Imaging Mitochondrial Ca²⁺ Uptake in Astrocytes and Neurons using Genetically Encoded Ca²⁺ Indicators (GECIs)

Demonstrating the procedure will be graduate student Zhe Zhang, research specialist Nannan Zhang, professors Illker Ozden and Shinghua Ding. The overall goal of this procedure is to develop cell type specific pair astrocytes and the neurons and the mitochondrial targeting method for in vitro and in vivo mitochondrial counts of imaging using genetically encoded calcium indicator, GCaMP5G and 6s. For in vitro or in vivo imaging, we will construct DNA plasmids containing astrocyte or neurons specific promoters gfaABC1D or CaMKII and genetically encoded calcium indicators, GCaMP5G/6s with mitochondrial targeting sequences.

For in vitro mitochondrial calcium imaging we transfected astrocytes neurons cultured on glass cover slips with the aforementioned DNA plasmids using the Lipofectamine method. Imaging can be started one to two days after transfection by transferring the glass cover slips to the profusion chamber under an epifluorescence or two-photon microscope. Here are examples of fluorescent signals from mitochondria and an astrocyte expressing GCaMP6s on the left, and for mitochondria in a neuron, expressing GCaMP5G on the right.

For in vivo imaging, we prepared serotype 5 associated adenovirus for expression of mito-GCaMP5G in astrocytes and serotype 9 associated adenovirus for expression of mito-GCaMP6s in neurons. For stereotaxic virus injection surgeries, we first anesthetized the mouse with 3%isoflurane. Later during the surgery, the isoflurane levels is reduced to 2%After the proper level of anesthesia is reached, the hair over the scalp is shaved and the mouse is positioned on a stereotax surgical instrument.

An ophthalmic ointment is applied to the eyes to protect them during the surgery. The injection surgeries are performed by using aseptic procedures, including sterile instruments and aseptic techniques. The surgical instruments are sterilized either by autoclaving or by a hot bead sterilizer.

After the mouse is mounted on the stereotax, the scalp is cleaned with betadine and 70%ethanol three times. Then the skin is cut open along the Bregma Lambda axis and the vertical is made by a high-speed drill over the target location. In this work, we targeted either the motor cortex or the paracortex.

A 33 gauge Hamilton syringe carrying an associated adenovirus vector is inserted through the hole into the brain and vectors are injected at the target area. For cortical delivery, we inject the virus solution at two depths in multiple steps. First, we insert the needle one millimeter deep into the cortex.

After allowing five minutes for the brain to recover, we move the needle up to 700 micrometers deep, inject 500 nano liters of virus solution. After injection is completed, we wait for five minutes to allow the virus to diffuse in the brain. And then we move the needle up to the second injection location at 300 micrometers deep.

Here, we inject another 500 nano liters of virus solution. After injection is completed, we wait for 10 minutes to allow the virus to diffuse and then we remove the needle from the brain and close the burr hole with bone wax or Kwik-Sil. Finally, the skin is sutured with Vetbond and the mouse is recovered on a heating pad before being sent back to the animal facility.

The mouse will be ready for use for in vivo imaging in a minimum of three weeks, which is the timeframe in which GCaMP expression matures. Cranial window implantation surgeries are also done by using aseptic conditions as described for injection surgeries. The cranial window surgical procedures are identical to the injection surgical procedures until the point of skull exposure.

Once the skull is exposed, quarters of a 2-3 mm diameter craniotomy over the virus injected area are marked with four shallow holes made by a high-speed drill attached to a manipulator. Then the section of the skull, at the gorges of the craniotomy are thinned by carefully and slowly drilling the bone around in a circle, connecting these four holes. Once the skull at the borders is thin enough, then the bone is lifted with sharp tweezers and removed.

The exposed dura mater can be removed or kept intact for implantation of the cranial window. Our cranial window consists of a glass cover slip of 3-5 millimeters in diameter, creating a silicone disc about 300 microns thick at the center. The mounting of the cranial window is accomplished by placing it over the craniotomy.

A piece of toothpick attached to a manipulator is used to push the cranial window gently onto the surface of the brain. Then, the edge of the cranial window is sealed with a small amount of silicone adhesive Kwik-Sil. Finally, the cranial window is fixed in the skull edges with dental acrylic.

Care should be given for applying the dental acrylic slightly over the edges of the cranial window for strong bonding. As many other research groups have implemented in the past, a 1.2%agarose gel can be used between the cover glass and the brain as an alternative to the silicone disc. After the cranial window is securely installed, a metal plate is attached to the skull.

This head plate will be used for fixing the head of the mouse on the stage of a two-photon microscope during imaging sessions. Mitochondrial calcium uptake in astrocytes can be elicited by ATD application while mitochondrial calcium uptake in neurons can be elicited by glutamate glycine application in artificial cerebral spinal fluid, with gravity through a perfusion system. Calcium uptake in individual mitochondria in astrocytes and neurons can then be observed.

The following movies show mitochondrial calcium uptake in an astrocyte elicited by ATP on the left and in a neuron elicited by glutamate and glycine on the right. The imaging is done by collecting time-lapsed in vivo two-photon images of mitochondrial fluorescent signals in astrocytes and neurons with an Ultima two-photon microscopy system from Prairie Technologies, which is equipped with a million Ti Sapphire Ultra one laser from Coherent. We use the excitation wavelengths of 880 to 910 nanometers, which are optimal for in vivo imaging with GCaMP5G and 6s.

Astrocytes and neuron-specific expression of mito-GCaMP5G and 6s and reconfirmed by co-localization of astrocyte-specific labeling of SR101 or neuron specific marker NeuN. Spontaneous mitochondrial calcium uptake in astrocytes and neurons in vivo can be observed by time-lapse imaging using two-photon microscopy. Images show spontaneous calcium increases in individual mitochondria in an astrocyte on the left and a neuron on the right.

Our approaches are useful for cell type specific mitochondrial calcium imaging in vitro and in vivo. After watching this video, you should have a good understanding of how to image mitochondrial calcium uptake in astrocytes and in neurons.