A Fine-Scale Functional Logic to Convergence from Retina to Thalamus.

Numerous well-defined classes of retinal ganglion cells innervate the thalamus to guide image-forming vision, yet the rules governing their convergence and divergence remain unknown. Using two-photon calcium imaging in awake mouse thalamus, we observed a functional arrangement of retinal ganglion cell axonal boutons in which coarse-scale retinotopic ordering gives way to fine-scale organization based on shared preferences for other visual features. Specifically, at the ∼6 µm scale, clusters of boutons from different axons often showed similar preferences for either one or multiple features, including axis and direction of motion, spatial frequency, and changes in luminance. Conversely, individual axons could "de-multiplex" information channels by participating in multiple, functionally distinct bouton clusters. Finally, ultrastructural analyses demonstrated that retinal axonal boutons in a local cluster often target the same dendritic domain. These data suggest that functionally specific convergence and divergence of retinal axons may impart diverse, robust, and often novel feature selectivity to visual thalamus.

Cortical reactivations predict future sensory responses.

Many theories of offline memory consolidation posit that the pattern of neurons activated during a salient sensory experience will be faithfully reactivated, thereby stabilizing the pattern1,2. However, sensory-evoked patterns are not stable but, instead, drift across repeated experiences3-6. Here, to investigate the relationship between reactivations and the drift of sensory representations, we imaged the calcium activity of thousands of excitatory neurons in the mouse lateral visual cortex. During the minute after a visual stimulus, we observed transient, stimulus-specific reactivations, often coupled with hippocampal sharp-wave ripples. Stimulus-specific reactivations were abolished by local cortical silencing during the preceding stimulus. Reactivations early in a session systematically differed from the pattern evoked by the previous stimulus-they were more similar to future stimulus response patterns, thereby predicting both within-day and across-day representational drift. In particular, neurons that participated proportionally more or less in early stimulus reactivations than in stimulus response patterns gradually increased or decreased their future stimulus responses, respectively. Indeed, we could accurately predict future changes in stimulus responses and the separation of responses to distinct stimuli using only the rate and content of reactivations. Thus, reactivations may contribute to a gradual drift and separation in sensory cortical response patterns, thereby enhancing sensory discrimination7.

Hypothalamic dopamine neurons motivate mating through persistent cAMP signalling.

Transient neuromodulation can have long-lasting effects on neural circuits and motivational states1-4. Here we examine the dopaminergic mechanisms that underlie mating drive and its persistence in male mice. Brief investigation of females primes a male's interest to mate for tens of minutes, whereas a single successful mating triggers satiety that gradually recovers over days5. We found that both processes are controlled by specialized anteroventral and preoptic periventricular (AVPV/PVpo) dopamine neurons in the hypothalamus. During the investigation of females, dopamine is transiently released in the medial preoptic area (MPOA)-an area that is critical for mating behaviours. Optogenetic stimulation of AVPV/PVpo dopamine axons in the MPOA recapitulates the priming effect of exposure to a female. Using optical and molecular methods for tracking and manipulating intracellular signalling, we show that this priming effect emerges from the accumulation of mating-related dopamine signals in the MPOA through the accrual of cyclic adenosine monophosphate levels and protein kinase A activity. Dopamine transients in the MPOA are abolished after a successful mating, which is likely to ensure abstinence. Consistent with this idea, the inhibition of AVPV/PVpo dopamine neurons selectively demotivates mating, whereas stimulating these neurons restores the motivation to mate after sexual satiety. We therefore conclude that the accumulation or suppression of signals from specialized dopamine neurons regulates mating behaviours across minutes and days.

Neurobiology of eating behavior, nutrition, and health.

Eating behavior and food-related decision making are among the most complex of the motivated behaviors, and understanding the neurobiology of eating behavior, and its developmental dynamics, is critical to advancing the nutritional sciences and public health. Recent advances from both human and animal studies are revealing that individual capacity to make health-promoting food decisions varies based on biological and physiological variation in the signaling pathways that regulate the homeostatic, hedonic, and executive functions; past developmental exposures and current life-stage; the food environment; and complications of chronic disease that reinforce the obese state. Eating rate drives increased calorie intake and represents an important opportunity to lower rates of food consumption and energy intake through product reformulation. Understanding human eating behaviors and nutrition in the context of neuroscience can strengthen the evidence base from which dietary guidelines are derived and can inform policies, practices, and educational programs in a way that increases the likelihood they are adopted and effective for reducing rates of obesity and other diet-related chronic disease.

Homeostatic circuits selectively gate food cue responses in insular cortex.

Physiological needs bias perception and attention to relevant sensory cues. This process is 'hijacked' by drug addiction, causing cue-induced cravings and relapse. Similarly, its dysregulation contributes to failed diets, obesity, and eating disorders. Neuroimaging studies in humans have implicated insular cortex in these phenomena. However, it remains unclear how 'cognitive' cortical representations of motivationally relevant cues are biased by subcortical circuits that drive specific motivational states. Here we develop a microprism-based cellular imaging approach to monitor visual cue responses in the insular cortex of behaving mice across hunger states. Insular cortex neurons demonstrate food-cue-biased responses that are abolished during satiety. Unexpectedly, while multiple satiety-related visceral signals converge in insular cortex, chemogenetic activation of hypothalamic 'hunger neurons' (expressing agouti-related peptide (AgRP)) bypasses these signals to restore hunger-like response patterns in insular cortex. Circuit mapping and pathway-specific manipulations uncover a pathway from AgRP neurons to insular cortex via the paraventricular thalamus and basolateral amygdala. These results reveal a neural basis for state-specific biased processing of motivationally relevant cues.

Network anatomy and in vivo physiology of visual cortical neurons.

In the cerebral cortex, local circuits consist of tens of thousands of neurons, each of which makes thousands of synaptic connections. Perhaps the biggest impediment to understanding these networks is that we have no wiring diagrams of their interconnections. Even if we had a partial or complete wiring diagram, however, understanding the network would also require information about each neuron's function. Here we show that the relationship between structure and function can be studied in the cortex with a combination of in vivo physiology and network anatomy. We used two-photon calcium imaging to characterize a functional property--the preferred stimulus orientation--of a group of neurons in the mouse primary visual cortex. Large-scale electron microscopy of serial thin sections was then used to trace a portion of these neurons' local network. Consistent with a prediction from recent physiological experiments, inhibitory interneurons received convergent anatomical input from nearby excitatory neurons with a broad range of preferred orientations, although weak biases could not be rejected.

Neurofibrillary tangle-bearing neurons are functionally integrated in cortical circuits in vivo.

Alzheimer's disease (AD) is pathologically characterized by the deposition of extracellular amyloid-ß plaques and intracellular aggregation of tau protein in neurofibrillary tangles (NFTs) (1, 2). Progression of NFT pathology is closely correlated with both increased neurodegeneration and cognitive decline in AD (3) and other tauopathies, such as frontotemporal dementia (4, 5). The assumption that mislocalization of tau into the somatodendritic compartment (6) and accumulation of fibrillar aggregates in NFTs mediates neurodegeneration underlies most current therapeutic strategies aimed at preventing NFT formation or disrupting existing NFTs (7, 8). Although several disease-associated mutations cause both aggregation of tau and neurodegeneration, whether NFTs per se contribute to neuronal and network dysfunction in vivo is unknown (9). Here we used awake in vivo two-photon calcium imaging to monitor neuronal function in adult rTg4510 mice that overexpress a human mutant form of tau (P301L) and develop cortical NFTs by the age of 7-8 mo (10). Unexpectedly, NFT-bearing neurons in the visual cortex appeared to be completely functionally intact, to be capable of integrating dendritic inputs and effectively encoding orientation and direction selectivity, and to have a stable baseline resting calcium level. These results suggest a reevaluation of the common assumption that insoluble tau aggregates are sufficient to disrupt neuronal function.

Imaging neuronal populations in behaving rodents: paradigms for studying neural circuits underlying behavior in the mammalian cortex.

Understanding the neural correlates of behavior in the mammalian cortex requires measurements of activity in awake, behaving animals. Rodents have emerged as a powerful model for dissecting the cortical circuits underlying behavior attributable to the convergence of several methods. Genetically encoded calcium indicators combined with viral-mediated or transgenic tools enable chronic monitoring of calcium signals in neuronal populations and subcellular structures of identified cell types. Stable one- and two-photon imaging of neuronal activity in awake, behaving animals is now possible using new behavioral paradigms in head-fixed animals, or using novel miniature head-mounted microscopes in freely moving animals. This mini-symposium will highlight recent applications of these methods for studying sensorimotor integration, decision making, learning, and memory in cortical and subcortical brain areas. We will outline future prospects and challenges for identifying the neural underpinnings of task-dependent behavior using cellular imaging in rodents.

Repeated stress triggers seeking of a starvation-like state in anxiety-prone female mice.

Elevated anxiety often precedes anorexia nervosa and persists after weight restoration. Patients with anorexia nervosa often describe self-starvation as pleasant, potentially because food restriction can be anxiolytic. Here, we tested whether repeated stress can cause animals to prefer a starvation-like state. We developed a virtual reality place preference paradigm in which head-fixed mice can voluntarily seek a starvation-like state induced by optogenetic stimulation of hypothalamic agouti-related peptide (AgRP) neurons. Prior to stress exposure, males but not females showed a mild aversion to AgRP stimulation. Strikingly, following multiple days of stress, a subset of females developed a strong preference for AgRP stimulation that was predicted by high baseline anxiety. Such stress-induced changes in preference were reflected in changes in facial expressions during AgRP stimulation. Our study suggests that stress may cause females predisposed to anxiety to seek a starvation state and provides a powerful experimental framework for investigating the underlying neural mechanisms.

Transient cAMP production drives rapid and sustained spiking in brainstem parabrachial neurons to suppress feeding.

Brief stimuli can trigger longer-lasting brain states. G-protein-coupled receptors (GPCRs) could help sustain such states by coupling slow-timescale molecular signals to neuronal excitability. Brainstem parabrachial nucleus glutamatergic (PBNGlut) neurons regulate sustained brain states such as pain and express Gs-coupled GPCRs that increase cAMP signaling. We asked whether cAMP in PBNGlut neurons directly influences their excitability and effects on behavior. Both brief tail shocks and brief optogenetic stimulation of cAMP production in PBNGlut neurons drove minutes-long suppression of feeding. This suppression matched the duration of prolonged elevations in cAMP, protein kinase A (PKA) activity, and calcium activity in vivo and ex vivo, as well as sustained, PKA-dependent increases in action potential firing ex vivo. Shortening this elevation in cAMP reduced the duration of feeding suppression following tail shocks. Thus, molecular signaling in PBNGlut neurons helps prolong neural activity and behavioral states evoked by brief, salient bodily stimuli.

Sensitization of meningeal afferents to locomotion-related meningeal deformations in a migraine model.

Migraine headache is hypothesized to involve the activation and sensitization of trigeminal sensory afferents that innervate the cranial meninges. To better understand migraine pathophysiology and improve clinical translation, we used two-photon calcium imaging via a closed cranial window in awake mice to investigate changes in the responses of meningeal afferent fibers using a preclinical model of migraine involving cortical spreading depolarization (CSD). A single CSD episode caused a seconds-long wave of calcium activation that propagated across afferents and along the length of individual afferents. Surprisingly, unlike previous studies in anesthetized animals with exposed meninges, only a very small afferent population was persistently activated in our awake mouse preparation, questioning the relevance of this neuronal response to the onset of migraine pain. In contrast, we identified a larger subset of meningeal afferents that developed augmented responses to acute three-dimensional meningeal deformations that occur in response to locomotion bouts. We observed increased responsiveness in a subset of afferents that were already somewhat sensitive to meningeal deformation before CSD. Furthermore, another subset of previously insensitive afferents also became sensitive to meningeal deformation following CSD. Our data provides new insights into the mechanisms underlying migraine, including the emergence of enhanced meningeal afferent responses to movement-related meningeal deformations as a potential neural substrate underlying the worsening of migraine headache during physical activity.

A spatially-resolved transcriptional atlas of the murine dorsal pons at single-cell resolution.

The "dorsal pons", or "dorsal pontine tegmentum" (dPnTg), is part of the brainstem. It is a complex, densely packed region whose nuclei are involved in regulating many vital functions. Notable among them are the parabrachial nucleus, the Kölliker Fuse, the Barrington nucleus, the locus coeruleus, and the dorsal, laterodorsal, and ventral tegmental nuclei. In this study, we applied single-nucleus RNA-seq (snRNA-seq) to resolve neuronal subtypes based on their unique transcriptional profiles and then used multiplexed error robust fluorescence in situ hybridization (MERFISH) to map them spatially. We sampled ~1 million cells across the dPnTg and defined the spatial distribution of over 120 neuronal subtypes. Our analysis identified an unpredicted high transcriptional diversity in this region and pinpointed the unique marker genes of many neuronal subtypes. We also demonstrated that many neuronal subtypes are transcriptionally similar between humans and mice, enhancing this study's translational value. Finally, we developed a freely accessible, GPU and CPU-powered dashboard ( http://harvard.heavy.ai:6273/ ) that combines interactive visual analytics and hardware-accelerated SQL into a data science framework to allow the scientific community to query and gain insights into the data.

Synaptic plasticity defect following visual deprivation in Alzheimer's disease model transgenic mice.

Amyloid-ß (Aß)-induced changes in synaptic function in experimental models of Alzheimer's disease (AD) suggest that Aß generation and accumulation may affect fundamental mechanisms of synaptic plasticity. To test this hypothesis, we examined the effect of APP overexpression on a well characterized, in vivo, developmental model of systems-level plasticity, ocular dominance plasticity. Following monocular visual deprivation during the critical period, mice that express mutant alleles of amyloid precursor protein (APPswe) and Presenilin1 (PS1dE9), as well as mice that express APPswe alone, lack ocular dominance plasticity in visual cortex. Defects in the spatial extent and magnitude of the plastic response are evident using two complementary approaches, Arc induction and optical imaging of intrinsic signals in awake mice. This defect in a classic paradigm of systems level synaptic plasticity shows that Aß overexpression, even early in postnatal life, can perturb plasticity in cerebral cortex, and supports the idea that decreased synaptic plasticity due to elevated Aß exposure contributes to cognitive impairment in AD.

Sensory cortical ensembles exhibit differential coupling to ripples in distinct hippocampal subregions.

Cortical neurons activated during recent experiences often reactivate with dorsal hippocampal CA1 sharp-wave ripples (SWRs) during subsequent rest. Less is known about cortical interactions with intermediate hippocampal CA1, whose connectivity, functions, and SWRs differ from those of dorsal CA1. We identified three clusters of visual cortical excitatory neurons that are excited together with either dorsal or intermediate CA1 SWRs, or suppressed before both SWRs. Neurons in each cluster were distributed across primary and higher visual cortices and co-active even in the absence of SWRs. These ensembles exhibited similar visual responses but different coupling to thalamus and pupil-indexed arousal. We observed a consistent activity sequence: (i) suppression of SWR-suppressed cortical neurons, (ii) thalamic silence, and (iii) activation of the cortical ensemble preceding and predicting intermediate CA1 SWRs. We propose that the coordinated dynamics of these ensembles relay visual experiences to distinct hippocampal subregions for incorporation into different cognitive maps.

Sensory cortical ensembles exhibit differential coupling to ripples in distinct hippocampal subregions.

Cortical neurons activated during recent experiences often reactivate with dorsal hippocampal CA1 ripples during subsequent rest. Less is known about cortical interactions with intermediate hippocampal CA1, whose connectivity, functions, and ripple events differ from dorsal CA1. We identified three clusters of putative excitatory neurons in mouse visual cortex that are preferentially excited together with either dorsal or intermediate CA1 ripples or suppressed before both ripples. Neurons in each cluster were evenly distributed across primary and higher visual cortices and co-active even in the absence of ripples. These ensembles exhibited similar visual responses but different coupling to thalamus and pupil-indexed arousal. We observed a consistent activity sequence preceding and predicting ripples: (1) suppression of ripple-suppressed cortical neurons, (2) thalamic silence, and (3) activation of intermediate CA1-ripple-activated cortical neurons. We propose that coordinated dynamics of these ensembles relay visual experiences to distinct hippocampal subregions for incorporation into different cognitive maps.

Transient cAMP production drives rapid and sustained spiking in brainstem parabrachial neurons to suppress feeding.

Brief stimuli can trigger longer lasting brain states. G protein-coupled receptors (GPCRs) could help sustain such states by coupling slow-timescale molecular signals to neuronal excitability. Brainstem parabrachial nucleus glutamatergic neurons (PBN Glut ) regulate sustained brain states such as pain, and express G s -coupled GPCRs that increase cAMP signaling. We asked whether cAMP directly influences PBN Glut excitability and behavior. Both brief tail shocks and brief optogenetic stimulation of cAMP production in PBN Glut neurons drove minutes-long suppression of feeding. This suppression matched the duration of prolonged elevations in cAMP, Protein Kinase A (PKA), and calcium activity in vivo and in vitro. Shortening this elevation in cAMP reduced the duration of feeding suppression following tail shocks. cAMP elevations in PBN Glut neurons rapidly lead to sustained increases in action potential firing via PKA-dependent mechanisms. Thus, molecular signaling in PBN Glut neurons helps prolong neural activity and behavioral states evoked by brief, salient bodily stimuli.

Sensitization of meningeal afferents to locomotion-related meningeal deformations in a migraine model.

Migraine headache is hypothesized to involve the activation and sensitization of trigeminal sensory afferents that innervate the cranial meninges. To better understand migraine pathophysiology and improve clinical translation, we used two-photon calcium imaging via a closed cranial window in awake mice to investigate changes in the responses of meningeal afferent fibers using a preclinical model of migraine involving cortical spreading depolarization (CSD). A single CSD episode caused a seconds-long wave of calcium activation that propagated across afferents and along the length of individual afferents. Surprisingly, unlike previous studies in anesthetized animals with exposed meninges, only a very small afferent population was persistently activated in our awake mouse preparation, questioning the relevance of this neuronal response to the onset of migraine pain. In contrast, we identified a larger subset of meningeal afferents that developed augmented responses to acute three-dimensional meningeal deformations that occur in response to locomotion bouts. We observed increased responsiveness in a subset of afferents that were already somewhat sensitive to meningeal deformation before CSD. Furthermore, another subset of previously insensitive afferents also became sensitive to meningeal deformation following CSD. Our data provides new insights into the mechanisms underlying migraine, including the emergence of enhanced meningeal afferent responses to movement-related meningeal deformations as a potential neural substrate underlying the worsening of migraine headache during physical activity.

Chronic stress triggers seeking of a starvation-like state in anxiety-prone female mice.

Elevated anxiety often precedes anorexia nervosa and persists after weight restoration. Patients with anorexia nervosa often describe hunger as pleasant, potentially because food restriction can be anxiolytic. Here, we tested whether chronic stress can cause animals to prefer a starvation-like state. We developed a virtual reality place preference paradigm in which head-fixed mice can voluntarily seek a starvation-like state induced by optogenetic stimulation of hypothalamic agouti-related peptide (AgRP) neurons. Prior to stress induction, male but not female mice showed mild aversion to AgRP stimulation. Strikingly, following chronic stress, a subset of females developed a strong preference for AgRP stimulation that was predicted by high baseline anxiety. Such stress-induced changes in preference were reflected in changes in facial expressions during AgRP stimulation. Our study suggests that stress may cause females predisposed to anxiety to seek a starvation state, and provides a powerful experimental framework for investigating the underlying neural mechanisms.

Competition between stochastic neuropeptide signals calibrates the rate of satiation.

We investigated how transmission of hunger- and satiety-promoting neuropeptides, NPY and αMSH, is integrated at the level of intracellular signaling to control feeding. Receptors for these peptides use the second messenger cAMP, but the messenger's spatiotemporal dynamics and role in energy balance are controversial. We show that AgRP axon stimulation in the paraventricular hypothalamus evokes probabilistic and spatially restricted NPY release that triggers stochastic cAMP decrements in downstream MC4R-expressing neurons (PVH MC4R ). Meanwhile, POMC axon stimulation triggers stochastic, αMSH-dependent cAMP increments. NPY and αMSH competitively control cAMP, as reflected by hunger-state-dependent differences in the amplitude and persistence of cAMP transients evoked by each peptide. During feeding bouts, elevated αMSH release and suppressed NPY release cooperatively sustain elevated cAMP in PVH MC4R neurons, thereby potentiating feeding-related excitatory inputs and promoting satiation across minutes. Our findings highlight how state-dependent integration of opposing, quantal peptidergic events by a common biochemical target calibrates energy intake.

Brainstem serotonin neurons selectively gate retinal information flow to thalamus.

Retinal ganglion cell (RGC) types relay parallel streams of visual feature information. We hypothesized that neuromodulators might efficiently control which visual information streams reach the cortex by selectively gating transmission from specific RGC axons in the thalamus. Using fiber photometry recordings, we found that optogenetic stimulation of serotonergic axons in primary visual thalamus of awake mice suppressed ongoing and visually evoked calcium activity and glutamate release from RGC boutons. Two-photon calcium imaging revealed that serotonin axon stimulation suppressed RGC boutons that responded strongly to global changes in luminance more than those responding only to local visual stimuli, while the converse was true for suppression induced by increases in arousal. Converging evidence suggests that differential expression of the 5-HT1B receptor on RGC presynaptic terminals, but not differential density of nearby serotonin axons, may contribute to the selective serotonergic gating of specific visual information streams before they can activate thalamocortical neurons.