Medical Hypotheses 82 (2014) 674–677

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Medical Hypotheses

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An in vitro model to study brain tissue recovery

http://dx.doi.org/10.1016/j.mehy.2014.03.0010306-9877/Published by Elsevier Ltd.

⇑ Corresponding author. Tel.: +1 347 407 4535.E-mail address: andrei.gourov@downstate.edu (A.V. Gourov).

Andrei V. Gourov ⇑, Bridget CurranSUNY 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 2014Accepted 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 functionin vitro. Here, we utilize this experimental approach to describe the recovery of functional connectivityin slices from the mouse hippocampus. Hippocampal slices were cut up bisecting the CA1 region (parietalcut) and each severed half placed adjacent to the other. Stimulation and recording electrodes were placedon 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 wasobserved 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 inthe adjacent hemi-slice. After 1 h excitatory postsynaptic potentials (EPSP) were detected. Based on thisobservation, we hypothesize that recovery of functional connectivity is enhanced by constant delivery ofelectrical pulses at low frequency to the damaged neural tissue. The described in vitro slice system maybecome a very suitable experimental method to investigate strategies to enhance the recovery of neuralconnectivity after brain injury.

Published by Elsevier Ltd.

Introduction

In vitro preparations that allow the incubation and recordingfrom brain tissue slices have been crucial to understanding thefunctional properties of neural systems [1,2,8]. A widely used braintissue slice preparation is obtained from transversal sections of thedissected rodent hippocampus; commonly known as the hippo-campal slice [17,19] The synaptic circuits within a hippocampalslice are well defined and allow detailed examination of synaptictransmission [3,11,14]. For instance, in the stratum radiatum re-gion of area CA1 of the hippocampal slice, one can examine theconduction of electrical activity between axon fibers from presyn-aptic neurons in the CA3 area that synapse to dendritic spines ofpostsynaptic neurons in the CA1 area [4,12,18].

Depending on incubation and experimental settings, there is aworkable time range (usually hours) that experimentation can bereliably carried out on hippocampal slices [13,16,20]. Slices thatare being electrically stimulated and recorded from can showunfaltering neural responses (e.g. EPSP) after hours of experimen-tation. Interestingly, there is evidence that slices that have beenincubated for the same amount of time but that have not beenelectrically 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 arebeing experimented on (i.e. stimulated and recorded) last longerthan 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 thenotion that electrical stimulation maintains the function of neuraltissue, there is experimental evidence indicating the usefulness ofelectrical stimulation to help the recovery of neural tissue [6,7,15].

Here, we speculated on the utilization of hippocampal brainslices from the mouse as experimental model to study the impactof electrical stimulation treatment on the recovery of functionalneural connectivity. We hypothesized that constant delivery ofpulses of electrical activity is key to the recovery process andreconnection 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 therecovery of functional synaptic connectivity.

Methods

Hippocampal slice preparation

Transverse hippocampal slices (400 lm) were obtained fromadult (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

Fig. 2. Representation of the parietal cut in a hippocampal slice. The arrows pointapproximate placement of electrodes on the slice. The distance between theelectrodes is about 100–150 microns.

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the State University of New York, Downstate Medical Center. Sliceswere cut in ice cold artificial cerebrospinal fluid (ACSF containing:(mM) 119 NaCl, 4.0 KCl, 1.5 MgSO4, 2.5 CaCl2, 26.2 NaHCO3, 1NaH2PO4 and 11 Glucose saturated with 95% O2, 5% CO2) and thenplaced into the incubation chamber bathed with oxygenated ACSFat 35 �C.

Slice stimulation and recording

Immediately after dissection slices were given a cut. The typicalheating 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 wereplaces in to the striatum radiatum area. A pair of stimulation (bipo-lar; FHC & Co., ME, USA) and recording (borosilicate glass pipettefilled with ACSF; 5–10 mX) electrodes were used to evoke and re-cord extracellular EPSP. They were placed in the slice in the CA1stratum radiatum area adjacent to each other on either side ofthe cut. Test pulse duration was 50 ls; test pulse intensity wasset 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 haveobserved that the tonicity and opacity of the tissue are good indi-cators and predictors of the functional state of the slice. Often, thevisual appearance of the area of tissue subjected to electrical stim-ulation and recording, which is functionally active, differs fromother regions within the same slice (Fig. 1A), as well as from tissueof un-stimulated slices (Fig. 1B).

Based on the assumption that electrical activity maintains neu-ral tissue functional, we tested the idea that a severed hippocampalslice can regain functional connectivity if subjected to regularpulses 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) andthe postsynaptic EPSP responses in the stratum radiatum regionof area CA1 [5,21]. A hippocampal slice from the mouse was cutalong its parietal axis bisecting the CA1 area (Fig. 2).

After being cut, both hemi-slices were placed adjacent to eachother. Stimulation and recording electrodes were placed in thestratum radiatum of area CA1of and cut slices. For the cut slice,the stimulation electrode was placed in the striatum radiatum areaof one hemi-slice and the recording electrode was placed about thesame region of the stratum radiatum in the other hemi-slice tocollect evoked responses. For control and cut slices electrical stim-ulation consisted of square pulses (50 ls) of 20 V at 0.033 Hz (once

Fig. 1. (A) Mice hippocampal slice after 2 h of electrical stimulation and EPSP recordingradiatum area (traced). Note the space between electrodes is more opaque than adjacenThe texture of the tissue looks more homogenous.

every 30 s). The experiments were performed 3 times. Initially therecording 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 stimulationthe neurons are able to accumulate the potential and dischargeit. 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 normalphysiological conditions about 2 h after dissection [9]. Consis-tently, intact (control) slices showed a fast exponential rise in theamplitude of the evoked EPSP over time; as expected for normalrecovery of slices (Fig. 4).

Remarkably, the cut slice also showed an increase in evokedEPSP amplitude, though about 10-fold weaker than the intact sliceand with a linear rather than an exponential rise. The expression ofEPSP responses in the intact slice was rapidly apparent shortlyafter the beginning of the stimulation and grew stronger with time.

Hypothesis

Our preliminary data suggest that severed connections acrossthe mouse hippocampal slice can regain functional connectivityafter delivery of regular pulses of electrical stimulation to the tis-sue. It is attractive to speculate that electrical activity maintainsthe normal functioning of excitable membranes and neuronal

. Recording (left) and stimulation (right) electrodes are placed in the CA1 stratumt areas (arrow). (B) Mice hippocampal slice without electrical stimulation after 2 h.

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). Ineach 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 atthe 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 discernibleresponses were observed during the first 30 min of recording (sweep 1–61), after the depolarization event (sweep 62) presynaptic fiber volley responses followed byincreasingly 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 webversion 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 sliceshows 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 werecarried out started after ‘‘depolarization signal’’.

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function; an idea that resonates with the common notion of ‘‘use itor lose it’’. It follows, therefore, that electrical treatments thatrecruit membrane activity and associated cellular functions mayplay 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 stimulationmay exacerbate the damage to the tissue (i.e. favoring apoptosis).Disrupted connectivity after parietal cut of the hippocampal slicemay 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 hippocampalslice, we speculate that regular but discrete pulses of electricalstimulation delivered at low frequency are fundamental to therecovery of functional connectivity. It would be important to testthis speculation and determine the biochemical (e.g. enzymatic)mechanisms that are modulated by electrical stimulation thatpromote tissue recovery. It is worth mentioning that the type ofstimulation electrode used in this study generates an ellipsoid-like

‘‘field potential’’ that emanates from the tip of the electrode andpropagates outwards over the tissue. This type of electrical fieldis likely to generate micro magnetic fields that might also playan important role in tissue recovery.

We speculated that at several mechanisms may activelyregulate the regaining of functional connectivity. On the one hand,electrical stimulation may favor the recovery and consolidation ofsynapses according to the ‘‘Hebbian’s postulate’’, that synapticcoactivity strengthens synaptic function [10]. On the other, theremay be a ‘‘top-to-bottom’’ mechanism that regulates synapticrecovery. We proposed that widespread activity in a neuronal pop-ulation generates oscillatory waves of neural activity that providerecurrent downstream activation of synapses to strengthen theirconnections between groups of neurons (Fig. 5).

In addition to our hypothesis, we like to highlight thedescription of an in vitro slice system that may be a very suitableexperimental method to investigate strategies to enhance therecovery of neural connectivity after brain injury.

Fig. 5. Recovery of functional connectivity after parietal cut of the hippocampal slice. (A) Connectivity in an intact hippocampal slice. (B) Disrupted connectivity is recoveredafter regular delivery of electrical stimuli that allows the hypothetical fusing of membranes from separate fibers. Insets represent the pattern of EPSP responses increaseduring 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.

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Conflict of interest

None declared.

Acknowledgements

I would like to thank Professor Juan Marcos Alarcon (SUNYDownstate, Department of Pathology) for his contribution to thework, equipment provided and theoretic correction of the conceptsproposed in the article.

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