Cortical layer 6 control of sensory responses in higher-order thalamus.

KEY POINTS: Thalamic activity is regulated by corticothalamic feedback from layers 5B and 6. To selectively study the importance of the layer 6 corticothalamic (L6 CT) projection, a transgenic mouse line was used in which layer 6 cells projecting to posterior medial thalamus (POm) were targeted for expression of channelrhodopsin-2. Repetitive optogenetic stimulation of this sub-type of L6 cells caused a rapid adaptation in POm spiking output, but had little effect on the spiking activity in the other cortical layers. L6 photoactivation increased POm spiking to the first, but not to subsequent whisker deflections in a 4 Hz train. A sub-population of L6 CT cells that can cause an initial increase in POm activity, that is not sustained with repetitive stimulation, could indicate that this L6 projection does not modulate ongoing sensory processing, but rather serves to briefly increase POm activity in specific behavioural contexts. ABSTRACT: Thalamic activity is regulated by corticothalamic feedback from layers 5B and 6. The nature of these feedback systems differs, one difference being that whereas layer 5 provides 'driver' input, the layer 6 input is thought to be 'modulatory'. To selectively study the importance of the layer 6 corticothalamic (L6 CT) projection, a transgenic mouse line was used in which layer 6 cells projecting to posterior medial thalamus (POm) were targeted for expression of channelrhodopsin-2 and in vivo electrophysiology recordings were done in urethane-anaesthetized mice. Pre- and postsynaptic targets were identified using tracing techniques and light-sheet microscopy in cleared intact brains. We find that optogenetic activation of this subtype of L6 CT cells (L6-Drd1) has little effect on cortical activity, but activates POm. Repetitive photoactivation of L6-Drd1 cells evoked a reliable response following every photoactivation, whereas in the connected POm area spiking was only initially increased. The response to repetitive whisker stimulation showed a similar pattern with only an initial increase in whisker-evoked spiking. Furthermore, the increase in whisker-evoked spiking with optogenetic activation of L6-Drd1 cells is additive, rather than multiplicative, causing even cells that in the absence of L6 activation produce relatively few spikes to increase their spiking substantially. We show that layer 6 corticothalamic cells can provide a strong, albeit rapidly depressing, input to POm. This type of cortical L6 activity could be important for rapid gain control in POm, rather than providing a modulation in phase with the whisking cycle.

A Nanobody-Based Proximity Ligation Assay Detects Constitutive and Stimulus-Regulated Native Arc/Arg3.1 Oligomers in Hippocampal Neuronal Dendrites.

Activity-regulated cytoskeleton-associated protein (Arc), the product of an immediate early gene, plays critical roles in synaptic plasticity and memory. Evidence suggests that Arc function is determined by its oligomeric state; however, methods for localization of native Arc oligomers are lacking. Here, we developed a nanobody-based proximity ligation assay (PLA) for detection, localization, and quantification of Arc-Arc complexes in primary rat hippocampal neuronal cultures. We used nanobodies with single, structurally defined epitopes in the bilobar Arc capsid domain. Nanobody H11 binds inside the N-lobe ligand pocket, while nanobody C11 binds to the C-lobe surface. For each nanobody, ALFA- and FLAG-epitope tags created a platform for antibody binding and PLA. Surprisingly, PLA puncta in neuronal dendrites revealed widespread constitutive Arc-Arc complexes. Treatment of cultures with tetrodotoxin or cycloheximide had no effect, suggesting stable complexes that are independent of recent neuronal activity and protein synthesis. To assess detection of oligomers, cultures were exposed to a cell-penetrating peptide inhibitor of the Arc oligomerization motif (OligoOFF). Arc-Arc complexes detected by H11 PLA were inhibited by OligoOff but not by control peptide. Notably, Arc complexes detected by C11 were unaffected by OligoOFF. Furthermore, we evaluated Arc complex formation after chemical stimuli that increase Arc synthesis. Brain-derived neurotrophic factor increased Arc-Arc signal detected by C11, but not H11. Conversely, dihydroxyphenylglycine (DHPG) treatment selectively enhanced H11 PLA signals. In sum, nanobody-based PLA reveals constitutive and stimulus-regulated Arc oligomers in hippocampal neuronal dendrites. A model is proposed based on detection of Arc dimer by C11 and higher-order oligomer by H11 nanobody.

Neural ensemble dynamics in trunk and hindlimb sensorimotor cortex encode for the control of postural stability.

The cortex has a disputed role in monitoring postural equilibrium and intervening in cases of major postural disturbances. Here, we investigate the patterns of neural activity in the cortex that underlie neural dynamics during unexpected perturbations. In both the primary sensory (S1) and motor (M1) cortices of the rat, unique neuronal classes differentially covary their responses to distinguish different characteristics of applied postural perturbations; however, there is substantial information gain in M1, demonstrating a role for higher-order computations in motor control. A dynamical systems model of M1 activity and forces generated by the limbs reveals that these neuronal classes contribute to a low-dimensional manifold comprised of separate subspaces enabled by congruent and incongruent neural firing patterns that define different computations depending on the postural responses. These results inform how the cortex engages in postural control, directing work aiming to understand postural instability after neurological disease.