Neuron-Astrocyte Metabolic Coupling Protects against Activity-Induced Fatty Acid Toxicity.

Metabolic coordination between neurons and astrocytes is critical for the health of the brain. However, neuron-astrocyte coupling of lipid metabolism, particularly in response to neural activity, remains largely uncharacterized. Here, we demonstrate that toxic fatty acids (FAs) produced in hyperactive neurons are transferred to astrocytic lipid droplets by ApoE-positive lipid particles. Astrocytes consume the FAs stored in lipid droplets via mitochondrial ß-oxidation in response to neuronal activity and turn on a detoxification gene expression program. Our findings reveal that FA metabolism is coupled in neurons and astrocytes to protect neurons from FA toxicity during periods of enhanced activity. This coordinated mechanism for metabolizing FAs could underlie both homeostasis and a variety of disease states of the brain.

The Anatomy and Physiology of Claustrum-Cortex Interactions.

The claustrum is one of the most widely connected regions of the forebrain, yet its function has remained obscure, largely due to the experimentally challenging nature of targeting this small, thin, and elongated brain area. However, recent advances in molecular techniques have enabled the anatomy and physiology of the claustrum to be studied with the spatiotemporal and cell type-specific precision required to eventually converge on what this area does. Here we review early anatomical and electrophysiological results from cats and primates, as well as recent work in the rodent, identifying the connectivity, cell types, and physiological circuit mechanisms underlying the communication between the claustrum and the cortex. The emerging picture is one in which the rodent claustrum is closely tied to frontal/limbic regions and plays a role in processes, such as attention, that are associated with these areas.

Social isolation produces a sex- and brain region-specific alteration of microglia state.

Social isolation is a profound form of psychological stress that impacts the mental health of a large proportion of society. Other experimental models of stress have demonstrated a microglia response that serves either a protective or pathological function. However, the effect of adult social isolation on microglia has not been thoroughly investigated. We measured microglia territory, branching, end points and phagocytic-lysosomal activity in group housed C57Bl/6 mice and mice that were socially isolated for 2 weeks. Our results show that the dorsomedial hypothalamus and hippocampal CA2 region of adult male mice undergo increased microglia volume, territory and endpoints following social isolation, whereas females exhibit this increase in the hypothalamus only. Males exhibited decreases in the phagocytic-lysosomal marker CD68 in microglia in these regions, whereas females showed an increase in CD68 in the hypothalamus suggesting sexually dimorphic and brain region-specific change in microglia state in response to social isolation. The prefrontal cortex, central amygdala, nucleus accumbens shell and visual cortex did not exhibit changes in microglia structure in either male or female mice. These data show that microglia in different brain regions undergo a distinct response to social isolation which may account for changes in cognition and behaviour associated with this prevalent form of psychological stress.

Major urinary protein excreted in rodent hindpaw sweat.

Alternative roles for sweat production beyond thermoregulation, considered less frequently, include chemical signaling. We identified the presence of a well-established rodent urinary pheromone, major urinary protein (MUP) in sweat ductules of the footpad dermal skin of mice. A hindpaw sweat proteomic analysis in hindpaw sweat samples collected in rats and generated by unmyelinated axon activation, identified seven lipocalin family members including MUP and 19 additional unique proteins. Behavioural responses to sniffing male mouse foot protein lysates suggested avoidance in a subset of male mice, but were not definitive. Rodent hindpaw sweat glands secrete a repertoire of proteins that include MUPs known to have roles in olfactory communication.

Cortical Control of Spatial Resolution by VIP+ Interneurons.

Neuronal tuning, defined by the degree of selectivity to a specific stimulus, is a hallmark of cortical computation. Understanding the role of GABAergic interneurons in shaping cortical tuning is now possible with the ability to manipulate interneuron classes selectively. Here, we show that interneurons expressing vasoactive intestinal polypeptide (VIP+) regulate the spatial frequency (SF) tuning of pyramidal neurons in mouse visual cortex. Using two-photon calcium imaging and optogenetic manipulations of VIP+ cell activity, we found that activating VIP+ cells elicited a stronger network response to stimuli of higher SFs, whereas suppressing VIP+ cells resulted in a network response shift toward lower SFs. These results establish that cortical inhibition modulates the spatial resolution of visual processing and add further evidence demonstrating that feature selectivity depends, not only on the feedforward excitatory projections into the cortex, but also on dynamic intracortical modulations by specific forms of inhibition. SIGNIFICANCE STATEMENT: We demonstrate that interneurons expressing vasoactive intestinal polypeptide (VIP+) play a causal role in regulating the spatial frequency (SF) tuning of neurons in mouse visual cortex. We show that optogenetic activation of VIP+ cells results in a shift in network preference toward higher SFs, whereas suppressing them shifts the network toward lower SFs. Several studies have shown that VIP+ cells are sensitive to neuromodulation and increase their firing during locomotion, whisking, and pupil dilation and are involved in spatially specific top-down modulation, reminiscent of the effects of top-down attention, and also that attention enhances spatial resolution. Our findings provide a bridge between these studies by establishing the inhibitory circuitry that regulates these fundamental modulations of SF in the cortex.

Opening Holes in the Blanket of Inhibition: Localized Lateral Disinhibition by VIP Interneurons.

Inhibitory interneurons in the neocortex often connect in a promiscuous and extensive fashion, extending a "blanket of inhibition" on the circuit. This raises the problem of how can excitatory activity propagate in the midst of this widespread inhibition. One solution to this problem could be the vasoactive intestinal peptide (VIP) interneurons, which disinhibit other interneurons. To explore how VIP interneurons affect the local circuits, we use two-photon optogenetics to activate them individually in mouse visual cortex in vivo while measuring their output with two-photon calcium imaging. We find that VIP interneurons have narrow axons and inhibit nearby somatostatin interneurons, which themselves inhibit pyramidal cells. Moreover, via this lateral disinhibition, VIP cells in vivo make local and transient "holes" in the inhibitory blanket extended by SOM cells. VIP interneurons, themselves regulated by neuromodulators, may therefore enable selective patterns of activity to propagate through the cortex, by generating a "spotlight of attention". SIGNIFICANCE STATEMENT: Most inhibitory interneurons have axons restricted to a nearby area and target excitatory neighbors indiscriminately, raising the issue of how neuronal activity can propagate through cortical circuits. Vasoactive intestinal peptide-expressing interneurons (VIPs) disinhibit cortical pyramidal cells through inhibition of other inhibitory interneurons, and they have very focused, "narrow" axons. By optogenetically activating single VIPs in live mice while recording the activity of nearby neurons, we find that VIPs break open a hole in blanket inhibition with an effective range of ∼120 µm in lateral cortical space where excitatory activity can propagate.

Spatial Reference Memory is Associated with Modulation of Theta-Gamma Coupling in the Dentate Gyrus.

Spatial reference memory in rodents represents a unique opportunity to study brain mechanisms responsible for encoding, storage and retrieval of a memory. Even though its reliance on hippocampal networks has long been established, the precise computations performed by different hippocampal subfields during spatial learning are still not clear. To study the evolution of electrophysiological activity in the CA1-dentate gyrus axis of the dorsal hippocampus over an iterative spatial learning paradigm, we recorded local field potentials in behaving mice using a newly designed appetitive version of the Barnes maze. We first showed that theta and gamma oscillations as well as theta-gamma coupling are differentially modulated in particular hippocampal subfields during the task. In addition, we show that dentate gyrus networks, but not CA1 networks, exhibit a transient learning-dependent increase in theta-gamma coupling specifically at the vicinity of the target area in the maze. In contrast to previous immediate early-gene studies, our results point to a long-lasting involvement of dentate networks in navigational memory in the Barnes maze. Based on these findings, we propose that theta-gamma coupling might represent a mechanism by which hippocampal areas compute relevant information.

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.

VIP+ interneurons control neocortical activity across brain states.

GABAergic interneurons are positioned to powerfully influence the dynamics of neural activity, yet the interneuron-mediated circuit mechanisms that control spontaneous and evoked neocortical activity remains elusive. Vasoactive intestinal peptide (VIP+) interneurons are a specialized cell class which synapse specifically on other interneurons, potentially serving to facilitate increases in cortical activity. In this study, using in vivo Ca(2+) imaging, we describe the interaction between local network activity and VIP+ cells and determine their role in modulating neocortical activity in mouse visual cortex. VIP+ cells were active across brain states including locomotion, nonlocomotion, visual stimulation, and under anesthesia. VIP+ activity correlated most clearly with the mean level of population activity of nearby excitatory neurons during all brain states, suggesting VIP+ cells enable high-excitability states in the cortex. The pharmacogenetic blockade of VIP+ cell output reduced network activity during locomotion, nonlocomotion, anesthesia, and visual stimulation, suggesting VIP+ cells exert a state-independent facilitation of neural activity in the cortex. Collectively, our findings demonstrate that VIP+ neurons have a causal role in the generation of high-activity regimes during spontaneous and stimulus evoked neocortical activity.

NMDA-dependent phase synchronization between septal and temporal CA3 hippocampal networks.

Increasing evidence suggests that synchronization between brain regions is essential for information exchange and memory processes. However, it remains incompletely known which synaptic mechanisms contribute to the process of synchronization. Here, we investigated whether NMDA receptor-mediated synaptic plasticity was an important player in synchronization between septal and temporal CA3 areas of the rat hippocampus. We found that both the septal and temporal CA3 regions intrinsically generate weakly synchronized δ frequency oscillations in the complete hippocampus in vitro. Septal and temporal oscillators differed in frequency, power, and rhythmicity, but both required GABAA and AMPA receptors. NMDA receptor activation, and most particularly the NR2B subunit, contributed considerably more to rhythm generation at the temporal than the septal region. Brief activation of NMDA receptors by application of extracellular calcium dramatically potentiated the septal-temporal coherence for long durations (>40 min), an effect blocked by the NMDA antagonist AP-5. This long-lasting NMDA-receptor-dependent increase in coherence was also associated with an elevated phase locking of spikes locally and across regions. Changes in coherence between oscillators were associated with increases in phase locking between oscillators independent of oscillator amplitude. Finally, although the septal CA3 rhythm preceded the oscillations in temporal regions in control conditions, this was reversed during the NMDA-dependent enhancement in coherence, suggesting that NMDA receptor activation can change the direction of information flow along the septotemporal CA3 axis. These data demonstrate that plastic changes in communication between septal and temporal hippocampal regions can arise from the NMDA-dependent phase locking of neural oscillators.

Brain-state-dependent constraints on claustrocortical communication and function.

Neural activity in the claustrum has been associated with a range of vigilance states, yet the activity patterns and efficacy of synaptic communication of identified claustrum neurons have not been thoroughly determined. Here, we show that claustrum neurons projecting to the retrosplenial cortex are most active during synchronized cortical states such as non-rapid eye movement (NREM) sleep and are suppressed during increased cortical desynchronization associated with arousal, movement, and REM sleep. The efficacy of claustrocortical signaling is increased during NREM and diminished during movement due in part to increased cholinergic tone. Finally, claustrum activation during NREM sleep enhances memory consolidation through the phase resetting of cortical delta waves. Therefore, claustrocortical communication is constrained to function most effectively during cognitive processes associated with synchronized cortical states, such as memory consolidation.

A simple and reliable method for claustrum localization across age in mice.

The anatomical organization of the rodent claustrum remains obscure due to lack of clear borders that distinguish it from neighboring forebrain structures. Defining what constitutes the claustrum is imperative for elucidating its functions. Methods based on gene/protein expression or transgenic mice have been used to spatially outline the claustrum but often report incomplete labeling and/or lack of specificity during certain neurodevelopmental timepoints. To reliably identify claustrum projection cells in mice, we propose a simple immunolabelling method that juxtaposes the expression pattern of claustrum-enriched and cortical-enriched markers. We determined that claustrum cells immunoreactive for the claustrum-enriched markers Nurr1 and Nr2f2 are devoid of the cortical marker Tle4, which allowed us to differentiate the claustrum from adjoining cortical cells. Using retrograde tracing, we verified that nearly all claustrum projection neurons lack Tle4 but expressed Nurr1/Nr2f2 markers to different degrees. At neonatal stages between 7 and 21 days, claustrum projection neurons were identified by their Nurr1-postive/Tle4-negative expression profile, a time-period when other immunolabelling techniques used to localize the claustrum in adult mice are ineffective. Finally, exposure to environmental novelty enhanced the expression of the neuronal activation marker c-Fos in the claustrum region. Notably, c-Fos labeling was mainly restricted to Nurr1-positive cells and nearly absent from Tle4-positive cells, thus corroborating previous work reporting novelty-induced claustrum activation. Taken together, this method will aid in studying the claustrum during postnatal development and may improve histological and functional studies where other approaches are not amenable.

Claustrum projections to the anterior cingulate modulate nociceptive and pain-associated behavior.

The anterior cingulate cortex (ACC) is critical for the perception and unpleasantness of pain.1,2,3,4,5,6 It receives nociceptive information from regions such as the thalamus and amygdala and projects to several cortical and subcortical regions of the pain neuromatrix.7,8 ACC hyperexcitability is one of many functional changes associated with chronic pain, and experimental activation of ACC pyramidal cells produces hypersensitivity to innocuous stimuli (i.e., allodynia).9,10,11,12,13,14 A less-well-studied projection to the ACC arises from a small forebrain region, the claustrum.15,16,17,18,19,20 Stimulation of excitatory claustrum projection neurons preferentially activates GABAergic interneurons, generating feed-forward inhibition onto excitatory cortical networks.21,22,23,24 Previous work has shown that claustrocingulate projections display altered activity in prolonged pain25,26,27; however, it remains unclear whether and how the claustrum participates in nociceptive processing and high-order pain behaviors. Inhibition of ACC activity reverses mechanical allodynia in animal models of persistent and neuropathic pain,1,9,28 suggesting claustrum inputs may function to attenuate pain processing. In this study, we sought to define claustrum function in acute and chronic pain. We found enhanced claustrum activity after a painful stimulus that was attenuated in chronic inflammatory pain. Selective inhibition of claustrocingulate projection neurons enhanced acute nociception but blocked pain learning. Inversely, chemogenetic activation of claustrocingulate neurons had no effect on basal nociception but rescued inflammation-induced mechanical allodynia. Together, these results suggest that claustrocingulate neurons are a critical component of the pain neuromatrix, and dysregulation of this connection may contribute to chronic pain.

Alterations in hippocampal network oscillations and theta-gamma coupling arise before Aß overproduction in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by memory impairments. Brain oscillatory activity is critical for cognitive function and is altered in AD patients. Recent evidence suggests that accumulation of soluble amyloid-beta (Aß) induces reorganization of hippocampal networks. However, whether fine changes in network activity might be present at very early stages, before Aß overproduction, remains to be determined. We therefore assessed whether theta and gamma oscillations and their cross-frequency coupling, which are known to be essential for normal memory function, were precociously altered in the hippocampus. Electrophysiological field potential recordings were performed using complete hippocampal preparations in vitro from young transgenic CRND8 mice, a transgenic mouse model of AD. Our results indicate that a significant proportion of 1-month-old TgCRND8 mice showed robust alterations of theta-gamma cross-frequency coupling in the principal output region of the hippocampus, the subiculum. In addition we showed that, compared to controls, these mice expressed negligible levels of Aß. Finally, these network alterations were not due to genetic factors as 15-day-old animals did not exhibit theta-gamma coupling alterations. Thus, initial alterations in hippocampal network activity arise before Aß accumulation and may represent an early biomarker for AD.

Fast and slow γ rhythms are intrinsically and independently generated in the subiculum.

Gamma rhythms are essential for memory encoding and retrieval. Despite extensive study of these rhythms in the entorhinal cortex, dentate gyrus, CA3, and CA1, almost nothing is known regarding their generation and organization in the structure delivering the most prominent hippocampal output: the subiculum. Here we show using a complete rat hippocampal preparation in vitro that the subiculum intrinsically and independently generates spontaneous slow (25-50 Hz) and fast (100-150 Hz) gamma rhythms during the rising phase and peak of persistent subicular theta rhythms. These two gamma frequencies are phase modulated by theta rhythms without any form of afferent input from the entorhinal cortex or CA1. Subicular principal cells and interneurons phase lock to both fast and slow gamma, and single cells are independently phase modulated by each form of gamma rhythm, enabling selective participation in neural synchrony at both gamma frequencies at different times. Fast GABAergic inhibition is required for the generation of fast gamma, whereas slow gamma is generated by excitatory and inhibitory mechanisms. In addition, the transverse subicular axis exhibits gamma rhythm topography with faster gamma coupling arising in the distal subiculum region. The subiculum therefore possesses a unique intrinsic circuit organization that can autonomously regulate the timing and topography of hippocampal output synchronization. These results suggest the subiculum is a third spontaneous gamma generator in the hippocampal formation (in addition to CA3 and the entorhinal cortex), and these gamma rhythms likely play an active role in mediating the flow of information between the hippocampus and multiple cortical and subcortical brain regions.

Dissociable pathways facilitate theta and non-theta states in the median raphe--septohippocampal circuit.

Hippocampal theta rhythms in vivo are modulated by a synchronizing projection from the medial septum (MS) and a desynchronizing input from the median raphe nucleus (MRn) of the brainstem. Inactivation of the MS suppresses theta rhythms while inactivation of the median raphe produces persistent theta. However, different pathways arise from within the MS and the median raphe and therefore different brain states could be facilitated by different forms of median raphe or septohippocampal inputs. Here, we found in urethane anesthetized rats that suppression of outputs from the MRn with procaine leads to persistent hippocampal theta as previously reported. The discharge properties of hippocampal theta-related cells recorded during both spontaneously occurring theta and MRn 8-OH-DPAT-induced theta did not differ significantly. This persistent theta was abolished by inactivation of the MS with either procaine or atropine sulfate. Selective inactivation of serotonergic median raphe outputs with the 5-HT-1A agonist (8-OH-DPAT) induced theta that was also abolished by medial septal inactivation using procaine. Thus, persistent theta following complete median raphe inactivation or selective serotonergic inactivation arises from a median raphe to MS pathway. However, 8-OH-DPAT infusions into the median raphe together with atropine infusions in the MS did not abolish theta activity. These data suggest that the non-serotonergic (possibly glutamatergic) median raphe projections to the MS can facilitate the generation of hippocampal theta in the absence of medial septal cholinergic tone. These results demonstrate that dissociable neuronal pathways in the median raphe-MS-hippocampal circuit promote different brain states (theta or non-theta) and a median raphe non-serotonergic (likely glutamatergic) system may serve a separate function from the ascending serotonergic raphe projection in the regulation of hippocampal network activity.

Premotor activity in the claustrum.

In this issue of Neuron, Chevée et al. (2022) performed extracellular electrophysiological recordings from claustrum neurons during a sensory selection task. They found that neural activity in the claustrum reflected future motor output rather than sensory inputs and that chemogenetic suppression of claustrum activity reduced motor impulsivity in this task.

Topographic gradients define the projection patterns of the claustrum core and shell in mice.

The claustrum is densely connected to the cortex and participates in brain functions such as attention and sleep. Although some studies have reported the widely divergent organization of claustrum projections, others describe parallel claustrocortical connections to different cortical regions. Therefore, the details underlying how claustrum neurons broadcast information to cortical networks remain incompletely understood. Using multicolor retrograde tracing we determined the density, topography, and co-projection pattern of 14 claustrocortical pathways, in mice. We spatially registered these pathways to a common coordinate space and found that the claustrocortical system is topographically organized as a series of overlapping spatial modules, continuously distributed across the dorsoventral claustrum axis. The claustrum core projects predominantly to frontal-midline cortical regions, whereas the dorsal and ventral shell project to the cortical motor system and temporal lobe, respectively. Anatomically connected cortical regions receive common input from a subset of claustrum neurons shared by neighboring modules, whereas spatially separated regions of cortex are innervated by different claustrum modules. Therefore, each output module exhibits a unique position within the claustrum and overlaps substantially with other modules projecting to functionally related cortical regions. Claustrum inhibitory cells containing parvalbumin, somatostatin, and neuropeptide Y also show unique topographical distributions, suggesting different output modules are controlled by distinct inhibitory circuit motifs. The topographic organization of excitatory and inhibitory cell types may enable parallel claustrum outputs to independently coordinate distinct cortical networks.

Spatially patterned excitatory neuron subtypes and projections of the claustrum.

The claustrum is a functionally and structurally complex brain region, whose very spatial extent remains debated. Histochemical-based approaches typically treat the claustrum as a relatively narrow anatomical region that primarily projects to the neocortex, whereas circuit-based approaches can suggest a broader claustrum region containing projections to the neocortex and other regions. Here, in the mouse, we took a bottom-up and cell-type-specific approach to complement and possibly unite these seemingly disparate conclusions. Using single-cell RNA-sequencing, we found that the claustrum comprises two excitatory neuron subtypes that are differentiable from the surrounding cortex. Multicolor retrograde tracing in conjunction with 12-channel multiplexed in situ hybridization revealed a core-shell spatial arrangement of these subtypes, as well as differential downstream targets. Thus, the claustrum comprises excitatory neuron subtypes with distinct molecular and projection properties, whose spatial patterns reflect the narrower and broader claustral extents debated in previous research. This subtype-specific heterogeneity likely shapes the functional complexity of the claustrum.

The claustrum.

The claustrum is a brain region that has been investigated for over 200 years, yet its precise function remains unknown. In the final posthumously released article of Francis Crick, written with Christof Koch, the claustrum was suggested to be critically linked to consciousness. Though the claustrum remained relatively obscure throughout the last half century, it has enjoyed a renewed interest in the last 15 years since Crick and Koch's article. During this time, the claustrum, like many other brain regions, has been studied with the myriad of modern systems neuroscience tools that have been made available by the intersection of genetic and viral technologies. This has uncovered new information about its anatomical connectivity and physiological properties and begun to reveal aspects of its function. From these studies, one clear consensus has emerged which supports Crick and Koch's primary interest in the claustrum: the claustrum has widespread extensive connectivity with the entire cerebral cortex, suggesting a prominent role in 'higher order processes'.