In Journal Club #5, Mac Hooks takes us through some recent results from Carl Petersen's lab, on the membrane potential dynamics of GABAergic neurons in the barrel cortex of behaving mice.

1. Membrane potential dynamics of GABAergic neurons in barrel cortex of behaving mice Gentet et al. (Petersen lab) Neuron 65: 422 (2010) Mac Hooks Journal Club #5

2. Methods GABAergic input suppresses AP firing in L2/3 pyramids All whiskers except C2 trimmed Awake head restrained GAD67-GFP knockin mouse; habituated to restraint Location of C2 barrel via intrinsic optical imaging In vivo 2p imaging of GFP+ GABAergic neurons Whole cell recording (K-gluconate; Alexa 594; biocytin) Recordings L2/3 (about 180-220µm deep) Sac after recording for anatomy

3. GABAergic input suppresses AP firing in L2/3 pyramids Slow, large amplitude Vm fluctuations Note AP frequency shift Quantification of AP frequency shift Mode shift to “bursting” (2 or more APs) in gabazine “ Quiet wakefulness” This shows that GABAergic transmission is capable of modulating firing, but not which circuits are engaged (note later that AP firing in excitatory is not affected by shift from quiet to active wakefulness)

4. GAD67-GFP knockin mouse Tamamaki et al. J Comp Neurol (2003) 467:60 Two lines: GAD67-GFP GAD67-GFP( Δ neo): mated to CAG Cre mouse to eliminate loxP-flanked PGK-neo cassette (in case it affects expression) (Almost) all GFP+ neurons are GABAergic Figure S1 from Petersen (left) Tamamaki (below ,neocortex) )gives GFP+ with GAD67 (156/196) and no GFP+/GAD- neurons

5. GAD67-GFP knockin mouse: Interneuron Subtypes (Motor cortex) Characterization of Interneuron Subtypes in Frontal/Motor Cortex All GFP+ cells are NeuN+ (about 19.5% of cortical neurons) L1/2 L2/3 L2/3 L5/6

6. GAD67-GFP knockin mouse: Interneuron Subtypes (S1 cortex) Figure S5 Tamamaki et al. marked for comparison

7. Whole-cell recordings of GABAergic neurons Classifying 3 cell types GFP+ GFP+ Ok. Should we ask for a more fine division of interneuron population? AP half width AP frequency Resting Vm Input resistance

8. Classifying 3 cell types, Quantified Whole-cell recordings of GABAergic neurons Fig S3 also adds more data ….

9. Behavioral modulation of Vm dynamics in GABAergic neurons Examples Tracking whisker angle (video; 20s limit) Vm (aligned) Expanded to show subthreshold Quantify: AP frequency Vm Variance of Vm 1-5Hz area (V dot Hz) of Vm fourier transform Divide time into (Q) and (W)

10. Behavioral modulation of Vm dynamics in GABAergic neurons 3 cell types, quantified

11. Behavioral modulation of Vm dynamics in GABAergic neurons Fast (subthreshold) membrane potential oscillations phase locked to whisking Whisker angle Average Vm (aligned to peak of protraction) Peak amplitude of fast (subthreshold) membrane potential oscillations, plotted at the time in the whisking cycle where it occurs NO DIFFERENCES BETWEEN CELL TYPES

12. Correlated activity of excitatory and inhibitory neurons during (Q) quiet wakefulness Two excitatory neurons (Dual in vivo patch: distance averaged 140±19 μ m; in a 300 μ m diameter barrel, this could put one in the middle and the other at the edge) Cross correlogram Essentially examining subthreshold correlation Does whisker trimming affect correlation across cortex?

13. Correlated activity of excitatory and inhibitory neurons during (Q) quiet wakefulness Highly synchronous slow oscillations Interneurons then can’t drive the hyperpolarized phase in pyramids …

14. Behavioral modulation of correlated activity

15. Behavioral modulation of correlated activity Why not subdivided for FS/non-FS?

16. Large brief specific events Action potentials in excitatory neurons are driven by large, brief, cell-specific depolarization (e.g. events in excitatory neurons are not correlated) Shuffled events Histogram of relative spike times (10ms bins) Data Essentially examining suprathreshold correlation

17. Action potentials in inhibitory neurons are driven by broadly synchronized depolarization Synchronized depolarizations

18. Summary of Vm prior to APs Spike triggered averages for … Spikes triggered in excitatory neurons include events not present in … … excitatory, or … FS interneuron Whereas these events are more similar (e.g. broad depolarizations) Vm slope in 20ms prior to spike initiation Is there any way this could be some artifact of the cell type?

19. A Model for Behavioral Modulation of Vm Dynamics

21. Feedforward inhibition engaged by thalamocortical input Cruikshank et al. (2010) Neuron 65:230 Connors’ data for VB feedforward to L4 is similar to what Petersen proposes for L3: -general principle of feedforward from layer to layer? -would POm input be more similar to ‘active’ wakefulness, while VB is more similar to ‘quiet’ wakefulness?