Optogenetic Control of Hippocampal Network Dynamics

Samenvatting

Source: Manseau, F., et al. Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit. J. Vis. Exp. (2017)

This video demonstrates the method to use optogenetics to modulate hippocampal network dynamics by activating genetically modified light-sensitive interneurons in a mouse brain slice, enhancing theta oscillation synchronization and studying neural circuit behavior.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Optogenetic control in the isolated hippocampus
   NOTE: For optogenetic control of hippocampal network activity, a cell-type specific strategy involving rhythmic activation of PV (parvalbumin-positive)-containing GABAergic cells is used here as these cells were shown to play a major role in synchronizing principal cell populations in the isolated hippocampus. Selective expression of the excitatory channelrhodopsin-2 (ChR2) in PV cells is achieved by crossing the PV-Cre mouse line with mice constitutively expressing ChR2 Cre-dependently (Ai32), thereby generating PV-ChY mice. Alternatively, viral vector constructs (e.g., AAVdj-ChETA-eYFP) can be injected into the hippocampus of PV-Cre or PV-TOM mice (P15-16) to drive the expression of the excitatory opsin in CA1/SUB interneurons (Figure 1A).
   1. Place an LFP electrode in the CA1/SUB (Cornu ammonis-1/subiculum) area and patch a nearby pyramidal cell in the isolated hippocampus of a mouse expressing the blue-light sensitive excitatory opsin ChR2 in PV interneurons.
   2. Place an optic fiber light guide (0.2 - 1 mm diameter) above the hippocampal preparation and center it on the recorded region. Use blue light (473 nm) from an LED source for optogenetic stimulation, consisting of light pulses (10 - 20 ms, triggered by a transistor-transistor logic signal) or sine wave voltage commands delivered at theta frequencies (4 - 12 Hz).
   3. Switch to the current clamp and characterize the activity of the pyramidal during spontaneous theta oscillations.
   4. Start the stimulation protocol and record the light responses. Observe that field oscillations and synaptic activity in the recorded neuron become increasingly synchronized during optogenetic stimulation and that rhythmic pacing of PV cells results in robust control of both frequency and power of theta oscillations (Figure 1).

Resultaten

Figure 1: Local Field Potential and Simultaneous Patch Clamp Recordings from Single CA1/SUB Pyramidal and PV Neurons during Spontaneous and Optogenetically-driven Theta Oscillations in the Isolated Hippoca...

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Reagents
Sodium Chloride	Sigma Aldrich	S9625
Sucrose	Sigma Aldrich	S9378
Sodium Bicarbonate	Sigma Aldrich	S5761
NaH2PO4 - sodium phosphate monobasic	Sigma Aldrich	S8282
Magnesium sulfate	Sigma Aldrich	M7506
Potassium Chloride	Sigma Aldrich	P3911
D-(+)-Glucose	Sigma Aldrich	G7528
Calcium chloride dihydrate	Sigma Aldrich	C5080
Sodium Ascorbate	Sigma Aldrich	A7631-25G
Equipment
95% O2/5% CO2 gas mixture (carbogen)	Vitalaire	SG466204A	perfusion system
Glass bottles/flasks (4 x 1 L)	n.a.	n.a.	perfusion system
Submerged recording Chamber	custom design (FM)	n.a.	Commercial alternative may be used
Multiclamp 700B patch-clamp amplifier	Molecular devices	MULTICLAMP	electrophysiology
Multiclamp 700B Commander Program	Molecular devices	MULTICLAMP	electrophysiology
Digital/Analogue converter	Molecular devices	DDI440	electrophysiology
Micromanipulators (manually operated )	Siskiyou	MX130	electrophysiology (LFP)
Micromanipulators (automated)	Siskiyou	MC1000e	electrophysiology (patch)
Audio monitor	A-M Systems	Model 3300	electrophysiology
Custom-built upright fluorescence microscope	Siskiyou	n.a.	Imaging
Analogue video camera	COHU	4912-2000/0000	Imaging
Digital frame grabber with imaging software	EPIX, Inc	PIXCI-SV7	Imaging
Olympus 2.5x objective	Olympus	MPLFLN	Imaging
Olympus 40x water immersion objective	Olympus	UIS2 LUMPLFLN	Imaging
Custom-made light-emitting diode (LED) system	custom	n.a.	optogenetic stimulation (Amhilon et al., 2015)
Animals
PV::Cre (KI) mice	Jackson Laboratory	stock number 008069	Allow Cre-directed gene expression in PV interneurons
Constitutive-conditional Ai9 mice (R26-lox-stop-lox-tdTomato (KI))	Jackson Laboratory	stock number 007905	Express TdTomato following Cre-mediated recombination
Ai32 mice (R26-lox-stop-lox-ChR2(H134R)-EYFP	Jackson Laboratory	stock number 012569	Express the improved channelrhodopsin-2/EYFP fusion protein following exposure to Cre recombinase
PVChY mice	In house breeding	n.a.	Offspring obtained from cross-breeding the PV-Cre line with Ai32 mice (R26-lox-stop-lox-ChR2(H134R)-EYFP