The thalamus receives input from 3 distinct cortical layers, but input from only 2 of these has been well characterized. We therefore investigated whether the third input, derived from layer 6b, is more similar to the projections from layer 6a or layer 5. We studied the projections of a restricted population of deep layer 6 cells (“layer 6b cells”) taking advantage of the transgenic mouse Tg(Drd1a-cre)FK164Gsat/Mmucd (Drd1a-Cre), that selectively expresses Cre-recombinase in a subpopulation of layer 6b neurons across the entire cortical mantle. At P8, 18% of layer 6b neurons are labeled with Drd1a-Cre::tdTomato in somatosensory cortex (SS), and some co-express known layer 6b markers. Using Cre-dependent viral tracing, we identified topographical projections to higher order thalamic nuclei. VGluT1+ synapses formed by labeled layer 6b projections were found in posterior thalamic nucleus (Po) but not in the (pre)thalamic reticular nucleus (TRN). The lack of TRN collaterals was confirmed with single-cell tracing from SS. Transmission electron microscopy comparison of terminal varicosities from layer 5 and layer 6b axons in Po showed that L6b varicosities are markedly smaller and simpler than the majority from L5. Our results suggest that L6b projections to the thalamus are distinct from both L5 and L6a projections.

Rodents extract information about nearby objects from the movement of their whiskers through dynamic computations that are carried out by a network of forebrain structures that includes the thalamus and the primary sensory (S1BF) and motor (M1wk) whisker cortices. The posterior nucleus (Po), a higher order thalamic nucleus, is a key hub of this network, receiving cortical and brainstem sensory inputs and innervating both motor and sensory whisker-related cortical areas. In a recent study in rats, we showed that Po inputs differently impact sensory processing in S1BF and M1wk. Here, in C57BL/6 mice, we measured Po synaptic bouton layer distribution and size, compared cortical unit response latencies to "in vivo" Po activation, and pharmacologically examined the glutamatergic receptor mechanisms involved. We found that, in S1BF, a large majority (56%) of Po axon varicosities are located in layer (L)5a and only 12% in L2-L4, whereas in M1wk this proportion is inverted to 18% and 55%, respectively. Light and electron microscopic measurements showed that Po synaptic boutons in M1wk layers 3-4 are significantly larger (~ 50%) than those in S1BF L5a. Electrical Po stimulation elicits different areaspecific response patterns. In S1BF, responses show weak or no facilitation, and involve both ionotropic and metabotropic glutamate receptors, whereas in M1wk, unit responses exhibit facilitation to repetitive stimulation and involve ionotropic NMDA glutamate receptors. Because of the different laminar distribution of axon terminals, synaptic bouton size and receptor mechanisms, the impact of Po signals on M1wk and S1BF, although simultaneous, is likely to be markedly different.

Thalamocortical posterior nucleus (Po) axons innervating the vibrissal somatosensory (S1) and motor (MC) cortices are key links in the brain neuronal network that allows rodents to explore the environment whisking with their motile snout vibrissae. Here, using fine-scale high-end 3D electron microscopy, we demonstrate in adult male C57BL/6 wild-type mice marked differences between MC versus S1 Po synapses in (1) bouton and active zone size, (2) neurotransmitter vesicle pool size, (3) distribution of mitochondria around synapses, and (4) proportion of synapses established on dendritic spines and dendritic shafts. These differences are as large, or even more pronounced, than those between Po and ventro-posterior thalamic nucleus synapses in S1. Moreover, using single-axon transfection labeling, we demonstrate that the above differences actually occur on the MC versus the S1 branches of individual Po cell axons that innervate both areas. Along with recently-discovered divergences in efficacy and plasticity, the synaptic structure differences reported here thus reveal a new subcellular level of complexity. This is a finding that upends current models of thalamocortical circuitry, and that might as well illuminate the functional logic of other branched projection axon systems.

Rodents move rhythmically their facial whiskers and compute differences between signals predicted and those resulting from the movement to infer information about objects near their head. These computations are carried out by a large network of forebrain structures that includes the thalamus and the primary somatosensory (S1BF) and motor (M1wk) cortices. Spatially and temporally precise mechanorreceptive whisker information reaches the S1BF cortex via the ventroposterior medial thalamic nucleus (VPM). Other whisker-related information may reach both M1wk and S1BF via the axons from the posterior thalamic nucleus (Po). However, Po axons may convey, in addition to direct sensory signals, the dynamic output of computations between whisker signals and descending motor commands. It has been proposed that this input may be relevant for adjusting cortical responses to predicted vs. unpredicted whisker signals, but the effects of Po input on M1wk and S1BF function have not been directly tested or compared in vivo. Here, using electrophysiology, optogenetics and pharmacological tools, we compared in adult rats M1wk and S1BF in vivo responses in the whisker areas of the motor and primary somatosensory cortices to passive multi-whisker deflection, their dependence on Po activity, and their changes after a brief intense activation of Po axons. We report that the latencies of the first component of tactile-evoked local field potentials in M1wk and S1BF are similar. The evoked potentials decrease markedly in M1wk, but not in S1BF, by injection in Po of the GABAA agonist muscimol. A brief high-frequency electrical stimulation of Po decreases the responsivity of M1wk and S1BF cells to subsequent whisker stimulation. This effect is prevented by the local application of omega-agatoxin, suggesting that it may in part depend on GABA release by fast-spiking parvalbumin (PV)-expressing cortical interneurons. Local optogenetic activation of Po synapses in different cortical layers also diminishes M1wk and S1BF responses. This effect is most pronounced in the superficial layers of both areas, known to be the main source and target of their reciprocal cortico-cortical connections.

A large part of the thalamus is not directly involved in relaying ascending sensory inputs to the cerebral cortex. The functions and wiring of such "non-primary" nuclei remain unclear. The Posterior nucleus, a representative "non-primary" nucleus of the rodent thalamus, receives heavy and powerful inputs from cortical layer 5 (L5) cells. In addition, Po receives also some trigeminal, spinal and superior colliculus (SC) inputs, as well as powerful inhibitory inputs from the zona incerta (ZI) and anterior pretectal nucleus (APT). To elucidate to which extent these input systems converge or remain separate within Po, we first mapped the distribution of terminals immunolabeled for markers of glutamatergic or GABAergic neurotransmission and L5 terminals constitutively labeled in Rbp4-Cre;Ai14 mice. Besides, we retrogradely traced and quantified the sources of brain and spinal cord input reaching different Po regions. In addition, we compared bouton sizes in axons anterogradely labeled from the above input sources. Our data delineate several domains within Po, each dominated by specific sets of inputs. Cortical L5 afferents predominate in central and ventral Po. In contrast, large glutamatergic terminals from the trigeminal complex and spinal cord as well as GABAergic terminals from APT and ZI prevail in rostral and dorsal Po, and along the border with the ventral posteromedial nucleus. SC inputs preferentially target dorsal and caudal Po. These findings reveal multiple partly overlapping domains within Po. Hence, thalamic neuron subpopulations in different parts of Po may integrate diverse combinations of tactile, motor and pain-related inputs and give rise to functionally diverse thalamocortical subnetworks. Significance statementMany nuclei of the thalamus are not involved in relaying to cortex new information about the world or the body. The functions and wiring of such "non-primary" nuclei remain unclear. Here, we first mapped across the brain and spinal cord the origin of the input pathways reaching the Posterior nucleus, a typical non-primary relay nucleus.Then, we analyzed input distribution within the nucleus and, as a proxy for synaptic strength, the size of their axon terminals. We report a complex tridimensional mosaic of partly overlapping input-specific domains and significant bouton size differences. This wiring may allow diverse inputs to converge in graded combinatorial fashion onto thalamic cell subpopulations, hence giving rise to emergent computations and functionally diverse thalamocortical subnetworks.