Selective laser ablation of interconnections between
neuronal sub-populations:
a test-bed for novel neuroprosthetic applications
Alberto Averna1#, Marta Bisio1#, Valentina Pasquale1, Paolo Bonifazi2, Francesco Difato1 and
Michela Chiappalone1*
1 Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, Genova, Italy
2 School of Physics and Astronomy, Tel Aviv University (TAU), Israel
#
Equal Contribution
* Corresponding author. E-mail address: [email protected]
Abstract
Cortical and hippocampal patterned networks, composed of inter-connected modules of different size, were grown
on commercially available Micro-Electrode Arrays to investigate neurodynamics. Such patterned networks, realized by means of multi-compartmental polydimethylsiloxane structures, represent an in vitro model to study electrophysiological activity of damaged neuronal networks in the central nervous system. A laser microdissector has
been used to selectively cut the neural connections between different modules, thus mimicking a pathological
condition. Spontaneous and evoked activity of modular neural networks has been characterized in both healthy
and damaged cultures.
1 Background
In the last decades, great efforts have been made to
focus on studying neuro-prostheses targeting lesions at
the level of the CNS and aimed at restoring lost cognitive functions [1, 2]. Simplified in vitro models of cell
assemblies can provide useful insights for the design of
future cognitive brain prostheses. In particular, studies
of healthy and lesioned in vitro neuronal networks can
be exploited as a test-bed for the development of innovative neuro-prostheses [3]. To investigate the inherent
properties of neuronal cell assemblies as a complement
to artificial computational models, engineered networks
of increasing structural complexity, from isolated finite-size networks up to inter-connected modules, were
grown on Multi Electrode Arrays (MEAs). Here, we
characterized the spontaneous and evoked activity of
2D neuronal cultures, before and after selective laser
dissection of physical connections between neuronal
sub-populations [4]. The quantitative characterization
of the lesion-induced changes in activity represents a
reproducible injury model to possibly test innovative
‘brain-prostheses‘.
2 Methods
All experimental procedures and animal care were
conducted in conformity with institutional guidelines,
in accordance with the European legislation (European
Communities Directive of 24 November 1986,
86/609/EEC).
Primary neuronal cultures were obtained from cerebral cortices and hippocampi of rat embryos at gestational day E18. In order to provide the physical confinement of neuronal cultures, a polymeric structure in
polydimethylsiloxane (PDMS), composed of different
modules, has been realized through soft lithography
[5]. The PDMS mask is positioned on the MEA substrate before the coating procedure, performed by putting a 100-µl drop of laminin and poly-D-lysine solution on the mask and leaving it in the vacuum chamber
for 20 minutes. Then, the mask is removed and cells
plated afterwards. The used MEAs (Multi Channel Systems, Reutlingen, Germany) can present three different
geometrical layouts: “4Q”, where 60 electrodes are organized in 4 separate quadrants; “8x8”, where electrodes are placed according to a square grid; 6x10, rectangular grid. The nominal cell density is around 500
cells/µl (~100 cells per network module).
Experiments on both cortical and hippocampal
modular networks, recorded between 20 and 25 Days
In Vitro (DIVs), have been carried out. Results have
been obtained from a dataset of 17 modular networks
(7 hippocampal and 10 cortical). The used experimental protocol consisted of 5 consecutive phases: i)
One-hour recording of spontaneous activity; ii) Stimulation session I, which consists of stimulating at least
two electrodes per cluster using a train of 50 positivethen-negative pulses (1.5 V peak-to-peak, duration 500
µs, duty cycle 50%) at 0.2 Hz; iii) Laser ablation of inter-cluster neural connections, whose aim is to isolate a
cluster which is physically and functionally connected
to at least another one; iv) One-hour recording of spontaneous activity after performing the lesion; v) Stimulation session II, from the same electrodes of phase ii.
The normal distribution of experimental data was
assessed using the Kolmogorov-Smirnov normality
test. According to the distribution of the data, we per-
formed either parametric or non-parametric tests and pvalues < 0.05 were considered significant.
ality between distinct brain areas, at the level of the
central nervous system.
Fig. 1. Optical micrograph of a patterned culture (hippocampal neurons, 20 DIV), composed of 3 cell clusters (or modules) over a 4Q
MEA. The same culture was monitored before (left) and after (right)
the optical lesion. It is visible that neural connections between the
upper left cluster and all the others were cut by the laser. Screen bar
200 µm.
3 Results
Depending on the distance between modules, the
module networks are able or not to establish spontaneous neuronal connections among them. Fig. 1 reports
optical images of a representative culture before (left)
and after the optical lesion (right).
We analysed both spontaneous and evoked activity
of modular networks before and after lesion. Regarding
spontaneous dynamics, the strong synchronization of
activity observed among the clusters before the lesion
(Fig. 2A), disappears almost completely after it (Fig.
2B). Moreover, a global decrease in the network mean
firing rate is observed after the lesion, both inside the
isolated cluster, and in all the other clusters that were
previously connected to the isolated one. Before performing the lesion, electrical stimulation was able to
evoke activity both within the cluster hosting the stimulating channel and in the connected modules (Fig. 2C,
black profiles). After laser dissection, the evoked activity remains confined within the isolated cluster without
spreading towards the other ones (Fig. 2C, red profiles). Figure 2D shows that a global decrease in the
evoked activity after the laser cut is present in both local and distal responses. Since lesion is aimed at reducing the amount of connections among clusters, intercluster responses are more affected than intra-cluster
ones.
4 Summary
We showed that selective laser dissection of interconnections among neural assemblies affects both
spontaneous and evoked activity of modular networks,
by inducing de-synchronization between the different
modules during spontaneous activity, and preventing
propagation of evoked responses among modules.
Those preliminary results present a reproducible experimental model, which can be used as a test-bed for
the design and development of innovative neuroprostheses aimed at restoring lost neuronal function-
2
Fig. 2. Comparison of spontaneous and evoked activity of healthy and
damaged modular cultures. A, B. 5-minute raster plot of spontaneous
activity of a representative patterned network before (A) and after (B)
lesion. The activity of different clusters (i.e. C1, C2 and C3) has been
highlighted in different colors. The blue one (C1) is the isolated cluster. C. Post-stimulus time histograms (PSTH) obtained when stimulating a channel belonging to the isolated cluster. The electrodes labelled
in blue belong to the isolated cluster, while the black cross indicates
the stimulated channel. Black curves report the evoked activity before
the lesion, while the red ones refer to the evoked activity after it. D.
Comparison between the statistical distributions of normalized PSTH
areas (i.e. ratio between post-lesion PSTH areas over pre-lesion ones),
either within the stimulated cluster (Intra Cluster) or in all the other
ones (Inter Cluster). Statistical analysis has been performed by using
the Mann-Whitney test [*p<0.05]).
Acknowledgement
The research leading to these results has received
funding from the European Union's Seventh Framework Programme (ICT-FET FP7/2007-2013, FET
Young Explorers scheme) under grant agreement n°
284772 BRAINBOW (www.brainbowproject.eu).
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