Distinct distribution of subplate neuron subtypes between the sensory cortices during the early postnatal period.

Subplate neurons (SpNs) are a heterogeneous neuronal population actively involved in early cortical circuit formation. In rodents, many SpNs survive and form layer 6b. The molecular heterogeneity of SpNs raises the question of whether different subpopulations of SpNs survive through the early postnatal period similarly and whether such diverse SpN populations in the auditory cortex (ACtx) share a common distribution pattern with other sensory systems. To address that, we investigated the expression pattern of multiple specific SpN markers in the ACtx, as well as in the visual (VCtx) and somatosensory (SCtx) cortices as controls, using complexin 3 (Cplx3) antibodies and different SpN-specific Cre-driver mice, such as connective tissue growth factor (CTGF), dopamine receptor D1 (Drd1a), and neurexophilin 4 (Nxph4). We focused on two early time windows in auditory development: (1) during the second postnatal week (PNW) before ear-canal opening and (2) during the third PNW after ear-canal opening. We compared the expression pattern of different SpN markers in ACtx with VCtx and SCtx. At both examined timepoints, Cplx3 and Nxph4 expressing SpNs form the largest and smallest population in the ACtx, respectively. Similar distribution patterns are observable in the VCtx and SCtx during the second PNW but not during the third PNW, for a higher proportion of Drd1a expressing SpNs is detected in the VCtx and CTGF expressing SpNs in the SCtx. This study suggests that different populations of SpNs might contribute differently to the development of individual sensory circuits.

Early retinal deprivation crossmodally alters nascent subplate circuits and activity in the auditory cortex during the precritical period.

Sensory perturbation in one modality results in the adaptive reorganization of neural pathways within the spared modalities, a phenomenon known as "crossmodal plasticity," which has been examined during or after the classic "critical period." Because peripheral perturbations can alter the auditory cortex (ACX) activity and functional connectivity of the ACX subplate neurons (SPNs) even before the critical period, called the precritical period, we investigated if retinal deprivation at birth crossmodally alters the ACX activity and SPN circuits during the precritical period. We deprived newborn mice of visual inputs after birth by performing bilateral enucleation. We performed in vivo widefield imaging in the ACX of awake pups during the first two postnatal weeks to investigate cortical activity. We found that enucleation alters spontaneous and sound-evoked activities in the ACX in an age-dependent manner. Next, we performed whole-cell patch clamp recording combined with laser scanning photostimulation in ACX slices to investigate circuit changes in SPNs. We found that enucleation alters the intracortical inhibitory circuits impinging on SPNs, shifting the excitation-inhibition balance toward excitation and this shift persists after ear opening. Together, our results indicate that crossmodal functional changes exist in the developing sensory cortices at early ages before the onset of the classic critical period.

Early retinal deprivation crossmodally alters nascent subplate circuits and activity in the auditory cortex during the precritical period.

Sensory perturbation in one modality results in adaptive reorganization of neural pathways within the spared modalities, a phenomenon known as "crossmodal plasticity", which has been examined during or after the classic 'critical period'. Because peripheral perturbations can alter auditory cortex (ACX) activity and functional connectivity of the ACX subplate neurons (SPNs) even before the classic critical period, called the precritical period, we investigated if retinal deprivation at birth crossmodally alters ACX activity and SPN circuits during the precritical period. We deprived newborn mice of visual inputs after birth by performing bilateral enucleation. We performed in vivo imaging in the ACX of awake pups during the first two postnatal weeks to investigate cortical activity. We found that enucleation alters spontaneous and sound-evoked activity in the ACX in an age-dependent manner. Next, we performed whole-cell patch clamp recording combined with laser scanning photostimulation in ACX slices to investigate circuit changes in SPNs. We found that enucleation alters the intracortical inhibitory circuits impinging on SPNs shifting the excitation-inhibition balance towards excitation and this shift persists after ear opening. Together, our results indicate that crossmodal functional changes exist in the developing sensory cortices at early ages before the onset of the classic critical period.

Cortical inhibitory but not excitatory synaptic transmission and circuit refinement are altered after the deletion of NMDA receptors during early development.

Neurons in the cerebral cortex form excitatory and inhibitory circuits with specific laminar locations. The mechanisms underlying the development of these spatially specific circuits is not fully understood. To test if postsynaptic N-methyl-D-aspartate (NMDA) receptors on excitatory neurons are required for the development of specific circuits to these neurons, we genetically ablated NMDA receptors from a subset of excitatory neurons in the temporal association cortex (TeA) through in utero electroporation and assessed the intracortical circuits connecting to L5 neurons through in vitro whole-cell patch clamp recordings coupled with laser-scanning photostimulation (LSPS). In NMDAR knockout neurons, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated connections were largely intact. In contrast both LSPS and mini-IPSC recordings revealed that γ-aminobutyric acid type A (GABAA) receptor-mediated connections were impaired in NMDAR knockout neurons. These results suggest that postsynaptic NMDA receptors are important for the development of GABAergic circuits.

Mesoscopic oblique plane microscopy with a diffractive light-sheet for large-scale 4D cellular resolution imaging.

Fundamental understanding of large-scale dynamic connectivity within a living organism requires volumetric imaging over a large field of view (FOV) at biologically relevant speed and resolution. However, most microscopy methods make trade-offs between FOV and axial resolution, making it challenging to observe highly dynamic processes at cellular resolution in 3D across mesoscopic scales (e.g., whole zebrafish larva). To overcome this limitation, we have developed mesoscopic oblique plane microscopy (Meso-OPM) with a diffractive light sheet. By augmenting the illumination angle of the light sheet with a transmission grating, we improved the axial resolution approximately sixfold over existing methods and approximately twofold beyond the diffraction limitation of the primary objective lens. We demonstrated a FOV up to 5.4 mm × 3.3 mm with resolution of 2.5 µm × 3 µm × 6 µm, allowing volumetric imaging of 3D cellular structures with a single scan. Applying Meso-OPM for in vivo imaging of zebrafish larvae, we report here in toto whole-body volumetric recordings of neuronal activity at 2 Hz volume rate and whole-body volumetric recordings of blood flow dynamics at 5 Hz with 3D cellular resolution.

Development of Auditory Cortex Circuits.

The ability to process and perceive sensory stimuli is an essential function for animals. Among the sensory modalities, audition is crucial for communication, pleasure, care for the young, and perceiving threats. The auditory cortex (ACtx) is a key sound processing region that combines ascending signals from the auditory periphery and inputs from other sensory and non-sensory regions. The development of ACtx is a protracted process starting prenatally and requires the complex interplay of molecular programs, spontaneous activity, and sensory experience. Here, we review the development of thalamic and cortical auditory circuits during pre- and early post-natal periods.

A characterization of laminar architecture in mouse primary auditory cortex.

Laminar architecture of primary auditory cortex (A1) has long been investigated by traditional histochemical techniques such as Nissl staining, retrograde and anterograde tracings. Uncertainty still remains, however, about laminar boundaries in mice. Here we investigated the cortical lamina structure by combining neuronal tracing and immunofluorochemistry for laminar specific markers. Most retrogradely labeled corticothalamic neurons expressed Forkhead box protein P2 (Foxp2) and distributed within the laminar band of Foxp2-expressing cells, identifying layer 6. Cut-like homeobox 1 (Cux1) expression in layer 2-4 neurons divided the upper layers into low expression layers 2/3 and high expression layers 3/4, which overlapped with the dense terminals of vesicular glutamate transporter 2 (vGluT2) and anterogradely labeled lemniscal thalamocortical axons. In layer 5, between Cux1-expressing layers 2-4 and Foxp2-defined layer 6, retrogradely labeled corticocollicular projection neurons mostly expressed COUP-TF interacting protein 2 (Ctip2). Ctip2-expressing neurons formed a laminar band in the middle of layer 5 distant from layer 6, creating a laminar gap between the two laminas. This gap contained a high population of commissural neurons projecting to contralateral A1 compared to other layers and received vGluT2-immunopositive, presumptive thalamocortical axon collateral inputs. Our study shows that layer 5 is much wider than layer 6, and layer 5 can be divided into at least three sublayers. The thalamorecipient layers 3/4 may be separated from layers 2/3 using Cux1 and can be also divided into layer 4 and layer 3 based on the neuronal soma size. These data provide a new insight for the laminar structure of mouse A1.

Laminar specific gene expression reveals differences in postnatal laminar maturation in mouse auditory, visual, and somatosensory cortex.

Proper formation of laminar structures in sensory cortexes is critical for sensory information processing. Previous studies suggested that the timing of neuronal migration and the laminar position of cortical neurons differ among sensory cortexes. How they differ during postnatal development has not been systematically investigated. Here, identifying laminas using transcription factors, we examined postnatal changes in neuronal density and distribution in presumptive primary auditory (ACx), visual (VCx), and somatosensory cortexes (SCx) in a strain of mice using immunofluorescence techniques. Development of laminar thickness and its cortical proportion differed among the sensory cortexes. Layers 2-4 defined by Cut-like homeobox 1 (Cux1)-expressing neurons were narrower, and layer 5 was wider in ACx compared to those in VCx or SCx, while Forkhead-box protein P2 (Foxp2)-defined layer 6 was wider in SCx than the other two sensory cortexes throughout postnatal development. Meanwhile, thalamocortical input layers identified by Cux1-expressing neurons formed later in ACx than in the other two cortical regions. The cell densities of ETS-related protein 81-expressing neurons increased in both lower and upper layers but at distinct timing, while those of COUP-TF-interacting protein 2 expressing neurons in the lower layers changed bidirectionally (i.e., increased or decreased) both in layer- and cortical region-specific manners. Foxp2-expressing cells in layer 6 distributed differently and declined at different timing among the sensory cortexes. Overall, we demonstrate that the maturational timing of lamina differs among the sensory cortexes and that postnatal age-dependent changes in neuronal distribution are unique to each of the sensory cortexes.