Layer 5 Intratelencephalic Neurons in the Motor Cortex Stably Encode Skilled Movement.

The primary motor cortex (M1) and the dorsal striatum play a critical role in motor learning and the retention of learned behaviors. Motor representations of corticostriatal ensembles emerge during motor learning. In the coordinated reorganization of M1 and the dorsal striatum for motor learning, layer 5a (L5a) which connects M1 to the ipsilateral and contralateral dorsal striatum, should be a key layer. Although M1 L5a neurons represent movement-related activity in the late stage of learning, it is unclear whether the activity is retained as a memory engram. Here, using Tlx3-Cre male transgenic mice, we conducted two-photon calcium imaging of striatum-projecting L5a intratelencephalic (IT) neurons in forelimb M1 during late sessions of a self-initiated lever-pull task and in sessions after 6 d of nontraining following the late sessions. We found that trained male animals exhibited stable motor performance before and after the nontraining days. At the same time, we found that M1 L5a IT neurons strongly represented the well-learned forelimb movement but not uninstructed orofacial movements. A subset of M1 L5a IT neurons consistently coded the well-learned forelimb movement before and after the nontraining days. Inactivation of M1 IT neurons after learning impaired task performance when the lever was made heavier or when the target range of the pull distance was narrowed. These results suggest that a subset of M1 L5a IT neurons continuously represent skilled movement after learning and serve to fine-tune the kinematics of well-learned movement.SIGNIFICANCE STATEMENT Motor memory persists even when it is not used for a while. IT neurons in L5a of the M1 gradually come to represent skilled forelimb movements during motor learning. However, it remains to be determined whether these changes persist over a long period and how these neurons contribute to skilled movements. Here, we show that a subset of M1 L5a IT neurons retain information for skilled forelimb movements even after nontraining days. Furthermore, suppressing the activity of these neurons during skilled forelimb movements impaired behavioral stability and adaptability. Our results suggest the importance of M1 L5a IT neurons for tuning skilled forelimb movements over a long period.

Variations in Commissural Input Processing Across Different Types of Cortical Projection Neurons.

To understand how incoming cortical inputs are processed by different types of cortical projection neurons in the medial prefrontal cortex, we compared intrinsic physiological properties of and commissural excitatory/inhibitory influences on layer 5 intratelencephalic (IT), layer 5 pyramidal tract (PT), and layers 2/3 IT projection neurons. We found that intrinsic physiological properties and commissural synaptic transmission varied across the three types of projection neurons. The rank order of intrinsic excitability was layer 5 PT > layer 5 IT > layers 2/3 IT neurons. Commissural connectivity was higher in layers 2/3 than layer 5 projection neurons, but commissural excitatory influence was stronger on layer 5 than layers 2/3 pyramidal neurons. Paired-pulse ratio was also greater in PT than IT neurons. These results indicate that commissural inputs activate deep layer PT neurons most preferentially and superficial layer IT neurons least preferentially. Deep layer PT neurons might faithfully transmit cortical input signals to downstream subcortical structures for reliable control of behavior, whereas superficial layer IT neurons might integrate cortical input signals from diverse sources in support of higher-order cognitive functions.

Amygdala-Cortical Control of Striatal Plasticity Drives the Acquisition of Goal-Directed Action.

In mammalian species, the capacity for goal-directed action relies on a process of cognitive-emotional integration, which melds the causal and incentive learning processes that link action-goal associations with the current value of the goal [1]. Recent evidence suggests that such integration depends on a cortical-limbic-striatal circuit centered on the posterior dorsomedial striatum (pDMS) [2]. Learning-related plasticity has been described at both classes of principal neuron in the pDMS, the direct (dSPNs) and indirect (iSPNs) pathway spiny projection neurons [3-5], and is thought to depend on inputs from prelimbic cortex (PL) [6] and the basolateral amygdala (BLA) [7]. Nevertheless, the relative contribution of these structures to the cellular changes associated with goal-directed learning has not been assessed, nor is it known whether any plasticity specific to the PL and BLA inputs to the pDMS is localized to dSPNs, iSPNs, or both cell types. Here, by combining instrumental conditioning with circuit-specific manipulations and ex vivo optogenetics in mice, we discovered that the PL and not the BLA input to pDMS is pivotal for goal-directed learning and that plasticity in the PL-pDMS pathway was bilateral and specific to dSPNs in the pDMS. Subsequent experiments revealed the BLA is critically but indirectly involved in striatal plasticity via its input to the PL; inactivation of the BLA projection to PL blocked goal-directed learning and prevented learning-related plasticity at dSPNs in pDMS.

Reconstruction of Intratelencephalic Neurons in the Mouse Secondary Motor Cortex Reveals the Diverse Projection Patterns of Single Neurons.

The secondary motor cortex (MOs) plays crucial roles in cognitive and executive processes and has reciprocal connections with numerous cortices in rodents. However, descriptions of the neuronal morphologies and projection patterns of the MOs at the level of a single neuron are lacking, severely hindering the comprehensive understanding of the wiring diagram of the MOs. Herein, we used a Cre-dependent adeno-associated virus (AAV) to fluorescently label ~80 pyramidal neurons nearby or in the MOs and acquired an uninterrupted whole-brain 3D dataset at a voxel resolution of 0.2 × 0.2 × 1 µm with a whole-brain fluorescence imaging system (fMOST). Based on our 3D dataset, we reconstructed the complete morphologies of 36 individual intratelencephalic (IT) neurons nearby or in the MOs and analyzed the projection patterns and projection strengths of these neurons at a single-neuron level based on several parameters, including the projection areas, the total number of branches, the fiber length, and the total number of terminal tips. We obtained a neuron with an axonal length of 318.43 mm, which is by far the longest reported axonal length. Our results show that all individual neurons in the MOs, regardless of whether they are located in layer 2/3 or layer 5, display diverse projection patterns and projection strengths, implying that these neurons might be involved in different brain circuits at different intensities. The results lay a solid foundation for exploring the relationship between neuronal morphologies and behavioral functions of the MOs at the level of a single neuron.

The Bilateral Prefronto-striatal Pathway Is Necessary for Learning New Goal-Directed Actions.

The acquisition of new goal-directed actions requires the encoding of action-outcome associations. At a neural level, this encoding has been hypothesized to involve a prefronto-striatal circuit extending between the prelimbic cortex (PL) and the posterior dorsomedial striatum (pDMS); however, no research identifying this pathway with any precision has been reported. We started by mapping the prelimbic input to the dorsal and ventral striatum using a combination of retrograde and anterograde tracing with CLARITY and established that PL-pDMS projections share some overlap with projections to the nucleus accumbens core (NAc) in rats. We then tested whether each of these pathways were functionally required for goal-directed learning; we used a pathway-specific dual-virus chemogenetic approach to selectively silence pDMS-projecting or NAc-projecting PL neurons during instrumental training and tested rats for goal-directed action. We found that silencing PL-pDMS projections abolished goal-directed learning, whereas silencing PL-NAc projections left goal-directed learning intact. Finally, we used a three-virus approach to silence bilateral and contralateral pDMS-projecting PL neurons and again blocked goal-directed learning. These results establish that the acquisition of new goal-directed actions depends on the bilateral PL-pDMS pathway driven by intratelencephalic cortical neurons.