Experience-dependent functional plasticity and visual response selectivity of surviving subplate neurons in the mouse visual cortex

Subplate neurons are early-born cortical neurons that transiently form neural circuits during perinatal development and guide cortical maturation. Thereafter, most subplate neurons undergo cell death, while some survive and renew their target areas for synaptic connections. However, the functional properties of the surviving subplate neurons remain largely unknown. This study aimed to characterize the visual responses and experience-dependent functional plasticity of layer 6b (L6b) neurons, the remnants of subplate neurons, in the primary visual cortex (V1). Two-photon Ca²⁺ imaging was performed in V1 of awake juvenile mice. L6b neurons showed broader tunings for orientation, direction, and spatial frequency than did layer 2/3 (L2/3) and L6a neurons. In addition, L6b neurons showed lower matching of preferred orientation between the left and right eyes compared with other layers. Post hoc 3D immunohistochemistry confirmed that the majority of recorded L6b neurons expressed connective tissue growth factor (CTGF), a subplate neuron marker. Moreover, chronic two-photon imaging showed that L6b neurons exhibited ocular dominance (OD) plasticity by monocular deprivation during critical periods. The OD shift to the open eye depended on the response strength to the stimulation of the eye to be deprived before starting monocular deprivation. There were no significant differences in visual response selectivity prior to monocular deprivation between the OD changed and unchanged neuron groups, suggesting that OD plasticity can occur in L6b neurons showing any response features. In conclusion, our results provide strong evidence that surviving subplate neurons exhibit sensory responses and experience-dependent plasticity at a relatively late stage of cortical development.

The mammalian cerebral cortex consists of six layers, with distinct roles in information processing (1, 2). At the bottom of the neocortex, on the boundary between the gray matter and white matter, there is a thin sheet of neurons called layer 6b (L6b) (3). Layer 6b neurons are thought to be remnants of subplate neurons based on their location and cell-type marker expression (4). During prenatal and early postnatal periods, subplate neurons form transient neuronal circuits that play key roles in cortical maturation (5–7). In the embryonic cortex, subplate neurons form short-lived synapses with early immature neurons to regulate radial migration (8). During perinatal development, subplate neurons transiently receive inputs from ingrowing thalamic axons and innervate layer 4 (L4) to guide thalamic inputs to the eventual target, L4 (5, 6). Thus, the circuits formed by subplate neurons at the perinatal developmental stage are essential to establish basic neuronal circuits before starting experience-dependent refinements (5–7). Subsequently, subplate neurons largely disappear due to programmed cell death, but some survive and reside in L6b (5, 6). In the adult cortex, L6b neurons form neuronal circuits with local and long-distance neurons, which are different from those formed during early development (9–12). Therefore, surviving subplate neurons may acquire a role in information processing after remodeling of neuronal connections. A recent study using three-photon Ca²⁺ imaging demonstrated that L6b neurons show visual responses with broad orientation/direction tuning in the adult mouse primary visual cortex (V1) (13). However, comparable evidence for L6b response properties with other layer neurons in V1 is lacking (14–20). Moreover, L6b neurons have diverse morphology and molecular expression (21–24). Neurons born during subplate neurogenesis show the different expression patterns of subplate markers in postnatal L6b (4). However, the response properties in each subtype of L6b neurons remain unknown.The sensory responsiveness of cortical neurons is considerably refined by sensory experience relatively late in development, referred to as the critical period (25, 26). Previous studies have demonstrated that sensory activities before the onset of the critical period affect the arrangement of subplate neuron neurites in the barrel cortex and local subplate circuits in the auditory cortex (27, 28). However, there is no direct evidence that the sensory responses of surviving subplate neurons are modified by sensory experience during the critical period. If experience-dependent plasticity occurs in subplate neuron responses, they will contribute to the experience-dependent development of sensory functions and possibly to the functions in the mature cortex. Ocular dominance (OD) plasticity in V1 is a canonical model used to examine experience-dependent refinement of sensory responses (25, 26, 29, 30). If one eye is occluded for several days during the critical period, neurons in V1 lose their response to the deprived eye. OD plasticity is robustly preserved across species and cell types. Therefore, OD plasticity is suitable for evaluating experience-dependent plasticity in L6b neurons.This study aimed to characterize the visual responses and OD plasticity of L6b neurons in V1. Toward this goal, two-photon Ca²⁺ imaging was performed in awake juvenile mice, followed by 3D immunohistochemistry with a subplate neuronal marker, connective tissue growth factor (CTGF) (4, 31). L6b neurons showed broader tuning to visual stimuli and lower binocular matching of orientation preference than did layer 2/3 (L2/3) and L6a neurons. Chronic two-photon imaging revealed significant OD plasticity in individual L6b neurons during the critical period. Our results provide strong evidence that L6b neurons, presumed to be subplate neuron remnants, exhibit sensory responses and experience-dependent functional plasticity at a relatively late stage of cortical development.