Neuroscience JoVE (Open Access) Citable · DOI

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Name: Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors
Uploaded: 2026-06-17
Duration: 7 min 51 s
Description: Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA ...

DOI: 10.3791/52115-v

What you'll learn

✓ Prepare primary embryonic mouse GABAergic medium spiny neuron cultures
✓ Establish co-cultures of neurons with HEK293 cells expressing GABAA receptors
✓ Assess inhibitory synapse formation and GABA receptor functionality in vitro

Protocol

The molecular mechanisms that co-ordinate the formation of inhibitory GABAergic synapses during ontogeny are largely unknown. To study these processes,we have developed a co-culture model system which incorporates embryonic medium spiny GABAergic neurons cultured together with stably transfected human embryonic kidney 293 (HEK293) cells expressing functional GABAA receptors.

Difficulty

advanced

Total time

~7–10 days (neuron isolation and culture maturation) plus ~24–48 hrs for co-culture establishment

Model organism

Mouse embryonic medium spiny neurons; HEK293 cells stably expressing GABAA receptors

Biosafety

BSL-1

Steps

Prepare mouse GABAergic medium spiny neuron culture

Isolate and culture embryonic medium spiny GABAergic neurons from mouse brain tissue. Establish primary neuronal cultures in appropriate growth media and allow neurons to mature in vitro.

▶ 01:22

Establish neuron-HEK293 cell co-culture

Combine matured GABAergic neurons with stably transfected HEK293 cells expressing functional GABAA receptors. Co-culture cells together to permit synapse formation and receptor-mediated interactions.

▶ 04:10

Analyze GABAA receptor-mediated synapse formation

Examine and quantify the formation of active inhibitory synaptic contacts between neurons and HEK293 cells. Assess GABAA receptor expression and functional integration at nascent synapses.

▶ 05:18

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