Differential Effects of Astrocyte Manipulations on Learned Motor Behavior and Neuronal Ensembles in the Motor Cortex.

Although motor cortex is crucial for learning precise and reliable movements, whether and how astrocytes contribute to its plasticity and function during motor learning is unknown. Here, we report that astrocyte-specific manipulations in primary motor cortex (M1) during a lever push task alter motor learning and execution, as well as the underlying neuronal population coding. Mice that express decreased levels of the astrocyte glutamate transporter 1 (GLT1) show impaired and variable movement trajectories, whereas mice with increased astrocyte Gq signaling show decreased performance rates, delayed response times, and impaired trajectories. In both groups, which include male and female mice, M1 neurons have altered interneuronal correlations and impaired population representations of task parameters, including response time and movement trajectories. RNA sequencing further supports a role for M1 astrocytes in motor learning and shows changes in astrocytic expression of glutamate transporter genes, GABA transporter genes, and extracellular matrix protein genes in mice that have acquired this learned behavior. Thus, astrocytes coordinate M1 neuronal activity during motor learning, and our results suggest that this contributes to learned movement execution and dexterity through mechanisms that include regulation of neurotransmitter transport and calcium signaling.SIGNIFICANCE STATEMENT We demonstrate for the first time that in the M1 of mice, astrocyte function is critical for coordinating neuronal population activity during motor learning. We demonstrate that knockdown of astrocyte glutamate transporter GLT1 affects specific components of learning, such as smooth trajectory formation. Altering astrocyte calcium signaling by activation of Gq-DREADD upregulates GLT1 and affects other components of learning, such as response rates and reaction times as well as trajectory smoothness. In both manipulations, neuronal activity in motor cortex is dysregulated, but in different ways. Thus, astrocytes have a crucial role in motor learning via their influence on motor cortex neurons, and they do so by mechanisms that include regulation of glutamate transport and calcium signals.

Cortical-subcortical interactions in goal-directed behavior.

Flexibly selecting appropriate actions in response to complex, ever-changing environments requires both cortical and subcortical regions, which are typically described as participating in a strict hierarchy. In this traditional view, highly specialized subcortical circuits allow for efficient responses to salient stimuli, at the cost of adaptability and context specificity, which are attributed to the neocortex. Their interactions are often described as the cortex providing top-down command signals for subcortical structures to implement; however, as available technologies develop, studies increasingly demonstrate that behavior is represented by brainwide activity and that even subcortical structures contain early signals of choice, suggesting that behavioral functions emerge as a result of different regions interacting as truly collaborative networks. In this review, we discuss the field's evolving understanding of how cortical and subcortical regions in placental mammals interact cooperatively, not only via top-down cortical-subcortical inputs but through bottom-up interactions, especially via the thalamus. We describe our current understanding of the circuitry of both the cortex and two exemplar subcortical structures, the superior colliculus and striatum, to identify which information is prioritized by which regions. We then describe the functional circuits these regions form with one another, and the thalamus, to create parallel loops and complex networks for brainwide information flow. Finally, we challenge the classic view that functional modules are contained within specific brain regions; instead, we propose that certain regions prioritize specific types of information over others, but the subnetworks they form, defined by their anatomical connections and functional dynamics, are the basis of true specialization.

Algebraic approach for subspace decomposition and clustering of neural activity.

We developed an approach to decompose neuronal signals into disjoint components, corresponding to task- or event-based epochs. This protocol describes how to project behavioral templates onto a low-dimensional subspace of neuronal responses to derive neuronal templates, then how to decompose and cluster neuronal responses using these derived templates. We outline these steps on complementary datasets of calcium imaging and spiking activity. Our approach relies on fundamental, linear algebraic principles and is adaptive to the temporal structure of the neural data. For complete details on the use and execution of this protocol, please refer to Adam et al. (2022).1.

Dynamic control of visually guided locomotion through corticosubthalamic projections.

Goal-directed locomotion requires control signals that propagate from higher order areas to regulate spinal mechanisms. The corticosubthalamic hyperdirect pathway offers a short route for cortical information to reach locomotor centers in the brainstem. We developed a task in which head-fixed mice run to a visual landmark and then stop and wait to collect the reward and examined the role of secondary motor cortex (M2) projections to the subthalamic nucleus (STN) in controlling locomotion. Our behavioral modeling, calcium imaging, and optogenetics manipulation results suggest that the M2-STN pathway can be recruited during visually guided locomotion to rapidly and precisely control the pedunculopontine nucleus (PPN) of the mesencephalic locomotor region through the basal ganglia. By capturing the physiological dynamics through a feedback control model and analyzing neuronal signals in M2, PPN, and STN, we find that the corticosubthalamic projections potentially control PPN activity by differentiating an M2 error signal to ensure fast input-output dynamics.

Brain-wide mapping of inputs to the mouse lateral posterior (LP/Pulvinar) thalamus-anterior cingulate cortex network.

The rodent homolog of the primate pulvinar, the lateral posterior (LP) thalamus, is extensively interconnected with multiple cortical areas. While these cortical interactions can span the entire LP, subdivisions of the LP are characterized by differential connections with specific cortical regions. In particular, the medial LP has reciprocal connections with frontoparietal cortical areas, including the anterior cingulate cortex (ACC). The ACC plays an integral role in top-down sensory processing and attentional regulation, likely exerting some of these functions via the LP. However, little is known about how ACC and LP interact, and about the information potentially integrated in this reciprocal network. Here, we address this gap by employing a projection-specific monosynaptic rabies tracing strategy to delineate brain-wide inputs to bottom-up LP→ACC and top-down ACC→LP neurons. We find that LP→ACC neurons receive inputs from widespread cortical regions, including primary and higher order sensory and motor cortical areas. LP→ACC neurons also receive extensive subcortical inputs, particularly from the intermediate and deep layers of the superior colliculus (SC). Sensory inputs to ACC→LP neurons largely arise from visual cortical areas. In addition, ACC→LP neurons integrate cross-hemispheric prefrontal cortex inputs as well as inputs from higher order medial cortex. Our brain-wide anatomical mapping of inputs to the reciprocal LP-ACC pathways provides a roadmap for understanding how LP and ACC communicate different sources of information to mediate attentional control and visuomotor functions.

Distinct prefrontal top-down circuits differentially modulate sensorimotor behavior.

Sensorimotor behaviors require processing of behaviorally relevant sensory cues and the ability to select appropriate responses from a vast behavioral repertoire. Modulation by the prefrontal cortex (PFC) is thought to be key for both processes, but the precise role of specific circuits remains unclear. We examined the sensorimotor function of anatomically distinct outputs from a subdivision of the mouse PFC, the anterior cingulate cortex (ACC). Using a visually guided two-choice behavioral paradigm with multiple cue-response mappings, we dissociated the sensory and motor response components of sensorimotor control. Projection-specific two-photon calcium imaging and optogenetic manipulations show that ACC outputs to the superior colliculus, a key midbrain structure for response selection, principally coordinate specific motor responses. Importantly, ACC outputs exert control by reducing the innate response bias of the superior colliculus. In contrast, ACC outputs to the visual cortex facilitate sensory processing of visual cues. Our results ascribe motor and sensory roles to ACC projections to the superior colliculus and the visual cortex and demonstrate for the first time a circuit motif for PFC function wherein anatomically non-overlapping output pathways coordinate complementary but distinct aspects of visual sensorimotor behavior.

Two-photon imaging in mice shows striosomes and matrix have overlapping but differential reinforcement-related responses.

Striosomes were discovered several decades ago as neurochemically identified zones in the striatum, yet technical hurdles have hampered the study of the functions of these striatal compartments. Here we used 2-photon calcium imaging in neuronal birthdate-labeled Mash1-CreER;Ai14 mice to image simultaneously the activity of striosomal and matrix neurons as mice performed an auditory conditioning task. With this method, we identified circumscribed zones of tdTomato-labeled neuropil that correspond to striosomes as verified immunohistochemically. Neurons in both striosomes and matrix responded to reward-predicting cues and were active during or after consummatory licking. However, we found quantitative differences in response strength: striosomal neurons fired more to reward-predicting cues and encoded more information about expected outcome as mice learned the task, whereas matrix neurons were more strongly modulated by recent reward history. These findings open the possibility of harnessing in vivo imaging to determine the contributions of striosomes and matrix to striatal circuit function.

Optogenetic astrocyte activation modulates response selectivity of visual cortex neurons in vivo.

Astrocytes play important roles in synaptic transmission and plasticity. Despite in vitro evidence, their causal contribution to cortical network activity and sensory information processing in vivo remains unresolved. Here we report that selective photostimulation of astrocytes with channelrhodopsin-2 in primary visual cortex enhances both excitatory and inhibitory synaptic transmission, through the activation of type 1a metabotropic glutamate receptors. Photostimulation of astrocytes in vivo increases the spontaneous firing of parvalbumin-positive (PV(+)) inhibitory neurons, while excitatory and somatostatin-positive (SOM(+)) neurons show either an increase or decrease in their activity. Moreover, PV(+) neurons show increased baseline visual responses and reduced orientation selectivity to visual stimuli, whereas excitatory and SOM(+) neurons show either increased or decreased baseline visual responses together with complementary changes in orientation selectivity. Therefore, astrocyte activation, through the dual control of excitatory and inhibitory drive, influences neuronal integrative features critical for sensory information processing.

Differential gene expression in the developing lateral geniculate nucleus and medial geniculate nucleus reveals novel roles for Zic4 and Foxp2 in visual and auditory pathway development.

Primary sensory nuclei of the thalamus process and relay parallel channels of sensory input into the cortex. The developmental processes by which these nuclei acquire distinct functional roles are not well understood. To identify novel groups of genes with a potential role in differentiating two adjacent sensory nuclei, we performed a microarray screen comparing perinatal gene expression in the principal auditory relay nucleus, the medial geniculate nucleus (MGN), and principal visual relay nucleus, the lateral geniculate nucleus (LGN). We discovered and confirmed groups of highly ranked, differentially expressed genes with qRT-PCR and in situ hybridization. A functional role for Zic4, a transcription factor highly enriched in the LGN, was investigated using Zic4-null mice, which were found to have changes in topographic patterning of retinogeniculate projections. Foxp2, a transcriptional repressor expressed strongly in the MGN, was found to be positively regulated by activity in the MGN. These findings identify roles for two differentially expressed genes, Zic4 and Foxp2, in visual and auditory pathway development. Finally, to test whether modality-specific patterns of gene expression are influenced by extrinsic patterns of input, we performed an additional microarray screen comparing the normal MGN to "rewired" MGN, in which normal auditory afferents are ablated and novel retinal inputs innervate the MGN. Data from this screen indicate that rewired MGN acquires some patterns of gene expression that are present in the developing LGN, including an upregulation of Zic4 expression, as well as novel patterns of expression which may represent unique processes of cross-modal plasticity.

Intrinsic patterning and experience-dependent mechanisms that generate eye-specific projections and binocular circuits in the visual pathway.

A defining feature of the mammalian nervous system is its complex yet precise circuitry. The mechanisms which underlie the generation of neural connectivity are the topic of intense study in developmental neuroscience. The mammalian visual pathway demonstrates precise retinotopic organization in subcortical and cortical pathways, together with the alignment and matching of eye-specific projections, and sophisticated cortical circuitry that enables the extraction of features underlying vision. New approaches employing molecular-genetic analyses, transgenic mice, novel recombinant probes, and high-resolution imaging are contributing to rapid progress and a new synthesis in the field. These approaches are revealing the ways in which intrinsic patterning mechanisms act in concert with experience-dependent mechanisms to shape visual projections and circuits.

Patterning and plasticity of the cerebral cortex.

The cerebral cortex of the human brain is a sheet of about 10 billion neurons divided into discrete subdivisions or areas that process particular aspects of sensation, movement, and cognition. Recent evidence has begun to transform our understanding of how cortical areas form, make specific connections with other brain regions, develop unique processing networks, and adapt to changes in inputs.

Acceleration of visually cued conditioned fear through the auditory pathway.

Defensive responses elicited by sensory experiences are critical for survival. Mice acquire a conditioned fear response rapidly to an auditory cue but slowly to a visual cue, a difference in learned behavior that is likely to be mediated by direct projections to the lateral amygdala from the auditory thalamus but mainly indirect ones from the visual thalamus. Here, we show that acquisition of visually cued conditioned fear is accelerated in 'rewired' mice that have retinal projections routed to the auditory thalamus. Visual stimuli induce expression of the immediate early gene Fos (also known as c-fos) in the auditory thalamus and the lateral amygdala in rewired mice, similar to the way auditory stimuli do in control mice. Thus, the rewired auditory pathway conveys visual information and mediates rapid activity-dependent plasticity in central structures that influence learned behavior.

Role of afferent activity in the development of cortical specification.

The surgical cross-modal rewiring paradigm is an experimental method for examining the physiological and anatomical consequences of exposing developing cortical subregions to specific types of patterned sensory inputs. Data from these experiments provide strong inferences about the role of extrinsic (subcortical) cortical inputs in shaping the local cortical networks that organize and process sensory information. Behavioral results from this work also suggest that such activity (and activity in general) is a profound organizer of cerebral connectivity. We discuss one future direction of these studies: the implication that extrinsic inputs regulate developmental genes that are responsible for refining the connectivity within local circuits, and a strategy to discover and characterize such genes.