intracellular recordings showed that repeated intra-BLA NPY injections resulted in hyperpolarization of BLA PNs at 2 weeks (2W) and 4 weeks (4W) after NPY treatment. At 2W, spontaneous IPSC frequencies were increased, whereas at 4W, resting Ih was markedly reduced and accompanied by decreased levels of HCN1 mRNA and protein expression in BLA. Knock-down of HCN1 channels in the BLA with targeted delivery of lentivirus containing HCN1-shRNA increased SI beginning 2W after injection and induced stress resilience. NPY treatment induced sequential, complementary changes in the inputs to BLA PNs and their postsynaptic properties that reduce excitability, a mechanism that contributes to less anxious behavior. Furthermore, HCN1 knock-down mimicked the increases in SI and stress resilience observed with NPY, indicating the importance of Ih in stress-related behavior.SIGNIFICANCE STATEMENT Resilience improves mental health outcomes in response to adverse situations. Neuropeptide Y (NPY) is associated with decreased stress responses and the expression of resilience in rodents and humans. Single or repeated injections of NPY into the basolateral amygdala (BLA) buffer negative behavioral effects of stress and induce resilience in rats, respectively. Here, we demonstrate that repeated administration of NPY into the BLA unfolds several cellular mechanisms that decrease the activity of pyramidal output neurons. One key mechanism is a reduction in levels of the excitatory ion channel HCN1. Moreover, shRNA knock-down of HCN1 expression in BLA recapitulates some of the actions of NPY and causes potent resilience to stress, indicating that this channel may be a possible target for therapy.

A Labeled-Line Neural Circuit for Pheromone-Mediated Sexual Behaviors in Mice.

In mice, various instinctive behaviors can be triggered by olfactory input. Despite growing knowledge of the brain regions involved in such behaviors, the organization of the neural circuits that convert olfactory input into stereotyped behavioral output remains poorly understood. Here, we mapped the neural circuit responsible for enhancing sexual receptivity of female mice by a male pheromone, exocrine gland-secreting peptide 1 (ESP1). We revealed specific neural types and pathways by which ESP1 information is conveyed from the peripheral receptive organ to the motor-regulating midbrain via the amygdala-hypothalamus axis. In the medial amygdala, a specific type of projection neurons gated ESP1 signals to the ventromedial hypothalamus (VMH) in a sex-dependent manner. In the dorsal VMH, which has been associated with defensive behaviors, a selective neural subpopulation discriminately mediated ESP1 information from a predator cue. Together, our data illuminate a labeled-line organization for controlling pheromone-mediated sexual behavioral output in female mice.

Awake dynamics and brain-wide direct inputs of hypothalamic MCH and orexin networks.

The lateral hypothalamus (LH) controls energy balance. LH melanin-concentrating-hormone (MCH) and orexin/hypocretin (OH) neurons mediate energy accumulation and expenditure, respectively. MCH cells promote memory and appropriate stimulus-reward associations; their inactivation disrupts energy-optimal behaviour and causes weight loss. However, MCH cell dynamics during wakefulness are unknown, leaving it unclear if they differentially participate in brain activity during sensory processing. By fiberoptic recordings from molecularly defined populations of LH neurons in awake freely moving mice, we show that MCH neurons generate conditional population bursts. This MCH cell activity correlates with novelty exploration, is inhibited by stress and is inversely predicted by OH cell activity. Furthermore, we obtain brain-wide maps of monosynaptic inputs to MCH and OH cells, and demonstrate optogenetically that VGAT neurons in the amygdala and bed nucleus of stria terminalis inhibit MCH cells. These data reveal cell-type-specific LH dynamics during sensory integration, and identify direct neural controllers of MCH neurons.

A temporal shift in the circuits mediating retrieval of fear memory.

Fear memories allow animals to avoid danger, thereby increasing their chances of survival. Fear memories can be retrieved long after learning, but little is known about how retrieval circuits change with time. Here we show that the dorsal midline thalamus of rats is required for the retrieval of auditory conditioned fear at late (24 hours, 7 days, 28 days), but not early (0.5 hours, 6 hours) time points after learning. Consistent with this, the paraventricular nucleus of the thalamus (PVT), a subregion of the dorsal midline thalamus, showed increased c-Fos expression only at late time points, indicating that the PVT is gradually recruited for fear retrieval. Accordingly, the conditioned tone responses of PVT neurons increased with time after training. The prelimbic (PL) prefrontal cortex, which is necessary for fear retrieval, sends dense projections to the PVT. Retrieval at late time points activated PL neurons projecting to the PVT, and optogenetic silencing of these projections impaired retrieval at late, but not early, time points. In contrast, silencing of PL inputs to the basolateral amygdala impaired retrieval at early, but not late, time points, indicating a time-dependent shift in retrieval circuits. Retrieval at late time points also activated PVT neurons projecting to the central nucleus of the amygdala, and silencing these projections at late, but not early, time points induced a persistent attenuation of fear. Thus, the PVT may act as a crucial thalamic node recruited into cortico-amygdalar networks for retrieval and maintenance of long-term fear memories.

Fear conditioning leads to alteration in specific genes expression in cortical and thalamic neurons that project to the lateral amygdala.

RNA transcription is needed for memory formation. However, the ability to identify genes whose expression is altered by learning is greatly impaired because of methodological difficulties in profiling gene expression in specific neurons involved in memory formation. Here, we report a novel approach to monitor the expression of genes after learning in neurons in specific brain pathways needed for memory formation. In this study, we aimed to monitor gene expression after fear learning. We retrogradely labeled discrete thalamic neurons that project to the lateral amygdala (LA) of rats. The labeled neurons were dissected, using laser microdissection microscopy, after fear conditioning learning or unpaired training. The RNAs from the dissected neurons were subjected to microarray analysis. The levels of selected RNAs detected by the microarray analysis to be altered by fear conditioning were also assessed by nanostring analysis. We observed that the expression of genes involved in the regulation of translation, maturation and degradation of proteins was increased 6 h after fear conditioning compared to unpaired or naïve trained rats. These genes were not expressed 24 h after training or in cortical neurons that project to the LA. The expression of genes involved in transcription regulation and neuronal development was altered after fear conditioning learning in the cortical-LA pathway. The present study provides key information on the identity of genes expressed in discrete thalamic and cortical neurons that project to the LA after fear conditioning. Such an approach could also serve to identify gene products as targets for the development of a new generation of therapeutic agents that could be aimed to functionally identified brain circuits to treat memory-related disorders.

The fear circuit of the mouse forebrain: connections between the mediodorsal thalamus, frontal cortices and basolateral amygdala.

A large forebrain circuit, including the thalamus, amygdala and frontal cortical regions, is responsible for the establishment and extinction of fear-related memories. Understanding interactions among these three regions is critical to deciphering the basic mechanisms of fear. With the advancement of molecular and optogenetics techniques, the mouse has become the main species used to study fear-related behaviours. However, the basic connectivity pattern of the forebrain circuits involved in processing fear has not been described in this species. In this study we mapped the connectivity between three key nodes of the circuit, i.e. the basolateral nucleus of the amygdala (BLA), the mediodorsal nucleus of the thalamus (MD) and the medial prefrontal cortex, which were shown to have closed triangular connectivity in rats. In contrast to rat, we found no evidence for this closed loop in mouse. There was no major input from the BLA to the MD and little overlap between medial prefrontal regions connected with both the BLA and MD. The common nodes in the frontal cortex, which displayed reciprocal connection with both the BLA and MD were the agranular insular cortex and the border zone of the cingulate and secondary motor cortex. In addition, the BLA can indirectly affect the MD via the orbital cortex. We attribute the difference between our results and earlier rat studies to methodological problems rather than to genuine species difference. Our data demonstrate that the BLA and MD communicate via cortical sectors, the roles in fear-related behaviour of which have not been extensively studied. In general, our study provides the morphological framework for studies of murine fear-related behaviours.