Niotrode Array for Rodent Brain Recording

Abstract

Micro-wire based microelectrodes are commonly used in extracellular electrophysiologic recordings. In ear-lier work, we pushed the limits in number of wires up to nine and introduced Niotrode called bundles. In this work, we present improvement steps in fabrication and post-processing by wet mechanical polishing, leading to very homogeneous front end recording sites with an impedance in the 1,5 MΩ range. We demonstrate the feasi-bility to use wire bundles in an array-arrangement and record from them in acute animal recordings.

Full text

Niotrode Array for Rodent Brain Recording
S. Hammad 1 a, S. Löffler 2, K. Mankodiya 1, D. Krapohl 1 b, M. Pohl1,4, A. Moser 2, V. Tronnier3
and U. G. Hofmann1
1Institute for Signal Processing, University of Luebeck, Germany.
2 Clinic for Neurology, University Medical Center Schleswig-Holstein, Campus Luebeck, Germany
3 Clinic for Neurosurgery, University Medical Center Schleswig-Holstein, Campus Luebeck, Germany
4Graduate School for Computing in Medicine and Life Sciences, University of Luebeck, Germany
Email: hofmann@isip.uni-luebeck.de
currently: a Centre for Sensory-Motor Interaction (SMI), Aalborg University, Aalborg, Denmark.
!b Department of Information Technology and Media, Mid Sweden University, Sundsvall, Sweden
Abstract
Micro-wire based microelectrodes are commonly used in extracellular electrophysiologic recordings. In ear-
lier work, we pushed the limits in number of wires up to nine and introduced Niotrode called bundles. In this
work, we present improvement steps in fabrication and post-processing by wet mechanical polishing, leading to
very homogeneous front end recording sites with an impedance in the 1,5 MΩ range. We demonstrate the feasi-
bility to use wire bundles in an array-arrangement and record from them in acute animal recordings.
!Keywords:!Niotrode,!fabrication,!polishing,!buckling!force,!impedance,!array.
1INTRODUCTION
Eavesdropping on brain‘s activity is nowadays
primarily done by extracellular neural recordings with
single microelectrodes or arrays in close proximity of
targeted neurons [3][4][5][6]. Despite the rapid pro-
gress of bulk micro-machined neural probes, wire
microelectrodes conserve remarkable inherited prop-
erties [7]. Their robustness, production simplicity,
low cost and ability of accessing deep brain structures
still attract neuroscientists to develop and use them
[8]. Recently demonstrated multisites bundle neural
probes showed a unique property of horizontal inves-
tigation of a single neuropil spot. Nevertheless, their
mechanical stability and volume was a critical com-
promise that hampered their assembly in array forms
[1]. Even more, the similarity of the cross section
area and distribution of their recording sites remains
an issue under discussion [7][9].
The following study was undertaken to develop an
array of bundle type neural probes with an internal
mechanical support concept to reduce the shafts’ vol-
ume, yet maintaining high channel count. The array is
planned to be used for recordings from rodent brain
with high selectivity and electrical stability. Moreo-
ver, we are investigating the pathways of developing
high order bundle microelectrode arrays that never
have been achieved before.
Niotrodes [1] have been redesigned using a simpli-
fied procedure. Sophisticated polishing process is
used to obtain flat cutting on the tip. Further, buckling
force test and impedance spectroscopy have been
performed , followed by acute recording from a rat
brain.
2MATERIALS & METHODS
2.1. Fabrication process
The current state-of-the-art Niotrode production
process was based on helically wrappping up to eight
NiChrome microwires around a ninth Pt/Ir one. A
simplified production method was described elswhere
[2] while using a tungsten strand (Ø 100µm) as inner
core to improve the mechanical stability of the Nio-
trodes. In brief, the desired number of microwires
including the tungsten stabiliser were inserted into a
hollow PTFE-shrinking tube, which on heating
clamped these wires in place as inner core. The strong
variation of the distance distribution of the NiChrome
wires around the center core was reduced by employ-
ing a wire guide during fabrication.
2.2. Post-processing
The method of choice to bring these microwires in
the desired length was by cutting them with a bone
cutter, which unfortunately was found to deforme the
Niotrode both in its cross-section area and on the
coating material (see Figure 1). A post-processing
system was developed to achieve a flat tip surface
and remove ridges on the wires‘ cut surfaces. The
first step was taken by a polishing system (see Figure
2.) composed of: a de-commissioned hard disc drive
(40GB, Maxtor, Singapore), a Niotrode micro-holder
made of the tip mechanics of a 0.5 mechanical pencil
(Top Action 0.5, G&m, Germany). The dimensional
difference between the Niotrode and the pencil is
bridged by inserting a piece of polyethene tubing in
the pencil’s chuck. A commercial DC motor (399-

9919, Mclennan Servo Supplies, UK) is used to pro-
duce another DOF for vertical rotation.
Figure 1: An electron micrograph showing the deformation in-
duced by using a bone cutter on the core‘s microwire bundle.
A raw Niotrode (after assembly, see Figure 3a) is
loaded in the Niotrode holder, which in turn is at-
tached to the DC motor. The supplied was chosen to
spin the Niotrode with 56rpm in the opposite direc-
tion as the hard drive top platter, which is coated with
fine grain commercial polishing paper. The Niotrode
is then lowered on the moving polishing paper and
mechanical bevelling/polishing (depending on the
paper grain size) is performed. Single drops of 0.9%
saline are added on the polishing film, prior to the
Niotrode tip to enhance removing leftoff particles.
In order to improve the cleaning of mechanically
polished tips, each Niotrode was immersed for 20h in
100% of specialized surface active cleaning agent for
cleaning non-ferrous metals and polymer surface
(Neutracon, Decon, UK). Rinsing with dd-water suc-
ceeded 10min ultrasound bathing in 80% Neutracon.
2.3. Mechanical testing
Buckling force measurement of probes was per-
formed by fixing them to a load cell (SS2, Althen,
Germany) itself mounted to a high precision com-
mercial 5DOF stereotactic frame (SASSU, pro-
medTEC GmbH, Lübeck) [13]. Two video cameras
(The Imaging Source, Charlotte, NC, USA) are fixed
perpendicular to each other in the probe‘s plane to
online monitor buckling of probes (free-fixed Euler
buckling [14]). The frames‘ z-motor moved the
mounted probe in discrete steps of 10µm downwards
towards a hard surface until buckling of the shaft was
visually noticed and the buckling force was deter-
mined.
2.4. Electrical testing
A common failure mode in hand-crafted microwire
probes is breach of electrical insulation and thus
shorting two or more wires. To test for short circuits
between the channels of a Niotrode we employed a
procedure described by D. Revision [10]. Hence, an
LCR meter (GWInstek LCR-821, Taiwan) was used
to measure the impedance between each channel of a
Niotrode and all of the other channels.
For characterization of the Niotrode’s impedance,
we utilized the above mentioned LCR-meter, in a
three point measurement setup in saline solution as is
described earlier [2].
Figure 2: Post-processing by polishing on a hard drive platter and
an opposite turning of a Niotrode fixture.
2.5. In vivo recording
A male Wistar rat (300g) was implanted with the as-
sembly for testing. All experimental procedures were
approved by the Ministry of Agriculture, Environ-
ment and Rural Affairs in Schleswig-Holstein, and
are performed under the guidelines of the University
of Luebeck Animal Welfare Office. The rat was ini-
tially anesthetized by a cocktail of 100mg/kg keta-
mine and 5mg/kg xylazine administered by intraperi-
toneal injection. The body temperature was main-
tained at 37°C by a circulating water-bath heating
pad. 30% of the initial anesthesia dose was adminis-
tered as the supplement when the rat showed toe-
twitch reflex.The Niotrode array was used to acutely
record from a brain of a 350g Wistar rat. Ketaminhy-
drochlorid (100mg/kgKG), Xylizinhydrochlorid
(20mg/kgKG) and were used as anaesethetic agents.
The Niotrode array was fixed by a clamp to a stereo-
taxic frame and connected to a head stage (ZIF-chip
Headstage, Tuck Davis Technology, USA) of a re-
cording system (RZ2 bioamp, Tuck Davis Technolo-
gies, USA).
3RESULTS & DISCUSSION
3.1. Fabrication of Niotrodes
Electron micrographs (Philips SEM 505, Philips,
Netherland) of Niotrodes immediately after heat
shrink bonding and initial cutting show ridges (data
not shown), irregular shaped tips and sometimes even
cast doubt to functionality, since seperate strands are
not visible. Consequently, post processing was a clear
requirement. Micrographs in Figure 3 illustrate ex-
emplary results after respective post-procesing steps.
Fig. 3 (a) shows a Niotrode after production. Fig. 3
(b) displays a manually grinded Niotrode. Note the
distortion of the original round diameter and the in-
homogeneous surface appaearance. Fig. 3 (c) and (d)
depict mechanically grinded Niotrodes using the pol-
ishing system with (d) and without (c) the DC motor
counter movement.

Figure 3: Light micrographs of Niotrode tips. (a) after
shrinking tube bonding and cutting, (b) after manual
polishing, (c) after unsupported mechanical and (d)
cross-movement supported polishing.
The following cleaning process in Neutracon re-
moved almost all grinding-caused adhering micro-
particles (Figure 4). Optical micrographs however
displayed polishing scratches on the tungsten wire,
which up till now did not seem to effect the function-
ality. Caution has to be taken, on how long any im-
mersion process in dd-water should take: Uncon-
trolled immersion causes the shrinking tube to expand
and thus to reduce mechanical integrity.
Figure 4: (a) A Niotrode tip after polishing. (b) A magnified view
of the same tip. (c) The tip surface after pronounced wet cleaning.
Note a change in wire distances before and after cleaning due to
water uptake of the shrinking tube.Images were taken by a digital
microscope (VHX 600, KEYENCE, Japan) with a magnification of
500, 2000, 1400 respectively.
3.2. Characterization of Niotrodes
Mechanical testing of different sized shanks of
Niotrodes (7, 8, 9 and 10mm, free-fixed Euler buck-
ling) displayed buckling force values in the range
from 94 to 47mN. Notably, 5mm shanks already ex-
ceeded the maximum scale value of our load cell
(300mN). Measured buckling force values clearly
exceeded those required for penetrating the pia mater
of a rats brain after removing the dura [11], or pene-
trating a peripheral nerve [12] .
The electrical short circuit test showed no shorts
occurring between the channels of the Niotrode .
The wet impedance of the Niotrode in saline at
1kHz was found to be in the range of 1.5 and 2 MΩ
with a relatively small deviation from the mean value.
This corroborates the similarity the of cross-section
area of the tips, obtained by the polishing process.
Figure 5: Assembly of two Niotrodes in a 1x2array equipped with
a standard multichannel cell phone plug. The inserts show micro-
graphs of the two Niotrode tips.
Figure 5 shows a 1x2 Niotrode array designed for
recording from rodent’s brain. The array was built by
fixing and soldering two single Niotrodes on a preci-
sion machined PCB carrier. This PCB may in the
future contain an active amplifier chip, but at the time
of this writing was only connected to a standard mul-
tichannel cell phone plug (AXR51228P, Panasonic ).
Figure 6: Recorded neural activities from the parietal associa-
tion cortex (PAC) 9 channels are shown.
3.3. Acute electrophysiological recordings
Niotrodes showed in acute animal experiments
satisfactory recording characteristics as can be seen in
Figure 6 with recordings from the parietal associative
cortex of rat.
The power spectrum analysis of the cortical signal
reveals peak activity at a frequency of 900 Hz in 0.75
to 2.0 mm depth. This can be attributed to the high
density of pyramidal cell firing in the respective ana-
tomical layers (here primary motor cortex, Figure 7).
Spike detection and clustering was performed from
filtered wave trains using a Matlab implementation of
the unsupervised spike detection and super-
paramagnetic clustering algorithm WaveClus [15].
Only one single cluster was obtained for all channels
and for all recordings. This, again, indicates the high
selectivity of the Niotrode array recording contact
sites.

Figure 7: Power spectrum of PMC recordings.
Figure 8: Results of wave clusterig in two different depth of PMC.
Moreover, cluster analysis reveals, that the spatial
resolution of one single Niotrode is small enough to
look at only one cell from different directions. The
spike shape differs from each other only for different
depth steps. Whisker toggling makes no change to the
prevalent cluster shape. Within one depth, only the
signal amplitude changes from channel to channel,
which accounts for the fact, that only one neuron is
monitored (see Figure 8).
4Conclusion
We could demonstrate an effective method for de-
veloping relatively small, robust and highly selective
neural probes. They show the potential to be assem-
bled in array form, which we demonstrate with a 1x2
array. Niotrodes thus may at one time enhance the
current microwire arrays and improve the understand-
ing the brain.
5Acknowledgement
We express our gratitude to M. Klinger from the In-
stitute of Anatomy, N. Koop, C. Cray, A. Nehme and
F. Beck from the Institute of Bio-Medical Optics,
both University of Lübeck. This work was in part
supported by the BMBF-grant 13N9190 “BiCIRTS”.
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