Melior neurophysiology models 13 mar13

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Melior neurophysiology models 13 mar13

  1. 1. Electrophysiology Models Nerve Conduction / Neuropathy Neuromuscular Reflex Function Spinal Reflex Excitability Cortical & Neuromuscular Evoked Potentials Auditory Sensory GatingSlide 1 Neurophysiology Models March, 2013
  2. 2. Neurophysiology Assays Nerve Conduction Evaluation of Neuropathy and NeurodegenerationSlide 2 Neurophysiology Models March, 2013
  3. 3. Chemo-neuropathy Evaluation Peripheral nerve amplitude and conduction velocity measurements •  Vincristine administered 2x / week (1.7 mg/kg sc) to mice for 10 weeks •  Caudal (tail) nerve conduction velocity is increased by treatment 75 µV 2 ms Bieri et al, 1997, J. Neurosci. Res. 50:821-8Slide 3 Neurophysiology Models March, 2013
  4. 4. IGF-I Protects Against Vincristine Reduction in Conduction Velocity (CV) Change in CV from pre- treatment baseline values Conclusion: •  Vehicle and veh/IGF treated animals showed a normal increase in caudal tail CV over 10 wks of treatment •  Vincristine (Vin/Veh) treatment caused a reduction in CV over this time •  The vincristine-induced decrease was ameliorated by IGF-I.Slide 4 Neurophysiology Models March, 2013 4
  5. 5. Behavioral and Morphological Protection by IGF-I in Vincristine Chemoneuropathy •  Gait measures (ipsi- and contralateral limb support) were reduced by vincristine treatment. IGF-I (1 mg/kg sc) reduced the effect of vincristine. •  Hot plate latency was increased by vincristine treatment. The increase was prevented by IGF-I (1 mg/kg sc). •  Axonal pathology (abnormal axons and myelin) produced by vincristine treatment was prevented by IGF-I (1 mg/kg sc). •  Body weight was not affected by vincristine or IGF-I.Slide 5 Neurophysiology Models March, 2013 5
  6. 6. Neurophysiological, Behavioral, and Morphological Evaluation of SOD-KO Mice American Journal of Pathology, Vol. 155, No. 2, August 1999 Copyright © American Society for Investigative Pathology •  Mice lacking cytoplasmic Cu/Zn superoxide dismutase (SOD) were used as a model of the neurodegenerative effects of familial ALS. •  Caudal (mixed, tail), sural (sensory), and tibial (motor) nerve conduction velocity and amplitudes were evaluated at 5 – 7 mos of age. •  Rod-running latency and stride length were evaluated at 4, 6, and 14 mos. •  Nerve histology and muscle histochemistry (SDH; red vs white fibers) were evaluated at 2 and 6 mos.Slide 6 Neurophysiology Models March, 2013
  7. 7. Conduction Velocity and Amplitude Changes distal Tibial (motor) SOD1 +/+ proximal SOD1 -/- Conduction latencies were increased in SOD Sural +/+ mice nerve SOD1 +/+ Sural (sensory) SOD1 -/- .05 ms SOD1 +/+ 10 mA Caudal (mixed) SOD1 -/-Slide 7 Neurophysiology Models March, 2013
  8. 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.Slide 8 Neurophysiology Models March, 2013
  9. 9. Nerve Conduction in Adult SD Rats Tibial (motor) nerve recording Sciatic notch Ave of 10 sweeps 50 µs ISI: 2 sec 10 mA 100 0 Sciatic Δx Amplitude (µV) -100 Tibial nerve 6.8 msec 250 Ankle 0 Tibial -250 -10 0 10 20 4.2 msec Latency difference: (6.8 – 4.2) msec = 2.6 msec Distance: 40 mm Conduction Velocity: 40 mm / 2.6 msec = 15.4 m/secSlide 9 Neurophysiology Models March, 2013
  10. 10. Nerve Conduction in Adult SD Rats Sural (sensory) nerve recording Ave of 50 10 sweeps response ISI: 2 sec Amplitude (µV) 0 Sural nerve 50 Stimulus artifact -100 -2 0 2 4 6 Δx 0.75 msec Latency difference: 0.75 msec 50 µs Distance: 23 mm 10 mA Conduction Velocity: 23 mm / 0.75 msec = 31 m/secSlide 10 Neurophysiology Models March, 2013
  11. 11. Nerve Conduction in Adult SD Rats Caudal (mixed) nerve recording Ave of 200 10 sweeps cm Proximal ISI: 2 sec 0 50 µs 0 Proximal 10 mA Amplitude (µV) Distal -200 5 5.5 msec 250 10 0 Distal -250 -10 0 10 20 30 3.0 msec Latency difference: (5.5 – 3.0) msec = 2.5 msec Distance: 50 mm Conduction Velocity: 50 mm / 2.5 msec = 20 m/secSlide 11 Neurophysiology Models March, 2013
  12. 12. Spinal Excitability: C-fiber Reflex – Pain SensitivitySlide 12 Neurophysiology Models March, 2013
  13. 13. Method for Recording Plantar Aδ, Aβ, and C-fiber Responses (CFR) C-fibers are small unmyelinated fibers transmitting diffuse pain signals Aδ & Aβ fibers are larger myelinated fibers transmitting pain and touch information Plantar nerve Spinal cord “Early” response Hind Aδ, Aβ fibers 2 ms foot Peroneal nerve Stimulus “Late” C-fiber Peroneus 0 100 200 response 300 400 l. muscle Time (msec) The integrated value of the CFR from 150 – 400 msec is a measure of the sensitivity to the stimulation and the excitability of the spinal neurons and muscle.Slide 13 Neurophysiology Models March, 2013
  14. 14. Characterization of C-fiber Reflex (CFR) “early” “late” 10 - 25 msec 150 - 400 msec Aδ/Aβ fiber response C-fiber response “C-fibers” are small unmyelinated axons mediating pain responses. They produce polysynaptic activation of spinal motoneurons and reflex muscle contractions – the “Late” response shown above. •  C-fiber response latency consistent with conduction in unyelinated C- fibers (0.5 - 1 m/sec) rather than myelinated Aδ/Aβ fibers (12-20 m/sec) •  Threshold of late response ~4x higher than early response •  Capsaicin causes desensitization of late response consistent w/ C-fiber activationSlide 14 Neurophysiology Models March, 2013
  15. 15. CFR Quantification CFR’s can be quantified by rectifying the responses between 150-400 msec EMG Stimulus 6 sec 2 msec x 10 mA Peroneal muscle EMG response Quantification of C-fiber reflex Rectified 150 (normalized %) Response 125 Amplitude 100 150 400 msec 75 Integrated LHL 50 400 10 Integrated RHL 25 Vi = ∫ V(t) dt CFR = (∑ Vi ) / 10 i=1 0 t = 150 0 20 40 60 Integrate over 250 msec Average over 1 min Time from start (min)Slide 15 Neurophysiology Models March, 2013
  16. 16. Verification of CFR Pathway Biceps femoris (isolated) Determination Tibialis anterior of muscle of origin Soleus The C-fiber response is Peroneus l. muscle produced by signals traveling in the plantar n. and activating 100 msec motoneurons of the Peroneus L. muscle Peroneus l. muscle response Determination After transection of sural nerve of afferent nerve pathway After transection of plantar nerve 100 msecSlide 16 Neurophysiology Models March, 2013
  17. 17. Effect of Capsaicin on CFR Capsaicin -5 s 30 µl x 0.4 mg/ml at stimulation site 6s 12 s Capsaicin initially enhances (6 & 12 sec) and then blocks the late 18 s response, consistent with desensitization of vanilloid receptors on C- 24 s fiber terminals. 30 s 36 s 42 s 48 s 54 sSlide 17 Neurophysiology Models March, 2013
  18. 18. Effect of Morphine on CFR Morphine (opoid-receptor antagonist) produces a biphasic dose response effect on the C-fiber reflex, enhancing it at 3 mg/kg and suppressing it at higher doses. Increased response at 3 mg/kg Morphine administered sc presumed to result from supra-spinal at time 0. N=3 rats per curve. disinhibition relative to spinal inhibition 180 Percent change in response 160 140 3 mg/kg 120 100 PBS 80 60 5.5 mg/kg 40 20 10 mg/kg 0 -25 -15 -5 0 5 15 25 35 Time relative to injection (min)Slide 18 Neurophysiology Models March, 2013
  19. 19. Morphine-Induced Inhibition of CFR is Reversed by Naloxone Naloxone (µ-opioid competitive agonist) reverses the effect of morphine. Average CFR’s from R & L hind limbs in 1 rat 400 CFR amplitude, % baseline 350 300 250 Morphine Naloxone 10 mg/kg sc 0.4 mg/kg sc 200 # 150 baseline 100 50 * 0 -20 -10 0 10 20 30 40 50 Time relative to first injection (min)Slide 19 Neurophysiology Models March, 2013
  20. 20. Determining the Site of Drug Action Four likely analgesic sites of action of a drug can be evaluated neurophysiologically: 1.  Action of the drug on sensory afferent nerve fibers. - Record the amplitude of the compound (plantar) nerve action potential and compare to the CFR amplitude. 2.  Action on motor efferent nerve fibers. - Integrity of the efferent axons from the spinal cord can be tested by stimulating the peroneal nerve and recording the peroneus muscle (“M”) response. 3.  Action on spinal cord interneurons in the dorsal horn. - Changes in the dorsal horn field potential (DHFP) reflect the ability of C-fiber afferents entering the cord to activate first-order interneurons. 4.  Action on descending supraspinal facilitatory / inhibitory pathways. - Assess changes in CFR amplitude following transection of the dorslal-lateral descending columns that modulate spinal excitability.Slide 20 Neurophysiology Models March, 2013
  21. 21. Evaluating Drug Effects on Afferent Nerve Conduction Integration Peroneal window Peroneus l. muscle EMG muscle Plantar nerve afferent volley 50 msec The amplitudes of the compound afferent nerve Spinal volley and the CFR cord are directly related Conduction velocity = 0.5 - 1.0 m/s once the afferent Stimulus-Response Recruitment volley exceeds Plantar nerve Integrated EMG / CAP 100 threshold for 80 motoneuron Plantar n. (% max.) 60 depolarization. 40 APV Hind foot Peroneal m. stimulation 20 EMG 2 ms 0 0 3 6 9 12 15 14 → 0 mA Stimulus current (mA)Slide 21 Neurophysiology Models March, 2013
  22. 22. Test Agent Does Not Inhibit Plantar Nerve C-fiber Afferent Volley Effect of test agent vs. time Mean effect of test agent 4000 120 Plantar n. volley p>0.05 Integrated activity 100 Percent change 3000 in response 80 2000 60 p=0.013 Test agent Peroneus 3 mg/kg i.v. 40 l. muscle 1000 EMG 20 N=4 N=4 0 0 -20 -10 0 10 20 Veh. Test agent Veh. Test agent Time relative to injection (min.) CFR Plantar n. APV 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.Slide 22 Neurophysiology Models March, 2013
  23. 23. Effect of Test Agent on the Efferent Peroneal Neuromuscular Pathway Spinal M-response C-Fiber Response cord Time (min) -42 relative to Plantar test agent -26 nerve injection (3 mg/kg iv) 4 Peroneal nerve 24 .05 ms 2 msec 100 msec 2 ms 10 mA 10 mA 600 Peroneal muscle amplitude Peroneal n. direct! 500 M-response! Peroneus 400 (mV, 25x) l. muscle 300 EMG Hind foot-! 200 stimulated! 100The direct M response is not C-fiber response!effected by the test drug => drug 0 (mv*msec)is not acting on the efferent path. -40 -20 0 20 40 Time (min) post injectionSlide 23 Neurophysiology Models March, 2013
  24. 24. Spinal Cord Dorsal Horn Field Potentials Plus CFR Recording Peroneus l. Peroneus muscle EMG l. muscle Peroneal nerve 10x gain L4 Spinal cord myelinated 100 msec afferent Plantar response C-fiber DHFP: nerve DHFP amplitude: Hind foot The dorsal spinal cord field potential stimulation (DHFP) amplitude is directly related to the CFR amplitude. 2 ms 1.4 mASlide 24 Neurophysiology Models March, 2013
  25. 25. Test Agent Does Not Inhibit Dorsal Horn Field Potential Effect of test agent vs. time Mean response inhibition 140 by test agent Dorsal horn % change in amplitude 120 field potential 0 Percent inhibition CFR vs. DHFP 100 20 N.S. 80 40 60 N=3 Test agent 60 40 3 mg/kg iv. C-fiber reflex p< 0.05 20 80 0 -10 0 10 20 30 40 50 CFR DHFP Time from injection (min) amplitude amplitude The test drug did not reduce the amplitude of the dorsal horn field potential => the drug did not impair transmission between primary efferent terminals and the first-order spinal interneurons in the dorsal horn.Slide 25 Neurophysiology Models March, 2013
  26. 26. Chronic Dorsal-lateral Funiculus T9 cord (DLF) Lesion and CFR DLF lesion 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). 8000 Integrated EMG activity The test agent blocked the CFR 6000 in normal animals (not shown), and also blocked it in animals with 4000 chronic DLF lesions. Test Agent 3 mg/kg iv. 2000 160 CFR Amplitude 0 (mv*ms/100) 120 -20 -10 0 10 20 30 40 62.5% 80 p<0.0001 Time post injection (min) 40 Lesion of the DLF pathway does not block CFR N= 10 inhibition produced by test agent => drug does not act 0 at supraspinal level. Pre 15’ Post injection injectionSlide 26 Neurophysiology Models March, 2013
  27. 27. Monosynaptic Spinal ReflexSlide 27 Neurophysiology Models March, 2013
  28. 28. Spinal Reflex Excitability: Spinal Monosynaptic (H-) Reflex The Hoffman or “H” reflex is the monosynaptic muscle reflex produced by DRG Spinal interneurons activating proprioceptive muscle afferents; Proprioceptive aka the common achilles tendon-tap reflex. afferents Spinal Stimulation of the tibial nerve activates cord axons innervating the plantar muscle, producing a direct “M” or muscle response, Tibial and also proprioceptive afferents traveling to 0.5  ms   nerve Motor neurons the spinal cord, which then activate spinal 1-­‐10  mA   motoneurons producing a second delayed “H” reflex response. Monosynaptic Plantar (“H”) response Unlike the CFR, the H-reflex does not muscle directly involve any excitatory or inhibitory interneurons. Thus drugs that affect e.g. GABA receptors or release should not affect Muscle Hind this reflex unless (like GABA-A agonists) foot (“M”) response they tonically increase GABAergic tone, whereas they do impair the C-fiber reflex.Slide 28 Neurophysiology Models March, 2013
  29. 29. Characterization of the Plantar H- (Monosynaptic) Reflex M-response H-reflex EMG (mV) stimulus -10 -5 0 5 10 15 Time (ms) •  Stimulation of the tibial nerve produces a direct muscle (M) response in the plantar muscle starting about 3 msec after the stimulation, followed by an H (monosynaptic) reflex response at about 10 msec. •  GABA-A receptor agonist drugs typically reduce this response, while antagonists facilitate it, assuming the drugs penetrate the blood-brain barrier. Benzodiazepines typically have no effect. •  A drug that directly affects peripheral axons or neuromuscular junctions (e.g. ssuccinylcholine) should inhibit this reflex.Slide 29 Neurophysiology Models March, 2013
  30. 30. Diazepam Does Not Alter H-Reflex M response 10 min before ß H- or monosynaptic reflex (MSR) responses Vehicle inject. H response from rat at various times before and after injection of either vehicle or 0.5 mg/kg IV Time of diazepam. Each waveform is the average of Vehicle inject. 10 successive responses obtained at 6 sec intervals. Red biphasic square wave at time 0 10 min before Drug inject. represents stimulus pulse. Scale at bottom right in mV applies to all recordings. Diazepam, a benzodiazepine, has no effect on MSR Amplitude Time of Drug monosynaptic reflexes. inject. 1400 10 min Peak-Peak Amplitude (µV) M response after 1200 Drug inject. 1000 20 min after 800 Diazepam Drug inject. Vehicle 0.5 mg/kg IV 600 4.0 2.0 400 H response 30 min after 0 Drug inject. -2.0 200 -4.0 -6.0 0 -20 0 20 40 60 80 100 -5 0 5 10 15 Time (min) Time (msec)Slide 30 Neurophysiology Models March, 2013
  31. 31. Cortical & Neuromuscular Evoked PotentialsSlide 31 Neurophysiology Models March, 2013
  32. 32. Assessment of Spinal Cord Function Magnetic Motor Stimulation: Basic Principles and Clinical Experience (EEG Suppl. 43; chapter 25, pps. 293-307Slide 32 Neurophysiology Models March, 2013
  33. 33. Evoked Potentials After SCI Somatosensory Auditory Stimulated Evoked Potentials Responses Motor function ASR 140 SEP 120 100 Cerebellar Myoelectric 80 Evoked Responses 60 40 20 0 1d 2d 7d 14d 21d 28d Sensory and motor evoked potentials provide a reliable and quantitative means of monitoring recovery after spinal injury.Slide 33 Neurophysiology Models March, 2013
  34. 34. Neuromuscular Electrophysiology Chronic electromyographic recording can be utilized to characterize neuromuscular disorders, e.g. spasticity and effects of muscle relaxants, myotonia, etc., as well as recovery of function. Rectified EMG activity Chronic EMG recording Iliacus during locomotion Biceps femoris Vastus lateralis Semi- tendinosus Stepping position Hindlimb footfallsSlide 34 Neurophysiology Models March, 2013
  35. 35. Auditory Sensory Gating ResponsesSlide 35 Neurophysiology Models March, 2013
  36. 36. Evaluation of Attention by Auditory Sensory Gating Response Paradigm: 1.  Electrodes implanted in rats under sodium pentobarbital anesthesia: •  Left frontal cortex - left sensory-motor cortex (above hippocampus) •  Depth electrode, right CA3 region of the HC, referenced to a skull screw •  Neck EMG 2.  One week after recovery, animal exposed to auditory tones as follows •  Pairs of 5 k Hz tones, 10 ms duration, 0.5 s apart •  10 s interval between pairs of tones 3.  Outcome: •  Amplitude = P1 - N1, mV (most robust effect) •  Outcome = ratio of amplitude of second (test) to first (conditioning) response. skull Stereotaxically placed electrodes 4.0 mm below dura in the hippocampal CA-3 regionSlide 36 Neurophysiology Models March, 2013
  37. 37. Effect of Amphetamine on Auditory Gating Responses •  Rats were chronically implanted with screw electrodes over frontal and sensory-motor cortices, and with a bipolar metal electrode into the CA3 region of the hippocampus (electrode tip separation ~ 1 mm). - Test tones were applied during surgery to optimize electrode placemnt •  Post surgical recovery, animals were placed into recording chamber and exposed to paired tones: - 3 k Hz, 10 ms duration - 0.5 s interval between test tones - 10 s between pairs of test tones •  Three sets of 30 stimulus tone pairs were delivered at ~ 6 min intervals while the rat was awake and resting •  Amphetamine (1 or 3 mg/kg ip) was then administered •  10’, 20’, and 30’ post drug administration, additional sets were recorded. •  Individual peak amplitudes were analyzed and compared as a function of “Conditioning” vs “Test” tone pulses, and drug: “Pre” vs “Amphetamine”.Slide 37 Neurophysiology Models March, 2013
  38. 38. Effect of Amphetamine on Auditory Gating Responses Typical Auditory Evoked Potentials Surface (EEG) recording CA-3 (depth) recording 1.5 1.5 F011_EEG F011_CA3 P1 P1 Cond. 1.0 Cond. EP Amp (mV) 1.0 Test Test 0.5 0.5 0.0 0.0 -0.5 N2 -0.5 N2 -1.0 N1 N1 -1.0 -1.5 0 0.05 0.1 0.15 0 0.05 0.1 0.15 Depth electrodes can be located in various cortical regions including frontal or auditory cortex, hippocampus, etc. either for continuous recording or for recording evoked potentials.Slide 38 Neurophysiology Models March, 2013
  39. 39. Effect of Amphetamine on Auditory Gating Responses •  N1 and P1 responses are well defined in EEG records •  In both surface and CA3 recordings, identified potentials occurred at similar latencies in both Conditioning and Test responses •  P1 and N1 responses showed similar latencies to surface and CA3 recording, but CA3 amplitudes were larger and used for evaluating the effect of amphetamine on auditory gating (below) Analysis of Peak-Peak Amplitudes of Auditory Evoked Potentials 2.5 (Cond vs Test): ANOVA, p= 0.02 Mean latencies (N= 3 Amplitide (mV) 2.0 responses) for the (P1 - N1) 1.5 VT P1-N1 amplitude difference as a VC function of (conditioning vs 1.0 Cond. Test test) and (Pre drug vs 0.5 Amphetamine). 0.0 Pre Drug Amphetamine 1 mg/kg IPSlide 39 Neurophysiology Models March, 2013
  40. 40. Effect of Amphetamine on Auditory Gating Responses Analysis of percent inhibition of the Test tone for various amplitude measures 100 p= 0.023 unpaired t-test, N= 3 Pre drug 80 Amphetamine p= 0.008 % Inhibition 60 % Inhibition = (VC – VT) * 100 * VC 40 100% = complete inhibition; * 0% = no effect 20 0 Amphetamine reduced inhibition of the P0-N1 P1-N1 Test evoked potential by all measures, Evoked Potential Peak-Peak Measure with P1-N1 and P0-N1+P1 showing the most robust effect.Slide 40 Neurophysiology Models March, 2013
  41. 41. Effect of Amphetamine on Auditory Gating Responses Pre Drug 0.8 P1 Conditioning Amplitude (mV) Test Pre dosing 0.4 100 Post dosing 0.0 Percent inhibition of Test Response 80 -0.4 p< 0.001 Tone -0.8 N1 60 p= 0.001 20 40 60 80 100 120 140 Time (ms) 40 Post Amphetamine 1 mg/kg IP 20 N=9 N=5 0.8 Conditioning Amplitude (mV) Test 0 0.4 P1 1.0 3.0 N = # of rats 0.0 Amphetamine (mg/kg ip) P1-N1 amplitudes -0.4 N1 Amphetamine at both 1 and 3 mg/kg IP reduced -0.8 inhibition of the auditory evoked gating responses. 20 40 60 80 100 120 140 Time (ms)Slide 41 Neurophysiology Models March, 2013
  42. 42. FiniSlide 42 Neurophysiology Models March, 2013

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