Patch and Puff: Target discovery in fresh brain sections

The “Patch and Puff” technique is a valuable method for studying neuronal excitability and synaptic function. Dr. Melissa Chee uses this approach—combining patch-clamp recording with localized drug application—to investigate neurotransmitter signaling in acute brain slices. Her work helps identify druggable targets in complex brain regions, especially within subcortical circuits.

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1. Why use this method?

The “Patch and Puff” technique is a helpful approach for studying neuronal excitability and synaptic function in response to drug application in acute brain slices. This method integrates patch-clamp electrophysiology with localized drug application (puffing) to assess the functional properties of neurons in real-time. By using fresh brain slices, researchers can preserve native cellular and synaptic physiology, allowing for high-resolution investigations of neurotransmitter receptor function, ion channel dynamics, and neuromodulatory signaling. This technique is useful for identifying druggable targets wihin heterogeneous cell populations, for example in subcortical structures.

2. What you’ll need

• Solutions and buffers
• Brain slicing equipment: vibratome for preparing acute brain slices (250–300 µm thick), carbogen taken to keep brain slices oxygenated, crushed ice to keep cutting solutions cold
• Upright microscope outfitted with: 10x and 40x objectives to visualize individual cells; fixed stage comprising a recording chamber to hold brain tissue; fluorescent imaging system to visualize pre-labeled cells, if applicable
• Patch-clamp electrophysiology instruments: micropipettes, amplifier, micromanipulators, signal acquisition software
• Puffing drug application system: micropipettes, tubing, pneumatic or pressure-based microejection system for local delivery of drugs

3. Step-by-step instructions

1. Prepare fresh brain slices. Extract rodent brains and rapidly section brain sections in ice-cold, oxygenated cutting solution using a vibratome. Maintain slice thickness at 250–300 µm to ensure optimal neuronal viability.
2. Transfer slices to recording chamber. Allow slices to recover in aCSF before beginning electrophysiological recordings
3. Prepare patch-clamp recording pipettes. Pull a glass micropipette then backfill it with intracellular solution to a resistance of 6–8 MΩ for whole-cell recordings.
4. Perform patch-clamp recordings. Visualize and identify healthy cells in your brain region of interest using infrared differential interference contrast (IR-DIC) or fluorescence microscopy. Use whole-cell or perforated patch recordings to assess membrane properties and synaptic currents.
5. Position the puff pipette. Prepare a second micropipette for drug application and place it about 50 µm away from the recorded cell to ensure localized delivery.
6. Data acquisition and analysis. Begin recording electrophysiological responses (e.g., excitatory/inhibitory post-synaptic currents, action potential firing) and analyze effects of puffed compounds on neuronal activity
7. Puff application of pharmacological agents. Deliver a controlled puff of desired pharmacological agent to measure receptor-specific responses. The puff is delivered by applying a very brief, less than two seconds, air pressure. Ensure minimal disruption to the surrounding environment.

4. Practical tips

• Optimize drug delivery by adjusting micropipette positioning and ejection pressure to ensure localized and reproducible drug exposure
• Use intracellular dyes to confirm cell identity and ensure accurate recordings
• Maintain consistent aCSF perfusion to avoid tissue degradation and ensure stable baseline recordings
• Check seal and access resistance throughout whole-cell recordings to maintain high-quality data acquisition

Pros

• Slice consistency
• Surveying multiple cells from the same slice
• Requires less animals
• Short(er) application or recording timeline
• Highly accessible and affordable to delivery drugs by puff application

Cons

• Drug may spread to immediately neighboring cells
• Puff may disrupt patch-clamp recording
• Cannot assess full receptor kinetics

5. Critical appraisal & implications for future research

• Drug testing at multiple cell types
• Pharmacology experiments possible
• Desensitization minimized
• Higher throughput screening

The “Patch and Puff” method enables precise dissection of receptor-mediated signaling in neural circuits governing metabolic and behavioral states but can be easily adapted to other research questions. Future research could integrate single-cell transcriptomics with patch-clamp profiling to link functional electrophysiological phenotypes with gene expression signatures. Additionally, coupling this technique with optogenetics and two-photon imaging will further elucidate how defined neuronal subpopulations differentially respond to neurotransmitter vs neuropeptide release. The development of high-throughput electrophysiological platforms could enhance drug discovery efforts targeting metabolic disorders and neurological diseases.

This protocol is licensed under a Creative Commons Attribution-NonCommercial (CC BY-NC) license, allowing sharing and adaptation for non-commercial purposes with proper attribution.