Melior neurophysiology models 13 mar13

Slide 1 Neurophysiology Models March, 2013

Nerve Conduction / Neuropathy Neuromuscular Reflex Function

Spinal Reflex Excitability Cortical & Neuromuscular Evoked Potentials

Auditory Sensory Gating

Electrophysiology Models


Neurophysiology Assays

Nerve Conduction Evaluation of Neuropathy

and Neurodegeneration


Chemo-neuropathy Evaluation

•  Vincristine administered 2x / week (1.7 mg/kg sc) to mice for 10 weeks •  Caudal (tail) nerve conduction velocity is increased by treatment

Bieri et al, 1997, J. Neurosci. Res. 50:821-8

75 µV

2 ms

Peripheral nerve amplitude and conduction velocity measurements

Slide 4 Neurophysiology Models March, 2013 4

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.

IGF-I Protects Against Vincristine Reduction in Conduction Velocity (CV)

Change in CV from pre-treatment baseline values

Slide 5 Neurophysiology Models March, 2013 5

•  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.

Behavioral and Morphological Protection by IGF-I in

Vincristine Chemoneuropathy


American Journal of Pathology, Vol. 155, No. 2, August 1999 Copyright © American Society for Investigative Pathology

Neurophysiological, Behavioral, and Morphological Evaluation of

SOD-KO Mice

•  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.


Conduction Velocity and Amplitude Changes

Sural nerve

.05 ms 10 mA

Tibial (motor)

distal

proximal

Sural (sensory)

Caudal (mixed)

SOD1 +/+

SOD1 -/-

SOD1 +/+

SOD1 -/-

SOD1 +/+

SOD1 -/-

Conduction latencies were increased in SOD

+/+ mice


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.


Nerve Conduction in Adult SD Rats

Sciatic notch

Ankle

50 µs 10 mA

Tibial (motor) nerve recording

Δ x

Tibial nerve

Ave of 10 sweeps ISI: 2 sec

Sciatic

100

0

-100

250

0

-250

Am

plitu

de (µ

V)

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


50 µs 10 mA

Sural (sensory) nerve recording

Sural nerve

Δ x

Am

plitu

de (µ

V)

0.75 msec

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


Stimulus artifact

response



Proximal

Distal

200

0

-200

250

0

-250

Am

plitu

de (µ

V)

0 10 20 30 -10 3.0 msec

5.5 msec

Latency difference: (5.5 – 3.0) msec = 2.5 msec Distance: 50 mm Conduction Velocity: 50 mm / 2.5 msec = 20 m/sec


50 µs 10 mA

0

5

10

cm Proximal

Distal

Caudal (mixed) nerve recording



C-fiber Reflex – Pain Sensitivity

Spinal Excitability:


“Early” response Aδ, Aβ fibers

“Late” C-fiber response 100 200 300 0 400

Stimulus

Time (msec)

Plantar nerve

Peroneus l. muscle

Spinal cord

Peroneal nerve

Hind foot 2 ms

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

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.


“early” 10 - 25 msec

Aδ/Aβ fiber response

“late” 150 - 400 msec C-fiber response

Characterization of C-fiber Reflex (CFR)

•  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

“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.


Quantification of C-fiber reflex

Peroneal muscle EMG response

Rectified Response

400 msec 150

∫ 400

t = 150

Vi = V(t) dt ∑ 10

i = 1 Vi

CFR = ( ) / 10

Average over 1 min Integrate over 250 msec Time from start (min)

Am

plitude (norm

alized %)

6 sec 2 msec x 10 mA

EMG Stimulus

0 25 50 75 100 125 150

0 20 40 60

Integrated LHL Integrated RHL Integrated LHL Integrated RHL

CFR Quantification CFR’s can be quantified by rectifying the responses between 150-400 msec


Peroneus l. muscle

Tibialis anterior

Biceps femoris (isolated)

Soleus

100 msec

Biceps (isolated)

100 msec

Determination of afferent

nerve pathway

Determination of muscle of origin

Peroneus l. muscle response

After transection of sural nerve

100 msec

After transection of plantar nerve

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


Capsaicin 30 µl x 0.4 mg/ml at stimulation site

Effect of Capsaicin on CFR

18 s

24 s

30 s

36 s

42 s 48 s

54 s

6 s

12 s

-5 s

Capsaicin initially enhances (6 & 12 sec) and then blocks the late response, consistent with desensitization of vanilloid receptors on C-fiber terminals.


Increased response at 3 mg/kg presumed to result from supra-spinal

disinhibition relative to spinal inhibition

Per

cent

cha

nge

in re

spon

se

Time relative to injection (min)

0

20

40

60

80

100

120

140

160

180

-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 (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.


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

ampl

itude

, % b

asel

ine

Morphine 10 mg/kg sc

Naloxone 0.4 mg/kg sc

Time relative to first injection (min)

baseline

*

#

Naloxone (µ-opioid competitive agonist) reverses the effect of morphine.


Determining the Site of Drug Action

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.

Four likely analgesic sites of action of a drug can be evaluated neurophysiologically:


0

20

40

60

80

100

0 3 6 9 12 15 Stimulus current (mA)

Inte

grat

ed E

MG

/ C

AP

(% m

ax.)

Peroneal m. EMG

Plantar n. APV

Stimulus-Response Recruitment

2 ms 14 → 0 mA

Peroneal muscle EMG

50 msec

Plantar nerve

Peroneus l. muscle

Hind foot stimulation

Spinal cord

Plantar nerve afferent volley

Conduction velocity = 0.5 - 1.0 m/s

Integration window

Evaluating Drug Effects on Afferent Nerve Conduction

The amplitudes of the compound afferent nerve volley and the CFR are directly related once the afferent volley exceeds threshold for motoneuron depolarization.


Test Agent Does Not Inhibit Plantar Nerve C-fiber Afferent Volley

Mean effect of test agent Effect of test agent vs. time

Per

cent

cha

nge

in

resp

onse

CFR Plantar n. APV

0

20

40

60

80

100

120

Veh. Test agent Veh. Test agent

p=0.013

p>0.05

N=4 N=4

0

1000

2000

3000

-20 -10 0 10 20

Inte

grat

ed a

ctiv

ity

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.


Effect of Test Agent on the Efferent Peroneal Neuromuscular Pathway

Plantar nerve

2 ms 10 mA

Spinal cord

Peroneus l. muscle

EMG

Peroneal nerve

.05 ms 10 mA

0

100

200

300

400

500

600

-40 -20 0 20 40 Pero

neal

mus

cle

ampl

itude

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.


Peroneus l. muscle

100 msec

Plantar nerve

2 ms 1.4 mA

L4 Spinal cord

Peroneus l. muscle EMG

myelinated afferent

response C-fiber DHFP:

DHFP amplitude: Hind foot stimulation

10x gain

Peroneal nerve

Spinal Cord Dorsal Horn Field Potentials Plus CFR Recording

The dorsal spinal cord field potential (DHFP) amplitude is directly related to the CFR amplitude.


Test Agent Does Not Inhibit Dorsal Horn Field 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

% c

hang

e in

am

plitu

de

CFR

vs.

DH

FP

C-fiber reflex

Dorsal horn field potential

Time from injection (min)

Test agent 3 mg/kg iv.

Perc

ent i

nhib

ition

80 60 40 20 0

CFR amplitude

DHFP amplitude

N=3

p< 0.05

N.S.

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.


Chronic Dorsal-lateral Funiculus (DLF) Lesion and CFR 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)

Inte

grat

ed E

MG

act

ivity

CFR

Am

plitu

de

(mv*

ms/

100)

0

40

80

120

160

Pre injection

15’ Post injection

62.5% p<0.0001

N= 10

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.


Monosynaptic Spinal Reflex


Spinal Reflex Excitability: Spinal Monosynaptic (H-) Reflex

The Hoffman or “H” reflex is the monosynaptic muscle reflex produced by activating proprioceptive muscle afferents; aka the common achilles tendon-tap reflex.

Stimulation of the tibial nerve activates axons innervating the plantar muscle, producing a direct “M” or muscle response, and also proprioceptive afferents traveling to the spinal cord, which then activate spinal motoneurons producing a second delayed “H” reflex response.

Unlike the CFR, the H-reflex does not directly involve any excitatory or inhibitory interneurons. Thus drugs that affect e.g. GABA receptors or release should not affect this reflex unless (like GABA-A agonists) they tonically increase GABAergic tone, whereas they do impair the C-fiber reflex.

Proprioceptive afferents

0.5 ms 1-‐10 mA

Spinal cord

Hind foot

Tibial nerve

Spinal interneurons

Motor neurons

Plantar muscle

Muscle (“M”)

response

Monosynaptic (“H”) response

DRG


Characterization of the Plantar H- (Monosynaptic) Reflex

-10 -5 0 5 10 15

Time (ms)

EMG (mV)

M-response H-reflex

stimulus

•  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.


ß 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.

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

Am

plitu

de

M response

H response

Diazepam Does Not Alter H-Reflex

0

200

400

600

800

1000

1200

1400

-20 0 20 40 60 80 100 Time (min)

Peak

-Pea

k A

mpl

itude

(µV)

M response

Vehicle

H response

Diazepam 0.5 mg/kg IV


Cortical & Neuromuscular Evoked Potentials


Magnetic Motor Stimulation: Basic Principles and Clinical Experience (EEG Suppl. 43; chapter 25, pps. 293-307

Assessment of Spinal Cord Function


1d 2d 7d 14d 21d 28d

140

120

100

80

60

40

20

0

SEP ASR

Motor function

Somatosensory Evoked Potentials Auditory Stimulated

Responses

Cerebellar Myoelectric Evoked Responses

Sensory and motor evoked potentials provide a reliable and quantitative means of monitoring recovery after spinal injury.

Evoked Potentials After SCI


Iliacus

Rectified EMG activity

Biceps femoris Vastus lateralis Semi- tendinosus

Stepping position

Hindlimb footfalls

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.

Neuromuscular Electrophysiology

Chronic EMG recording during locomotion


Auditory Sensory Gating Responses


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.

Evaluation of Attention by Auditory Sensory Gating Response

Stereotaxically placed electrodes 4.0 mm below dura in the hippocampal CA-3 region

skull


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”.


Typical Auditory Evoked Potentials

F011_EEG

-1.0

-0.5

0.0

0.5

1.0

1.5

0 0.05 0.1 0.15

EP

Am

p (m

V) Cond.

Test

N1

P1

N2

Surface (EEG) 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

CA-3 (depth) recording


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.



•  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

Mean latencies (N= 3 responses) for the (P1 - N1) amplitude difference as a function of (conditioning vs test) and (Pre drug vs Amphetamine).

P1-N1

0.0

0.5

1.0

1.5

2.0

2.5

Pre Drug Amphetamine 1 mg/kg IP

Am

pliti

de (m

V)

Cond. Test

(Cond vs Test): ANOVA, p= 0.02

VC VT


Analysis of percent inhibition of the Test tone for various amplitude measures

0

20

40

60

80

100

P0-N1 P1-N1

% In

hibi

tion

*

*

p= 0.023

p= 0.008

unpaired t-test, N= 3

% Inhibition = (VC – VT) * 100 VC

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.


Evoked Potential Peak-Peak Measure

Pre drug Amphetamine



Pre Drug

-0.8

-0.4

0.0

0.4

0.8

20 40 60 80 100 120 140 Time (ms)

Am

plitu

de (m

V)

N1

P1 Conditioning Test

Tone

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 0

20

40

60

80

100

1.0 3.0

Amphetamine (mg/kg ip)

Perc

ent i

nhib

ition

of

Tes

t Res

pons

e

Pre dosing

Post dosing

p< 0.001

p= 0.001

N=9 N=5

N = # of rats P1-N1 amplitudes

Conditioning Test

Am

plitu

de (m

V)

Amphetamine at both 1 and 3 mg/kg IP reduced inhibition of the auditory evoked gating responses.


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Melior neurophysiology models 13 mar13