1. Techniques in Electrophysiology What you are expected to gain from this lecture: 1. Approaches 2. In-vivo vs. in-vitro preparations 3. Advantages & Pitfalls 4. Types of Measures

2. 5 Common Ephys Approaches: 1.EEG 2.Extracellular/Local Field Potentials 3.Intracellular – Sharp Electrode 4.Patch-Clamp Configurations 5.Multi-Unit Array Recordings

3. EEGs Recording spontaneous brain (voltage volume conductance) activity from the scalp, described in rhythmic activity: Delta (<4 Hz), theta (4-7 Hz), gamma (30- 100 Hz) Clinical Neuroscience: epilepsy, coma, tumors, stroke, focal brain damage, depth of anesthesia Coordinate cortical activity = high contribution Deep structure activity = low contribution Application to Cognitive Psychology: Evoked Potentials: time lock of EEG to presentation of stimuli Event Related Potentials: average of EEG over many trials of higher processing conditions (e.g., memory, attention) N1 or P3 = coma recovery

4. Typical Slice/Culture Ephys Rig

5. Patch-Clamp Electrophysiology Apply positive pressure (2-6 MΩ) Clear tissue as you move down Near cell membrane > ‘bubble’ Apply negative pressure > suction until 1 GΩ seal

6. 4 Common Patch-Clamp Configurations Cell-Attached Inside-Out Outside-Out Whole-Cell Suction Pull Quickly Pull Slowly >1 GΩ seal – going ‘whole-cell’ does not compromise the seal: prevents leak current & extracellular buffer from entering the neuron Binding Site?

7. Perforated Patch Recording Back-filling – nystatin, gramicidin, or amphotericin B (antibiotic/antifungal) – creates pores for select ions to pass Pros: Prevent dialysis of the intracellular contents & current run-down, used for hard to patch cells Cons: slow, high access resistance, weak membrane which leads to whole-cell configuration start ~10-15 min ~20-30 min

8. Voltage Clamp: holding the cell at a predetermined value (e.g., -70 mV) the amount of current (e.g., mA) required to maintain that value is recorded voltage-dependent K + channels, spontaneous EPSCs Cons: Space Clamp (i.e., inability to adequately maintain holding command in distal dendrites) & washout of cytosolic factors in whole-cell Current Clamp: can be used to measure the ‘resting membrane potential’ current is injected into the cell to maintain a predetermined membrane potential (e.g., -80 mV) the injected current is constant and free fluctuations in the membrane potential are recorded AP waveform, plasticity of EPSPs, intrinsic excitability Voltage vs. Current Clamp sEPSC Somatic current injection producing AP firing

9. A. B. Stimulation Local Field Potentials - fEPSPs SA = stimulus artifact * = presynaptic fiber volley – presynaptic activity generated by stimulation fEPSP = field excitatory postsynaptic potential PS = somatic population spike – coordinated spiking activity The initial slope of the fEPSP (mV/ ms) in the s.r. is a widely used measure in LTP studies SA * fEPSP PS A. B.

10. Intracellular/Sharp Recording Intracellular recording – used ‘sharp’ glass electrodes with > 25 MΩ resistance (#1) records the change in membrane potential that the incoming current causes (#2) fEPSP without a clear presynaptic fiber volley

11. Single Channel Recordings Cell-attached (CA), inside-out (IO), and outside-out (OO) patches Patch typically contains one or a few channels Measure channel open probability, open time at different voltages or in the presence of a test compound CA: stable (>20GΩ seal), low- background but less control over holding potential IO: access to intracellular sites & signaling pathways, difficult to obtain, must replace bath solution from external to internal OO: repetitive & different doses, but less stable, disruption of cytoskeleton

12. Preparations 1. Acute slices 2. Organotypic cultures 3. Dissociated cultures 4. Cell Lines 5. In vivo

13. Acute Slices Widely used technique Usually from adolescent rodents, coronal sections Used the day they are made Best to do cardiac perfusion to maintain slice viability Buffer must be oxygenated and at the correct pH/osmolarity Pros: treatments can be done in vivo, numerous brain regions can be prepared, slices are not too excitable, can combine ephys with confocal imaging, versatile (voltage or current clamp, fields, intracellular, plasticity, etc) Cons: difficult to get viable slices in adult rodents, confound of recordings in adolescents …translatation to adults, afferents are severed, there are changes in instrinsic excitability over the day of recording, bath application of drugs

14. Organotypic Slice Cultures Helios Gene Gun – can be used to load gold particles coated with cDNA into cells on the day of culturing to change protein expression Multiple brain regions (hpc, co-cultures) grown on porous membrane inserts Prepared from 2-8 day old rodent pups Maintained for months

15. Dissociated Cultures Autaptic/Microisland Cultured Primary Dissociated Neurons Acutely Dissociated Neurons - the neurons preserve their dendritic structure proximal to the soma, maintain intact synaptic boutons, and are largely devoid of glial ensheathments. Typically prepared in low- or high-density from embryonic or <24 h old pups Hippocampal, Cortical, Striatal cultures are common

16. Pros: Self-cleaning after insult during preparation, highly controllable experimental conditions, ease & success of growing & maintaining, can be used almost anytime, gene gun & lentiviral expression is easy, combine with imaging, focal drug application & whole-cell currents in dissociated neurons, glutamate uncaging/calcium transients in dendritic spines (dissociated neurons), versatile (current & voltage clamp, fEPSPs, etc) Cons: Thin over time, loss of afferents (except hpc), developmental differences, contamination, highly excitable (transections), dissociated neurons don’t have intrinsic networks or glial cells, de novo expression of excitatory connections Cultures

17. Cell Lines HEK 293 Cells Xenopus oocytes PC-12 Adrenal Cells Pros: excellent for answering certain ?’s Express select proteins Point mutation studies Model system for neuronal differentiation Cons: Non-mammalian, non-CNS cells Lack complete neuronal constituents (e.g., signaling complexes)

18. Performed under anesthesia or in freely-moving rodents In Vivo Recordings Intra- & extra-cellular, whole-cell, single or multi-unit array recordings Network Properties: Can stimulate in one region and record in another (e.g., mPFC influence on NAc plasticity) Phase locking to brain rhythms (e.g., mPFC neurons & hippocampal theta)

19. In Vivo Recordings Lee et al., 2006, Neuron, v51, p399

20. In Vivo Recordings

21. Multi-unit Array Recordings Pros: recording from an in vivo situation, network activity, population & single cell activity, phase locking of gamma & theta rhythms, correlation of neuronal or network activity with ongoing behavior, becoming more common Cons: Technically difficult, confound of anesthesia, application of mathematics to isolate data, probes are time-consuming to fabricate

22. Data, data, data AP: waveform, peak, half-width, AHP, frequency, back-propagating AP Subthreshold excitatory postsynaptic potentials: LTP, LTD Current-Voltage relationships: Mg unblock of NMDA receptors, shifts in voltage activation & inactivation curves Paired-pulse facilitation: second event that follows is up to 5X as large due to increased probability of presynaptic vesicle release miniEPSPs – recorded in presence of TTX: changes in amplitude: postsynaptic event changes in frequency: presynaptic release

23. Spike Sorting – used in multi-array recording to assign spikes to different neurons based on their spike properties Pharmacological & Electrical Isolation of distinct currents Data, data, data