Targeted Evaluation of Age Related 

Pharmacodynamics Using Mammalian 

CNS Neurolemma Preparations 

J. Marshall Clark, Ph.D.

Professor and Director, MPAL

Department of Veterinary and Animal Sciences 

University of Massachusetts, Amherst MA 01003
Project Contributors

Project funded by the Council for the Advancement of 

Pyrethroid Human Risk Assessment (CAPHRA)

University of Massachusetts-Amherst

• J. Marshall Clark, PhD, Professor and Director MPAL (Study Director)

• Edwin Murenzi, PhD Candidate, MCB

• Kyong Sup Yoon, PhD, Research Assistant Professor

Salve Regina University

• Steven B. Symington, PhD, Associate Professor (Principal Investigator)

• Abigail Toltin, PhD Candidate, BioMed Eng & Biotech 
Assay in the Context of the Adverse 

Outcome Pathway (AOP)
Overview of the experimental procedure to analyze the effect of pyrethroids 

on rat brain neurolemma tissue microtransplanted into Xenopus laevis oocytes

Goal 1: To determine the 

relative levels of VSSC 

isoform expression in 

juvenile and adult 

rat brain

Goal 2: Validate the 

physical and 

functional presence of 

rat brain channels in 

Xenopus oocytes 

microinjected with 

adult and juvenile 

neurolemma

Goal 3: Perform concentrationdependent response curves to evaluate 

age-dependent pharmacodynamics 

differences on TTX-sensitive current 

from juvenile and adult rat brain 

neurolemma injected into Xenopus

oocytes
Goal 1: To determine the relative levels of VSSC 

isoform expression in juvenile and adult rat brain

(Automated Western Blotting)

Characterization of Test System 
VSSC isoform expression in PND15 and PND90 rat brain tissues
Goal 2: Validate the physical presence of rat brain 

channels in Xenopus oocytes microinjected with 

adult and juvenile neurolemma

(Immunohistochemistry)

Characterization of Test System 
Incorporation of rat brain tissue into the plasma membrane 

of Xenopus laevis oocytes

A B

White Light Fluorescent Light White Light Fluorescent Light

Whole Oocyte Tissue Sliced Oocyte
Functional Electrophysiology Analysis 

Goal 3: Perform pyrethroid concentration-dependent TTXsensitive response curves to evaluate age-dependent 

pharmacodynamics differences between juvenile and adult 

rat brain neurolemma injected into Xenopus oocytes

High-Throughput Electrophysiology (TEVC)
Characteristics of Neurolemma-injected Oocytes

Strengths

• Receptors are in their native state within the appropriate lipid environments

• Proper aged-related isoform expression patterns and post-translational modifications

• In vivo relevant analysis of ionotropic and metabotropic receptors

• Functional preparation (e.g., ion channel function and neurotransmitter release)

• More rapid assessment of currents post injection

• Small amounts of tissue are required

• Works with fresh or frozen tissue

• Brain regional differences can be determined

• Species differences (including the use of human brain tissue) can be determined

Limitations

• Preparation results in complex current involving multiple channels and transporters

• Variability in the level of P2 incorporation into the oocyte plasma membrane

• Relatively uncharacterized in terms of the proportion of outside-out versus inside-out 

neurolemma fragment incorporation
Effect of ion channel antagonists on the total ion current (outward) measured 

during depolarization of Xenopus oocytes microtransplanted with rat brain neurolemma 

tissue using two electrode voltage clamp (TEVC) electrophysiological technique
Na+

Na+

Na Ca2+ +

Ca2+

ClAleu, Blasi, Solsona, and Marsal. Eur J Neurosci. 2002

Cao, Shafer, and & Murray. J Pharmacol Exp Ther. 2011 

Membrane

Extracellular

Intracellular

KB-R7943

TTX

Niflumic Acid 

or NPPB

VSSC

CaCl

SOURCE OF OUTWARD CURRENT

Ca2+

Ca2+

VSCC

MVIIC
Sample traces of TTX-sensitive currents illustrating the 

regions where area under the curve (AUC), 

inactivation tau and deactivation tau values are obtained
Experimental Setup - Electrophysiology Assay

Subtract TTX treatment 

trace from insecticide 

trace (gives TTXSensitive trace) 

Average TTX-sensitive 

traces 

Calculate area under the 

curve of averaged traces

Obtain normalized 

percent difference over 

control

Plot dose response curve

Control

1x10-9M

5x10-9M

1x10-8M

5x10-8M

1x10-7M

5x10-7M

1x10-6M

Sham 

Injected

P2 Injected 

(0.5mg/ml)

Biological Rep 1

Step 2: Perfusing

oocytes with 

pyrethroid for 5 

min at 0.45 ml/min

Step 4: Plot the 

normalized response 

vs insecticide 

concentration

Each data point 

is an average of 

18-30 individual 

oocytes

(n > 3)

Biological Rep 2 Biological Rep 3

Step 1: Determine TTX-sensitive current for each treatment group 

using automated TEVC 

-10 -8 -6 -4

0

200

400

600

800

1000

1200

1400

Con

Log [Permethrin, M]

TTX-Sensitive Current

(% Over Control)

Step 3: Average the 

TTX-sensitive 

current from each 

biological rep

Step 1

•Perfusion 1: ND96 (+ Additions)

•Duration: 5 Minutes

Step 2

•Perfusion 2: Treatment (+ Additions)

•Duration: 5 Minutes

Step 3

•Perfusion 3: 1x10-5M TTX (+ Additions)

•Duration: 5 Minutes

Log [Insecticide], M
Indices of 

Toxicity DDT DDE

βMax 1028 N/A

EC50 1.6x10-8 M N/A

R2 0.11 N/A

n 5 1

Positive Control Compounds: DDT vs. DDE

Log [Treatment, M]

Normalized TTX-Sensitive AUC

(% Over Control)

-9 -8 -7 -6 -5

-1000

-500

0

500

1000

1500

2000

2500

3000

NFA

DDT

DDE
No Additions

Effect of increasing concentrations of permethrin (A) or deltamethrin (B) on 

depolarization-evoked, TTX-sensitive outward current associated with rat brain neurolemma

microtransplanted into Xenopus oocytes
With NFA

Effect of increasing concentrations of permethrin (A) or deltamethrin (B) on 

depolarization-evoked, TTX-sensitive inward current associated with rat brain neurolemma

microtransplanted into Xenopus oocytes
With NFA + NPPB

Effect of increasing concentrations of permethrin (A) or deltamethrin (B) on 

depolarization-evoked, TTX-sensitive inward current associated with rat brain neurolemma

microtransplanted into Xenopus oocytes
Key Parameters from Electrophysiology Assay

• EC50 - “Potency” value determined from the plot of Log Pyrethroid

Concentration versus the Area Under the Curve (AUC), which is measured

from the TTX-sensitive current that occurs during oocyte depolarization

• βmax - “Efficacy” value determined as the maximum AUC value

• Inactivation Tau Ratio- τ0.5 value determined from the exponential decay

rate of the late current during inactivation following depolarization in the

presence of 10-6 M pyrethroid / τ0.5 value determined from the exponential

decay rate of the late current during inactivation following depolarization in

the absence of pyrethroid

• Deactivation Tau Ratio-τ0.5 value determined from the exponential decay

rate of the tail current during deactivation following repolarization in the

presence of 10-6 M pyrethroid / τ0.5 value determined from the exponential

decay rate of the tail current during deactivation following repolarization in

the absence of pyrethroid
Potency (EC50)

1x10-10

1x10-9

1x10-8

1x10-7

1x10-6

EC50 (M)

EC50s for either permethrin or deltamethrin are not significantly different

when juvenile versus adult neurolemma are compared

7-fold

less

42-fold

less
Efficacy (β Max) 

 * 

*

*

βmax values for both permethrin and deltamethrin are greater

in adult versus juvenile neurolemma
Tau Value Ratios

Tau value ratios for either permethrin or deltamethrin are not 

significantly different when juvenile and adult neurolemma are compared
Conclusions – Age Related 

Differences? 

• No statistically significant differences in the relative 

potency estimates for permethrin and deltamethrin on 

VSSCs from adult versus juvenile rat brain tissues.

• In each of the assays, both pyrethroids appeared to be 

more efficacious on the adult versus the juvenile 

neurolemma-injected oocytes. 

• The increase in efficacy observed with adult versus 

juvenile rats appears to be due to the increased level of 

expression of VSSCs in adult rat brain.
In vivo versus in vitro relevance 

Literature Research Neurolemma Research

Test

Pyrethroid Bifenthrin Bifenthrin Bifenthrin Bifenthrin Permethrin Permethrin Permethrin

Potency 

Estimate 200 nM 500 nM 853 nM 196 nM 15 nM 200 nM 170 nM

Measured 

Response

Blood 

Concentration

Brain 

Concentration

Blood 

Concentration

Brain 

Concentration

TTX-Sensitive

Current

(No additions)

TTX-Sensitive

Current

(+ NFA)

TTX-Sensitive

Current

(+NFA + 

NPPB)

Reference Scollon et al. 

2011

Scollon et al. 

2011

Gammon et al.

2014

Gammon et al. 

2014
END
Summary of the age-dependent effects (PND 15 vs PND 90) of pyrethroids on the rat 

brain neurolemma microtransplanted into Xenopus oocytes.