In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Summary

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

Abstract

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders^1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons^1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons^3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience.

One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)^5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity^3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity⁷. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors³. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated.

In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain^8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter¹⁰. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes⁴.

Protocol

The experimental procedures described below were approved by the National Institute of Mental Health Animal Care and Use Committee and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

1. Pre-operative Preparation

1. Clean all tools in a hot bead sterilizer before aseptic surgery, clean the surgery site with 70% ethanol, and put down clean drop cloths. Wear sterile gloves. Anesthetize the animal with freshly prepared 1.2% avertin solution, given at 0.02 ml/g, intraperitonially. Alternatively, anesthetize with isoflurane gas through a nose cone, 5% for induction, 1.5% for maintenance, with a passive scavenger circuit. Check the anesthesia level using tail or toe pinches to ensure full sedation.
2. Cover the animal's eyes with sterile ophthalmic ointment, and inject freshly prepared dexamethasone (0.2 mg/kg) and carprofen (5 mg/kg) subcutaneously to prevent brain swelling and inflammation¹¹. Shave the hair over the skull between the ears, and sterilize the skin with betadine scrub and three alternating swabs of 70% ethanol.
3. Mount the animal in a stereotaxic surgery stage with earbars, with a water circulation heating pad below the animal set at 37 °C. Regularly check the animal's anesthesia levels, and supplement the original anesthetic dose as needed.
4. Inject 200 μl of 0.5% Marcaine under the scalp skin to numb the area. Incise the skin and remove the skin flap over the skull. Remove the periosteum and dry the area with clean cotton swabs.

2. Chronic Cranial Window Surgery

1. Use a high-speed dental drill with a 0.5 mm burr to gently outline a 3-5 mm diameter circle over the brain region of interest. Periodically wet the drilling site with sterile 0.9% saline and clear away bone dust with clean cotton swabs. If the bone bleeds, use gelfoam presoaked with sterile saline to blot the bleed and wait for it to stop.
2. When the last bone layer is reached, lift and remove the bone island with fine-tipped forceps. Dura attachments to the bottom shelf of the bone may be encountered if the window crosses a bone suture. These should be gently removed as the bone flap is lifted.
3. Roll moistened gel foam gently over the exposed dura to clean its surface and wait for any dural bleeding to stop. Critical step: do not proceed until all dural bleeding has stopped. Blood that becomes trapped inside the cranial window usually leads to an opaque window.
4. If the region of interest is under a location where the dura is particularly thick, removing the dura above it may be necessary. If this is the case, gently separate the dura from the pia below with very fine forceps, make a small incision in the lifted dura with another pair of fine forceps, then grasp the cut edges and gently split the dura. The dura is thin but strong, so be careful not to slice the brain with the intact edges of the dura.
5. Rinse the area with sterile ACSF or sterile saline.
6. Lay a sterile glass coverslip (3-5 mm) over the dura or pia. Critical step: If the surrounding skull curves significantly, use Kwiksil¹² adhesive to fill in spaces where the dura or pia would not make close contact with the coverslip in regions that will not be imaged.
7. Use cyanoacrylate gel to cover the elastomer adhesive and the edges of the glass coverslip. Cover the entire exposed skull with cyanoacrylate gel, or a krazyglue and dental cement mixture. Critical step: Do not allow any cyanoacrylate gel to touch the brain surface.
8. Embed a custom-made metal head fixation bar at the opposite end of the skull.
9. Return the animal to a warm recovery chamber, after an intraperitonial injection of ketoprofen, 5 mg/kg, for pain management. Continue the analgesic for two more days post-operatively.
10. After two weeks of post-operative recovery, anesthetize the animal with isoflurane (5% for induction, 1.5% for maintenance), mount it in a custom-made microscope stage with a head-fixation frame, and check the cranial window for optical clarity under illumination with blue light. Brain surface blood vessel clarity is highly indicative of cranial window quality. If the blood vessel edges are sharply defined, the window is likely to be useable. The two-week post-operative recovery time is recommended to allow sufficient time for the animal to recover from surgery. Cranial windows typically clear and improve during this time, and remain optically transparent for additional weeks to months until regrowth of skull or thickening of the dura degrades window quality.

3. Behavioral Protocol and Laser Scanning Two-photon Microscopy

1. Animals with clear cranial windows will be exposed to an environmental stimulus or subjected to a behavioral training session, and then imaged afterwards at the time of maximal Arc-GFP expression. Before beginning a behavioral protocol, the animal should be kept in a consistent home cage environment to minimize day-to-day variation in baseline Arc-GFP expression levels.
2. Begin behavioral training or environmental stimulation paradigms, depending on the brain region of interest. For example, animals can be exposed to different visual environments over consecutive days, and imaged each day after visual stimulation to identify stimulus-specific responses in the visual cortex¹⁰.
3. Arc-GFP fluorescence in neurons typically reaches its peak level two hours after stimulation. The experimental timeline may be optimized to facilitate the detection of Arc-GFP expression in neurons at their peak fluorescence levels.
4. After a behavioral training session or environmental stimulation is completed, anesthetize the animal with isoflurane (5% for induction, 1.5% for maintenance) and mount the animal in the custom-made microscope stage with head-fixation frame under the two-photon microscope.
5. The animal's head position is fixed to the head-fixation frame that connects directly to the stage, using the implanted metal bar on the skull. Isoflurane and oxygen are continuously supplied to the mouse through a nose cone. Body temperature is maintained using a heating pad.
6. Ensure that the microscope detectors are protected from ambient light by conducting two-photon laser scanning in a dark room. Use a 20x or 25x (1.05 numerical aperture) water immersion lens for imaging. First, under epi-fluorescence illumination, acquire an image of the surface blood vessel patterns over the brain region of interest with a CCD camera for future image alignment.
7. Start two-photon laser scanning to acquire a 3-D image stack. An Olympus FV1000MPE multi-photon microscope is used in our setup. The excitation wave-length of the two-photon laser is set at 920 nm, and the power of the laser emitted from the objective is set at approximately 50 mW. Emitted fluorescence is simultaneously detected in green and red channels (dichroic mirror at 570 nm, barrier filters at 495-540 nm and 570-625 nm), using an external two-channel photomutiplier detection system placed close to the specimen. Arc-GFP fluorescence only appears in the green channel, whereas tissue auto-fluorescence appears in both channels^{13, 14}.
8. Typical image stacks have dimensions of approximately 320x320x100 μm (width x length x depth), a horizontal resolution of 0.5 μm/pixel, and a vertical resolution of 3 μm/pixel.
9. After acquiring the image stack, return the animal to its home cage. Do not disturb the animal until the next behavioral and imaging session. Repeat the behavior and imaging procedure across days as desired. Use the previously acquired brain surface blood vessel image to orient back to the same imaging location.

Results

This protocol describes a method to track experience-dependent molecular changes in individual cortical neurons in live animals. A chronic cranial window is first created over a cortical region of interest in a mouse carrying a fluorescent reporter of gene expression. Two-photon microscopy can then be coupled with various behavioral paradigms to observe behaviorally induced molecular changes in individual neurons and track such changes in the same sets of neurons over multiple days (Figure 1).

Discussion

The in vivo imaging method described here enables repeated examination of Arc gene expression changes in the same sets of neurons over multiple days in the live animal. It is an efficient and versatile method to obtain information about neural plasticity-related molecular dynamics in individual neurons in response to various behavioral experiences. Standard histochemical methods such as in situ hybridization and immunostaining can achieve single-cell resolution^3, but lack the ability to tra...

Materials

Name	Company	Catalog Number	Comments
FV1000 multi-photon laser scanning microscope	Olympus	FV1000MPE	Imaging
Dissection microscope	Omano	555V107	Surgery
Stereotaxis surgery stage for mice	Harvard Apparatus	726335	Surgery
20X or 25X water immersion objective	Olympus	XLPL25XWMP	Imaging
Microscope stage with head-fixation frame	Custom made	N/A	Imaging
Fine forceps	Fine Science Tools	11251-20	Surgery
Dental drill burr	Fine Science Tools	19007-05	Surgery
CCD camera	QImaging	QICAM 12-bit	Imaging