UT Southwestern - NSF SURF Poster Session - Aug 7, 2015 Juan Pascual Rare Brain Disorder Clinic, Department of Neurology and Neuropediatrics

1. -10 -5 5 10 15 20
-20
20
40
60
80
100
Time(min)
%change
-10 0 10 20 30 40 50 60 70 80 90 100
-0.5
0
0.5
Volts(mv)
Milliseconds
Evoked action potential
Sensory Evoked Cortical Function
in Pyruvate Dehydrogenase Deficient Mice
Sandra Field1,2, Vikram Jakkamsetti2, Qian Ma2 and Juan Pascual2
Neuroscience Department, University of Texas at Dallas, Richardson, TX1
Department of Neurology and Neuropediatrics, UT Southwestern Medical Center, Dallas, TX2
Introduction
• Brain function requires glucose.
• Glucose is converted into glutamate in the
Krebs cycle.
Neurophysiology Data Analysis:
Analysis was done offline using
programs written in MATLAB.
Discussion and Significance
Methods
Neurons appear to lag in
time when firing after
stimuli in PDHD.
Supported by:
NSF SURF grant (SRF)
NIH grant 5R01NS077015-04 (JMP)
Evoked Action Potentials
Time Profile of Acetate Modulation
The effect of acetate is
projected to peak at 45
min and last about an
hour. Research still
being done in order to
add more numbers.
References
1. Lioudmila P, Hausknecht K, Stachowiak M, Dlugos C, Richards J, Patel M, Cerebral developmental
abnormalities in a mouse with systemic pyruvate dehydrogenase deficiency. PLoS One. 2013 Jun
26;8(6):e67473.
2. Marin-Valencia I, Good L, Ma Q, Malloy C, Patel M, Pascual J, Cortical metabolism in pyruvate
dehydrogenase deficiency revealed by ex vivo multiplet C-NMR of the adult mouse brain. Neurochemistry
International. 2012 Dec;61(7).
3.Möhler H, Pathophysiological aspects of diversity in neuronal inhibition:
a new benzodiazepine pharmacology. Dialogues Clinical Neuroscience. 2002 Sep;4(3):261-9.
4. Sun Q, Huguenard J, Prince D, Barrel cortex microcircuits: thalamocortical feedforward inhibition in spiny
stellate cells is mediated by a small number of fast-spiking interneurons. Journal of Neuroscience. 2006
Jan 25;26(4):1219-30.
5. Gabernet L, Jadhav S, Feldman D, Carandini M, Scanziani M., Somatosensory integration controlled by
dynamic thalamocortical feed-forward inhibition. Neuron. 2005 Oct 20;48(2):315-27.
Acetate Modulation
Acetate decreases lag
time of neural firing in
PDHD mice.
• Pyruvate dehydrogenase deficiency (PDHD)
consequences include cortical atrophy, seizures,
and intellectual disability.
Pyruvate
Dehydrogenase
Pyruvate
Glucose
Citrate
α-ketoglutarate
Succinate
Oxaloacetate
Acetyl-CoA
Krebs Cycle
Peak Latency
Glutamate
Acknowledgements
First and foremost I thank my mentor Dr. Juan Pascual for all
his support and direction, and my postdoc Vikram Jakamsetti
for all his advice. Thanks to Qian Ma for her work in
designing our mice, and to the other Pascual Lab members
who helped me learn, Karthik Rajasekaran, and Levi Good.
Thanks to Nancy Street, Vanessa Powell, and the NSF SURF
program. Additionally, thanks to all my teachers and mentors
in the past without whom this would not have been possible.
GFAP-Cre-Lox knockdown mice: A transgenic mouse that
expresses a chromosome containing Lox flanking the DNA
sequence which codes for PDH was employed. This system
reduces PDH activity in cortical neurons and astroglia by at
least 50%. Male and female C57BL6 mice of each
genotype aged P21-P27. For each assay, control or PDH
mutant groups were recorded from, and control mice utilized
were littermate controls. Each mouse contributed to one
recorded site.
Acute Surgery: Mice were anesthetized using a
combination of ketamine, xylazine, and acepromazine
followed by exposure of left barrel cortex via craniotomy
posterior to the bregma.
Electrophysiology: Stimulation was delivered at whisker
pad, and local field potential and action potential recordings
were made in layer IV at 500 µm using an activated iridium
oxide electrode with impedance 1.0 – 1.5 MΩ. Whisker pad
stimulation (2 – 3 mA) was delivered by programmable
pulse Master-8 stimulator receiving instructions via
programs written in MATLAB. Each stimuli was 2
milliseconds and was followed by the next at 1, 2, 5 or 15
second interval on a semi-random basis. Responses were
amplified and filtered with a MultiClamp 700B
programmable amplifier and digitized with Digidata 1440A.
Objectives
• Investigate cortical function in mouse model of
PDHD. Deliver physiologically relevant whisker
stimulation and compare cortical responses.
• Observe changes in response when acetate is
used to drive the Krebs cycle.
Acetate – A baseline of 10 to 12
minutes was recorded for each
animal, after which intraperitoneal
acetate was delivered and
response monitored.
0 20 40 60 80 100 120
0
2
4
6
8
10
12
14
0 20 40 60 80 100
0
50
100
150
200
Trial#
0 20 40 60 80 100
0
50
100
150
200Control n=1
Neuronal jitter - spike-timing dispersion.
Interneurons and inhibition affect jitter.
Neural precision = 1/jitter
Lag in peak latency.
Erratic and inconsistent.
Spike times are time-locked to stimuli
0 20 40 60 80 100 120
0
2
4
6
8
10
12
14
Peak latency
Control PDHD
ActionPotentialsBaseline
0
0.1
0.2
0.3
0.4
0.5
0.6
Response Strength
ActionPotentialsBaseline
Control n=2
PDHD n=3
Acetate increases
response strength in
PDHD mice
Response strength was
calculated as total number
of responding action
potentials per stimuli.
This shows PDHD
animals might have less
response than control
animals.
ActionPotentials
%∆fromBaseline
Time (Min)
Control n=1
PDHD n=1
0
10
20
30
40
50
milliseconds
p=0.2
Control n=5
PDHD n=2
Control n=2
PDHD n=2
W
T
K
O
0
10
20
30
milliseconds
*
Control n=5
PDHD n=2
Milliseconds
Control PDHD
Standard Deviation of Spike-Timing (Jitter)
PDHD n=1
Neuron firing is more
erratic in time (i.e. less
time-locked to stimulus)
in PDHD mice which
show about 150% more
jitter than control..
Low response strength: The baseline in PDHD mice shows
low response strength. The weakness is linked to the
deficiency of pyruvate dehydrogenase which prevents
pyruvate from entering the Krebs Cycle and generating
glutamate. Lack of glutamate available for release at
synapses could cause low response strength.
Acetate improves function: Even with a thinner cortex and
a 50% reduction in PDH activity in cortical neurons and
astroglia, there was improvement in cortical response
strength and speed with the introduction of acetate. This
could be helpful in reducing seizures and enhancing
intellectual ability.
Feed-forward
inhibition
Feed-back inhibition
Abnormal spike timing and
precision: Aberrant feed-forward
inhibition could contribute to
problems in neuronal firing times.
Neural precision in PDHD is lost
and can contribute to abnormal
processing of sensory stimuli.
Future Directions
 Increase numbers for statistical significance
 Compare intracortical action potential response to fast
repeated stimuli (temporal response properties)
Baseline
Baseline
Acetate Modulation
Control n=2
PDHD n=2
Time (min)
Milliseconds
Control PDHD
Jitter increases from
baseline as anesthetic
wears off. It is much
less than the control, so
is considered an
improvement.
Stimulus artifact
Baseline
Acetate Modulation
-10 -5 5 10 15 20
-50
50
100
150
Time (mins)
%change
frombaseline
p=0.1
Time (Min)
ActionPotentials
%∆fromBaseline
p=0.1
Control n=2
PDHD n=3
#ofactionpotentials
Results
30
0
20
10
50
100
150
-50
-10 -5 5 10 15 20
Milliseconds
-10 -5 5 10 15 20
-60
-40
-20
20
40
Time(min)
%change%Change
Time (min)