Neuronal Populations Research Articles

Enhanced In Situ Spatial Proteomics by Effective Combination of MALDI Imaging and LC-MS/MS

Highly specialized cells are fundamental for the proper functioning of complex organs. Variations in cell-type-specific gene expression and protein composition have been linked to a variety of diseases. Investigation of the distinctive molecular makeup of these cells within tissues is therefore critical in biomedical research. Although several technologies have emerged as valuable tools to address this cellular heterogeneity, most workflows lack sufficient in situ resolution and are associated with high costs and extremely long analysis times. Here, we present a combination of experimental and computational approaches that allows a more comprehensive investigation of molecular heterogeneity within tissues than by either shotgun LC-MS/MS or MALDI imaging alone. We applied our pipeline to the mouse brain, which contains a wide variety of cell types that not only perform unique functions but also exhibit varying sensitivities to insults. We explored the distinct neuronal populations within the hippocampus, a brain region crucial for learning and memory that is involved in various neurological disorders. As an example, we identified the groups of proteins distinguishing the neuronal populations of the dentate gyrus (DG) and the cornu ammonis (CA) in the same brain section. Most of the annotated proteins matched the regional enrichment of their transcripts, thereby validating the method. As the method is highly reproducible, the identification of individual masses through the combination of MALDI-IMS and LC-MS/MS methods can be used for the much faster and more precise interpretation of MALDI-IMS measurements only. This greatly speeds up spatial proteomic analyses and allows the detection of local protein variations within the same population of cells. The method’s general applicability has the potential to be used to investigate different biological conditions and tissues and a much higher throughput than other techniques making it a promising approach for clinical routine applications.

Innate responses to numerousness reveal neural activation in different brain regions in newly-hatched visually naïve chicks

Whether non-symbolic encoding of quantity is predisposed at birth with dedicated hard-wired neural circuits is debated. Here we presented newly-hatched visually naive chicks with stimuli (flashing dots) of either identical or different numerousness (with a ratio 1:3) with their continuous physical appearance (size, contour length, density, convex hull) randomly changing. Chicks spontaneously tell apart the stimuli on the basis of the number of elements. Upon presentation of either fixed or changing numerousness chicks showed different expression of early gene c-fos in the visual Wulst, the hippocampal formation, the intermediate medial mesopallium, and the caudal part of the nidopallium caudolaterale. The results support the hypothesis that the ability to discriminate quantities does not require any specific instructive experience and involves a neural network with several populations of number-selective neurons. Evidence for innateness of non-symbolic numerical cognition have implications for both neurobiology and philosophy of mathematics.

Making Ramón y Cajal proud: Development of cell identity and diversity in the cerebral cortex.

Since the beautiful images of Santiago Ramón y Cajal provided a first glimpse into the immense diversity and complexity of cell types found in the cerebral cortex, neuroscience has been challenged and inspired to understand how these diverse cells are generated and how they interact with each other to orchestrate the development of this remarkable tissue. Some fundamental questions drive the field's quest to understand cortical development: what are the mechanistic principles that govern the emergence of neuronal diversity? How do extrinsic and intrinsic signals integrate with physical forces and activity to shape cell identity? How do the diverse populations of neurons and glia influence each other during development to guarantee proper integration and function? The advent of powerful new technologies to profile and perturb cortical development at unprecedented resolution and across a variety of modalities has offered a new opportunity to integrate past knowledge with brand new data. Here, we review some of this progress using cortical excitatory projection neurons as a system to draw out general principles of cell diversification and the role of cell-cell interactions during cortical development.

An Unpredictable Brain Is a Conscious, Responsive Brain.

Severe traumatic brain injuries typically result in loss of consciousness or coma. In deeply comatose patients with traumatic brain injury, cortical dynamics become simple, repetitive, and predictable. We review evidence that this low-complexity, high-predictability state results from a passive cortical state, represented by a stable repetitive attractor, that hinders the flexible formation of neuronal ensembles necessary for conscious experience. Our data and those from other groups support the hypothesis that this cortical passive state is because of the loss of thalamocortical input. We identify the unpredictability and complexity of cortical dynamics captured by local field potential as a sign of recovery from this passive coma attractor. In this Perspective article, we discuss how these electrophysiological biomarkers of the recovery of consciousness could inform the design of closed-loop stimulation paradigms to treat disorders of consciousness.

Central amygdala CRF+ neurons promote heightened threat reactivity following early life adversity in mice

Failure to appropriately predict and titrate reactivity to threat is a core feature of fear and anxiety-related disorders and is common following early life adversity (ELA). A population of neurons in the lateral central amygdala (CeAL) expressing corticotropin releasing factor (CRF) have been proposed to be key in processing threat of different intensities to mediate active fear expression. Here, we use in vivo fiber photometry to show that ELA results in sex-specific changes in the activity of CeAL CRF+ neurons, yielding divergent mechanisms underlying the augmented startle in ELA mice, a translationally relevant behavior indicative of heightened threat reactivity and hypervigilance. Further, chemogenic inhibition of CeAL CRF+ neurons selectively diminishes startle and produces a long-lasting suppression of threat reactivity. These findings identify a mechanism for sex-differences in susceptibility for anxiety following ELA and have broad implications for understanding the neural circuitry that encodes and gates the behavioral expression of fear.

Adult Human Brain Tissue Cultures to Study NeuroHIV.

HIV-associated neurocognitive disorders (HAND) persist under antiretroviral therapy as a complex pathology that has been difficult to study in cellular and animal models. Therefore, we generated an ex vivo human brain slice model of HIV-1 infection from surgically resected adult brain tissue. Brain slice cultures processed for flow cytometry showed >90% viability of dissociated cells within the first three weeks in vitro, with parallel detection of astrocyte, myeloid, and neuronal populations. Neurons within brain slices showed stable dendritic spine density and mature spine morphologies in the first weeks in culture, and they generated detectable activity in multi-electrode arrays. We infected cultured brain slices using patient-matched CD4+ T-cells or monocyte-derived macrophages (MDMs) that were exposed to a GFP-expressing R5-tropic HIV-1 in vitro. Infected slice cultures expressed viral RNA and developed a spreading infection up to 9 days post-infection, which were significantly decreased by antiretrovirals. We also detected infected myeloid cells and astrocytes within slices and observed minimal effect on cellular viability over time. Overall, this human-centered model offers a promising resource to study the cellular mechanisms contributing to HAND (including antiretroviral toxicity, substance use, and aging), infection of resident brain cells, and new neuroprotective therapeutics.

Serotonergic and dopaminergic neurons in the dorsal raphe are differentially altered in a mouse model for parkinsonism

Parkinson’s disease (PD) is characterized by motor impairments caused by degeneration of dopamine neurons in the substantia nigra pars compacta. In addition to these symptoms, PD patients often suffer from non-motor comorbidities including sleep and psychiatric disturbances, which are thought to depend on concomitant alterations of serotonergic and noradrenergic transmission. A primary locus of serotonergic neurons is the dorsal raphe nucleus (DRN), providing brain-wide serotonergic input. Here, we identified electrophysiological and morphological parameters to classify serotonergic and dopaminergic neurons in the murine DRN under control conditions and in a PD model, following striatal injection of the catecholamine toxin, 6-hydroxydopamine (6-OHDA). Electrical and morphological properties of both neuronal populations were altered by 6-OHDA. In serotonergic neurons, most changes were reversed when 6-OHDA was injected in combination with desipramine, a noradrenaline (NA) reuptake inhibitor, protecting the noradrenergic terminals. Our results show that the depletion of both NA and dopamine in the 6-OHDA mouse model causes changes in the DRN neural circuitry.

Serotonergic and dopaminergic neurons in the dorsal raphe are differentially altered in a mouse model for parkinsonism.

Parkinson's disease (PD) is characterized by motor impairments caused by degeneration of dopamine neurons in the substantia nigra pars compacta. In addition to these symptoms, PD patients often suffer from non-motor comorbidities including sleep and psychiatric disturbances, which are thought to depend on concomitant alterations of serotonergic and noradrenergic transmission. A primary locus of serotonergic neurons is the dorsal raphe nucleus (DRN), providing brain-wide serotonergic input. Here, we identified electrophysiological and morphological parameters to classify serotonergic and dopaminergic neurons in the murine DRN under control conditions and in a PD model, following striatal injection of the catecholamine toxin, 6-hydroxydopamine (6-OHDA). Electrical and morphological properties of both neuronal populations were altered by 6-OHDA. In serotonergic neurons, most changes were reversed when 6-OHDA was injected in combination with desipramine, a noradrenaline (NA) reuptake inhibitor, protecting the noradrenergic terminals. Our results show that the depletion of both NA and dopamine in the 6-OHDA mouse model causes changes in the DRN neural circuitry.

Neuroimmune Interactions in the Intestine

Recent advances have contributed to a mechanistic understanding of neuroimmune interactions in the intestine and revealed an essential role of this cross talk for gut homeostasis and modulation of inflammatory and infectious intestinal diseases. In this review, we describe the innervation of the intestine by intrinsic and extrinsic neurons and then focus on the bidirectional communication between neurons and immune cells. First, we highlight the contribution of neuronal subtypes to the development of colitis and discuss the different immune and epithelial cell types that are regulated by neurons via the release of neuropeptides and neurotransmitters. Next, we review the role of intestinal inflammation in the development of visceral hypersensitivity and summarize how inflammatory mediators induce peripheral and central sensitization of gut-innervating sensory neurons. Finally, we outline the importance of immune cells and gut microbiota for the survival and function of different neuronal populations at homeostasis and during bacterial and helminth infection.

A potentiation of REM sleep-active neurons in the lateral habenula may be responsible for the sleep disturbance in depression

Psychiatric disorders with dysfunction of the lateral habenula (LHb) show sleep disturbance, especially a disinhibition of rapid eye movement (REM) sleep in major depression. However, the role of LHb in physiological sleep control and how LHb contributes to sleep disturbance in major depression remain elusive. Here, we found that functional manipulations of LHb glutamatergic neurons bidirectionally modulated both non-REM (NREM) sleep and REM sleep. Activity recording revealed heterogeneous activity patterns of LHb neurons across sleep/wakefulness cycles, but LHb neurons were preferentially active during REM sleep. Using an activity-dependent tagging method, we selectively labeled a population of REM sleep-active LHb neurons and demonstrated that these neurons specifically promoted REM sleep. Neural circuit studies showed that LHb neurons regulated REM sleep via projections to the ventral tegmental area but not to the rostromedial tegmental nucleus. Furthermore, we found that the increased REM sleep in a depression mouse model was associated with a potentiation of REM sleep-active LHb neurons, including an increased proportion, elevated spike firing, and altered activity mode. Importantly, inhibition of REM sleep-active LHb neurons not only attenuated the increased REM sleep but also alleviated depressive-like behaviors in a depression mouse model. Thus, our results demonstrated that REM sleep-active LHb neurons selectively promoted REM sleep, and a potentiation of these neurons contributed to depression-associated sleep disturbance.

Acetylcholine Neurons Become Cholinergic during Three Time Windows in the Developing Mouse Brain.

Acetylcholine (ACh) neurons in the central nervous system are required for the coordination of neural network activity during higher brain functions, such as attention, learning, and memory, as well as locomotion. Disturbed cholinergic signaling has been described in many neurodevelopmental and neurodegenerative disorders. Furthermore, cotransmission of other signaling molecules, such as glutamate and GABA, with ACh has been associated with essential roles in brain function or disease. However, it is unknown when ACh neurons become cholinergic during development. Thus, understanding the timeline of how the cholinergic system develops and becomes active in the healthy brain is a crucial part of understanding brain development. To study this, we used transgenic mice to selectively label ACh neurons with tdTomato. We imaged serial sectioned brains and generated whole-brain reconstructions at different time points during pre- and postnatal development. We found three crucial time windows-two in the prenatal and one in the postnatal brain-during which most ACh neuron populations become cholinergic in the brain. We also found that cholinergic gene expression is initiated in cortical ACh interneurons, while the cerebral cortex is innervated by cholinergic projection neurons from the basal forebrain. Taken together, we show that ACh neuron populations are present and become cholinergic before postnatal day 12, which is the onset of major sensory processes, such as hearing and vision. We conclude that the birth of ACh neurons and initiation of cholinergic gene expression are temporally separated during development but highly coordinated by brain anatomical structure.

Corticostriatal ensemble dynamics across heroin self-administration to reinstatement.

Corticostriatal projection neurons from prelimbic medial prefrontal cortex to the nucleus accumbens core critically regulate drug-seeking behaviors, yet the underlying encoding dynamics whereby these neurons contribute to drug seeking remain elusive. Here we use two-photon calcium imaging to visualize the activity of corticostriatal neurons in mice from the onset of heroin use to relapse. We find that the activity of these neurons is highly heterogeneous during heroin self-administration and seeking, with at least 8 distinct neuronal ensembles that display both excitatory and inhibitory encoding dynamics. These neuronal ensembles are particularly apparent during relapse, where excitatory responses are amplified compared to heroin self-administration. Moreover, we find that optogenetic inhibition of corticostriatal projection neurons attenuates heroin seeking regardless of the relapse trigger. Our results reveal the precise corticostriatal activity dynamics underlying drug-seeking behaviors and support a key role for this circuit in mediating relapse to drug seeking.

Regulator of G Protein Signaling 14 protein expression profile in the adult mouse brain.

Regulator of G protein signaling 14 (RGS14) is a multifunctional signaling protein that serves as a natural suppressor of synaptic plasticity in the mouse brain. Our previous studies showed that RGS14 is highly expressed in postsynaptic dendrites and spines of pyramidal neurons in hippocampal area CA2 of the developing mouse brain. However, our more recent work with adult rhesus macaque brain shows that RGS14 is found in multiple neuron populations throughout hippocampal area CA1 and CA2, caudate nucleus, putamen, globus pallidus, substantia nigra, and amygdala in the adult rhesus monkey brain. In the mouse brain, we also have observed RGS14 protein in discrete limbic regions linked to reward behavior and addiction, including the central amygdala and the nucleus accumbens, but a comprehensive mapping of RGS14 protein expression in the adult mouse brain is lacking. Here, we report that RGS14 is more broadly expressed in mouse brain than previously known. Intense RGS14 staining is observed in specific neuron populations of the hippocampal formation, amygdala, septum, bed nucleus of stria terminalis and ventral striatum/nucleus accumbens. RGS14 is also observed in axon fiber tracts including the dorsal fornix, fimbria, stria terminalis, and the ventrohippocampal commissure. Moderate RGS14 staining is observed in various other adjacent regions not previously reported. These findings show that RGS14 is expressed in brain regions that govern aspects of core cognitive functions such as sensory perception, emotion, memory, motivation, and execution of actions, and suggests that RGS14 may serve to suppress plasticity and filter inputs in these brain regions to set the overall tone on experience-to-action processes.

The stabilized supralinear network accounts for the contrast dependence of visual cortical gamma oscillations.

When stimulated, neural populations in the visual cortex exhibit fast rhythmic activity with frequencies in the gamma band (30-80 Hz). The gamma rhythm manifests as a broad resonance peak in the power-spectrum of recorded local field potentials, which exhibits various stimulus dependencies. In particular, in macaque primary visual cortex (V1), the gamma peak frequency increases with increasing stimulus contrast. Moreover, this contrast dependence is local: when contrast varies smoothly over visual space, the gamma peak frequency in each cortical column is controlled by the local contrast in that column's receptive field. No parsimonious mechanistic explanation for these contrast dependencies of V1 gamma oscillations has been proposed. The stabilized supralinear network (SSN) is a mechanistic model of cortical circuits that has accounted for a range of visual cortical response nonlinearities and contextual modulations, as well as their contrast dependence. Here, we begin by showing that a reduced SSN model without retinotopy robustly captures the contrast dependence of gamma peak frequency, and provides a mechanistic explanation for this effect based on the observed non-saturating and supralinear input-output function of V1 neurons. Given this result, the local dependence on contrast can trivially be captured in a retinotopic SSN which however lacks horizontal synaptic connections between its cortical columns. However, long-range horizontal connections in V1 are in fact strong, and underlie contextual modulation effects such as surround suppression. We thus explored whether a retinotopically organized SSN model of V1 with strong excitatory horizontal connections can exhibit both surround suppression and the local contrast dependence of gamma peak frequency. We found that retinotopic SSNs can account for both effects, but only when the horizontal excitatory projections are composed of two components with different patterns of spatial fall-off with distance: a short-range component that only targets the source column, combined with a long-range component that targets columns neighboring the source column. We thus make a specific qualitative prediction for the spatial structure of horizontal connections in macaque V1, consistent with the columnar structure of cortex.

Reelin differentially shapes dendrite morphology of medial entorhinal cortical ocean and island cells.

The function of medial entorhinal cortex layer II (MECII) excitatory neurons has been recently explored. MECII dysfunction underlies deficits in spatial navigation and working memory. MECII neurons comprise two major excitatory neuronal populations, pyramidal island and stellate ocean cells, in addition to the inhibitory interneurons. Ocean cells express reelin and surround clusters of island cells that lack reelin expression. The influence of reelin expression by ocean cells and interneurons on their own morphological differentiation and that of MECII island cells has remained unknown. To address this, we used a conditional reelin knockout (RelncKO) mouse to induce reelin deficiency postnatally in vitro and in vivo. Reelin deficiency caused dendritic hypertrophy of ocean cells, interneurons and only proximal dendritic compartments of island cells. Ca2+ recording showed that both cell types exhibited an elevation of calcium frequencies in RelncKO, indicating that the hypertrophic effect is related to excessive Ca2+ signalling. Moreover, pharmacological receptor blockade in RelncKO mouse revealed malfunctioning of GABAB, NMDA and AMPA receptors. Collectively, this study emphasizes the significance of reelin in neuronal growth, and its absence results in dendrite hypertrophy of MECII neurons.

Perceptual Resolution of Ambiguity: Can Tuned, Divisive Normalization Account for both Interocular Similarity Grouping and Difference Enhancement.

Our visual system usually provides a unique and functional representation of the external world. At times, however, the visual system has more than one compelling interpretation of the same retinal stimulus; in this case, neural populations compete for perceptual dominance to resolve ambiguity. Spatial and temporal context can guide perceptual experience. Recent evidence shows that ambiguous retinal stimuli are sometimes resolved by enhancing either similarity or differences among multiple percepts. Divisive normalization is a canonical neural computation that enables context-dependent sensory processing by attenuating a neuron's response by other neurons. Experiments here show that divisive normalization can account for perceptual representations of either similarity enhancement (so-called grouping) or difference enhancement, offering a unified framework for opposite perceptual outcomes.

Optimal control of oscillatory neuronal models with applications to communication through coherence

Macroscopic oscillations in the brain are involved in various cognitive and physiological processes, yet their precise function is not completely understood. Communication through coherence (CTC) theory proposes that these rhythmic electrical patterns might serve to regulate the information flow between neural populations. Thus, to communicate effectively, neural populations must synchronize their oscillatory activity, ensuring that input volleys from the presynaptic population reach the postsynaptic one at its maximum phase of excitability. We consider an Excitatory–Inhibitory (E–I) network whose macroscopic activity is described by an exact mean-field model. The E–I network receives periodic inputs from either one or two external sources, for which effective communication will not be achieved in the absence of control. We explore strategies based on optimal control theory for phase–amplitude dynamics to design a periodic control that sets the target population in the optimal phase to synchronize its activity with a specific presynaptic input signal and establish communication. The control mechanism resembles the role of a higher cortical area in the context of selective attention. To design the control, we use the phase–amplitude reduction of a limit cycle and leverage recent developments in this field in order to find the most effective control strategy regarding a defined cost function. Furthermore, we present results that guarantee the local controllability of the system close to the limit cycle.

Dopamine neurons drive spatiotemporally heterogeneous striatal dopamine signals during learning

Environmental cues, through Pavlovian learning, become conditioned stimuli that invigorate and guide animals toward rewards. Dopamine (DA) neurons in the ventral tegmental area (VTA) and substantia nigra (SNc) are crucial for this process, via engagement of a reciprocally connected network with their striatal targets. Critically, it remains unknown how dopamine neuron activity itself engages dopamine signals throughout the striatum, across learning. Here, we investigated how optogenetic Pavlovian cue conditioning of VTA or SNc dopamine neurons directs cue-evoked behavior and shapes subregion-specific striatal dopamine dynamics. We used a fluorescent biosensor to monitor dopamine in the nucleus accumbens (NAc) core and shell, dorsomedial striatum (DMS), and dorsolateral striatum (DLS). We demonstrate spatially heterogeneous, learning-dependent dopamine changes across striatal regions. Although VTA stimulation-evoked robust dopamine release in NAc core, shell, and DMS, predictive cues preferentially recruited dopamine release in NAc core, starting early in training, and DMS, late in training. Negative prediction error signals, reflecting a violation in the expectation of dopamine neuron activation, only emerged in the NAc core and DMS. Despite the development of vigorous movement late in training, conditioned dopamine signals did not emerge in the DLS, even during Pavlovian conditioning with SNc dopamine neuron activation, which elicited robust DLS dopamine release. Together, our studies show a broad dissociation in the fundamental prediction and reward-related information generated by VTA and SNc dopamine neuron populations and signaled by dopamine across the striatum. Further, they offer new insight into how larger-scale adaptations across the striatal network emerge during learning to coordinate behavior.

Mixed Representations of Sound and Action in the Auditory Midbrain.

Linking sensory input and its consequences is a fundamental brain operation. During behavior, the neural activity of neocortical and limbic systems often reflects dynamic combinations of sensory and task-dependent variables, and these "mixed representations" are suggested to be important for perception, learning, and plasticity. However, the extent to which such integrative computations might occur outside of the forebrain is less clear. Here, we conduct cellular-resolution two-photon Ca2+ imaging in the superficial "shell" layers of the inferior colliculus (IC), as head-fixed mice of either sex perform a reward-based psychometric auditory task. We find that the activity of individual shell IC neurons jointly reflects auditory cues, mice's actions, and behavioral trial outcomes, such that trajectories of neural population activity diverge depending on mice's behavioral choice. Consequently, simple classifier models trained on shell IC neuron activity can predict trial-by-trial outcomes, even when training data are restricted to neural activity occurring prior to mice's instrumental actions. Thus, in behaving mice, auditory midbrain neurons transmit a population code that reflects a joint representation of sound, actions, and task-dependent variables.

A high-density 1,024-channel probe for brain-wide recordings in non-human primates.

Large-scale neural population recordings with single-cell resolution across the primate brain remain challenging. Here we introduce the Neuroscroll probe that isolates single neuronal activities simultaneously from 1,024 densely spaced channels that are flexibly distributed across the shank of the probe. The Neuroscroll probe length is easily tunable for individual probes from 10 mm to 90 mm, covering the brain size of non-human primates and humans, and the probes remain intact and functional after repeated bending deformations. The Neuroscroll probes provided reliable recordings from large neural populations with high chronic stability up to 105 weeks in rats. Recording with each Neuroscroll probe yielded hundreds of well-isolated single units simultaneously from multiple brain regions distributed across the entire depth of the rhesus macaque brain. With the thousand simultaneously recorded channels, unprecedented probe length, excellent mechanical stability and flexible recording site distribution, the Neuroscroll probes enable a wide range of new experimental paradigms in system neuroscience studies with great versatility.