# 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 O utcome P athway (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 concentration dependent 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 N eurolemma- injected O ocytes 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 incorporationEffect 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 + 

## Ca 2+



## Ca 2+ 

## Cl Aleu , Blasi , S olsona , 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
Ca 2+



## Ca 2+ VSCC
MVIIC

# Sample traces of TTX-sensitive currents illustrating the regions where a rea u nder the c urve (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 - 9 M 5x10 - 9 M 1x10 - 8 M 5x10 - 8 M 1x10 -7 M 5x10 -7 M 1x10 - 6 M 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 - 5 M TTX (+ Additions) • Duration: 5 Minutes

Log [Insecticide], MIndices of Toxicity DDT DDE β Max 1028 N/A EC 50 1.6x10 - 8 M N/A

R 2 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 i nward 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

• EC 50 - “Potency” value determined from the plot of Log Pyrethroid Concentration versus the A rea U nder the C urve (AUC), which is measured from the TTX- sensitive current that occurs during oocyte depolarization

• β max - “Efficacy” value determined as the maximum AUC value

• Inactivation Tau Ratio - τ 0 . 5 value determined from the exponential decay rate of the late current during inactivation following depolarization in the presence of 10 - 6 M pyrethroid / τ 0 . 5 value determined from the exponential decay rate of the late current during inactivation following depolarization in the absence of pyrethroid

• Deactivation Tau Ratio - τ 0 . 5 value determined from the exponential decay rate of the tail current during deactivation following repolarization in the presence of 10 - 6 M pyrethroid / τ 0 . 5 value determined from the exponential decay rate of the tail current during deactivation following repolarization in the absence of pyrethroid

# Potency (EC 50

# )

1x10 - 10 1x10 -9 1x10 -8 1x10 -7 1x10 -6EC50(M)

EC 50s 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)

*   

## * *

β max 

## 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

# ENDSummary of the age - dependent effects (PND 15 vs PND 90) of pyrethroids on the rat brain neurolemma microtransplanted into Xenopus oocytes.