Multiscale model of primary motor cortex circuits predicts in vivo cell-type-specific, behavioral state-dependent dynamics.

Understanding cortical function requires studying multiple scales: molecular, cellular, circuit, and behavioral. We develop a multiscale, biophysically detailed model of mouse primary motor cortex (M1) with over 10,000 neurons and 30 million synapses. Neuron types, densities, spatial distributions, morphologies, biophysics, connectivity, and dendritic synapse locations are constrained by experimental data. The model includes long-range inputs from seven thalamic and cortical regions and noradrenergic inputs. Connectivity depends on cell class and cortical depth at sublaminar resolution. The model accurately predicts in vivo layer- and cell-type-specific responses (firing rates and LFP) associated with behavioral states (quiet wakefulness and movement) and experimental manipulations (noradrenaline receptor blockade and thalamus inactivation). We generate mechanistic hypotheses underlying the observed activity and analyzed low-dimensional population latent dynamics. This quantitative theoretical framework can be used to integrate and interpret M1 experimental data and sheds light on the cell-type-specific multiscale dynamics associated with several experimental conditions and behaviors.

Untangling the cortico-thalamo-cortical loop: cellular pieces of a knotty circuit puzzle.

Functions of the neocortex depend on its bidirectional communication with the thalamus, via cortico-thalamo-cortical (CTC) loops. Recent work dissecting the synaptic connectivity in these loops is generating a clearer picture of their cellular organization. Here, we review findings across sensory, motor and cognitive areas, focusing on patterns of cell type-specific synaptic connections between the major types of cortical and thalamic neurons. We outline simple and complex CTC loops, and note features of these loops that appear to be general versus specialized. CTC loops are tightly interlinked with local cortical and corticocortical (CC) circuits, forming extended chains of loops that are probably critical for communication across hierarchically organized cerebral networks. Such CTC-CC loop chains appear to constitute a modular unit of organization, serving as scaffolding for area-specific structural and functional modifications. Inhibitory neurons and circuits are embedded throughout CTC loops, shaping the flow of excitation. We consider recent findings in the context of established CTC and CC circuit models, and highlight current efforts to pinpoint cell type-specific mechanisms in CTC loops involved in consciousness and perception. As pieces of the connectivity puzzle fall increasingly into place, this knowledge can guide further efforts to understand structure-function relationships in CTC loops.

Cortico-Thalamo-Cortical Circuits of Mouse Forelimb S1 Are Organized Primarily as Recurrent Loops.

Cortical projections to the thalamus arise from corticothalamic (CT) neurons in layer 6 and pyramidal tract-type (PT) neurons in layer 5B. We dissected the excitatory synaptic connections in the somatosensory thalamus formed by CT and PT neurons of the primary somatosensory (S1) cortex, focusing on mouse forelimb S1. Mice of both sexes were studied. The CT neurons in S1 synaptically excited S1-projecting thalamocortical (TC) neurons in subregions of both the ventral posterior lateral and posterior (PO) nuclei, forming a pair of recurrent cortico-thalamo-cortical (C-T-C) loops. The PT neurons in S1 also formed a recurrent loop with S1-projecting TC neurons in the same subregion of the PO. The PT neurons in the adjacent primary motor (M1) cortex formed a separate recurrent loop with M1-projecting TC neurons in a nearby subregion of the PO. Collectively, our results reveal that C-T-C circuits of mouse forelimb S1 are primarily organized as multiple cortical cell-type-specific and thalamic subnucleus-specific recurrent loops, with both CT and PT neurons providing the strongest excitatory input to TC neurons that project back to S1. The findings, together with those of related studies of C-T-C circuits, thus suggest that recurrently projecting thalamocortical neurons are the principal targets of cortical excitatory input to the mouse somatosensory and motor thalamus.SIGNIFICANCE STATEMENT Bidirectional cortical communication with the thalamus is considered an important aspect of sensorimotor integration for active touch in the somatosensory system, but the cellular organization of the circuits mediating this process is not well understood. We used an approach combining cell-type-specific anterograde optogenetic excitation with single-cell recordings targeted to retrogradely labeled thalamocortical neurons to dissect these circuits. The findings reveal a consistent pattern: cortical projections to the somatosensory thalamus target thalamocortical neurons that project back to the same cortical area. Commonalities of these findings to previous descriptions of related circuits in other areas suggest that cortico-thalamo-cortical circuits may generally be organized primarily as recurrent loops.

Reduction of thalamic and cortical Ih by deletion of TRIP8b produces a mouse model of human absence epilepsy.

Absence seizures occur in several types of human epilepsy and result from widespread, synchronous feedback between the cortex and thalamus that produces brief episodes of loss of consciousness. Genetic rodent models have been invaluable for investigating the pathophysiological basis of these seizures. Here, we identify tetratricopeptide-containing Rab8b-interacting protein (TRIP8b) knockout mice as a new model of absence epilepsy, featuring spontaneous spike-wave discharges on electroencephalography (EEG) that are the electrographic hallmark of absence seizures. TRIP8b is an auxiliary subunit of the hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels, which have previously been implicated in the pathogenesis of absence seizures. In contrast to mice lacking the pore-forming HCN channel subunit HCN2, TRIP8b knockout mice exhibited normal cardiac and motor function and a less severe seizure phenotype. Evaluating the circuit that underlies absence seizures, we found that TRIP8b knockout mice had significantly reduced HCN channel expression and function in thalamic-projecting cortical layer 5b neurons and thalamic relay neurons, but preserved function in inhibitory neurons of the reticular thalamic nucleus. Our results expand the known roles of TRIP8b and provide new insight into the region-specific functions of TRIP8b and HCN channels in constraining cortico-thalamo-cortical excitability.

Synaptic circuit organization of motor corticothalamic neurons.

Corticothalamic (CT) neurons in layer 6 constitute a large but enigmatic class of cortical projection neurons. How they are integrated into intracortical and thalamo-cortico-thalamic circuits is incompletely understood, especially outside of sensory cortex. Here, we investigated CT circuits in mouse forelimb motor cortex (M1) using multiple circuit-analysis methods. Stimulating and recording from CT, intratelencephalic (IT), and pyramidal tract (PT) projection neurons, we found strong CT↔ CT and CT↔ IT connections; however, CT→IT connections were limited to IT neurons in layer 6, not 5B. There was strikingly little CT↔ PT excitatory connectivity. Disynaptic inhibition systematically accompanied excitation in these pathways, scaling with the amplitude of excitation according to both presynaptic (class-specific) and postsynaptic (cell-by-cell) factors. In particular, CT neurons evoked proportionally more inhibition relative to excitation (I/E ratio) than IT neurons. Furthermore, the amplitude of inhibition was tuned to match the amount of excitation at the level of individual neurons; in the extreme, neurons receiving no excitation received no inhibition either. Extending these studies to dissect the connectivity between cortex and thalamus, we found that M1-CT neurons and thalamocortical neurons in the ventrolateral (VL) nucleus were remarkably unconnected in either direction. Instead, VL axons in the cortex excited both IT and PT neurons, and CT axons in the thalamus excited other thalamic neurons, including those in the posterior nucleus, which additionally received PT excitation. These findings, which contrast in several ways with previous observations in sensory areas, illuminate the basic circuit organization of CT neurons within M1 and between M1 and thalamus.

Reciprocal interareal connections to corticospinal neurons in mouse M1 and S2.

Primary motor (M1) and secondary somatosensory (S2) cortices, although anatomically and functionally distinct, share an intriguing cellular component: corticospinal neurons (CSP) in layer 5B. Here, we investigated the long-range circuits of CSPs in mouse forelimb-M1 and S2. We found that interareal projections (S2 → M1 and M1 → S2) monosynaptically excited pyramidal neurons across multiple layers, including CSPs. Area-specific differences were observed in the relative strengths of inputs to subsets of CSPs and other cell types, but the general patterns were similar. Furthermore, subcellular mapping of the dendritic distributions of these corticocortical excitatory synapses onto CSPs in both areas also showed similar patterns. Because layer 5B is particularly thick in M1, but not S2, we studied M1-CSPs at different cortical depths, quantifying their dendritic morphology and mapping inputs from additional cortical (M2, contralateral M1, and local layer 2/3) and thalamic (VL nucleus) sources. These results indicated that CSPs exhibit area-specific modifications on an otherwise conserved synaptic organization, and that different afferents innervate M1-CSP dendritic domains in a source-specific manner. In the cervical spinal cord, CSP axons from S2 and M1 partly converged on middle layers, but S2-CSP axons extended further dorsally, and M1-CSP axons ventrally. Thus, our findings identify many shared features in the circuits of M1 and S2 and show that these areas communicate via mutual projections that give each area monosynaptic access to the other area's CSPs. These interareally yoked CSP circuits may enable M1 and S2 to operate in a coordinated yet differentiated manner in the service of sensorimotor integration.

A genuine layer 4 in motor cortex with prototypical synaptic circuit connectivity.

The motor cortex (M1) is classically considered an agranular area, lacking a distinct layer 4 (L4). Here, we tested the idea that M1, despite lacking a cytoarchitecturally visible L4, nevertheless possesses its equivalent in the form of excitatory neurons with input-output circuits like those of the L4 neurons in sensory areas. Consistent with this idea, we found that neurons located in a thin laminar zone at the L3/5A border in the forelimb area of mouse M1 have multiple L4-like synaptic connections: excitatory input from thalamus, largely unidirectional excitatory outputs to L2/3 pyramidal neurons, and relatively weak long-range corticocortical inputs and outputs. M1-L4 neurons were electrophysiologically diverse but morphologically uniform, with pyramidal-type dendritic arbors and locally ramifying axons, including branches extending into L2/3. Our findings therefore identify pyramidal neurons in M1 with the expected prototypical circuit properties of excitatory L4 neurons, and question the traditional assumption that motor cortex lacks this layer.

Organization of cortical and thalamic input to pyramidal neurons in mouse motor cortex.

Determining how long-range synaptic inputs engage pyramidal neurons in primary motor cortex (M1) is important for understanding circuit mechanisms involved in regulating movement. We used channelrhodopsin-2-assisted circuit mapping to characterize the long-range excitatory synaptic connections made by multiple cortical and thalamic areas onto pyramidal neurons in mouse vibrissal motor cortex (vM1). Each projection innervated vM1 pyramidal neurons with a unique laminar profile. Collectively, the profiles for different sources of input partially overlapped and spanned all cortical layers. Specifically, orbital cortex (OC) inputs primarily targeted neurons in L6. Secondary motor cortex (M2) inputs excited neurons mainly in L5B, including pyramidal tract neurons. In contrast, thalamocortical inputs from anterior motor-related thalamic regions, including VA/VL (ventral anterior thalamic nucleus/ventrolateral thalamic nucleus), targeted neurons in L2/3 through L5B, but avoided L6. Inputs from posterior sensory-related thalamic areas, including POm (posterior thalamic nuclear group), targeted neurons only in the upper layers (L2/3 and L5A), similar to inputs from somatosensory (barrel) cortex. Our results show that long-range excitatory inputs target vM1 pyramidal neurons in a layer-specific manner. Inputs from sensory-related cortical and thalamic areas preferentially target the upper-layer pyramidal neurons in vM1. In contrast, inputs from OC and M2, areas associated with volitional and cognitive aspects of movements, bypass local circuitry and have direct monosynaptic access to neurons projecting to brainstem and thalamus.

Corticospinal-specific HCN expression in mouse motor cortex: I(h)-dependent synaptic integration as a candidate microcircuit mechanism involved in motor control.

Motor cortex is a key brain center involved in motor control in rodents and other mammals, but specific intracortical mechanisms at the microcircuit level are largely unknown. Neuronal expression of hyperpolarization-activated current (I(h)) is cell class specific throughout the nervous system, but in neocortex, where pyramidal neurons are classified in various ways, a systematic pattern of expression has not been identified. We tested whether I(h) is differentially expressed among projection classes of pyramidal neurons in mouse motor cortex. I(h) expression was high in corticospinal neurons and low in corticostriatal and corticocortical neurons, a pattern mirrored by mRNA levels for HCN1 and Trip8b subunits. Optical mapping experiments showed that I(h) attenuated glutamatergic responses evoked across the apical and basal dendritic arbors of corticospinal but not corticostriatal neurons. Due to I(h), corticospinal neurons resonated, with a broad peak at ∼4 Hz, and were selectively modulated by α-adrenergic stimulation. I(h) reduced the summation of short trains of artificial excitatory postsynaptic potentials (EPSPs) injected at the soma, and similar effects were observed for short trains of actual EPSPs evoked from layer 2/3 neurons. I(h) narrowed the coincidence detection window for EPSPs arriving from separate layer 2/3 inputs, indicating that the dampening effect of I(h) extended to spatially disperse inputs. To test the role of corticospinal I(h) in transforming EPSPs into action potentials, we transfected layer 2/3 pyramidal neurons with channelrhodopsin-2 and used rapid photostimulation across multiple sites to synaptically drive spiking activity in postsynaptic neurons. Blocking I(h) increased layer 2/3-driven spiking in corticospinal but not corticostriatal neurons. Our results imply that I(h)-dependent synaptic integration in corticospinal neurons constitutes an intracortical control mechanism, regulating the efficacy with which local activity in motor cortex is transferred to downstream circuits in the spinal cord. We speculate that modulation of I(h) in corticospinal neurons could provide a microcircuit-level mechanism involved in translating action planning into action execution.

Circuit-specific intracortical hyperconnectivity in mice with deletion of the autism-associated Met receptor tyrosine kinase.

Local hyperconnectivity in the neocortex is a hypothesized pathophysiological state in autism spectrum disorder (ASD). MET, a receptor tyrosine kinase that regulates dendrite and spine morphogenesis, has been established as a risk gene for ASD. Here, we analyzed the synaptic circuit organization of identified pyramidal neurons in the anterior frontal cortex of mice with a dorsal pallium-derived, conditional knock-out (cKO) of Met. Synaptic mapping by glutamate uncaging identified layer 2/3 as the main source of local excitatory input to layer 5 projection neurons in controls. In both cKO and heterozygotes, this pathway was stronger by a factor of approximately 2. This increase was both sublayer and projection-class specific, restricted to corticostriatal neurons in upper layer 5B and not neighboring corticopontine neurons. Paired recordings in cKO slices demonstrated increased unitary connectivity. We propose that excitatory hyperconnectivity in specific neocortical microcircuits constitutes a physiological basis for Met-mediated ASD risk.

Top-down laminar organization of the excitatory network in motor cortex.

Cortical layering is a hallmark of the mammalian neocortex and a major determinant of local synaptic circuit organization in sensory systems. In motor cortex, the laminar organization of cortical circuits has not been resolved, although their input-output operations are crucial for motor control. Here, we developed a general approach for estimating layer-specific connectivity in cortical circuits and applied it to mouse motor cortex. From these data we computed a laminar presynaptic --> postsynaptic connectivity matrix, W(post,pre), revealing a complement of stereotypic pathways dominated by layer 2 outflow to deeper layers. Network modeling predicted, and experiments with disinhibited slices confirmed, that stimuli targeting upper, but not lower, cortical layers effectively evoked network-wide events. Thus, in motor cortex, descending excitation from a preamplifier-like network of upper-layer neurons drives output neurons in lower layers. Our analysis provides a quantitative wiring-diagram framework for further investigation of the excitatory networks mediating cortical mechanisms of motor control.