Project Details
Description
TASKS:
Task 1. To determine the cellular connectivity of PVN inputs to GPe neurons
The majority of neurons in the PVN are glutamatergic, including CRF neurons. To examine the synaptic connectivity between the PVN and the GPe, credependent ChR2eYFP encoding adenoassociated virus (AAV) will be injected into the PVN of CRFcre mice 3–5 week prior to ex vivo electrophysiological recordings. Identified PV+ GPe neurons and Npas1+ GPe neurons in ex vivo slices will be voltage clamped with a cesium methanesulphonate internal solution at –70 mV. PVN inputs will be optogenetically activated using blue light delivered to the GPe. The glutamatergic nature of the synaptic responses will be confirmed by their sensitivity to AMPA and NMDA receptor blockers, NBQX and CPP. AMPANMDA, pairedpulse, and kinetic properties of synaptic responses will be measured as additional biophysical signatures of the PVN input. A subset of mice will be perfused fixed to examine the expression of vesicular glutamate transporter (vGluT2) in ChR2eYFP labelled axons using confocal immunofluorescence analysis.
Task 2. To determine the cellular connectivity of CeA inputs to GPe neurons
CRF neurons in the CeA are GABAergic. To examine the synaptic connectivity between the CeA and the GPe, credependent ChR2eYFP encoding AAV will be injected into the CeA of CRFcre mice 3–5 weeks prior to ex vivo electrophysiological recordings. Identified PV+ GPe neurons and Npas1+ GPe neurons in ex vivo slices will be voltage clamped with a cesium chloride internal solution at –70 mV. CeA inputs will be optogenetically activated using blue light delivered to the GPe. The GABAergic nature of the synaptic responses will be confirmed by their sensitivity to GABAA receptor blockers, SR95531. Pairedpulse and kinetic properties of synaptic responses will be measured as additional biophysical signatures of the CeA input. A subset of mice will be perfused fixed to examine the expression of vesicular GABA transporter (vGAT) in ChR2eYFP labelled axons using confocal immunofluorescence analysis.
Task 3. To determine the effects of CRF on intrinsic properties of GPe neurons
To examine CRF release, PVN or CeA inputs will be targeted using approaches similar to those outlined in Task 1–2. In the presence of fast synaptic blockers (i.e. NBQX, CPP, and SR95531), CRF release will be optogenetically evoked using a train of light pulses at 20 Hz in ex vivo slices. The cellular impact on identified GPe neurons will be first examined by monitoring the autonomous activity of identified PV+ GPe neurons and Npas1+ GPe neurons in cellattached configuration. In addition, intrinsic excitability of PV+ GPe neurons and Npas1+ GPe neurons will be characterized using a series of current steps in wholecell current clamp configuration. The number of action potential evoked with current injections will be used as an outcome measure. Biophysical and pharmacological properties of CRFresponsive conductances will be determined in voltageclamp. Effects of exogenously applied CRF on GPe neurons will be compared to optogenetically evoked
responses. Lastly, the effects of CRFR1 antagonist (R121919) on (both endogenous and exogenous) CRFinduced changes in firing and membrane properties will be examined.
Task 4. To determine the effects of CRF on synaptic inputs to GPe neurons
To investigate if CRF effects major synaptic inputs to GPe neurons, GABAergic inputs from the dStr or the glutamatergic inputs from the STN will be examined. In ex vivo slices, dStr inputs and STN inputs will be activated using op
Status | Finished |
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Effective start/end date | 6/5/17 → 3/31/22 |
Funding
- University of Texas Health Science Center at Houston (SA0000539 // 5R01MH112768-05)
- National Institute of Mental Health (SA0000539 // 5R01MH112768-05)
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