High Glucose Effects on the Electrical Properties of Hippocampal

Pyramidal Neurons Rylan Urban, and Verónica A. Campanucci

Neural Systems and Plasticity Research Group, Department of Physiology, University of Saskatchewan

ABSTRACT

ACKNOWLEDGMENTS

This study was supported by a NSERC Research Grant to V.C. and a NSERC-USRA scholarship to R.U. Thank you Andrew for all your help, your diverse knowledge and support was much appreciated.

INTRODUCTION

CONCLUSION AND FUTURE DIRECTIONS

RESULTS

Receptor for Advanced Glycation

End Products (RAGE)

3. High glucose did not affect NMDA-evoked currents

Histogram and example traces show the comparison of NMDA-evoked currents

recorded in neurons from wild type mice exposed to control (WT; n=9) or high

glucose (WT + HG; n=6), and from RAGE KO mice exposed to control (RAGE KO;

n=7) or high glucose (RAGE KO + HG; n=10). Further experiments are required to

increase n values for statistical purposes.

METHODS

WT + HG

RAGE KO + HG

More than 285 million people worldwide are affected by diabetes including more

than 9 million Canadians. Diabetes mellitus is characterised by either insulin

deficiency (type 1) or insulin resistance (type 2) causing a rise in plasma glucose

levels. Diabetes causes damage to many bodily systems including the central

nervous systems (CNS). Diabetes has been linked to white matter lesions, brain

atrophy and infarcts, global brain volume loss, and decline in cognitive function

increasing the risk of dementia and Alzheimer’s disease by up to 65%. Diabetes

negatively affects memory and learning suggesting the hippocampus as a target of

CNS pathology.

Primary Hippocampal Cultures

• Hippocampi were cultured from male wild type or RAGE KO

mice at 0-3 days postnatal (Falzone et al. 2009).

• Cells were plated on laminin coated cover slips and kept in a

humidified 5% CO2 incubator at 37⁰C.

• Cultures were fed supplemented Neurobasal media and

replaced every 3-4 days. To imitate hyperglycemic conditions,

25mM glucose was added to the culture media 7 days after culture

for a additional week.

Whole Cell Patch Clamp Recordings

• Electrophysiological recordings were performed 14-17 days after

culturing.

• Action potentials were invoked by injecting a series of 100pA

depolarizing currents steps for 500ms. Resting membrane

potentials were recorded 60 seconds after whole cell attachment.

to study cellular responses to specific stimuli in high glucose and normal

physiological conditions. We also take advantage of a RAGE knock-out (RAGE KO)

mouse model lacking RAGE expression in order to test its potential role in cellular

hippocampal abnormalities caused by high glucose conditions.

The receptor for advanced glycation end products

(RAGE) is a multi-ligand receptor that has been

shown to play a central role in the pathology of the

CNS in diabetes, particularly in cognitive impairment.

RAGE activation correlates with the generation of

oxidative stress and inflammation in the hippocampus

of rodents leading to brain damage.

In the present study we look at the effects of high

glucose on cultured mouse hippocampal pyramidal

neurons. We use patch clamp electrophysiology RAGE structure (DeLano Scientific LLC.)

Preliminary findings summarized here show that exposure of cultured neurons to

high glucose conditions caused a decrease in excitability, which correlates with an

increased in the resting membrane potential. Both changes mediated by high

glucose conditions were prevented in neurons from RAGE KO mice.

Our preliminary findings also suggest that decreased neuronal excitability could

have a direct impact on memory and learning impairment associated with diabetes.

It should be noted that cells lacking functional RAGE under normal physiological

conditions were less excitable than wild type cells exposed to high glucose. More

work in this area is needed to fully understand these results.

NMDA receptor currents seemed to be unaffected by high glucose conditions at

50µM concentration. Since NMDA receptors play a pivotal role in memory and

learning it is curious as to why little difference was detected. It is possible that not all

cellular NMDA receptors were sampled due to insufficient NMDA concentration

needed for saturation. More work in this area is needed.

HYPOTHESIS

High glucose causes abnormalities in NMDA-evoked currents and action potential

generation in cultured pyramidal neurons through a RAGE-mediated pathway.

A

B

C

Patch Pipette

Pyramidal Neuron

3 Barrel Perfusion Pipette

500µm Glial Cells

200µm

• During NMDA recordings, cells

were clamped at -60mV and let

to stabilize for 5 minutes before

NMDA application.

• 50µM NMDA was applied for 1

second every 15 seconds using

a pressurized Fast Step

Perfusion System set for 2.0 PSI.

• Control solution was washed

over the cell at all other times

during the recordings.

Control Solution

1. Hippocampal pyramidal neurons exposed to high glucose conditions seem to show decreased excitability in neurons from wild type mice but less so in neurons from RAGE KO Mice

Injection of depolarizing current for 500ms at 100pA increasing steps (10 steps) induced the generation of action potentials. Action potential generation peaked at 150pA and declined with further injection of current (A). 1 week of high glucose conditions caused a reduction in action potential generation during the injection of current >150pA and 50pA, this effect was much smaller in neurons from RAGE KO mice. Histogram (B) and example traces (C) show the comparison of action potentials generated in cultures from wild type mice exposed to control conditions (WT; n=15), wild type mice exposed to high glucose (WT + HG; n=11), and RAGE KO mice exposed to high glucose (RAGE KO + HG; n=17) by the injection of 250pA of current. Further experiments are required to increase n values for statistical purposes.

50µM NMDA

Diabetes has been link to cognitive impairments, however the molecular mechanisms underlying this deleterious effect is poorly known. Here we studied electrical properties of cultured hippocampal pyramidal neurons exposed to high glucose or normal physiological conditions using the whole cell patch clamp technique. Neurons exposed to high glucose conditions had significantly more negative resting membrane potentials than control neurons. This effect was prevented when using neurons from RAGE knock-out (KO) mice were used. Quantification of neuronal excitability by injection of depolarizing current shows that neurons in high glucose were slightly less excitable than those in control conditions. We also found that RAGE-KO neurons were slightly more excitable in high glucose than wild type neurons in high glucose. In addition, quantification of NMDA-evoked currents from wild type and RAGE-KO neurons did not show differences between the two experimental conditions. Our preliminary data indicates that high glucose may effect neuronal excitability by a RAGE-mediated pathway.

500pA

2.0s

WT

RAGE KO + HG

WT + HG

-60mV

-60mV

-60mV

250pA

2. High glucose conditions cause a more negative resting membrane potential in neurons from wild type mice but not

in neurons from RAGE KO mice

We recorded the resting membrane potential of pyramidal neurons from wild type or RAGE KO mice in control or high glucose

conditions. High glucose induced a more negative resting membrane potential in neurons from wild type but not in neurons from

RAGE KO mice.

References

[1] Manschot, S.M.; Brands, A.M.A.; Van Der Grond, J; Kessels, R.P.C.; Algra, A.;

Kappelle, L.J.; Biessels, G.J. (2006) Brain magnetic resonance imaging correlates

of impaired cognition in patients with type 2 diabetes. Diabetes. Vol.55(4), p.1106(8)

[2] Ramasamy, R.; Vannuci, S.J.; Du Yan, S.S.; Herold, K.; Yan, S.F.; Schmidt,

A.M. (2005). Glycation end products and RAGE: a common thread in aging,

diabetes, neurodegeneration, and inflammation. Glycobiology. Vol.15(7), p. 16(12)

[3] http://www.diabetes.ca/

[4] Falzone, T.; Stokin, G.B.; Lillo, C.; Rodrigues, E.M.; Westerman, E.L.; Williams,

D.S.; Goldstein, L.S.B. (2009) Axonal Stress Kinase Activation and Tau Misbehavior

Induced by Kinesin-1 Transport Defect. J. of Neurosci. Vol 29(18), p.5758(9) Statistical comparison by ANOVA; * p < 0.05; ** p < 0.01

WT WT + HG RAGE KO RAGE KO + HG

Resting Membrane Potential -53.9mV** -58.8mV -55.3mV -55.2mV*

N Value 17 13 14 18