Hindbrain Double-Negative Feedback Mediates Palatability-Guided Food and Water Consumption.

Hunger and thirst have distinct goals but control similar ingestive behaviors, and little is known about neural processes that are shared between these behavioral states. We identify glutamatergic neurons in the peri-locus coeruleus (periLCVGLUT2 neurons) as a polysynaptic convergence node from separate energy-sensitive and hydration-sensitive cell populations. We develop methods for stable hindbrain calcium imaging in free-moving mice, which show that periLCVGLUT2 neurons are tuned to ingestive behaviors and respond similarly to food or water consumption. PeriLCVGLUT2 neurons are scalably inhibited by palatability and homeostatic need during consumption. Inhibition of periLCVGLUT2 neurons is rewarding and increases consumption by enhancing palatability and prolonging ingestion duration. These properties comprise a double-negative feedback relationship that sustains food or water consumption without affecting food- or water-seeking. PeriLCVGLUT2 neurons are a hub between hunger and thirst that specifically controls motivation for food and water ingestion, which is a factor that contributes to hedonic overeating and obesity.

Dynamic Reorganization of Neuronal Activity Patterns in Parietal Cortex.

Neuronal representations change as associations are learned between sensory stimuli and behavioral actions. However, it is poorly understood whether representations for learned associations stabilize in cortical association areas or continue to change following learning. We tracked the activity of posterior parietal cortex neurons for a month as mice stably performed a virtual-navigation task. The relationship between cells' activity and task features was mostly stable on single days but underwent major reorganization over weeks. The neurons informative about task features (trial type and maze locations) changed across days. Despite changes in individual cells, the population activity had statistically similar properties each day and stable information for over a week. As mice learned additional associations, new activity patterns emerged in the neurons used for existing representations without greatly affecting the rate of change of these representations. We propose that dynamic neuronal activity patterns could balance plasticity for learning and stability for memory.

A pharynx-to-brain axis controls pharyngeal inflammation-induced anxiety.

Anxiety is a remarkably common condition among patients with pharyngitis, but the relationship between these disorders has received little research attention, and the underlying neural mechanisms remain unknown. Here, we show that the densely innervated pharynx transmits signals induced by pharyngeal inflammation to glossopharyngeal and vagal sensory neurons of the nodose/jugular/petrosal (NJP) superganglia in mice. Specifically, the NJP superganglia project to norepinephrinergic neurons in the nucleus of the solitary tract (NTSNE). These NTSNE neurons project to the ventral bed nucleus of the stria terminalis (vBNST) that induces anxiety-like behaviors in a murine model of pharyngeal inflammation. Inhibiting this pharynx→NJP→NTSNE→vBNST circuit can alleviate anxiety-like behaviors associated with pharyngeal inflammation. This study thus defines a pharynx-to-brain axis that mechanistically links pharyngeal inflammation and emotional response.

Arginine-vasopressin-expressing neurons in the murine suprachiasmatic nucleus exhibit a circadian rhythm in network coherence in vivo.

The suprachiasmatic nucleus (SCN) is composed of functionally distinct subpopulations of GABAergic neurons which form a neural network responsible for synchronizing most physiological and behavioral circadian rhythms in mammals. To date, little is known regarding which aspects of SCN rhythmicity are generated by individual SCN neurons, and which aspects result from neuronal interaction within a network. Here, we utilize in vivo miniaturized microscopy to measure fluorescent GCaMP-reported calcium dynamics in arginine vasopressin (AVP)-expressing neurons in the intact SCN of awake, behaving mice. We report that SCN AVP neurons exhibit periodic, slow calcium waves which we demonstrate, using in vivo electrical recordings, likely reflect burst firing. Further, we observe substantial heterogeneity of function in that AVP neurons exhibit unstable rhythms, and relatively weak rhythmicity at the population level. Network analysis reveals that correlated cellular behavior, or coherence, among neuron pairs also exhibited stochastic rhythms with about 33% of pairs rhythmic at any time. Unlike single-cell variables, coherence exhibited a strong rhythm at the population level with time of maximal coherence among AVP neuronal pairs at CT/ZT 6 and 9, coinciding with the timing of maximal neuronal activity for the SCN as a whole. These results demonstrate robust circadian variation in the coordination between stochastically rhythmic neurons and that interactions between AVP neurons in the SCN may be more influential than single-cell activity in the regulation of circadian rhythms. Furthermore, they demonstrate that cells in this circuit, like those in many other circuits, exhibit profound heterogenicity of function over time and space.

Principles of nociceptive coding in the anterior cingulate cortex.

The perception of pain is a multidimensional sensory and emotional/affective experience arising from distributed brain activity. However, the involved brain regions are not specific for pain. Thus, how the cortex distinguishes nociception from other aversive and salient sensory stimuli remains elusive. Additionally, the resulting consequences of chronic neuropathic pain on sensory processing have not been characterized. Using in vivo miniscope calcium imaging with cellular resolution in freely moving mice, we elucidated the principles of nociceptive and sensory coding in the anterior cingulate cortex, a region essential for pain processing. We found that population activity, not single-cell responses, allowed discriminating noxious from other sensory stimuli, ruling out the existence of nociception-specific neurons. Additionally, single-cell stimulus selectivity was highly dynamic over time, but stimulus representation at the population level remained stable. Peripheral nerve injury-induced chronic neuropathic pain led to dysfunctional encoding of sensory events by exacerbation of responses to innocuous stimuli and impairment of pattern separation and stimulus classification, which were restored by analgesic treatment. These findings provide a novel interpretation for altered cortical sensory processing in chronic neuropathic pain and give insights into the effects of systemic analgesic treatment in the cortex.

Combined Experiments with in Vivo Fiber Photometry and Behavior Tests Can Facilitate the Measurement of Neuronal Activity in the Primary Somatosensory Cortex and Hyperalgesia in an Inflammatory Pain Mice Model.

The pain matrix, which includes several brain regions that respond to pain sensation, contribute to the development of chronic pain. Thus, it is essential to understand the mechanism of causing chronic pain in the pain matrix such as anterior cingulate (ACC), or primary somatosensory (S1) cortex. Recently, combined experiment with the behavior tests and in vivo calcium imaging using fiber photometry revealed the interaction between the neuronal function in deep brain regions of the pain matrix including ACC and the phenotype of chronic pain. However, it remains unclear whether this combined experiment can identify the interaction between neuronal activity in S1, which receive pain sensation, and pain behaviors such as hyperalgesia or allodynia. In this study, to examine whether the interaction between change of neuronal activity in S1 and hyperalgesia in hind paw before and after causing inflammatory pain was detected from same animal, the combined experiment of in vivo fiber photometry system and von Frey hairs test was applied. This combined experiment detected that amplitude of calcium responses in S1 neurons increased and the mechanical threshold of hind paw decreased from same animals which have an inflammatory pain. Moreover, we found that the values between amplitude of calcium responses and mechanical thresholds were shifted to negative correlation after causing inflammatory pain. Thus, the combined experiment with fiber photometry and the behavior tests has a possibility that can simultaneously consider the interaction between neuronal activity in pain matrix and pain induced behaviors and the effects of analgesics or pain treatments.

Catecholamines modulate the hypoxic ventilatory response of larval zebrafish (Danio rerio).

The hypoxic ventilatory response (HVR) in fish is an important reflex that aids O2 uptake when low environmental O2 levels constrain diffusion. In developing zebrafish (Danio rerio), the acute HVR is multiphasic, consisting of a rapid increase in ventilation frequency (fV) during hypoxia onset, followed by a decline to a stable plateau phase above fV under normoxic conditions. In this study, we examined the potential role of catecholamines in contributing to each of these phases of the dynamic HVR in zebrafish larvae. We showed that adrenaline elicits a dose-dependent ß-adrenoreceptor (AR)-mediated increase in fV that does not require expression of ß1-ARs, as the hyperventilatory response to ß-AR stimulation was unaltered in adrb1-/- mutants, generated by CRISPR/Cas9 knockout. In response to hypoxia and propranolol co-treatment, the magnitude of the rapidly occurring peak increase in fV during hypoxia onset was attenuated (112±14 breaths min-1 without propranolol to 68±17 breaths min-1 with propranolol), whereas the increased fV during the stable phase of the HVR was prevented in both wild type and adrb1-/- mutants. Thus, ß1-AR is not required for the HVR and other ß-ARs, although not required for initiation of the HVR, are involved in setting the maximal increase in fV and in maintaining hyperventilation during continued hypoxia. This adrenergic modulation of the HVR may arise from centrally released catecholamines because adrenaline exposure failed to activate (based on intracellular Ca2+ levels) cranial nerves IX and X, which transmit O2 signals from the pharyngeal arch to the central nervous system.

Optogenetic therapy restores retinal activity in primate for at least a year following photoreceptor ablation.

All retina-based vision restoration approaches rely on the assumption that photoreceptor loss does not preclude reactivation of the remaining retinal architecture. Whether extended periods of vision loss limit the efficacy of restorative therapies at the retinal level is unknown. We examined long-term changes in optogenetic responsivity of foveal retinal ganglion cells (RGCs) in non-human primates following localized photoreceptor ablation by high-intensity laser exposure. By performing fluorescence adaptive optics scanning light ophthalmoscopy (AOSLO) of RGCs expressing both the calcium indicator GCaMP6s and the optogenetic actuator ChrimsonR, it was possible to track optogenetic-mediated calcium responses in deafferented RGCs over time. Fluorescence fundus photography revealed a 40% reduction in ChrimsonR fluorescence from RGCs lacking photoreceptor input over the 3 weeks following photoreceptor ablation. Despite this, in vivo imaging revealed good cellular preservation of RGCs 3 months after the loss of photoreceptor input, and histology confirmed good structural preservation at 2 years. Optogenetic responses of RGCs in primate persisted for at least 1 year after the loss of photoreceptor input, with a sensitivity index similar to optogenetic responses recorded in intact retina. These results are promising for all potential therapeutic approaches to vision restoration that rely on preservation and reactivation of RGCs.

Modality-Specific Modulation of Temperature Representations in the Spinal Cord after Injury.

Different types of tissue injury, such as inflammatory and neuropathic conditions, cause modality-specific alternations on temperature perception. There are profound changes in peripheral sensory neurons after injury, but how patterned neuronal activities in the CNS encode injury-induced sensitization to temperature stimuli is largely unknown. Using in vivo calcium imaging and mouse genetics, we show that formalin- and prostaglandin E2-induced inflammation dramatically increase spinal responses to heating and decrease responses to cooling in male and female mice. The reduction of cold response is largely eliminated on ablation of TRPV1-expressing primary sensory neurons, indicating a crossover inhibition of cold response from the hyperactive heat inputs in the spinal cord. Interestingly, chemotherapy medication oxaliplatin can rapidly increase spinal responses to cooling and suppress responses to heating. Together, our results suggest a push-pull mechanism in processing cold and heat inputs and reveal a synergic mechanism to shift thermosensation after injury.SIGNIFICANCE STATEMENT In this paper, we combine our novel in vivo spinal cord two-photon calcium imaging, mouse genetics, and persistent pain models to study how tissue injury alters the sensation of temperature. We discover modality-specific changes of spinal temperature responses in different models of injury. Chemotherapy medication oxaliplatin leads to cold hypersensitivity and heat hyposensitivity. By contrast, inflammation increases heat sensitivity and decreases cold sensitivity. This decrease in cold sensitivity results from the stronger crossover inhibition from the hyperactive heat inputs. Our work reveals the bidirectional change of thermosensitivity by injury and suggests that the crossover inhibitory circuit underlies the shifted thermosensation, providing a mechanism to the biased perception toward a unique thermal modality that was observed clinically in chronic pain patients.

Deficiency of Inositol Monophosphatase Activity Decreases Phosphoinositide Lipids and Enhances TRPV1 Function In Vivo.

Membrane remodeling by inflammatory mediators influences the function of sensory ion channels. The capsaicin- and heat-activated transient receptor potential vanilloid 1 (TRPV1) channel contributes to neurogenic inflammation and pain hypersensitivity, in part because of its potentiation downstream of phospholipase C-coupled receptors that regulate phosphoinositide lipid content. Here, we determined the effect of phosphoinositide lipids on TRPV1 function by combining genetic dissection, diet supplementation, and behavioral, biochemical, and functional analyses in Caenorhabditis elegans As capsaicin elicits heat and pain sensations in mammals, transgenic TRPV1 worms exhibit an aversive response to capsaicin. TRPV1 worms with low levels of phosphoinositide lipids display an enhanced response to capsaicin, whereas phosphoinositide lipid supplementation reduces TRPV1-mediated responses. A worm carrying a TRPV1 construct lacking the distal C-terminal domain features an enhanced response to capsaicin, independent of the phosphoinositide lipid content. Our results demonstrate that TRPV1 activity is enhanced when the phosphoinositide lipid content is reduced, and the C-terminal domain is key to determining agonist response in vivo.

Spatial organization and transitions of spontaneous neuronal activities in the developing sensory cortex.

The sensory cortex underlies our ability to perceive and interact with the external world. Sensory perceptions are controlled by specialized neuronal circuits established through fine-tuning, which relies largely on neuronal activity during the development. Spontaneous neuronal activity is an essential driving force of neuronal circuit refinement. At early developmental stages, sensory cortices display spontaneous activities originating from the periphery and characterized by correlated firing arranged spatially according to the modality. The firing patterns are reorganized over time and become sparse, which is typical for the mature brain. This review focuses mainly on rodent sensory cortices. First, the features of the spontaneous activities during early postnatal stages are described. Then, the developmental changes in the spatial organization of the spontaneous activities and the transition mechanisms involved are discussed. The identification of the principles controlling the spatial organization of spontaneous activities in the developing sensory cortex is essential to understand the self-organization process of neuronal circuits.

Changing Functional Signatures of Microglia along the Axis of Brain Aging.

Microglia, the innate immune cells of the brain, are commonly perceived as resident macrophages of the central nervous system (CNS). This definition, however, requires further specification, as under healthy homeostatic conditions, neither morphological nor functional properties of microglia mirror those of classical macrophages. Indeed, microglia adapt exceptionally well to their microenvironment, becoming a legitimate member of the cellular brain architecture. The ramified or surveillant microglia in the young adult brain are characterized by specific morphology (small cell body and long, thin motile processes) and physiology (a unique pattern of Ca2+ signaling, responsiveness to various neurotransmitters and hormones, in addition to classic "immune" stimuli). Their numerous physiological functions far exceed and complement their immune capabilities. As the brain ages, the respective changes in the microglial microenvironment impact the functional properties of microglia, triggering further rounds of adaptation. In this review, we discuss the recent data showing how functional properties of microglia adapt to age-related changes in brain parenchyma in a sex-specific manner, with a specific focus on early changes occurring at middle age as well as some strategies counteracting the aging of microglia.

BACE inhibition-dependent repair of Alzheimer's pathophysiology.

Amyloid-ß (Aß) is thought to play an essential pathogenic role in Alzheimer´s disease (AD). A key enzyme involved in the generation of Aß is the ß-secretase BACE, for which powerful inhibitors have been developed and are currently in use in human clinical trials. However, although BACE inhibition can reduce cerebral Aß levels, whether it also can ameliorate neural circuit and memory impairments remains unclear. Using histochemistry, in vivo Ca2+ imaging, and behavioral analyses in a mouse model of AD, we demonstrate that along with reducing prefibrillary Aß surrounding plaques, the inhibition of BACE activity can rescue neuronal hyperactivity, impaired long-range circuit function, and memory defects. The functional neuronal impairments reappeared after infusion of soluble Aß, mechanistically linking Aß pathology to neuronal and cognitive dysfunction. These data highlight the potential benefits of BACE inhibition for the effective treatment of a wide range of AD-like pathophysiological and cognitive impairments.

Relationship between the fluorescence intensity of rhodamine-labeled orexin A and the calcium responses in cortical neurons: An in vivo two-photon calcium imaging study.

Neural responses to a ligand vary widely between neurons; however, the mechanisms underlying this variation remain unclear. One possible mechanism is a variation in the number of receptors expressed in each neural membrane. Here, we synthesized a rhodamine-labeled orexin A compound, enabling us to quantify the amount of orexin binding to its receptors, OX1 and OX2, which principally couple to the Gq/11 protein. The rhodamine intensity and calcium response were measured under tetrodotoxin application from insular cortical glutamatergic neurons in Thy1-GCaMP6s transgenic mice using an in vivo two-photon microscope. Applying rhodamine-labeled orexin A (10 µM) to the cortical surface gradually and heterogeneously increased both the intensity of the rhodamine fluorescence and [Ca2+]i. Calcium responses started simultaneously with the increase in rhodamine-labeled orexin fluorescence and reached a plateau within several minutes. We classified neurons as high- and low-responding neurons based on the peak amplitude of the [Ca2+]i increase. The rhodamine fluorescence intensity was larger in the high-responding neurons than the low-responding neurons. Preapplication of SB334867 and TCS-OX2-29, OX1 and OX2 antagonists, respectively, decreased the proportion of high-responding neurons. These results suggest that the diverse receptor expression level in neural membranes is involved in mechanisms underlying varied neural responses, including [Ca2+]i increases.

Endogenous sequential cortical activity evoked by visual stimuli.

Although the functional properties of individual neurons in primary visual cortex have been studied intensely, little is known about how neuronal groups could encode changing visual stimuli using temporal activity patterns. To explore this, we used in vivo two-photon calcium imaging to record the activity of neuronal populations in primary visual cortex of awake mice in the presence and absence of visual stimulation. Multidimensional analysis of the network activity allowed us to identify neuronal ensembles defined as groups of cells firing in synchrony. These synchronous groups of neurons were themselves activated in sequential temporal patterns, which repeated at much higher proportions than chance and were triggered by specific visual stimuli such as natural visual scenes. Interestingly, sequential patterns were also present in recordings of spontaneous activity without any sensory stimulation and were accompanied by precise firing sequences at the single-cell level. Moreover, intrinsic dynamics could be used to predict the occurrence of future neuronal ensembles. Our data demonstrate that visual stimuli recruit similar sequential patterns to the ones observed spontaneously, consistent with the hypothesis that already existing Hebbian cell assemblies firing in predefined temporal sequences could be the microcircuit substrate that encodes visual percepts changing in time.

Sparse, reliable, and long-term stable representation of periodic whisker deflections in the mouse barrel cortex.

The rodent whisker system is a preferred model for studying plasticity in the somatosensory cortex (barrel cortex). Contrarily, only a small amount of research has been conducted to characterize the stability of neuronal population activity in the barrel cortex. We used the mouse whisker system to address the neuronal basis of stable perception in the somatosensory cortex. Cortical representation of periodic whisker deflections was studied in populations of neurons in supragranular layers over extended time periods (up to 3 months) with long-term two-photon Ca(2+) imaging in anesthetized mice. We found that in most of the neurons (87%), Ca(2+) responses increased sublinearly with increasing number of contralateral whisker deflections. The imaged population of neurons was activated in a stereotypic way over days and for different deflection rates (pulse frequencies). Thus, pulse frequencies are coded by response strength rather than by distinct neuronal sub-populations. A small population of highly responsive neurons (~3%) was sufficient to decode the whisker stimulus. This conserved functional map, led by a small set of highly responsive neurons, might form the foundation of stable sensory percepts.

Cadmium inhibits calcium activity in hippocampal CA1 neurons of freely moving mice.

Cadmium (Cd) is a ubiquitous toxic heavy metal and a potential neurotoxicant due to its wide use in industrial manufacturing processes and commercial products, including fertilizers. The general population is exposed to Cd through food and smoking due to high transfer rates of Cd from contaminated soil. Cd has been shown to mimic calcium ions (Ca2+) and interfere with intracellular Ca2+ levels and Ca2+ signaling in in vitro studies. However, nothing is known about Cd's effects on Ca2+ activity in neurons in live animals. This study aimed to determine if Cd disrupts Ca2+ transients of neurons in CA1 region of the hippocampus during an associative learning paradigm. We utilized in vivo Ca2+ imaging in awake, freely moving C57BL/6 mice to measure Ca2+ activity in CA1 excitatory neurons expressing genetically encoded Ca2+ sensor GCaMP6 during an associative learning paradigm. We found that a smaller proportion of neurons are activated in Cd-treated groups compared with control during fear conditioning, suggesting that Cd may contribute to learning and memory deficit by reducing the activity of neurons. We observed these effects at Cd exposure levels that result in blood Cd levels comparable with the general U.S. population levels. This provides a possible molecular mechanism for Cd interference of learning and memory at exposure levels relevant to U.S. adults. To our knowledge, our study is the first to describe Cd effects on brain Ca2+ activity in vivo in freely behaving mice. This study provides evidence for impairment of neuronal calcium activity in hippocampal CA1 excitatory neurons in freely moving mice following cadmium exposure.

Midbrain dopaminergic degeneration differentially modulates primary motor cortex activity and motor behavior in hemi-parkinsonian rats.

Parkinson's disease (PD) is marked by degeneration in the nigrostriatal dopaminergic pathway, affecting motor control via complex changes in the cortico-basal ganglia-thalamic motor network, including the primary motor cortex (M1). The modulation of M1 neuronal activity by dopaminergic inputs, particularly from the ventral tegmental area (VTA) and substantia nigra pars compacta (SNc), plays a crucial role in PD pathophysiology. This study investigates how nigrostriatal dopaminergic degeneration influences M1 neuronal activity in rats using in vivo calcium imaging. Histological analysis confirmed dopaminergic lesion severity, with high lesion level rats showing significant motor deficits. Levodopa treatment improved fine motor abilities, particularly in high lesion level rats. Analysis of M1 calcium signals based on dopaminergic lesion severity revealed distinct M1 activity patterns. Animals with low dopaminergic lesion showed increased calcium events, while high lesion level rats exhibited decreased activity, partially restored by levodopa. These findings suggest that M1 activity is more sensitive to transient fluctuations in dopaminergic transmission, rather than to chronic high or low dopaminergic signaling. This study underscores the complex interplay between dopaminergic signaling and M1 neuronal activity in PD symptoms development. Further research integrating behavioral and calcium imaging data can elucidate mechanisms underlying motor deficits and therapeutic responses in PD.

The NKCC1 inhibitor bumetanide restores cortical feedforward inhibition and lessens sensory hypersensitivity in early postnatal fragile X mice.

BACKGROUND: Exaggerated responses to sensory stimuli, a hallmark of Fragile X syndrome (FXS), contribute to anxiety and learning challenges. Sensory hypersensitivity is recapitulated in the Fmr1 knockout (KO) mouse model of FXS. Recent studies in Fmr1 KO mice have demonstrated differences in activity of cortical interneurons and a delayed switch in the polarity of GABA signaling during development. Previously, we reported that blocking the chloride transporter NKCC1 with the diuretic bumetanide, could rescue synaptic circuit phenotypes in primary somatosensory cortex (S1) of Fmr1 KO mice. However, it remains unknown whether bumetanide can rescue earlier circuit phenotypes or sensory hypersensitivity in Fmr1 KO mice. METHODS: We used acute and chronic systemic administration of bumetanide in Fmr1 KO mice and performed in vivo 2-photon calcium imaging to record neuronal activity, while tracking mouse behavior with high-resolution videos. RESULTS: We demonstrate that layer (L) 2/3 pyramidal neurons in S1 of Fmr1 KO mice show a higher frequency of synchronous events at postnatal day (P) 6 compared to wild-type controls. This was reversed by acute administration of bumetanide. Furthermore, chronic bumetanide treatment (P5-P14) restored S1 circuit differences in Fmr1 KO mice, including reduced neuronal adaptation to repetitive whisker stimulation, and ameliorated tactile defensiveness. Bumetanide treatment also rectified the reduced feedforward inhibition of L2/3 neurons in S1 and boosted the circuit participation of parvalbumin interneurons. CONCLUSIONS: This further supports the notion that synaptic, circuit, and sensory behavioral phenotypes in Fmr1 KO can be mitigated by inhibitors of NKCC1, such as the FDA-approved diuretic bumetanide.

Endocannabinoid-mediated rescue of somatosensory cortex activity, plasticity and related behaviors following an early in life concussion.

Due to the assumed plasticity of immature brain, early in life brain alterations are thought to lead to better recoveries in comparison to the mature brain. Despite clinical needs, how neuronal networks and associated behaviors are affected by early in life brain stresses, such as pediatric concussions, have been overlooked. Here we provide first evidence in mice that a single early in life concussion durably increases neuronal activity in the somatosensory cortex into adulthood, disrupting neuronal integration while the animal is performing sensory-related tasks. This represents a previously unappreciated clinically relevant mechanism for the impairment of sensory-related behavior performance. Furthermore, we demonstrate that pharmacological modulation of the endocannabinoid system a year post-concussion is well-suited to rescue neuronal activity and plasticity, and to normalize sensory-related behavioral performance, addressing the fundamental question of whether a treatment is still possible once post-concussive symptoms have developed, a time-window compatible with clinical treatment.