Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Summary

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Abstract

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Introduction

Proper cortical function requires a balance between excitatory projection neurons and inhibitory GABAergic interneurons, an extremely heterogeneous population with distinct morphologies, electrophysiological properties, connectivity and neurochemical markers. Abnormal development and function of interneurons (and specific interneuron subgroups) has been linked to the pathobiology of psychiatric disorders such as schizophrenia, autism and epilepsy^1,2,3. Furthermore, many genes implicated in these brain disorders are strongly enriched in young interneurons⁴

Protocol

All experimental procedures were conducted in accordance with the National Institutes of Health guidelines and were approved by the NICHD Animal Care and Use Committee (ACUC). The protocol described below utilizes Nkx2.1-Cre^C/+;Ai9^+/- pups to harvest MGE-derived interneuron precursors, but can be performed on any desired fluorescent reporter mouse line. Both male and female early postnatal mice (P0-P2) were used indiscriminately for donor and host tissue.

1. Solution Preparation

1. Prepare sucrose artificial cerebrospinal fluid (sACSF) according to the following recipe (unit in mM): 87 NaCl, 26 NaHCO<....

Results

This protocol demonstrates how to harvest specific brain regions from early postnatal brains (Figure 1-2), collect single cell dissociations of interneuron precursors, and transplant these cells into various brain regions in naive WT postnatal pups (Figure 3). For posthoc analysis, brains that received interneuron precursor grafts were harvested between P30-35 to characterize cell morphology, neurochemical marker.......

Discussion

One critical aspect of this protocol is maximizing the survivability of the cells. Ensuring that the tissue and cells are always in ice cold carboxygenated sACSF is necessary to promote cell survival. This requires an efficient dissection and dissociation strategy to minimize the length of time that the cells spend in various solution and outside of the brain environment. Depending on the number of brain regions being dissected and transplanted, it can be beneficial to have a partner aid in the dissection and/or transpla.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium chloride	Sigma	S7653
Sodium bicarbonate	Sigma	S6297
Potassium chloride	Sigma	P9541
Sodium phosphate monobasic	Sigma	S0751
Calcium chloride	Sigma	C5080
Magnesium chloride	Sigma	M2670
Glucose	Sigma	G7528
Sucrose	Sigma	S7903
Brain Matrices	Roboz	SA-2165	Only needed if harvesting striatum
Fine point Dumont Forceps	Roboz	RS-4978
Microdissecting scissors	Roboz	RS-5940
Razor blades	ThermoFisher	12-640
Pasteur pipettes	ThermoFisher	1367820C
Nanoject III	Drummond	3-000-207
Manual Manipulator w/ stand	World Precision Instruments	M3301R/M10
5 ml round bottom plastic tubes	ThermoFisher	149591A
60 mm Petri dishes	ThermoFisher	12556001
100 mm Petri dishes	ThermoFisher	12565100
Pronase	Sigma	10165921001
Fetal Bovine Serum (FBS)	ThermoFisher	16140063
DNase I	Sigma	4716728001
Celltrics 50um filters	Sysmex	04-0042327
Trypan blue	ThermoFisher	15-250-061
Hemocytometer	ThermoFisher	02-671-6