Thalamocortical axons control the cytoarchitecture of neocortical layers by area-specific supply of VGF.

Neuronal abundance and thickness of each cortical layer are specific to each area, but how this fundamental feature arises during development remains poorly understood. While some of area-specific features are controlled by intrinsic cues such as morphogens and transcription factors, the exact influence and mechanisms of action by cues extrinsic to the cortex, in particular the thalamic axons, have not been fully established. Here, we identify a thalamus-derived factor, VGF, which is indispensable for thalamocortical axons to maintain the proper amount of layer 4 neurons in the mouse sensory cortices. This process is prerequisite for further maturation of the primary somatosensory area, such as barrel field formation instructed by a neuronal activity-dependent mechanism. Our results provide an actual case in which highly site-specific axon projection confers further regional complexity upon the target field through locally secreting signaling molecules from axon terminals.

Thalamic NMDA receptor function is necessary for patterning of the thalamocortical somatosensory map and for sensorimotor behaviors.

NMDARs play a major role in patterning of topographic sensory maps in the brain. Genetic knock-out of the essential subunit of NMDARs in excitatory cortical neurons prevents whisker-specific neural pattern formation in the barrel cortex. To determine the role of NMDARs en route to the cortex, we generated sensory thalamus-specific NR1 (Grin1)-null mice (ThNR1KO). A multipronged approach, using histology, electrophysiology, optical imaging, and behavioral testing revealed that, in these mice, whisker patterns develop in the trigeminal brainstem but do not develop in the somatosensory thalamus. Subsequently, there is no barrel formation in the neocortex yet a partial afferent patterning develops. Whisker stimulation evokes weak cortical activity and presynaptic neurotransmitter release probability is also affected. We found several behavioral deficits in tasks, ranging from sensorimotor to social and cognitive. Collectively, these results show that thalamic NMDARs play a critical role in the patterning of the somatosensory thalamic and cortical maps and their impairment may lead to pronounced behavioral defects.

NMDAR-regulated dynamics of layer 4 neuronal dendrites during thalamocortical reorganization in neonates.

Thalamocortical (TC) connectivity is reorganized by thalamic inputs during postnatal development; however, the dynamic characteristics of TC reorganization and the underlying mechanisms remain unexplored. We addressed this question using dendritic refinement of layer 4 (L4) stellate neurons in mouse barrel cortex (barrel cells) as a model; dendritic refinement of L4 neurons is a critical component of TC reorganization through which postsynaptic L4 neurons acquire their dendritic orientation toward presynaptic TC axon termini. Simultaneous labeling of TC axons and individual barrel cell dendrites allowed in vivo time-lapse imaging of dendritic refinement in the neonatal cortex. The barrel cells reinforced the dendritic orientation toward TC axons by dynamically moving their branches. In N-methyl-D-aspartate receptor (NMDAR)-deficient barrel cells, this dendritic motility was enhanced, and the orientation bias was not reinforced. Our data suggest that L4 neurons have "fluctuating" dendrites during TC reorganization and that NMDARs cell autonomously regulate these dynamics to establish fine-tuned circuits.

Cortical adenylyl cyclase 1 is required for thalamocortical synapse maturation and aspects of layer IV barrel development.

Experimental evidence from mutant or genetically altered mice indicates that the formation of barrels and the proper maturation of thalamocortical (TC) synapses in the primary somatosensory (barrel) cortex depend on mechanisms mediated by neural activity. Type 1 adenylyl cyclase (AC1), which catalyzes the formation of cAMP, is stimulated by increases in intracellular Ca(2+) levels in an activity-dependent manner. The AC1 mutant mouse, barrelless (brl), lacks typical barrel cytoarchitecture, and displays presynaptic and postsynaptic functional defects at TC synapses. However, because AC1 is expressed throughout the trigeminal pathway, the barrel cortex phenotype of brl mice may be a consequence of AC1 disruption in cortical or subcortical regions. To examine the role of cortical AC1 in the development of morphological barrels and TC synapses, we generated cortex-specific AC1 knock-out (CxAC1KO) mice. We found that neurons in layer IV form grossly normal barrels and TC axons fill barrel hollows in CxAC1KO mice. In addition, whisker lesion-induced critical period plasticity was not impaired in these mice. However, we found quantitative reductions in the quality of cortical barrel cytoarchitecture and dendritic asymmetry of layer IV barrel neurons in CxAC1KO mice. Electrophysiologically, CxAC1KO mice have deficits in the postsynaptic but not in the presynaptic maturation of TC synapses. These results suggest that activity-dependent postsynaptic AC1-cAMP signaling is required for functional maturation of TC synapses and the development of normal barrel cortex cytoarchitecture. They also suggest that the formation of the gross morphological features of barrels is independent of postsynaptic AC1 in the barrel cortex.

Exuberant thalamocortical axon arborization in cortex-specific NMDAR1 knockout mice.

Development of whisker-specific neural patterns in the rodent somatosensory system requires NMDA receptor (NMDAR)-mediated activity. In cortex-specific NR1 knockout (CxNR1KO) mice, while thalamocortical afferents (TCAs) develop rudimentary whisker-specific patterns in the primary somatosensory (barrel) cortex, layer IV cells do not develop barrels or orient their dendrites towards TCAs. To determine the role of postsynaptic NMDARs in presynaptic afferent development and patterning in the barrel cortex, we examined the single TCA arbors in CxNR1KO mice between postnatal days (P) 1-7. Sparsely branched TCAs invade the cortical plate on P1 in CxNR1KO mice as in control mice. In control animals, TCAs progressively elaborate patchy terminals, mostly restricted to layer IV. In CxNR1KO mice, TCAs develop far more extensive arbors between P3-7. Their lateral extent is twice that of controls from P3 onwards. By P7, CxNR1KO TCAs have significantly fewer branch points and terminal endings in layers IV and VI but more in layers II/III and V than control mouse TCAs. Within expansive terminal arbors, CxNR1KO TCAs develop focal terminal densities in layer IV, corresponding to the rudimentary whisker-specific patches. Given that thalamic NMDARs are spared in CxNR1KO mice, the present results show that postsynaptic NMDARs play an important role in refinement of presynaptic afferent arbors and whisker-specific patterning in the developing barrel cortex.

NMDA receptor-dependent pattern transfer from afferents to postsynaptic cells and dendritic differentiation in the barrel cortex.

N-Methyl-D-aspartate receptors (NMDARs) are important for synaptic refinement during development. In CxNR1KO mice, cortical excitatory neurons lack NR1, the essential subunit of the NMDAR, and in their primary somatosensory (S1) cortex whisker-specific cellular patterns, "barrels," are absent. Despite this cytoarchitectural defect, thalamocortical axons (TCAs) representing the mystacial vibrissae form topographically organized patterns and undergo critical period plasticity. This region-specific knockout mouse model allows for dissection of the mechanisms underlying patterning of the pre- and postsynaptic neural elements in the S1 cortex. In the absence of functional NMDARs, layer IV cell numbers are unaltered, but these cells fail to segregate into barrels. Furthermore, the dendritic fields of spiny stellate cells do not orient toward TCA terminal patches as in normal mice. Instead, they radiate in all directions covering larger territories, exhibiting profuse branching with increased spine density. Comparison of TCA patches with serotonin transporter (5-HTT) immunohistochemistry or Dil labeling also indicates that in the CxNR1KO cortex TCAs form smaller patches and individual axon terminal branching is not as well developed as in control cortex. Our results suggest that postsynaptic NMDAR activation is critical in communicating periphery-related sensory patterns from TCAs to barrel cells. When postsynaptic NMDAR function is disrupted, layer IV spiny stellate cells remain imperceptive to patterning of their presynaptic inputs and elaborate exuberant dendritic specializations.

Lesion-induced thalamocortical axonal plasticity in the S1 cortex is independent of NMDA receptor function in excitatory cortical neurons.

Neural activity plays an important role in refinement and plasticity of synaptic connections in developing vertebrate sensory systems. The rodent whisker-barrel pathway is an excellent model system to investigate the role of activity in formation of patterned neural connections and their plasticity. When whiskers on the snout or the sensory nerves innervating them are damaged during a critical period in development, whisker-specific patterns are altered along the trigeminal pathway, including the primary somatosensory (S1) cortex. In this context, NMDA receptor (NMDAR)-mediated activity has been implicated in patterning and plasticity of somatosensory maps. Using CxNR1KO mice, in which NMDAR1 (NR1), the essential NMDAR subunit gene, is disrupted only in excitatory cortical neurons, we showed that NMDAR-mediated activity is essential for whisker-specific patterning of barrel cells in layer IV of the S1 cortex. In CxNR1KO mice, thalamocortical axons (TCAs) representing the large whiskers segregate into rudimentary patches, but barrels as cellular modules do not develop. In this study, we examined lesion-induced TCA plasticity in CxNR1KO mice. TCA patterns underwent normal structural plasticity when their peripheral inputs were altered after whisker lesions during the critical period. The extent of the lesion-induced morphological plasticity and the duration of the critical period were quantitatively indistinguishable between CxNR1KO and control mice. We conclude that TCA plasticity in the neocortex is independent of postsynaptic NMDAR activity in excitatory cortical neurons, and that non-NMDAR-mediated cortical activity and/or subcortical mechanisms must be operational in this process.