Name: homochronic transplantation of interneuron precursors into early postnatal mouse brains
Uploaded: 2018-06-08T14:30:00
Description: Watch this Scientific Journal Video about Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains at JoVE.com

This research was supported by the National Institutes of Health (K99MH104595) and the NICHD intramural research program to T.J.P. We thank Gord Fishell, in whose lab this approach was originally established.

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-CreC/+;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. Soluti...

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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...

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 epilepsy1,2,3. Furthermore, many genes implicated in these brain disorders are strongly enriched in young interneurons4. Thus, a greater understanding of the mechanisms that regulate interneuron fate determination and maturation is needed to understand normal development and potential etiologies of numerous brain diseases.
Forebrain interneurons are born primarily from two transient embryonic structures, the medial and caudal ganglionic eminences (MGE and CGE, respectively). These postmitotic cells (interneuron precursors) then undergo a protracted tangential migration phase to disperse throughout the telencephalon where they integrate into a wide variety of circuits. MGE-derived interneurons consist of three largely non-overlapping, neurochemically defined subgroups: fast spiking parvalbumin (PV+) interneurons, non-fast spiking somatostatin (SST+) interneurons, and late spiking neuronal nitric oxide synthase (nNOS+) interneurons that constitute hippocampal neurogliaform and ivy cells. Numerous labs have identified several mechanisms within the MGE that regulate initial fate decisions into PV+ or SST+ interneurons, including spatial gradients of morphogens, birthdate of interneuron precursors, and the mode of neurogenic division5,6,7,8,9,10. It has been proposed that interneurons initially differentiate into &#39;cardinal classes&#39; and then progressively mature into &#39;definitive classes&#39; as they interact with their environment11. Recent evidence indicates that some mature interneuron subtypes may be genetically hardwired as these cells become postmitotic in the ganglionic eminences, indicating that early defined intrinsic genetic programs may play a larger role than previously appreciated12,13. However, the key question of how the intrinsic genetic programs interact with environmental cues to drive differentiation into distinct interneuron subtypes remains largely unexplored.
Numerous studies have transplanted embryonic MGE cells directly into a variety of brain regions, with the consensus results that grafted cells mature and release GABA to generally inhibit the local endogenous circuitry14,15,16,17,18,19. These promising observations have generated significant interest in using human induced pluripotent stem cell (hIPSC)-derived interneurons to treat a variety of brain diseases. However, very few of these studies assess if these grafted cells mature into the expected types of mature interneurons, a critical component when one thinks about translational approaches.
To address how environment influences interneuron differentiation and maturation, a strategy was devised to transplant immature interneuron precursors into new brain environments in order to examine whether grafted interneurons adopt features of the host environment or retain features from the donor environment20. MGE transplants are not suitable to address this question because the MGE contains a mixed population of interneuron and GABAergic projection cells that disperse throughout numerous brain regions21. Without knowing where these MGE cells would have migrated, one can not fully assess how these transplantations are affected by the brain environment. By harvesting interneuron precursors at early postnatal timepoints, this problem is circumvented by obtaining immature cells that have completed their migration and reached their target brain region but have minimal interaction with the environment. By focusing on specific features of interneurons that are differentially expressed between distinct brain regions, one can then determine how the host environment changes interneuron properties. The general approach outlined in this protocol should be applicable to any investigator that wants to examine how young neurons behave when challenged in a new environment.

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			<td style="width:136px;">C5080</td>
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			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">M2670</td>
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			<td style="width:136px;">SA-2165</td>
			<td>Only needed if harvesting striatum</td>
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			<td style="width:136px;">1367820C</td>
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			<td height="21" style="height:21px;width:247px;">5 ml round bottom plastic tubes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">149591A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">60 mm Petri dishes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">12556001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">100 mm Petri dishes</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">12565100</td>
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			<td height="21" style="height:21px;width:247px;">Pronase</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">10165921001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:203px;">ThermoFisher</td>
			<td style="width:136px;">16140063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">DNase I</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:136px;">4716728001</td>
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			<td height="21" style="height:21px;width:247px;">Trypan blue</td>
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			<td height="21" style="height:21px;width:247px;">Hemocytometer</td>
			<td style="width:203px;">ThermoFisher</td>
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homochronic transplantation of interneuron precursors into early postnatal mouse brains

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.

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...

Watch this Scientific Journal Video about Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains at JoVE.com

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

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.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

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