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soma. The axon region was rapidly stretched via mechanical param-
eters based on clinical TBI. Taxol was applied either 30 mins before
injury, 5 or 15 mins post-injury. Immunocytochemical analysis was
performed at 1, 24, 48 hrs post-injury for Ankyrin-G, total-tau and
phospho-tau AT8, AT270 and S404. In addition, calcium influx using
the calcium indicator Fluo-4 and the number of surviving axons was
assessed.
Results:
Microtubule stabilization with Taxol prior to TAI resulted
in: 1) apparent maintenance of the axon initial segment (AIS) diffu-
sion barrier, 2) decrease in somal total-tau and phospho-tau accu-
mulation, beginning within 1 hr post injury and continuing though
48 hrs, 3) a substantial mitigation of intra-axonal increases in calcium
concentration. While post-injury treatment did not dramatically in-
fluence these outcomes, like pre-treatment, it did decrease in the
number of degenerating axons observed at 24 hrs.
Conclusions:
These in-vitro data demonstrate that enhancing ax-
onal microtubule stability can substantially improve outcome and
axon survival after TAI. Taxol protection of the AIS barrier may
account for reduced accumulation of tau in the cell-soma. In addi-
tion, Taxol treatment mitigates progressive calcium influx into axons
after injury, potentially by maintaining cytoskeletal structure and
reducing ion-channel dysregulation. Moreover, the observation that
microtubule stabilization after injury promotes axon survival, sug-
gests that taxane treatment may have potential therapeutic value for
clinical TBI.
Acknowledgments
Supported by DOD grant, PT110785 and NIH grant NS056202.
Keywords: traumatic axonal injury, phosphorylated tau, calcium
B3-09
FUNCTIONAL ALTERATIONS IN INTRINSIC AND SYNAP-
TIC PROPERTIES 1 MONTH AFTER MILD TRAUMATIC
BRAIN INJURY
Kimberle Jacobs
, Jianli Sun
Virginia Commonwealth University, Anatomy & Neurobiology,
Richmond, USA
Mild traumatic brain injury (mTBI) produces long lasting cognitive
dysfunction without cell death but with diffuse axonal injury. Using
YFPh mice allows identification of the axonal condition of layer V
pyramidal neurons in somatosensory cortex after central fluid per-
cussion injury. Using this paradigm with 1–2 day survival period, we
have previously shown alterations in both intrinsic and synaptic
properties of intact (IT) and axotomized (AX) neurons. Here we
examined these properties at 32–40 days. Excitatory post synaptic
currents (EPSCs) and inhibitory synaptic currents (IPSCs) were re-
corded in voltage-clamp mode at
-
50 mV and
+
10 mV respectively
QX-314 in the patch solution). Measures are reported as mean
–
SEM, and compared with t-tests, with n
‡
9 cells and significance at
p
<
0.05.
The action potential amplitude was increased in both AX and IT
neurons at 32–40 days. The percentage of intrinsically bursting (IB)
neurons was previously shown to be decreased at 2 days in the
IT population, with this longer survival period the percentage
of IB neurons showed a trend toward being increased in the IT
(50%, z-test, p
=
0.054) compared to sham (17%) with AX neurons
at 36%.
The frequency of spontaneous (s) EPSCs was significantly de-
creased in the IT population compared to both control and TBI AX
neurons. Neither area nor amplitude of sEPSCs were altered. The
miniature (1 mM TTX in bath) EPSCs showed the same trend,
suggesting that there may be a compensatory reduction in the
number of excitatory synapses on intact neurons. There was no
change in the frequency, amplitude or area of sIPSCs in either AX or
IT compared to controls. However, the frequency of mIPSCs was
significantly decreased in IT neurons compared to controls. The ratio
of the frequency of excitatory to inhibitory events (E/I
R
) for spon-
taneous events was significantly lower in IT neurons compared to
control cells. The E/I
R
for miniature events was not different from
control.
These data suggest that some recovery occurs in the network after
the initial hyperexcitability and that compensatory activity-lowering
mechanisms take place in IT neurons.
Supported by NIH-NS077675.
Keywords: EPSCs, IPSCs, Intrinsic properties, action potential
B3-10
ALTERED GABAERGIC SYNAPTIC TRANSMISSION IN
LAYER V PYRAMIDAL NEURONS AFTER MILD TRAU-
MATIC BRAIN INJURY
Xiaotao Jin
, Kimberle Jacobs
Virginia Commonwealth University, Anatomy & Neurobiology,
Richmond, USA
Traumatic brain injury (TBI) is a major cause of disability and
sometimes produces epilepsy. We recently demonstrated epilepti-
form fields after a mild injury in mouse in which layer V pyramidal
neurons had an increased frequency of miniature-excitatory-post-
synaptic-currents. Since network excitability is also affected by in-
hibition, here we examined overall inhibition and that due to
optogenetically selective activation of somatostatin-containing GA-
BAergic interneurons(SS). Mice selectively expressing channelrho-
dopsin in SS were given a mild, central fluid percussion or sham
injury. Whole cell patch clamp was used to record spontaneous,
electrically evoked and light-evoked inhibitory postsynaptic currents
(s-, e-, l- IPSCs) in layer V pyramidal neurons, 1–2 days after injury
with glutamate receptor antagonists in the bathing medium. The s-
IPSC frequency was significantly increased (9.7
–
1.0 for 6 TBI and
6.0
–
1.6 Hz for 6 control neurons, t-test, p
<
0.05), while amplitude
was unchanged. The e- and l-IPSCs were recorded across a series of
5 intensities, induced with increasing duration of stimulation located
*
100
l
m lateral to the recorded neuron. For evoked IPSC com-
parisons, repeated measures ANOVAs were employed. The peak e-
IPSC was not significantly different between control and TBI (n
=
11
control and 9 TBI). Light was applied to activate channelrhodopsin
through the 60X objective, positioned over the electrical-stimulating
electrode. The l-IPSC was significantly greater in TBI compared to
controls (n
=
16 control and 9 TBI, p
<
0.05). In some of the same
neurons, light and electrical stimulation were applied simulta-
neously. While in controls the addition of the light did not signifi-
cantly change the peak IPSC, in TBI neurons light significantly
suppressed the IPSC. Direct comparison between control and TBI
neurons for the condition of electrical
+
light stimulation showed a
significantly reduced IPSC peak in TBI neurons (n
=
5 neurons each
in control and TBI, p
<
0.05). Together these results suggest that SS
interneuron output is enhanced after TBI, but that the network
consequence of this may be to produce disinhibition through the
suppression of output from other inhibitory cell types. Supported by
NIH grant NS077675.
Keywords: Optogenetics, channelrhodopsin, somatostatin inter-
neurons, cortical interneurons, IPSCs
A-53