Appetite suppression occurs following a meal and also during conditions when it is unfavorable to eat, such as during illness or exposure to toxins. A brain region hypothesized to play a role in appetite suppression is the parabrachial nucleus (PBN)1-3, a heterogeneous population of neurons surrounding the superior cerebellar peduncle in the brainstem. The PBN is thought to mediate the suppression of appetite induced by the anorectic hormones amylin and cholecystokinin, as well as lithium chloride and lipopolysaccharide, compounds that mimic the effects of toxic foods and bacterial infections, respectively4-6. Hyperactivity of the PBN is also thought to cause starvation following ablation of orexigenic agouti-related peptide (AgRP) neurons in adult mice1,7. However, the identities of PBN neurons that regulate feeding are unknown, as are the functionally relevant downstream projections. Here we identify calcitonin gene-related peptide (CGRP)-expressing neurons in the outer external lateral subdivision of the PBN that project to the laterocapsular division of the central nucleus of the amygdala (CeAlc) as forming a functionally important circuit for the suppression of appetite. Using genetically-encoded anatomical, optogenetic8, and pharmacogenetic9 tools, we demonstrate that activation of PBelo CGRP neurons projecting to the CeAlc suppresses appetite. In contrast, inhibition of these neurons increases food intake in circumstances when mice do not normally eat and prevents starvation in adult AgRP neuron-ablated mice. Taken together, our data demonstrate that this neural circuit from the PBN to CeAlc mediates appetite suppression in conditions when it is unfavorable to eat. This neural circuit may provide targets for therapeutic intervention to overcome or promote appetite.

SUMMARY Animals learn to avoid harmful situations by associating a neutral stimulus with a painful one, resulting in a stable threat memory. In mammals, this form of learning requires the amygdala. Although pain is the main driver of aversive learning, the mechanism that transmits pain signals to the amygdala is not well resolved. Here we show that neurons expressing calcitonin gene-related peptide (CGRP) in the parabrachial nucleus are critical for relaying pain signals to the central nucleus of amygdala, and that this pathway may transduce the affective motivational aspects of pain. Genetic silencing of CGRP neurons blocks pain responses and memory formation, while their optogenetic stimulation produces defensive responses and a threat memory. The pain-recipient neurons in the central amygdala expressing CGRP receptors are also critical for establishing a threat memory. The identification of the neural circuit conveying affective pain signals may be pertinent for treating pain conditions with psychiatric comorbidities.

In the face of starvation animals will engage in high-risk behaviors that would normally be considered maladaptive. Starving rodents for example will forage in areas that are more susceptible to predators and will also modulate aggressive behavior within a territory of limited or depleted nutrients. The neural basis of these adaptive behaviors likely involves circuits that link innate feeding, aggression, and fear. Hypothalamic AgRP neurons are critically important for driving feeding and project axons to brain regions implicated in aggression and fear. Using circuit-mapping techniques, we define a disynaptic network originating from a subset of AgRP neurons that project to the medial nucleus of the amygdala and then to the principle bed nucleus of the stria terminalis, which plays a role in suppressing territorial aggression and reducing contextual fear. We propose that AgRP neurons serve as a master switch capable of coordinating behavioral decisions relative to internal state and environmental cues.

Homeostatic synaptic plasticity adjusts the strength of synapses during global changes in neural activity, thereby stabilizing the overall activity of neural networks. Suppression of synaptic activity increases synaptic strength by inducing synthesis of retinoic acid (RA), which activates postsynaptic synthesis of AMPA-type glutamate receptors (AMPARs) in dendrites and promotes synaptic insertion of newly synthesized AMPARs. Here, we show that fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates dendritic protein synthesis, is essential for increases in synaptic strength induced by RA or by blockade of neural activity in the mouse hippocampus. Although activity-dependent RA synthesis is maintained in Fmr1 knock-out neurons, RA-dependent dendritic translation of GluR1-type AMPA receptors is impaired. Intriguingly, FMRP is only required for the form of homeostatic plasticity that is dependent on both RA signaling and local protein synthesis. Postsynaptic expression of wild-type or mutant FMRP(I304N) in knock-out neurons reduced the total, surface, and synaptic levels of AMPARs, implying a role for FMRP in regulating AMPAR abundance. Expression of FMRP lacking the RGG box RNA-binding domain had no effect on AMPAR levels. Importantly, postsynaptic expression of wild-type FMRP, but not FMRP(I304N) or FMRP⌬RGG, restored synaptic scaling when expressed in knock-out neurons. Together, these findings identify an unanticipated role for FMRP in regulating homeostatic synaptic plasticity downstream of RA. Our results raise the possibility that at least some of the symptoms of fragile X syndrome reflect impaired homeostatic plasticity and impaired RA signaling.

Summary Fear is a graded central motive state ranging from mild to intense. As threat intensity increases, fear transitions from discriminative to generalized. The circuit mechanisms that process threats of different intensity are not well resolved. Here, we isolate a unique population of locally projecting neurons in the central nucleus of the amygdala (CeA) that produce the neuropeptide corticotropin-releasing factor (CRF). CRF-producing neurons and CRF in the CeA are required for discriminative fear, but both are dispensable for generalized fear at high US intensities. Consistent with a role in discriminative fear, CRF neurons undergo plasticity following threat conditioning and selectively respond to threat-predictive cues. We further show that excitability of genetically isolated CRF-receptive (CRFR1) neurons in the CeA is potently enhanced by CRF and that CRFR1 signaling in the CeA is critical for discriminative fear. These findings demonstrate a novel CRF gain-control circuit and show separable pathways for graded fear processing.

Memories become less precise and generalize over time as memory traces re-organize in hippocampal-cortical networks. Increased time-dependent loss of memory precision characterizes overgeneralization of fear in post-traumatic stress disorder (PTSD) and age-related cognitive impairments. In the hippocampal dentate gyrus (DG), memories are thought to be encoded by so-called “engram-bearing” dentate granule cells (eDGCs). Here we show using rodents that contextual fear conditioning increases connectivity between eDGCs and inhibitory interneurons in the downstream hippocampal CA3 region. We identify actin-binding LIM protein 3 (abLIM3) as a mossy fiber terminal localized cytoskeletal factor, whose levels decrease upon learning. Downregulation of abLIM3 in DGCs was sufficient to increase connectivity with CA3 stratum lucidum interneurons (SLINs), promote parvalbumin (PV) SLIN activation, enhance feed-forward inhibition onto CA3, and maintain a fear memory engram in the dentate gyrus (DG) over time. Furthermore, abLIM3 downregulation in DGCs conferred conditioned context-specific reactivation of memory traces in hippocampal-cortical and amygdalar networks and decreased fear memory generalization at remote time points. Consistent with age-related hyperactivity of CA3, learning failed to increase DGC-SLIN connectivity in 17 month-old mice, whereas abLIM3 downregulation was sufficient to restore DGC-SLIN connectivity, increase PV-SLIN activation and improve remote memory precision. These studies exemplify a connectivity-based strategy targeting a molecular brake of feedforward inhibition in DG-CA3 that may be harnessed to decrease time-dependent memory generalization in PTSD and improve memory precision in aging.

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Highlights d Single viral vector CRISPR/SaCas9 for efficient in vivo mutagenesis d Cre-and Flp-dependent cell type-specific gene mutagenesis