1. An in vitro model to study brain tissue recovery
Andrei V. Gourov ⇑
, Bridget Curran
SUNY Downstate Medical Center, 450 Clarkson Avenue, Box 25, Brooklyn, NY 11203, USA
a r t i c l e i n f o
Article history:
Received 5 January 2014
Accepted 2 March 2014
a b s t r a c t
Brain tissue slices can be maintained within metabolically stable conditions for long periods of time
(hours). This experimental setting has been productive for investigating long-term neural function
in vitro. Here, we utilize this experimental approach to describe the recovery of functional connectivity
in slices from the mouse hippocampus. Hippocampal slices were cut up bisecting the CA1 region (parietal
cut) and each severed half placed adjacent to the other. Stimulation and recording electrodes were placed
on each side of the cut; with one electrode stimulating one hemi-slice (20 V, 0.033 Hz) and the other elec-
trode recording the evoked response from the adjacent hemi-slice. As expected, no evoked response was
observed shortly after the beginning of stimulation. However, 20–40 min after the initiation of stimula-
tion a large depolarization signal was detected. Right after that, fiber volley potentials were observed in
the adjacent hemi-slice. After 1 h excitatory postsynaptic potentials (EPSP) were detected. Based on this
observation, we hypothesize that recovery of functional connectivity is enhanced by constant delivery of
electrical pulses at low frequency to the damaged neural tissue. The described in vitro slice system may
become a very suitable experimental method to investigate strategies to enhance the recovery of neural
connectivity after brain injury.
Published by Elsevier Ltd.
Introduction
In vitro preparations that allow the incubation and recording
from brain tissue slices have been crucial to understanding the
functional properties of neural systems [1,2,8]. A widely used brain
tissue slice preparation is obtained from transversal sections of the
dissected rodent hippocampus; commonly known as the hippo-
campal slice [17,19] The synaptic circuits within a hippocampal
slice are well defined and allow detailed examination of synaptic
transmission [3,11,14]. For instance, in the stratum radiatum re-
gion of area CA1 of the hippocampal slice, one can examine the
conduction of electrical activity between axon fibers from presyn-
aptic neurons in the CA3 area that synapse to dendritic spines of
postsynaptic neurons in the CA1 area [4,12,18].
Depending on incubation and experimental settings, there is a
workable time range (usually hours) that experimentation can be
reliably carried out on hippocampal slices [13,16,20]. Slices that
are being electrically stimulated and recorded from can show
unfaltering neural responses (e.g. EPSP) after hours of experimen-
tation. Interestingly, there is evidence that slices that have been
incubated for the same amount of time but that have not been
electrically stimulated show poor or short-lasting responses.
Though there is a selection bias, as one makes recording from slices
showing the strongest responses, the idea is that slices that are
being experimented on (i.e. stimulated and recorded) last longer
than un-stimulated slices. In addition, as time progresses, it is com-
monly observed a different change in tonicity and opacity of stim-
ulated versus non stimulated hippocampal slices. Supporting the
notion that electrical stimulation maintains the function of neural
tissue, there is experimental evidence indicating the usefulness of
electrical stimulation to help the recovery of neural tissue [6,7,15].
Here, we speculated on the utilization of hippocampal brain
slices from the mouse as experimental model to study the impact
of electrical stimulation treatment on the recovery of functional
neural connectivity. We hypothesized that constant delivery of
pulses of electrical activity is key to the recovery process and
reconnection of damaged neural tissue. Our preliminary data sug-
gest this may be so by showing that regular low frequency electri-
cal stimulation across a cut hippocampal slice promotes the
recovery of functional synaptic connectivity.
Methods
Hippocampal slice preparation
Transverse hippocampal slices (400 lm) were obtained from
adult (C57BL/6NTac, 2 months old) mice. We used one experimen-
tal group contains 8 mice. All procedures were performed in com-
pliance with the Institutional Animal Care and Use Committee of
http://dx.doi.org/10.1016/j.mehy.2014.03.001
0306-9877/Published by Elsevier Ltd.
⇑ Corresponding author. Tel.: +1 347 407 4535.
E-mail address: andrei.gourov@downstate.edu (A.V. Gourov).
Medical Hypotheses 82 (2014) 674–677
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Medical Hypotheses
journal homepage: www.elsevier.com/locate/mehy

2. the State University of New York, Downstate Medical Center. Slices
were cut in ice cold artificial cerebrospinal fluid (ACSF containing:
(mM) 119 NaCl, 4.0 KCl, 1.5 MgSO4, 2.5 CaCl2, 26.2 NaHCO3, 1
NaH2PO4 and 11 Glucose saturated with 95% O2, 5% CO2) and then
placed into the incubation chamber bathed with oxygenated ACSF
at 35 °C.
Slice stimulation and recording
Immediately after dissection slices were given a cut. The typical
heating and equilibration were not performed and slices were di-
rectly placed into the interface chamber with O2/CO2 (95%/5%,
respectively) aeration. Stimulation and recording electrodes were
places in to the striatum radiatum area. A pair of stimulation (bipo-
lar; FHC & Co., ME, USA) and recording (borosilicate glass pipette
filled with ACSF; 5–10 mX) electrodes were used to evoke and re-
cord extracellular EPSP. They were placed in the slice in the CA1
stratum radiatum area adjacent to each other on either side of
the cut. Test pulse duration was 50 ls; test pulse intensity was
set at 20 V and test sampling was at 0.033 Hz (once every 30 s).
Results
The functional properties of hippocampal slices can be main-
tained for many hours under our experimental setting. We have
observed that the tonicity and opacity of the tissue are good indi-
cators and predictors of the functional state of the slice. Often, the
visual appearance of the area of tissue subjected to electrical stim-
ulation and recording, which is functionally active, differs from
other regions within the same slice (Fig. 1A), as well as from tissue
of un-stimulated slices (Fig. 1B).
Based on the assumption that electrical activity maintains neu-
ral tissue functional, we tested the idea that a severed hippocampal
slice can regain functional connectivity if subjected to regular
pulses of electrical stimulation.
We examined this possibility by electrically stimulating axon fi-
bers from presynaptic CA3 neurons and measuring the evoked pre-
synaptic fiber activation response (presynaptic fiber volleys) and
the postsynaptic EPSP responses in the stratum radiatum region
of area CA1 [5,21]. A hippocampal slice from the mouse was cut
along its parietal axis bisecting the CA1 area (Fig. 2).
After being cut, both hemi-slices were placed adjacent to each
other. Stimulation and recording electrodes were placed in the
stratum radiatum of area CA1of and cut slices. For the cut slice,
the stimulation electrode was placed in the striatum radiatum area
of one hemi-slice and the recording electrode was placed about the
same region of the stratum radiatum in the other hemi-slice to
collect evoked responses. For control and cut slices electrical stim-
ulation consisted of square pulses (50 ls) of 20 V at 0.033 Hz (once
every 30 s). The experiments were performed 3 times. Initially the
recording showed artifacts that reflects impulse of stimulation.
After 20–40 min huge depolarization signals have been observed.
Next sweep after this signal express fiber volley responses with fol-
lowing EPSP recovering (Fig. 3).
We make a proposition that during the electrical stimulation
the neurons are able to accumulate the potential and discharge
it. This phenomenon possibly shows support for the fusion of pre-
viously disrupted membranes followed by the recovery of connec-
tions between neurons.
As we reported previously, hippocampal slices recover normal
physiological conditions about 2 h after dissection [9]. Consis-
tently, intact (control) slices showed a fast exponential rise in the
amplitude of the evoked EPSP over time; as expected for normal
recovery of slices (Fig. 4).
Remarkably, the cut slice also showed an increase in evoked
EPSP amplitude, though about 10-fold weaker than the intact slice
and with a linear rather than an exponential rise. The expression of
EPSP responses in the intact slice was rapidly apparent shortly
after the beginning of the stimulation and grew stronger with time.
Hypothesis
Our preliminary data suggest that severed connections across
the mouse hippocampal slice can regain functional connectivity
after delivery of regular pulses of electrical stimulation to the tis-
sue. It is attractive to speculate that electrical activity maintains
the normal functioning of excitable membranes and neuronal
Fig. 1. (A) Mice hippocampal slice after 2 h of electrical stimulation and EPSP recording. Recording (left) and stimulation (right) electrodes are placed in the CA1 stratum
radiatum area (traced). Note the space between electrodes is more opaque than adjacent areas (arrow). (B) Mice hippocampal slice without electrical stimulation after 2 h.
The texture of the tissue looks more homogenous.
Fig. 2. Representation of the parietal cut in a hippocampal slice. The arrows point
approximate placement of electrodes on the slice. The distance between the
electrodes is about 100–150 microns.
A.V. Gourov, B. Curran / Medical Hypotheses 82 (2014) 674–677 675

3. function; an idea that resonates with the common notion of ‘‘use it
or lose it’’. It follows, therefore, that electrical treatments that
recruit membrane activity and associated cellular functions may
play an important role in the repair of damaged neural tissue.
However, perhaps not all kinds of electrical stimulation will work.
For instance, continuous or too strong an electrical stimulation
may exacerbate the damage to the tissue (i.e. favoring apoptosis).
Disrupted connectivity after parietal cut of the hippocampal slice
may be recovered upon selective electrical stimulation that pro-
motes processes that allow the fusing of membranes.
Based on our knowledge of the physiology of the hippocampal
slice, we speculate that regular but discrete pulses of electrical
stimulation delivered at low frequency are fundamental to the
recovery of functional connectivity. It would be important to test
this speculation and determine the biochemical (e.g. enzymatic)
mechanisms that are modulated by electrical stimulation that
promote tissue recovery. It is worth mentioning that the type of
stimulation electrode used in this study generates an ellipsoid-like
‘‘field potential’’ that emanates from the tip of the electrode and
propagates outwards over the tissue. This type of electrical field
is likely to generate micro magnetic fields that might also play
an important role in tissue recovery.
We speculated that at several mechanisms may actively
regulate the regaining of functional connectivity. On the one hand,
electrical stimulation may favor the recovery and consolidation of
synapses according to the ‘‘Hebbian’s postulate’’, that synaptic
coactivity strengthens synaptic function [10]. On the other, there
may be a ‘‘top-to-bottom’’ mechanism that regulates synaptic
recovery. We proposed that widespread activity in a neuronal pop-
ulation generates oscillatory waves of neural activity that provide
recurrent downstream activation of synapses to strengthen their
connections between groups of neurons (Fig. 5).
In addition to our hypothesis, we like to highlight the
description of an in vitro slice system that may be a very suitable
experimental method to investigate strategies to enhance the
recovery of neural connectivity after brain injury.
Fig. 3. (A) Amplitude of evoked responses (voltage in logarithmical scale) recorded from cut slices over a period of 150 min. Three different examples are shown (runs 1–3). In
each run, an increase in response amplitude occurred between 20 and 40 min of recording (arrows). (B) Representative trace of a big depolarization event (red) observed at
the times indicated by arrows in A. Prior traces are shown in black (examples are from run 1). In all runs, the depolarization event preceded EPSP appearance. (C)
Representative traces from run 1 at different recording times: 0.5 min (sweep 1), 31 min (sweep 62), 31.5 min (sweep 63) and 129 min (sweep 258). While no discernible
responses were observed during the first 30 min of recording (sweep 1–61), after the depolarization event (sweep 62) presynaptic fiber volley responses followed by
increasingly bigger EPSP responses were observed (sweeps 63 and 258). (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)
Fig. 4. (A) Normal recovery of an intact hippocampal slice shows an exponential rising of the EPSP amplitude in the stratum radiatum. (B) Recovery in a cut hippocampal slice
shows a modest linear increase in EPSP amplitude in the stratum radiatum. The recovery was measured by calculation of percent bios between two equal parts of the graphs.
A-table shows significant increase EPSP amplitude (343.99%) on intact slice. B-table shows that recovery EPSP cut slices is 22.54%. The measurements of the cut slice were
carried out started after ‘‘depolarization signal’’.
676 A.V. Gourov, B. Curran / Medical Hypotheses 82 (2014) 674–677

4. Conflict of interest
None declared.
Acknowledgements
I would like to thank Professor Juan Marcos Alarcon (SUNY
Downstate, Department of Pathology) for his contribution to the
work, equipment provided and theoretic correction of the concepts
proposed in the article.
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Fig. 5. Recovery of functional connectivity after parietal cut of the hippocampal slice. (A) Connectivity in an intact hippocampal slice. (B) Disrupted connectivity is recovered
after regular delivery of electrical stimuli that allows the hypothetical fusing of membranes from separate fibers. Insets represent the pattern of EPSP responses increase
during slice recovery for normal (frame A) and cut (frame B) conditions. Note the oscillatory versus linear rise pattern in the intact and cut slice, respectively.
A.V. Gourov, B. Curran / Medical Hypotheses 82 (2014) 674–677 677