In Vivo Visualization of Spontaneous Activity in Neonatal Mouse Sensory Cortex at a Single-Neuron Resolution

Summary

Primary sensory areas in the neocortex exhibit unique spontaneous activities during development. This article describes how to visualize individual neuron activities and primary sensory areas to analyze area-specific synchronous activities in neonatal mice in vivo.

Abstract

The mammalian brain undergoes dynamic developmental changes at both the cellular and circuit levels throughout prenatal and postnatal periods. Following the discovery of numerous genes contributing to these developmental changes, it is now known that neuronal activity also substantially modulates these processes. In the developing cerebral cortex, neurons exhibit synchronized activity patterns that are specialized to each primary sensory area. These patterns markedly differ from those observed in the mature cortex, emphasizing their role in regulating area-specific developmental processes. Deficiencies in neuronal activity during development can lead to various brain diseases. These findings highlight the need to examine the regulatory mechanisms underlying activity patterns in neuronal development. This paper summarizes a series of protocols to visualize primary sensory areas and neuronal activity in neonatal mice, to image the activity of individual neurons within the cortical subfields using two-photon microscopy in vivo, and to analyze subfield-related activity correlations. We show representative results of patchwork-like synchronous activity within individual barrels in the somatosensory cortex. We also discuss various potential applications and some limitations of this protocol.

Introduction

The cerebral cortex contains several sensory areas with distinct functions. The areas receive inputs originating from their corresponding sensory organs, mostly conveyed through the spinal cord or brainstem and relayed via the thalamus^1,2. Notably, neurons in each primary sensory area exhibit uniquely synchronized activity during early developmental stages, which also originate from sensory organs or the lower nervous centers, but essentially differ from the activities observed in the mature cortex³.

In neonatal rodents, for example, the primary visual area (V1) displays wave-like activity, which originates in the retina (retinal wave) and propagates through the entire visual pathway while conserving retinotopy⁴. The primary auditory area (A1) exhibits synchronous activity organized in band-shaped subregions that correspond to the isofrequency bands in the mature brain. The activity emanates from the cochlea's inner hair cells^5,6. The barrel cortex in the primary somatosensory area (S1) shows a patchwork-like activity pattern in which layer 4 neurons within individual barrels, namely, neurons responsive to individual whiskers, are synchronously activated⁷. Although proposed to originate from the trigeminal ganglion, the source of the activity remains unknown⁷. Consequently, neonatal activity patterns are specialized both within each primary sensory area and within intra-areal subfields. The simultaneous visualization of neuronal activity and structure of primary sensory areas may facilitate an inquiry into the contribution of these activity patterns to the development of sensory systems.

In this article, we summarized a series of protocols: (1) to visualize individual neuronal activities using sparse labeling of GCaMP and primary sensory areas using TCA-RFP mice that express red fluorescent protein in thalamocortical axons⁷, (2) to image single cell-level activity in neonatal mice using two-photon microscopy in vivo, and (3) to analyze the activity correlations within S1 barrel cortex. The representative results show patchwork-like synchronized activity within individual barrels of a postnatal day (P)6 mouse. Despite some limitations, this technique can be used for chronic imaging, wide-field imaging across multiple sensory areas, and various manipulation experiments. The multifaceted analysis of neuronal activity during development will enrich our comprehension of brain circuit formation mechanisms.

Protocol

All the experiments were conducted in accordance with the guidelines for animal experimentation of Kumamoto University and the National Institute of Genetics and approved by the animal experimentation committees. 1. In utero electroporation (IUE) Mate male TCA-RFP mice of ICR background with female wild-type ICR mice. Observe the vaginal plug to check for mating early morning of the next day. Observe the abdomen to check for pregnancy 2 weeks later. …

Representative Results

Figure 1 shows the representative results of layer 4 neuron activities in the barrel cortex of a P6 pup visualized using the present protocol. Two-photon images of the green channel (GCaMP) and red channel (TCA-RFP) were temporally averaged and shown in Figure 1A. Because TCA-RFP fluorescence was much weaker than GCaMP fluorescence, the GCaMP signal leaked into the red channel (Figure 1A1,A2). Fourteen ROIs were dra…

Discussion

Given that the spontaneous activities emerge from the sensory organ or lower nervous system and travel to the primary sensory area through a pathway equivalent to that of a mature nervous system³, it is crucial to define the primary sensory area and the location of imaged neurons within the area. In this protocol, we addressed this requirement by employing transgenic mice that visualize thalamocortical axons and the Supernova system that expresses GCaMP sparsely⁸. These tec…

Materials

20× objective lens (water immersion)
250 mL Vacuum Filter/Storage Bottle System	Corning	431096
4%-paraformaldehyde phosphate buffer solution (4% PFA)	Nacalai	09154-85
Acrylic resin (UNIFAST II)	GC	N/A
Agarose	Sigma	A9793
Aspirator tube assembly	Drummond	2-040-000
CaCl2•2H2O	Nacalai	06731-05
Electroporator	BEX	GEB14
Eye drop (Scopisol)	Senju Pharmaceutical	N/A
Fluorescence stereo microscope	Leica	M165FC
Glucose	Nacalai	16806-25
Heating pad	Muromachi Kikai	FHC-HPS
HEPES	Gibco	15630-080
Isoflurane	Pfizer	N/A
KCl	Nacalai	28514-75
MgSO4•7H2O	Wako	131-00405
Micropipette puller	Narishige	PC-100
Multiphoton laser	Spectra-Physics	Mai Tai eHP DeepSee
Multiphoton microscope	Zeiss	LSM 7MP
NaCl	Nacalai	31320-05
Non-woven fabric (Kimwipe)	Kimberly Clark	S-200
Phosphate buffered saline (PBS)	Nacalai	27575-31
Plasmid: CAG-loxP-STOP-loxP-GCaMP6s-ires-tTA-WPRE	Addgene	pK175
Plasmid: TRE-nCre	Addgene	pK031
Precision calibrated micropipets	Drummond	2-000-050
Razor blade	Feather	FA-10
Rimadyl (50 mg/mL Carprofen)	Zoetis JP	N/A
Round cover glass, 3-mm-diameter	Matsunami	CS01078
Saline	Otsuka	035175315
Sodium pentobarbital	Nacalai	26427-72
Stage for imaging living pup (two single-axis translation stage for XY positioning, two-axis goniometer, base plate, adjustable pillar for z positioning)	ThorLabs	LT1/M, GN2/M, BM2060/M, MLP01/M
TCA-RFP mouse	N/A	N/A	Mizuno et al., 2018a
Tissue adhesive (Vetbond)	3M	1469SB
Titanium bar	Endo Scientific Instrument	N/A	Custom made (Mizuno et al., 2018b)
Titanium bar fixing plate		N/A	Custom made (Mizuno et al., 2018b)
Trypan blue	Sigma	T8154
Tweezers with platinum plate electrode, 5 mm diameter	BEX	CUY650P5
Wild-type ICR mouse	Nihon SLC	Slc:ICR