Design of a Flexible Parylene-based Multi-electrode Array for
Multi-region Recording from the Rat Hippocampus
Huijing Xu IEEE Student Member, Ahuva Weltman, Min-Chi Hsiao IEEE Member, Kee Scholten, Ellis
Meng IEEE Senior Member, Theodore W. Berger IEEE Fellow and Dong Song IEEE Member
Abstract — The hippocampus is a critical deep brain
structure in several aspects. It is directly related to theformation of new long-term declarative memory. The
malfunction of the hippocampus closely relates to various
disease and pathological conditions. It is also a model
structure for the study of cortical function and synaptic
plasticity in general because of its special
neuro-anatomical structure and intrinsic connections
within the hippocampus formation. Both theunderstanding of roles that the hippocampus plays inrecognition memory and the study of neural plasticity
require simultaneously recording of neural activities frommultiple sub-regions of the hippocampus from behavioral
animals. However the distribution of cells in thehippocampus make the recording from multiple
sub-regions a big challenge with the traditional uni-length
micro-wire arrays. Well-designed electrode arrays are
required to reach multiple regions simultaneously becauseof the distinctive double C shape of the hippocampus cellbody layers. In this work, we designed a multi-shankselectrode which uses Parylene C, a highly biocompatible
and flexible polymer, as a base and has multiple recordingsites specially positioned along the longitudinal axis to fit
the curvy shape of the rat hippocampus.
I. INTRODUCTION
The hippocampus and the surrounding anatomicallyassociated cortical areas together compose the hippocampus
formation which have been widely studied for the past fewdecades. Through study of its anatomical characteristics,
researchers noticed the unique distribution of cells in thehippocampus. It was quite different from that found anywhere
else in the cortex [1]. The majority of cells in the hippocampus
are packed densely into a thin layer which curve into a double
C structure. Based on the cytoarchitectural distinction ofprinciple cells in the hippocampus, it was divided into threesubdivisions as the dentate gyrus (DG), the CA3 and the CA1.
Intrinsic connections between hippocampal sub-regions andthe entorhinal cortex (EC) form a closed feedback loop [2]. It
is widely believed that sensory inputs from cortical regions areThis work was funded by NSF INSPIRE.
H. J. Xu, M. C. Hsiao, D. Song and T. W. Berger are with the Center of
Neural Engineering, Department of Biomedical Engineering, University ofSouthern California, Los Angeles, CA9089 USA (email: huijingx@usc.edu).
A. Weltman, K. Scholten and E. Meng are with the Biomedical
Microsystems Laboratory at Department of Biomedical Engineering,
University of Southern California, Los Angeles, CA90089 USA.

propagated through the excitatory connections within thehippocampus and are processed by hippocampal sub-regions
into output signals for long-term storage in the cortex [3].

Figure 1. Diagrammatic representation of the rat brain and the relative
location of the hippocampal formation on the left side of the brain. Pictured
on the top right is a transverse slice of the hippocampus stained with crystal
violet to show the cell body layers and hippocampal sub-regions. Intrinsic
connections are illustrated with lines superimposed on the histology and areshown in the bottom diagram.

Lesion studies in the rat have indicated that damage that
occurs along the longitudinal axis of the hippocampus affects
the process of spatial and non-spatial information differently[4]. Neurotoxic lesions made in different sub-regions of thedorsal hippocampus of rats resulted in different impairment in
detecting spatial novelty [5]. Based on neural activities
recorded from ensembles of CA3 and CA1 neurons from rats,
researchers were able to build a mathematical model which
can simulate the function of the hippocampus [6] [7]. Thesestudies indicate that simultaneous recordings of neuralactivities from multiple hippocampal sub-regions not only
help the understanding of the mechanisms underlying
cognitive function related to the hippocampus but also thedevelopment of computational models to mimic the functionof the hippocampus. This also benefits the study of how
information is processed in the hippocampus. Although
micro-wire arrays have been widely used for in vivo
recordings from the hippocampus, micro-wires withcomplicated arrangements are required in order to reachmultiple sub-regions [8]. Parylene C is a micro-machinable
and USP class VI biocompatible polymer commonly used inFDA-approved implants. Compared with other commonlyused materials such as metal wires and silicon, the highflexibility and mechanical strength of the Parylene C make it

an ideal structural material for cortical recording electrodes.
Parylene devices can be easily fabricated and the placement ofrecording sites can be easily manipulated using conventional
micromachining techniques [9]. These advantages make theuse of Parylene C probes for chronic recordings frombehavioral animals have great potential.
II. MATERIALS AND METHODS
A rat brain atlas, histology results together with in vivo
recordings were used to decide the layout of Parylene shanks
and the placement of recording sites.
A. Brain Atlas and Brain Slices
The parameter for electrodes implantation was initiallydecided based on the brain atlas [10]. The most distal edges of
the hippocampus and depth differences between the CA1 andthe CA3, the CA1 and the DG cell body layers at locations
between 2.45 - 4.00 mm posterior to the Bregma weremeasured. One rat was deeply anesthetized with an injectionof ketamine and xylazine mixture and perfused with 10%
formalin (PFA). Its dissected brain was fixed with 10% PFA
overnight and dehydrated with 18% sucrose solution for one
day. 50 µm coronal sections were sliced and stained withcresyl violet to identify the cell body layers (Figure 2).

Figure 2. Comparison of histology results (top) with values obtained from thebrain atlas (bottom). Histology results were coronal sections obtained from a)
2.45, b) 3.25 mm and c) 3.90 mm from the Bregma.

B. Surgery
With the information from the brain atlas reference andhistology results, we further tested and modified the parameterof recording site placement with neural activities observed
during surgery and in vivo recording from rats chronicallyimplanted with micro-wire electrodes. Both uni-length
micro-wire arrays and specially designed multi-length
electrode arrays were used. All animal preparation and animalcare were in accordance with the National Institute of Healthand Institute of Animal Care and Use Committee guidelines
and the Department of Animal Resources at the University ofSouthern California.
Male Sprague-Dawley (SD) rats greater than threemonths and with weights between 350 to 450g were housed in
12/12hr light and dark cycle, all surgery were conductedduring the light cycle. All animals were given ad libitum
access to food and water. Rats were pre-anesthetized with anintraperitoneal (IP) injection of a ketamine and xylazine
mixture. Animals were anesthetized intra-operatively with amaintenance dose of a mixture of isoflurane and oxygen.
Toe-pinch withdrawal reflexes were evaluated frequently toensure proper dosing. Animal body temperatures weremaintained with a warm pad. Surgeries were performed on astereotactic frame. Each animal was fixed on the frame by ear
bars holding its skull in place. A 2x4 mm bone window wasdrilled out of the cranium above the dorsal hippocampus andthe dura was carefully removed. Three small holes were also
drilled for anchor screws. One screw contacted thecerebrospinal fluid to serve as a ground for the electrode. The
cranium was sealed with bone wax and dental cement after theelectrode reached desired locations.
The detection of complex spikes (CS), a group of action
potentials fired with less than 5 msec inter-spike intervals(shown in Figure 4), was used as the indicator that themicro-wire was approaching the CA1 and the CA3 cell bodylayers. The observation of CS in the depth between 2.20 - 3.20mm from the skull was considered as the recording from theCA1 cell body layer and the detection of CS in the deeperregion (3.60 - 4.40 mm from the skull) was considered as therecording from the CA3 cell body layer.
Two types of surgery were performed on three animals.
One animal was sacrificed immediately after the implantationof a 3x5 uni-length micro-wire array to verify that the CS weobserved during implantation were generated by cells locatedin the CA1 and CA3 cell body layers, respectively. For thisnon-survival experiment, a brief DC current of 300 µA wasapplied to the micro-wires right after implantation to makeelectrical lesions at the tip. The animal was then perfused with
10% PFA and 3% Kfe (Potassium Ferrocyanide into 10%PFA). Brain slices were evaluated to verify tip locations.
Micro-wire arrays were also implanted chronically into twoanimals. One animal was chronically implanted with a 3x5
uni-length micro-wire array and another animal waschronically implanted with a special designed multi-length
electrode array (as shown in Figure 5a) to measure the depth
difference between the CA1 and the CA3 regions. For
chronically implanted animals, scalp wounds were sutured
and bacitracin zinc ointment was applied to the cut to prevent
infection. Bupropion (BUP) was injected to relief pain. In vivo
recording were taken after the animal fully recovered. After
long-term recording completed animals were sacrificed asdescribed above.
C. Data Acquisition
Neural activities were monitored during the operation ofboth the non-survival surgery and chronically implantations.
During the surgery, signals were differentially amplified 1000
times and band pass filtered from 300 to 3000 Hz to check foraction potentials. Filtered neural activities were displayedwith an oscilloscope and played through a speaker. In vivo
data was recorded with an on-line data acquisition system(OmniPlex, Plexon, Inc) and digitized at 40 kHz for storage.

Spike sorting was done by performing a principle component
analysis (PCA) to wave forms with an off-line sorting system(Offline Sorter, Plexon, Inc).
D. Parylene Probe Fabrication
The fabrication steps of a single-metal-layer Parylene C
based electrode array is illustrated in Figure 3. A layer of 10µm Parylene C was vapor-deposited on the silicon wafer in aPDS2010 system. Platinum electrode recording sites, traces
and contact pads were lithographically patterned on top of thebase layer using e-beam deposition at a thickness around 2000
Å. The metal layer was then encapsulated by another layer of
10 µm thick Parylene C. Electrodes and contact pad weresubsequently exposed by deep reactive iron etching (DRIE)
with an oxygen plasma and the probes was cut out from thesubstrate. The device was released from the silicon carrier
wafer in a water bath.

using the detection of CS as the criteria for cell body layer
localization is accurate. The depth from which CS wererecorded from both the non-survival experiment and thechronically implantation of a uni-length micro-wire array arelisted in Table II.

Figure 4. Complex spikes recorded during implantation (left) and thecorresponding histology results (middle). Diagram on the right illustrated thechannel from which CS were recorded and its relative location along the APaxis.
TABLE II.

Figure 3. Fabrication processes of the Parylene C based micro-electrode arraywith a single layer of metal.

III. RESULTS
A. Depth Difference Measurement
The measurement of distances between the CA1 and theCA3 cell body layers in different regions along theanterior-posterior (AP) axis from both the brain atlas andbrain slices are listed in Table I. It is obvious that the depth
differences are not uniform along the AP axis. In addition, thedifferences measured from the brain atlas matches that fromthe histology results. On average, the CA3 cell body layer isabout 1.00 to 1.30 mm more ventral than the cell body layer of
the CA1 region.
TABLE I.

HIPPOCAMPUS MEASURE FROM BRAIN ATLAS AND BRAINSLICES

Rat1

Rat2

AP(mm)

CA1

CA3

CA1

CA3-2.45

3.55

3.95

3.65

4.25

-2.70

3.35

4.20

3.65

4.20

-2.95

3.30

4.35

3.60

3.85

-3.20

3.05

4.35

3.60

4.35

-3.45

3.05

4.40

3.35

4.35

-3.70

2.80

4.40
3.10
4.35
Rat1: the animal used in non-survival experiment.
Rat2: the animal used in chronically implantation.

The special designed multi-length electrode array has alength difference of 1.3 mm between the CA1 group with theCA3 and DG group respectively. With this difference, certain
channels in each set was capable of recording unitaryactivities from the cell body layer. The histology results also
verified that the recording was from desired region. However,with a uniformed length difference, only a small portion of thewires in each group were able to reach the cell body layer
accurately.

HIPPOCAMPUS MEASURE FROM BRAIN ATLAS AND BRAINSLICESAP(mm)Brain Atlas (mm)Brain Slices (mm)MLML-2.45-3.25
-3.90

3.50
4.40
4.70

CA1-CA3

CA1-CA3

1.10
2.25
1.00
1.30
3.00
1.25
1.20
3.50
1.20
AP: anterior to posterior, ML: medial to lateral.

Neural activities together with histology results obtained
from the non-survival experiment (Figure 4) verified that

Figure 5. a) Diagrammatic representation of the multi-length electrode array.
b), c), d) action potential recorded from the CA1, the CA3 and DG region,
respectively, and its corresponding histology result.

Figure 6. Diagrammatic representation of the layout of the CA1-CA3 Parylene probe (left) and the CA1-DG Parylene probe (right). The arrangement ofrecording sites is designed base on measurements obtained from histology results and the brain atlas. Each recording site has a diameter of 30 µm and thespacing between recording sites within each recording group is 40 µm.

B. Design of the Parylene Probe
By combining the in vivo recording results, histology
results and the information attracted from the brain atlas, two64 channels Parylene probes are designed for the recordingfrom the CA1 and the CA3, the CA1 and the DG sub-region of
the rat hippocampus respectively. For the CA1-CA3 probe,
two groups of four recording sites are placed on each shank.
The spacing between each recording site in one group is 40µm which make the recording group span a wide enough depth(240 µm) to cover the CA1 and the CA3 cell body layers. Thedistance between two recording group is specially arranged tomatch the measurement we obtained above. The CA1-DG
Parylene probe has a similar geometry as the CA1-CA3 probe.
Since the DG cell body layer has two blades which spans amuch larger region than the CA3 ones, five recording sites areplaced in the DG group. These five recording sites are further
divided into two sub-sites which target at both blades
separately. The spacing between recording sites in theCA1group is also 40 µm.
C. Insertion Tool
Since Parylene C is a flexible material, we also designed ainsertion tool to guide the Parylene probe during surgicalplacement into the brain. The insertion tool is composed ofeight micro-wires 75 µm in diameter. The Parylene probe will
be attached to the insertion tool with polyethylene glycol, awater-soluble biodegradable adhesive. After the Parylene
probe is inserted into desired depth, the insertion tool will beretrieved.

Figure 7. A close look at the geometry of the insertion tool. Eight notches are
etched out on the silicon wafer. The spacing between each notch is 250 µm
and the diameter of each notch is 90 µm. Pyrex is used to cover the notches
and also will hold the micro-wires in place.

IV. FUTURE WORKThe Parylene probe will be implanted chronically into therat hippocampus. The placement of recording sites will befurther optimized to best fit the anatomical distribution ofprinciple cells in the hippocampus. The stability of neuralactivities recorded with the Parylene probe and theperformance of the Parylene probe in long-term implantation
will also be evaluated.
ACKNOWLEDGMENT
The authors would like to thank Brain Kim and Lawrence
Yu for their expert guidance and insight suggestions. We also
thank Dr. Donghai Zhu of the Keck Photonics Laboratory forhelp with fabrication.
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