1. William Buchser Neuroscience Journal Club April 28 th 2004 Timing of neural responses in cortical organotypic slices

2. Background Timing Events which occur with timing greater than a few seconds is probably grasped by out frontal cortex – a component of complex thought processing. Timing in the shorter range (less than a second or two) is thought of as automatic – the brain has a system that deals with it unconsciously. “The neural representation of time.” Ivry RB, Spencer RM

3. Background “The neural representation of time.” Ivry RB, Spencer RM Ordinal Metric Parkinson’s Disease LR W O R D Duration Discrimination

4. Timing Examination LR LR 400 ms600 ms200 ms300 ms http://www.neurobio.ucla.edu/~dbuono/InterThr.htm

5. Background “The neural representation of time.” Ivry RB, Spencer RM Our temporal resolution in the auditory system is much higher than in other sensory systems Timing has been studied using psychological, behavioral, and neuro-imaging approaches. Few in vitro studies exist  this paper! Can we observe a physiological ‘memory trace’ in brain slices?

6. This paper uses organotypic slices to try determine if reliably timed ‘late’ responses can be observed in vitro, and what mechanisms lead to their existence.

7. http://caddlab.rad.unc.edu Methods - Adult Sprague Dawley Rats - Remove Brain and make slices - Use coronal slice, 350- 400μm thick slices containing auditory cortex

8. Organotypic Slices Millipore: Millicell®-CM Coronal slices are placed on special membranes used for culture, they are bathed constantly in artificial cerebrospinal fluid.

9. Recording Paradigm LateralMedial Stimulating Electrode Recording Pipette Ventral Dorsal A single pulse was applied every 15-30 s to elicit synaptic responses. Stimulation intensity ranged from 30 to 100 µAmpere. Slice from: Redefining the tonotopic core of rat auditory cortex: Physiological evidence for a posterior field Neot N. Doron, Joseph E. Ledoux, Malcolm N. Semple

10. Horizontal Voltage Sweeps

11. Timing of the Late Response Average response of 60 trials below -80mV -50mV Stimulus Figure 1A

12. -80mV -30mV Time (min) Action Potentials Timing of the Late Response Figure 1B

13. Timing of the Late Response We now know what the ‘late’ response looks like. A monosynaptic PSP is observed within the first few milliseconds after stimulus. After more time (between 50 and 400 ms) more EPSPs occur (probably polysynaptic in nature), and are sometimes integrated into action potentials (all of this is the ‘late’ response). Summary - Figure 1

14. What does mono and polysynaptic mean? Stimulus

15. Latency vs. Accuracy What is the latency and accuracy of the timed responses? By examining the mean and the SD for the latency of the first spike, we can make some conclusions about relationships therein. Preface - Figure 2

16. Mean latency of the first spike versus the SD Correlation r = 0.94 Figure 2 High Accuracy High Variability Short Latency Long Latency

17. Latency vs. Standard Deviation Strong correlation between the latency and the SD (accuracy) of firing. Summary - Figure 2

18. Electrode Distance and First Spike Latency Does the distance from the stimulating electrode correlate to the observed latency? Others have made the observation that with a monosynaptic connection, distance is highly correlated with latency. Polysynaptic connections on the other hand, may or may not be correlated with spatial relationships. Preface - Figure 3

19. Recording Paradigm LateralMedial Stimulating Electrodes Recording Pipette Ventral Dorsal 650µm 1200µm Figure 3

20. Figure 3B A cell that fires sooner to more distant stimulation

21. Distance vs. Mono and Poly Response Latency Figure 3A

22. Electrode Distance and First Spike Latency Distance from stimulating electrode affects the latency of the sub-threshold monosynaptic response. Distance from the electrode has no simple relationship with the latency in neuron firing (polysynaptic integration leading to response) Summary - Figure 3

23. The Network nature of the Late Response The complex nature of the ‘late’ response of a single cell implies polysynaptic network input. To test this, we will determine the extent that the late response is reliant on NMDA receptors. We will use the NMDA blocker APV to determine if NMDA receptors play a role in the late response. Preface - Figure 4

24. Figure 4A -80mV -10mV Late responses are dependent on NMDA receptors

25. Network nature of the Late Response NMDA receptors are indicated in the late response due to an abolition of polysynaptic potential during APV’s application Therefore, neurons are likely to get excitatory polysynaptic input which needs to be integrated before a supra-threshold response occurs. Summary - Figure 4

26. Paired Recordings To determine whether different cells exhibit similar responses to the same stimulus, we recorded simultaneously from two neurons. Preface - Figure 5

27. Recording Paradigm LateralMedial Stimulating Electrode Recording Pipettes Ventral Dorsal 750µm 50µm Figure 5

28. Figure 5A Different cells respond to same stimulus Time (min) 50350 750µm Stimulus

29. Figure 5A Average sub-threshold response of two cells 750µm AP Red cell firing elicited an EPSP in blue cell Stimulus

30. Figure 5C Two cells compared by correlating their sub-threshold output. Mean = 0.84

31. Figure 5 demonstrates that two different neurons have different responses to the same stimulus Although the action potentials are different, a lot of the subthreshold response is similar between them, reflecting shared input from the network. Regardless of shared inputs, there is significant difference such to produce different supra- threshold responses. Summary - Figure 5 Paired recordings and shared input

32. Figure 6A Cross-Correlation between two neurons

33. Figure 6B First-spike latency between neurons that fire at different intervals is correlated.

34. Other correlations of paired recordings Summary - Figure 6 The scatter plot indicates that there is a strong correlation between the first spike in the red and green neuron Activity in the green cell is contributing to the firing of the red cell.

35. To find if hard-wired ‘delay lines’ exist in the network, we can vary different stimulus parameters and see if the latency changes. For the final experiment, they test whether intensity has an effect on the latency of the late response Preface - Figure 7 Not in Printed Copy Stimulus intensity and first spike latency

36. Figure 7A -80mV -20mV Stimulus intensity and first spike latency Low (5-10% below high) High

37. Figure 7B Stimulus intensity and first spike latency

38. Latencies are stimulus-level (intensity) dependent. Therefore the latency is not hardwired into a particular neuron. Stimulus intensity may be involved in the mechanism in which timing is learned. Summary - Figure 7 Not in Printed Copy Stimulus intensity and first spike latency

39. Summary Action potentials occurring up to 300 ms after stimulus onset are observed in organotypic slices from rat auditory cortex. These late responses are attributed to dynamics of the neural network Buonomano hypothesizes that timing in this range could be a local process, reflected in the dynamics of neurons recruited in a task-specific manner. “The neural representation of time.” Ivry RB, Spencer RM