2. Neurophysiology Assays
Nerve Conduction
Evaluation of Neuropathy
and Neurodegeneration
Slide 2 Neurophysiology Models March, 2013
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. 50:821-8
Slide 3 Neurophysiology Models March, 2013
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. 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
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. 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
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. 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
activation
Slide 14 Neurophysiology Models March, 2013
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. 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 msec
Slide 16 Neurophysiology Models March, 2013
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 s
Slide 17 Neurophysiology Models March, 2013
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. 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. 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. 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. 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. 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!
100
The 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 injection
Slide 23 Neurophysiology Models March, 2013
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 mA
Slide 24 Neurophysiology Models March, 2013
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. 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 injection
Slide 26 Neurophysiology Models March, 2013
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. 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. 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
32. Assessment of Spinal Cord Function
Magnetic Motor Stimulation: Basic Principles and Clinical Experience
(EEG Suppl. 43; chapter 25, pps. 293-307
Slide 32 Neurophysiology Models March, 2013
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. 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
footfalls
Slide 34 Neurophysiology Models March, 2013
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 region
Slide 36 Neurophysiology Models March, 2013
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. 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. 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 IP
Slide 39 Neurophysiology Models March, 2013
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. 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
