1. Nerve Conduction / Neuropathy Neuromuscular Reflex Function Spinal Reflex Excitability Cortical & Neuromuscular Evoked Potentials Auditory Sensory Gating Electrophysiology Models

2. NEUROPHYSIOLOGY ASSAY Nerve Conduction

7. Conduction Velocity & Amplitude Changes Sural nerve .05 ms 10 mA Tibial (motor) distal proximal Sural (sensory) Caudal (mixed)

8. Nerve Conduction Velocities and Amplitudes at 5–7 Months of Age in SOD -/- Mice Wild type KO * * * Conclusion : SOD KO mice showed significant reductions in the conduction velocity of the caudal (tail) and tibial nerves, and in the latency of the plantar muscle response to tibial nerve stimulation.

9. Nerve Conduction in Adult SD Rats Sciatic notch Ankle 50 µ s 10 mA Tibial (motor) nerve recording  x Tibial nerve  Ave of 10 sweeps ISI: 2 sec Sciatic 100 0 -100 250 0 -250 Amplitude ( µV) 4.2 msec 6.8 msec Latency difference: (6.8 – 4.2) msec = 2.6 msec Distance: 40 mm Conduction Velocity: 40 mm / 2.6 msec = 15.4 m/sec Tibial 0 20 -10 10  Actual Data, Adult Rat

10. Nerve Conduction in Adult SD Rats 50 µ s 10 mA Sural (sensory) nerve recording Sural nerve  x Amplitude ( µV) 0.75 msec  Actual Data, Adult Rat Latency difference: 0.75 msec Distance: 23 mm Conduction Velocity: 23 mm / 0.75 msec = 31 m/sec 50 0 -100 50 0 2 6 -2 4  Ave of 10 sweeps ISI: 2 sec Stimulus artifact response

11. Nerve Conduction in Adult SD Rats Proximal Distal 200 0 -200 250 0 -250 Amplitude ( µV) 0 10 20 30 -10 3.0 msec 5.5 msec  Actual Data, Adult Rat Latency difference: (5.5 – 3.0) msec = 2.5 msec Distance: 50 mm Conduction Velocity: 50 mm / 2.5 msec = 20 m/sec  Ave of 10 sweeps ISI: 2 sec 50 µ s 10 mA 0 5 10 cm Proximal Distal Caudal (mixed) nerve recording

12. Spinal Reflex Excitability: C-fiber and Monosynaptic Reflexes

13. “ Early” response A  /A β fibers “ Late” response C-fibers 100 200 300 0 400 Stimulus Time (msec) Plantar nerve Peroneus l. muscle Spinal cord Peroneal nerve Method for Recording Plantar A  /A β and C-fiber Responses (CFR) C-fibers are small unmyelinated fibers transmitting diffuse pain signals A  /A β fibers are larger myelinated fibers transmitting pain and touch information Hind foot 2 ms

15. Quantification of C-fiber reflex Average over 1 min Integrate over 225 msec Time from start (min) Amplitude (normalized %) 0 25 50 75 100 125 150 0 20 40 60 Integrated LHL Integrated RHL Integrated LHL Integrated RHL CFR Quantification Peroneal muscle EMG response Rectified Response 375 msec 150  375 t = 150 V i = V(t) dt  10 i = 1 V i CFR =   / 10 6 sec 2 msec x 10 mA EMG Stimulus

16. Determination of afferent nerve pathway Determination of muscle of origin Verification of CFR Pathway The C-fiber response is produced by signals traveling in the plantar n. and activating motoneurons of the Peroneus L. muscle Peroneus l. muscle Tibialis anterior Biceps femoris (isolated) Soleus 100 msec Biceps (isolated) 100 msec Peroneus l. muscle response After transection of sural nerve 100 msec After transection of plantar nerve

17. Capsaicin 30 µl x 0.4 mg/ml at stimulation site Effect of Capsaicin on C-fiber Response Capsaicin initially enhances (6 & 12 sec) and then blocks the late response, consistent with desensitization of vanilloid receptors on C-fiber terminals. 18 s 24 s 30 s 36 s 42 s 48 s 54 s 6 s 12 s -5 s

18. Increased response at 3 mg/kg presumed to result from supra-spinal disinhibition relative to spinal inhibition Percent change in response Time relative to injection (min) -25 -15 -5 0 5 15 25 35 10 mg/kg 5.5 mg/kg 3 mg/kg PBS Morphine administered sc at time 0. N=3 rats per curve. Effect of Morphine on CFR Morphine produces a biphasic dose response effect on the C-fiber reflex, enhancing it at 3 mg/kg and suppressing it at higher doses. 0 20 40 60 80 100 120 140 160 180

19. Morphine-Induced Inhibition of CFR is Reversed by Naloxone Average CFR’s from R & L hind limbs in 1 rat 0 50 100 150 200 250 300 350 400 -20 -10 0 10 20 30 40 50 CFR amplitude, % baseline Morphine 10 mg/kg sc Naloxone 0.4 mg/kg sc Time relative to first injection (min) baseline * #

21. Peroneal muscle EMG Plantar nerve Peroneus l. muscle Hind foot stimulation 2 ms 14  0 mA Spinal cord Plantar nerve afferent volley Conduction velocity = 0.5 - 1.0 m/s Integration window Plantar n. Afferent Volley versus CFR 50 msec 0 20 40 60 80 100 0 3 6 9 12 15 Stimulus current (mA) Integrated EMG / CAP (% max.) Peroneal m. EMG Plantar n. APV Stimulus-Response Recruitment

22. Test Agent Does Not Inhibit Plantar Nerve C-fiber Afferent Volley Mean effect of test agent Effect of test agent vs. time Percent change in response CFR Plantar n. APV 0 1000 2000 3000 -20 -10 0 10 20 Integrated activity Peroneus l. muscle EMG Plantar n. volley 4000 Time relative to injection (min.) Test agent 3 mg/kg i.v. The C-fiber response but not the amplitude of the plantar n. volley is reduced by the test drug => the drug is not acting on the efferent pathway. 0 20 40 60 80 100 120 Veh. Test agent Veh. Test agent p=0.013 p>0.05 N=4 N=4

23. Effect of Test Agent on the Efferent Peroneal Neuromuscular Pathway 0 100 200 300 400 500 600 -40 -20 0 20 40 Peroneal muscle amplitude Hind foot stimulated C-fiber response (mv*msec) Peroneal n. direct M-response (mV, 25x) Time (min) post injection -42 4 -26 24 2 msec Time (min) relative to test agent injection (3 mg/kg iv) 100 msec M-response C-Fiber Response The direct M response is not effected by the test drug => drug is not acting on the efferent path. Plantar nerve 2 ms 10 mA Spinal cord Peroneus l. muscle EMG Peroneal nerve .05 ms 10 mA

24. Chronic Dorsal-lateral Funiculus (DLF) Lesion and CFR The test agent blocked the CFR in normal animals (not shown), and also blocked it in animals with chronic DLF lesions. Chronic DLF lesions were made in rats ~4 weeks prior to evaluation of a test agent on the CFR. Spinal lesions did not block the response to morphine or naloxone (not shown). Lesion of the DLF pathway does not block CFR inhibition produced by test agent => drug does not act at supraspinal level. T9 cord DLF lesion Test Agent 3 mg/kg iv. 0 2000 4000 6000 8000 -20 -10 0 10 20 30 40 Time post injection (min) Integrated EMG activity CFR Amplitude (mv*ms/100) 0 40 80 120 160 Pre injection 15’ Post injection 62.5% p<0.0001 N= 10

25. Peroneus l. muscle 100 msec Plantar nerve 2 ms 14 mA L4 Spinal cord Peroneus l. muscle EMG myelinated afferent response C-fiber DHEP: DHEP amplitude: Hind foot stimulation 10x gain Peroneal nerve Spinal Cord Dorsal Horn Field Potentials Plus CFR Recording

26. Test Agent Does Not Inhibit Dorsal Horn Evoked Potential Effect of test agent vs. time Mean response inhibition by test agent 0 20 40 60 80 100 120 140 -10 0 10 20 30 40 50 % change in amplitude CFR vs. DHEP C-fiber refles Dorsal horn evoked potential Time from injection (min) Test agent 3 mg/kg iv. Percent inhibition 80 60 40 20 0 CFR amplitude DHEP amplitude N=3 p< 0.05 N.S. The test drug does not reduce the amplitude of the dorsal horn evoked potential => the drug does not impair transmission between primary efferent terminals and the first-order spinal interneurons in the dorsal horn.

27. Monosynaptic Spinal Reflex

30. H- or monosynaptic reflex (MSR) responses from rat at various times before and after injection of either vehicle or 0.5 mg/kg IV diazepam. Each waveform is the average of 10 successive responses obtained at 6 sec intervals. Red biphasic square wave at time 0 represents stimulus pulse. Scale at bottom right in mV applies to all recordings. Diazepam, a benzodiazepine, has no effect on monosynaptic reflexes. (Lack of) Effect of Diazepam on H-Response 0 200 400 600 800 1000 1200 1400 -20 0 20 40 60 80 100 Time (min) Peak-Peak Amplitude ( µV) M response Vehicle H response Diazepam 0.5 mg/kg IV 10 min before Vehicle inject. Time of Vehicle inject. 10 min before Drug inject. Time of Drug inject. 10 min after Drug inject. 20 min after Drug inject. 30 min after Drug inject. Time (msec) -5 0 5 10 15 0 2.0 4.0 -4.0 -2.0 -6.0 MSR Amplitude M response H response

31. Cortical and Neuromuscular Evoked Potentials

32. Assessment of Spinal Cord Function Magnetic Motor Stimulation: Basic Principles and Clinical Experience (EEG Suppl. 43; chapter 25, pps. 293-307

33. SEP ASR Motor function Somatosensory Evoked Potentials Auditory Stimulated Responses Cerebellar Myoelectric Evoked Responses Conclusion : Sensory and motor evoked potentials provide a reliable means of monitoring recovery after spinal injury.

34. Auditory Sensory Gating Responses Effect of Amphetamine

37. Typical Auditory Evoked Potentials Effect of Amphetamine on Auditory Gating Responses F011_EEG -1.0 -0.5 0.0 0.5 1.0 1.5 0 0.05 0.1 0.15 EP Amp (mV) Cond. Test N1 P1 N2 P0 Surface recording F011_CA3 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 0 0.05 0.1 0.15 Cond. Test N1 P1 N2 P0 CA-3 Recording

39. Analysis of percent inhibition of the test tone for various amplitude measures 0 20 40 60 80 100 P0-N1 P1-N1 P0-N1+P1 % Inhibition Pre drug Amphet. * * * p= 0.023 p= 0.009 p= 0.008 unpaired t-test, N= 3 100% = complete inhibition; 0% = no effect Amphetamine reduced inhibition of the Test evoked potential by all measures, with P1-N1 and P0-N1+P1 showing the most robust effect. Effect of Amphetamine on Auditory Gating Responses Evoked Potential Peak-Peak Measure % Inhibition = (V C – V T ) * 100 V C

40. Effect of Amphetamine on Gating Responses Post Amphetamine 1 mg/kg IP -0.8 -0.4 0.0 0.4 0.8 20 40 60 80 100 120 140 Time (ms) N1 P1 Amplitude (mV) Amphetamine at both 1 and 3 mg/kg IP reduced inhibition of the auditory evoked gating responses. Pre Drug -0.8 -0.4 0.0 0.4 0.8 20 40 60 80 100 120 140 Time (ms) Amplitude (mV) N1 P1 Conditioning Test Tone 0 20 40 60 80 100 1.0 3.0 Amphetamine (mg/kg ip) Percent inhibition of Test Response Pre dosing Post dosing p< 0.001 p= 0.001 N=9 N=5 N = # of rats; P1-N1 amplitudes Conditioning Test

41. Fini