In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Summary

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Abstract

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

Introduction

The precise wiring of neuronal circuits in the cerebral cortex is essential for higher brain functions including perception, cognition, and learning and memory. Cortical circuits are dynamically refined during postnatal development. Studies have investigated the process of cortical circuit formation using histological and in vitro culture analyses. However, the dynamics of circuit formation in living mammals has remained mostly unexplored.

Two-photon microscopy has been widely used for the in vivo analyses of neuronal circuits in the adult mouse brain^1,2. However, owing to technical challenges, only a limited number of studies have addressed neuronal circuit formation in newborn mice. For example, Carrillo et al. performed the time-lapse imaging of climbing fibers in the cerebellum in the second postnatal week³. Portera-Cailliau et al. reported the imaging of axons in cortical layer 1 in the first postnatal week⁴. In the present study, we summarize a protocol for the observation of layer 4 cortical neurons and their dendrites in newborn mice. Results obtained by applying this protocol, which includes two methodologies, are reported in our recent publication⁵. First, we use the Supernova vector system^5,6 for labeling individual neurons in the neonatal brain. In the Supernova system, the fluorescent proteins used for neuronal labeling are exchangeable and labeled cell-specific gene knockdown and editing/knockout analyses are also possible. Second, we describe a surgical procedure for cranial window preparation in fragile neonatal mice. Together, these methodologies allow the in vivo observation of individual neurons in neonatal brains.

Protocol

Experiments should be performed in accordance with the animal welfare guidelines prescribed by the experimenter's institution.

1. Preparation of Pups for Imaging

NOTE: Pups with sparsely labeled cortical neurons can be obtained by in utero electroporation (IUE) of Supernova vectors^5,6. The Supernova system consists of the following two vectors: TRE-Cre and CAG-loxP-STOP-loxP-Gene X-ires-tTA-WPRE. In this system, sparse labeling relies on TRE leakage. In a sparse population of transfected neurons, TRE drives the weak expression of Cre and tTA. Subsequently, only in these cells, the expression of gene X is facilitated by a positive feedback of the tTA-TRE cycles. The achieved sparse and bright labeling allows the visualization of morphological details of individual neurons in vivo. Details of the IUE procedure are not described in this protocol since they have been described elsewhere^7,8,9,10,11.

1. Prepare timed-pregnant mice for IUE.
2. Prepare a DNA solution for IUE. For sparse labeling with RFP, use a solution containing pK031:TRE-Cre (5 ng/µL) and pK029:CAG-loxP-STOP-loxP-RFP-ires-tTA-WPRE (1 µg/µL) or a solution containing pK031:TRE-Cre (5 ng/µL) and pK273:CAG-loxP-STOP-loxP-CyRFP-ires-tTA-WPRE (1 µg/µL).
   NOTE: Various proteins can be expressed in the labeled neurons using different combinations of vectors. Also, various genes can be knocked-down or knocked-out specifically in labeled cells^5,6 (e.g., a series of vectors for the Supernova system are available from RIKEN BioResource Research Center and from Addgene).
3. Perform regular IUE^7,8,9,10,11 to label cortical neurons. For the labeling of layer 4 neurons, use embryonic day-14 embryos.
4. Wait for pup delivery and growth.

2. Surgery

1. Anesthetize the postnatal day-5 (P5) pup using isoflurane gas (1.0%). Perform a tail-pinch test to check the level of anesthesia. If the pup responds to the pinch, increase the isoflurane concentration (up to 2.0%) or wait until the response disappears. Maintain the pup's body temperature during surgery using a heating pad.
2. Subcutaneously inject an analgesic (carprofen, 5 mg/kg).
3. Sterilize the pup's skin covering the skull by wiping it with 70% ethanol three times.
4. Remove approximately 20 mm² of the skin covering the skull using scissors sterilized with 70% ethanol (Figure 1A).
5. Remove the fascia of the skull using sterilized forceps and a clean, sterile cotton swab (Figure 1A).
6. Apply tissue adhesive using loading tips to the incised skin surface to stop the bleeding. Do not apply tissue adhesive to the imaging area (Figure 1B) because this makes the opening of the skull more difficult.
7. Place the pup on a heating pad (37 °C) and allow it to recover from anesthesia. Wait until the tissue adhesive has dried and solidified (approximately 30 min).
8. If necessary, apply more tissue adhesive and wait for it to dry and solidify.

3. Cranial Window Preparation

1. Anesthetize the pup using isoflurane (1.0% - 2.0%) and check the anesthesia level by a tail-pinch test.
2. Carefully open the skull with a sterilized razor blade leaving the dura intact (1 mm in diameter) (Figure 1C). Use a gelatin sponge (cut into small pieces, approximately < 2 mm³, using sterilized scissors, and apply them using tweezers) soaked in cortex buffer¹² (125 mmol/L NaCl, 5 mmol/L KCl, 10 mmol/L glucose, 10 mmol/L HEPES, 2 mmol/L CaCl₂, and 2 mmol/L MgSO₄; pH 7.4; 300 mOsm/L; room temperature) to stop the bleeding. When opening the skull, apply a cortex buffer to keep the brain surface moist.
3. Remove any buffer and blood from the dural surface using a gelatin sponge. Apply a thin layer of 1.0% low-melting-point agarose (dissolved in cortex buffer) using yellow tips. Using a heat block machine, maintain the temperature of the agarose solution at 42°C until application.
   NOTE: The draining of buffer and blood must be performed from the side of the craniotomy while taking care that the dry gelatin sponge does not come in contact with the dura. Failure to do so may damage the dura.
4. Apply a round glass coverslip (No. 1, 3 mm in diameter) onto the agarose gel layer. Remove all bubbles between the coverslip and the agarose gel layer by pouring an excess of agarose gel between them. Remove the excess gel protruding from under the coverslip using tweezers (Figure 1D and 1E).
5. Secure the coverslip using dental cement (Figure 1F).
6. Mix the cement powder and the cement liquid. Apply the mixture using yellow tips before it becomes solidified. Do not apply dental cement onto the dura, because this may damage the brain.
7. Attach a sterile titanium bar (custom made, approximately 30 mg, see Figure 1G) on the cranial bone using dental cement. Align the titanium bar and the coverslip (on the surface of the dura) in parallel to easily capture images.
8. Cover the exposed skull with dental cement (Figure 1H).
9. Recover the pup from anesthesia. Keep it on a heater (37 °C) until the dental cement has solidified (1 h).

4. Two-photon Imaging

NOTE: The in vivo images in Figure 2 were acquired using a two-photon microscope with a titanium-sapphire laser (beam diameter [1/e²]²: 1.2 mm).

1. Set the two-photon laser wavelength. For RFP excitation, use 1,000 nm (450 mW/mm² at 400 µm of depth).
   NOTE: The laser power should be reduced as the z-position moves up.
2. Wipe the surface of the coverslip with 70% ethanol.
3. Anesthetize the pup using isoflurane (1.5% - 2.0%) and check the anesthesia level using a tail-pinch test.
4. Attach the pup to the titanium plate on the imaging stage using a titanium bar on the pup's head (Figure 2A and 2B). Adjust the head such that the coverslip is parallel to the objective lens using the goniometer stage (Figure 2B). Maintain body temperature of the pup using a heating pad (37 °C).
5. Set the isoflurane concentration to 0.7% - 1.0%.
   NOTE: A very high isoflurane concentration may cause accidental death of the pup during imaging.
6. Place the imaging stage under the objective lens (20X, NA 1.0) of the two-photon microscope (Figure 2B and 2C).
7. Apply one drop of water onto the coverslip. Use epi-fluorescence to locate the fluorescent protein-labeled neurons in the area where the dura has been exposed.
8. Acquire z-stack images at 1.4-µm intervals. For layer 4 neuron imaging, set the z-width to 150 - 300 µm to image the entire dendritic morphology (Figure 2D and 2E). Use slow scanning and averaging to get clear images showing the neuronal morphology (it usually takes > 20 min to acquire the entire dendritic morphology).
   NOTE: The following parameters are recommended for imaging. Excitation wavelength: 1,000 nm, scanner: galvanometer type, dichroic mirror: 690 nm, emission filter: 575 - 620 nm bandpass, detector: GaAsP type, gain setting > 100, image size > 512 x 512 µm, field of view > 600 x 600 µm, pixel resolution < 1.2 µm.

5. Recovery and Nursing

1. Detach the pup from the imaging stage.
2. Place the pup on a heater (37 °C) and allow it to recover (15 min).
3. Feed the pup warm milk using a micropipette at 2-h intervals and gently stimulate the stomach to allow excretion. Confirm that the pup is drinking milk by measuring its body weight.

6. Re-imaging

1. Anesthetize the pup with isoflurane (1.5% - 2.0%), and check the anesthesia level using a tail-pinch test. Attach it to the imaging stage.
2. Locate the previously imaged neurons and acquire a z-stack image. The identification of neurons is easy owing to their sparse labeling with Supernova.
3. Repeat steps 5.1 - 6.2 until imaging is completed. NOTE: The pup can be imaged up to 18 h without losing weight.
   

Results

Figures 2D - 2F show representative results of two-photon time-lapse imaging of layer 4 cortical neurons using the present protocol. For the purpose of analysis, select neurons with clear dendritic morphology throughout the imaging periods. We analyzed the dendritic morphology of imaged neurons using morphological analysis software. Representative dendritic morphology reconstruction is shown in Figure 2F. Neurons showing disc...

Discussion

Critical Steps in the Protocol and Troubleshooting:

The most critical step of the protocol is the removal of the skull (Protocol step 3.2). Upon insertion, the razor blade often adheres to the dura, causing dural bleeding and damage to the brain. This can be avoided by adding a drop of cortex buffer on the skull and removing the skull in cortex buffer.

Bleeding from the dura and the skin after cranial window preparation leads to occlusion of the window. To avoid this, ...

Materials

Name	Company	Catalog Number	Comments
pK031. TRE-Cre	Authors	-	Available from RIKEN BRC and Addgene
pK029. CAG-loxP-STOP-loxP-RFP-ires-tTA-WPRE	Authors	-	Available from RIKEN BRC and Addgene
pK273. CAG-loxP-STOP-loxP-CyRFP-ires-tTA-WPRE	Authors	-	Available from authors
Isoflurane	Wako	099-06571
410 Anaesthesia Unit (isoflurane gas machine)	Univentor	8323101
Vetbond (tissue adhesive)	3M	084-1469SB
MµltiFlex Round (loading tip)	Sorenson	13810
Gelfoam (gelatin sponge)	Pfizer	09-0353-01
Agarose	Sigma	A9793	Low melting point
Round-shaped coverslip	Matsunami	-	Custom made
Unifast 2 (dental cement)	GC	-
Titanium bar	Authors	-	Custom made (see Figure 1G)
Rimadyl (carprofen)	Zoetis	-	Injectable
2-photon microscope	Zeiss	LSM7MP
Titanium-sapphire laser	Spertra-Physics	Mai-Tai eHPDS
Titanium plate	Authors	-	Custom made (see Figure 2A)
IMARIS, FilamentTracer, MeasurementPro	BITPLANE
Goniometer stage	Thorlabs	GN2/M