Preparation of Acute Subventricular Zone Slices for Calcium Imaging

Summary

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Abstract

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.

Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.

Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

Protocol

1. Preparation of Solutions, Dissection, and Vibratome

1. Solutions must be prepared at the correct osmolarity and pH (Table 1). Compared to artificial cerebrospinal fluid (ACSF), dissection solution is prepared with lower concentrations of sodium and calcium, and higher concentrations of magnesium. This is to minimize excitotoxicity effects during slicing.
2. Both dissection and ACSF solutions must be saturated with 95% O₂/5% CO₂ by bubbling for at least 10 min to reach the desired pH of 7.3.
3. Prepare an ice bath in a tray. Place a dissection plate in the ice bath and fill with dissection solution. B....

Discussion

Calcium imaging of SVZ cells has been used to study patterns in spontaneous activity in neuroblasts¹⁰, receptor channel expression in both neuroblasts and astrocytes^4,6,8 and astrocytic calcium waves³. Since cells in the SVZ are either immature or have glial properties, they do not fire action potentials^11,12, meaning that millisecond changes in voltage potential indicative of activity in mature networks is not applicable in this region. Therefore, capturing the slower calcium .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sucrose	Sigma	S0389	Dissection: 147 mM ACSF: 0 mM
NaCl	Sigma	S9888	Dissection: 42 mM ACSF: 126 mM
KCl	Sigma	P3911	Dissection: 2.5 mM ACSF: 2.5 mM
MgCl2.6H2O	Sigma	M9272	Dissection: 4.33 mM ACSF: 1 mM
NaH2PO4.H2O	Sigma	S8282	Dissection: 1.25 mM ACSF: 1.25 mM
Glucose	Sigma	G8270	Dissection: 10 mM ACSF: 10 mM
NaHCO3	Sigma	S6014	Dissection: 26 mM ACSF: 26 mM
CaCl2.2H2O	Sigma	C3306	Dissection: 1.33 mM ACSF: 2 mM
Table 1. Chemical list and recipes of dissection solution and ACSF.
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Vibratome	Leica	VT 1000S
Super Glue	Surehold or 3M	Surehold 3G Super Glue or 3M Vet-Bond
Dissection tools	Roboz or Ted Pella
Fluo-4 AM calcium-sensitive dye	Invitrogen	F14201
Oregon Green BAPTA-1 AM calcium-sensitive dye	Invitrogen	O6807
Pluronic F-127 20% solution in DMSO	Invitrogen	P3000MP
Upright confocal microscope	Olympus	FV300 or FV1000
Water-immersion objectives	Olympus	LUMPlanFl 40 x W/IR (NA 0.80); LUMPlanFl 60 x W/I (NA 0.90)
Micromanipulators	Sutter	MPC-200/MPC-325/MPC-385
Pressure controller	Parker Hannifin	Picospritzer	<3 PSI during application
Pipette puller	Sutter or Narshige	Sutter P-97 or Narshige PP-830
Glass pipettes	Sutter	BF150-110-10	I.D.:1.10, O.D.: 1.50
Peristaltic pump	Harvard Apparatus	Model 720	flow rate: 1 ml/min
Chamber bath	Warner Instruments	RC-26 GLP	Low profile allows for objective clearance
Tubing	Tygon
Temperature Controller	Warner Instruments	TC-324B/344B
Table 2. Materials/equipment list.