A watertight acrylic-free titanium recording chamber for electrophysiology
in behaving monkeys
Daniel L. Adams,1John R. Economides,1Cristina M. Jocson,1John M. Parker,2
and Jonathan C. Horton1
1Program in Neuroscience and2Laboratory Animal Resource Center University of California, San Francisco,
San Francisco, California
Submitted 3 May 2011; accepted in final form 9 June 2011
Adams DL, Economides JR, Jocson CM, Parker JM, Horton
JC. A watertight acrylic-free titanium recording chamber for electro-
physiology in behaving monkeys. J Neurophysiol 106: 1581–1590,
2011. First published June 15, 2011; doi:10.1152/jn.00405.2011.—
Neurophysiological recording in alert monkeys requires the creation
of a permanent aperture in the skull for repeated insertion of micro-
electrodes. Most laboratories use polymethyl methacrylate to attach a
recording chamber over the skull opening. Here, we describe a
titanium chamber that fastens to the skull with screws, using no
polymethyl methacrylate. The gap between the base of the chamber
and the skull is filled with hydroxyapatite, forming a watertight
gasket. As the chamber base osseointegates with the skull, the hy-
droxyapatite is replaced with bone. Rather than having a finite
lifetime, the recording chamber becomes more firmly anchored the
longer it is in place. It has a small footprint, low profile, and needs
little maintenance to control infection. Toilette consists of occasional
application of betadine to clean the scalp margin, followed by appli-
cation of neomycin, polymyxin, and bacitracin ointment. Antibiotic is
also placed inside the chamber to suppress bacterial proliferation.
Thickening of the dura within the chamber can be prevented by
regular application of mitocycin C and/or bevacizumab, an antibody
against vascular endothelial growth factor. By conducting an e-mail
survey, this protocol for chamber maintenance was compared with
procedures used in 37 other vision research laboratories. Refinement
of appliances and techniques used for recordings in awake monkeys
promises to increase the pace of scientific discovery and to benefit
primate; macaque; physiology; awake behaving; alert; polymethyl
methacrylate; dental acrylic; hydroxyapatite
THE ABILITY TO MAKE DAILY extracellular recordings in alert
monkeys has yielded a wealth of valuable data about the
function of the primate brain. It has also reduced the number of
animals required for neuroscience research, because a single
animal can continue to provide data for months or even years.
To perform such recordings, two devices are necessary: a
stabilizing headpost and a recording chamber. Previously, we
described a biocompatible titanium headpost, which attaches
with screws alone and requires minimal maintenance (Adams
et al. 2007). Here we report a titanium recording chamber,
which also attaches with screws and forms a permanent,
watertight seal with the skull.
A major advantage of both of these appliances is that they
dispense with the acrylic polymer, polymethyl methacrylate
(PMMA; also known as Plexiglas, Lucite, Perspex, and Acry-
lite). PMMA is also known as dental cement, a name that stems
from its use in the manufacture of prosthetic dentures and as a
“luting agent” to attach prostheses to tooth stumps.
PMMA use in cranioplasty began in Germany (Kleinschmidt
1941; Zander 1963), where it rapidly replaced metal for cranio-
facial reconstructive surgery following trauma or craniotomy
(Sanan and Haines 1997). Lacking adhesive properties, it acts
primarily as a filler or grout, and screws are required to anchor
it to external bone surfaces like the skull (Nikolis et al. 2009;
Smith et al. 1999). In human surgery, PMMA reconstructions
are sealed under the scalp, protecting them from infection.
PMMA was first used to secure implanted electrodes in a
primate by Delgado (1952). He noted that, “the small hole in
the skull is closed with dental cement, which holds the elec-
trodes firmly in position. Only a minute quantity should be
used to avoid the formation of an excessive protuberance on
the skull.” Reporting on a technique for chronic neuronal
recording, Sheatz (1961) emphasized that “quantities [of
PMMA] should always be minimal, especially in those loca-
tions where cement will be adjacent to subcutaneous tissue.” In
these early experiments, PMMA was used to secure implanted
electrodes under the skull for recordings in alert animals. The
only exposed appliance was the electrical connector, minimiz-
ing the potential for bacterial infection.
To record from multiple sites in the alert animal, one must
devise a method to withdraw electrodes and move them to a
new location (Wurtz 1968). The first permanent recording
chamber was developed by Evarts (1966; 1968). It provided an
open window to the dura mater to allow repeated electrode
penetrations over a small region. A stainless steel cylinder was
fastened to the skull with three bolts. The bolt heads were
inserted between the bone and dura along slots extending from
the trephined skull margin. Nuts were placed on the protruding
bolt stems to trap the outer edge of a flange at the cylinder’s
base. A small amount of dental cement was applied around the
base of the cylinder to seal it to the skull.
As Evarts’ technique became popular, many laboratories
integrated the chamber and the head fixation device in a large
dome of PMMA, held in place with slot bolts, bone screws, or
machine screws. The covering of a wide area of skull with
acrylic is not ideal, for both aesthetic and practical reasons.
Bacteria flourish at the margin between the scalp and acrylic,
requiring regular cleaning, debridement of granulation tissue,
antiseptics, and application of antibiotics. If the seal between
the chamber and skull is not perfect, exudate will leak under
the implant. The moist interface between implant and skull
provides an environment for proliferation of microorganisms.
Address for reprint requests and other correspondence: D. L. Adams,
Beckman Vision Center, 10 Koret Way, Univ. of California, San Francisco,
San Francisco, California 94143-0730 (e-mail: email@example.com).
J Neurophysiol 106: 1581–1590, 2011.
First published June 15, 2011; doi:10.1152/jn.00405.2011.
15810022-3077/11 Copyright © 2011 the American Physiological Society www.jn.org
Eventually, infection can spread to the chamber interior, caus-
ing a dural abscess or meningitis. Figure 1 shows the skull
from a macaque that had two chambers and a headpost im-
planted for 6 mo in an acrylic headcap. Widespread thinning of
the skull occurred from bone resorption, and a large gap
developed around the base of each chamber.
The problems associated with acrylic-based implants are
familiar, yet this compound remains widely used, perhaps
because the known drawbacks of acrylic are feared less than
the unknown risks of new materials. Individual laboratories
learn to cope with the challenges posed by recording chambers
through trial and error; experience is shared largely by word of
mouth because few publications address the subject. To facil-
itate the exchange of information, we surveyed vision research
laboratories engaged in daily recordings from alert monkeys to
identify key problems associated with recording chambers, and
to compare standard operating procedures. Here we review the
collective experience of colleagues in the field and describe our
development of an acrylic-free, watertight titanium recording
chamber designed to reduce skin margin toilette and to pro-
mote internal sterility.
The objective was to design, manufacture, and test a recording
chamber that attaches securely to the skull and forms a watertight seal
with the bone surface. This was achieved by using a titanium chamber
fastened to the skull with titanium orthopedic screws. To reduce
chronic infection and ensuing bone loss, we opted to eliminate PMMA
entirely. Instead, we used hydroxyapatite paste to seal the gap be-
tween the base of the chamber and the skull. The chamber was
constructed from unalloyed, commercially pure (CP) titanium, be-
cause this material is easier to machine than titanium alloys, and its
superior ductility permits bending of the feet without fracture. CP
titanium has osseointegrative properties and corrosion resistance in a
saline environment, making it suitable for use as a bone-anchored
implant (Pohler 2000; Rubo de Rezende and Johansson 1993).
The chamber was machined from a single piece of 2-in.-diameter
CP titanium bar stock. Six evenly spaced feet surrounded the chamber
base, each with a single screw hole. The outer surface and base of the
chamber were grit-blasted (mesh size 50), roughening the finish to
encourage osseointegration (Buser et al. 1991). Three chambers with
this design were implanted into male rhesus monkeys, two adult and
Following these three implantations, the design was improved by
reducing the number of feet from six to five (Fig. 2). This change was
intended to reduce scalp retraction from the chamber wall. The feet
separate the scalp from the skull, allowing it to pull back from the
chamber. To lower the screw profiles, we ground 1 mm off their heads
before insertion, and countersank the holes more deeply. The weight
of the chamber was 12.7 g (not including the lid or the 5 titanium
screws). A digital three-dimensional model is available upon request.
The chamber drawing was sent to a computer numerical control
workshop for manufacture.
Implantation surgery. All procedures were approved by the Uni-
versity of California, San Francisco Institutional Animal Care and Use
Committee. Animals were sedated with ketamine (10 mg/kg im) and
prepared for sterile surgery. An endotracheal tube was inserted, and
ventilation provided with 2% isoflurane in 50:50 N2O/O2to maintain
surgical anesthesia. ECG, rectal temperature, inspired and expired
anesthetic agents, end-tidal CO2, and oxygen were monitored contin-
uously. The animal was placed on a heated blanket in a stereotaxic
apparatus. The scalp was shaved and swabbed with 5% povidone-
iodine. An important factor in promoting good wound closure and
long-term implant health is to avoid circulatory compromise of the
scalp. Care was taken to preserve the major tributaries of the occipital
arteries and the superficial temporal arteries by tracing their course
with a miniature ultrasonic Doppler flow detector (model 811-BTS,
Parks Medical Electronics, Aloha, OR).
To implant the chamber over the operculum of striate cortex, an
incision was made starting at the midline, extending ?40 mm poste-
rior laterally, avoiding the arteries identified by ultrasound. Bone was
Fig. 1. A: lateral view of a Macaca mulatta skull
from an animal that developed an infection
between the polymethyl methacrylate (PMMA)
headcap and the skull. Postmortem revealed
extensive thinning of bone that was covered by
PMMA. Many screw holes had enlarged suffi-
ciently to release the screws. The two trephine
holes were enlarged, with thinned edges. In
contrast, excessive bone proliferation was ob-
served at the perimeter of the headcap (arrow).
B: view of the chamber interior from inside the
skull in another macaque. The base of a stain-
less steel chamber and the surrounding mass of
blue and green PMMA is visible through the
trephination. Bone resorption occurred around
the chamber base, opening a gap.
Fig. 2. A: orthographic drawing of the acrylic-free chamber. Inner diameter of
the chamber bore is 19 mm. B: rendering of cross section through the chamber
and lid mounted on a trephined bone surface. The cross-sectioned lid has been
rotated slightly to show the silicone o-ring (orange) and the V-shaped groove
that mates with a nylon-tipped set screw to hold the lid in place. The lid’s vent
is shown, sealed with a countersunk screw. A ring of hydroxyapatite seals the
chamber to the skull. C: detail cross section of the chamber base, showing the
2 ? 2.5 mm chamfer that is packed with hydroxyapatite following implanta-
tion. The screw hole is countersunk with a spherical profile of radius 2.5 mm
to match the shape of “Synthes style” bone screw heads.
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exposed over a region measuring ?30 ? 30 mm by elevating the
galea and periosteum. It is sometimes necessary to disinsert the
temporalis muscle from the superior temporalis line to widen the skull
exposure. This is done best with a small bone curette to avoid bleeding
and muscle damage.
The chamber was matched to the curvature of the skull by placing
it in a vise and gently bending the feet. It was immersed in a sterilizing
solution before placing it back on the skull to test the fit. Once the feet
were curved suitably to conform to the skull surface (maximum ?1
mm gap between skull surface and underside of foot), a 2-mm
orthopedic drill-bit (part no. 2.0 QCK, Veterinary Orthopedics Im-
plants, South Burlington, VT) was used to make a pilot hole (Fig. 3A).
To prevent accidental perforation of the dura, we sheathed the drill bit
in 12-G hypodermic tubing. A series of tubes were precut in lengths
ranging from 2 to 5 mm shorter than the drill bit so that the maximum
depth of the hole could be limited precisely. The screws were 2.7
mm-diameter ? 8 mm-long titanium cortex screws (part no. T270.08,
Veterinary Orthopedics Implants). Further details have been published
previously (Adams et al. 2007).
Once all the screws were in place, the chamber was sealed to the
skull surface using hydroxyapatite paste. Mimix QS (Biomet Micro-
fixation, Jacksonville, FL) is a fully synthetic apatite calcium phos-
phate material formed by tetracalcium and tricalcium phosphate.
Supplied as a sterile, nonpyrogenic, hydrophilic white powder, it is
mixed with citric acid to form a moldable paste. It was mixed and
pushed into the circular cavity between base of the chamber and the
bone surface using a spatula. A gloved finger was swept around the
ring to push the paste further into the gap, forming a smooth-surfaced
hydroxyapatite gasket. This maneuver must be performed quickly
because the paste begins to set after only 2–3 min. When complete,
the paste formed a continuous bead, caulking the space between the
chamfered chamber base and the bone surface (Fig. 3B).
After irrigation with physiological saline, the galea was sutured
over the top of the chamber feet using 5-0 or 6-0 polyglactin (Vicryl,
Ethicon, Somerville, NJ) absorbable suture. The skin incision was
closed with 4-0 silk, drawing the skin tightly up to the chamber wall.
It was necessary to suture only on one side of the chamber; the scalp
incision on the other side abutted the chamber wall (Fig. 3C). Topical
antibiotic ointment was applied to the incision (neomycin sulphate,
polymyxin sulfate and bacitracin zinc, Vetro-biotic, Pharmaderm,
NY). The chamber was filled with the antibiotic ointment prior to
closing the lid. After surgery, an opiate analgesic was administered
every 8 h for 2 days (buprenorphine HCl, 0.3 mg/kg im).
Trephination. In one animal, the skull was trephined at the time of
chamber implantation, while in two animals the procedure was carried
out weeks later. The delay was intended to allow bone remodeling to
reinforce the screws, so that the chamber was securely fastened before
trephination. It also provided extra time for the hydroxyapatite seal to
mature before opening the skull.
Using the anesthetic technique described above, a circular disk of
bone was removed using a 19 mm (3/4 in) diameter carbide grit hole
saw (Lenox Tools item no. 2991212CG). We modified the hole saw
by removing the abrasive grit from its outer surface with a grinding
wheel to prevent abrasion of the inside wall of the chamber. Since the
hole saw was guided by the chamber alone, its central drill bit was
withdrawn. A standard variable-speed electric drill was used to cut out
the bone disk. Rotation speed was kept below 200 rpm, and the
chamber interior was flushed frequently with sterile saline to mini-
mize heat buildup and to remove bone dust. Periodic checking of the
cutting progress allowed us to bias the pressure on different quadrants
of the circular cut to achieve an even trephination. Inevitably, how-
ever, one portion of the circular hole reached the dura first. The
advantage of using an abrasive hole saw, rather than a tool with sharp
teeth is that the dura is pushed away by the cutting surface and not
After the skull disk was removed, the chamber interior was allowed
to dry until the dura became translucent. This made it possible to
identify the lunate sulcus, and therefore to know the approximate
locations of V1, the V2 border, and V4 (Fig. 4). Photographs were
taken so that future electrode penetrations could be targeted to
the desired cortical area, and to avoid puncturing vessels in the
lunate sulcus. The chamber was then refilled with antibiotic oint-
ment, and the lid was closed. After surgery, buprenorphine was
administered as described above.
Assessment of performance. After 16 mo of daily extracellular
recordings, fluoroscopy was performed in one animal to evaluate the
seal between the chamber and skull (Phillips Allura Xper FD-10).
Images were obtained before and 45 min after filling the chamber with
a liquid contrast agent (lohexol 350, Omnipaque, GE Healthcare). The
next day the animal was euthanized for histological analysis. A lethal
dose of pentobarbital sodium (150 mg/kg iv) was administered,
followed by perfusion with normal saline and 1% paraformaldehyde.
Fig. 3. Chamber implantation procedure. A: one screw has been placed, and the
pilot hole for the second is being drilled. The scalp is retracted with a muscle
hook, and the drill bit is sheathed in a 12-G tube that leaves a limited length
of drill bit exposed. Holes are drilled slowly by hand while holding the drill in
a tap-wrench. B: once all screws are in place but prior to trephination, the
hydroxyapatite gasket is formed. It is visible as a white ring around the floor
of the chamber. C: following implantation, the scalp is sutured tightly around
the sealed chamber.
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The skull cap was removed, with dura and chamber attached. After
decalcification, thin sections of bone were stained with hematoxylin
and eosin to assess the bone/titanium interface and the hydroxyapatite
The recording chamber can be implanted at the same time as
the headpost, or in a subsequent surgical procedure. Simulta-
neous implantation has the advantage of sparing the animal a
second operation. However, if the monkey will require a
prolonged training period before recordings, it may be prefer-
able to install the headpost first.
Previously, we described a titanium headpost with a foot-
plate that attaches to the cranium without acrylic. A survey
revealed that 3/107 titanium headposts of similar design failed
because the footplate fractured (Adams et al. 2007). As a
precaution, our headpost design has been modified by increas-
ing the thickness of the footplate from 1.0 to 1.5 mm. Other
minor improvements have also been made (contact authors for
details). This headpost osseointegrates with the skull, forming
a permanent attachment (Fig. 5).
The recording chambers we designed were implanted in
three monkeys, and used for 6 mo, 16 mo, and 4 yr. After
implantation, photographs were taken at regular intervals to
assess performance (Fig. 6A). Wound healing was rapid, with
the circular scalp margin initially remaining apposed to the
chamber wall, covering the feet and screws entirely. With time,
scalp retraction sometimes occurred over chamber feet (Fig.
6B). To prevent scalp retraction, the chamber design was
modified slightly (Fig. 2). It is preferable for the scalp to cover
the feet, but exposure of the chamber feet did not cause screw
attachment to the skull to loosen. With normal fur regrowth,
the chambers appeared unobtrusive.
Chamber performance. The chamber lid was designed to fit
inside, rather than outside the chamber bore, to give it a flush
profile. A silicone o-ring in a circumferential groove in the lid
provided a watertight seal against the chamber bore. A vent
hole in the center of the lid that sealed with a countersunk
machine screw allowed excess liquids (e.g., antibiotic oint-
ment) to escape from the chamber during lid insertion.
Fig. 5. Postmortem view of titanium headpost implantation in an adult
macaque. The headpost was in place for 3 yr and 8 mo.
Fig. 6. A: view of the chamber 16 mo after implantation. Fur surrounding the
chamber has been trimmed. B: animal in Fig. 3, photographed 4 yr after
implantation. The scalp has receded from the chamber, exposing the anterior
foot and its screw head. However, the scalp remains healthy with scant exudate
along the margin around the chamber.
Fig. 4. Chamber interior immediately following trephination, 2 wk following
chamber implantation. When allowed to dry slightly, the dura becomes
semitransparent, and the large blood vessels that run along sulci can be
visualized. Photographs taken at this point are useful for planning electrode
penetrations to avoid surface vessels. V1, striate cortex; LS, lunate sulcus; V4,
visual area 4.
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If not opened regularly, the lid sometimes became difficult
to remove from the chamber. Pulling hard on the lid when the
animal is stabilized is not advisable because it stresses both
chamber and headpost attachments. A lid extraction tool that
exploited the thread of the vent hole was invented to pull the lid
into a cup-shaped chamber (Fig. 7). It pushed on the shoulder
of the chamber’s outer wall, applying no force to the bone
screws. Lid adhesion occurs because fluid dries between the
chamber bore and lid; lubrication with antibiotic ointment prior
to lid replacement helps to avoid this problem.
The lid was secured by a 4–40 stainless steel set screw,
tipped with nylon to avoid marring the surface. The screw tip
inserted into a V-shaped groove, pulling the lid down to its
fully seated position when tight. With daily tightening and
loosening, these retaining set screws were prone to wear. If the
hexagonal cup became rounded, removal of a tightened set
screw was difficult. Galvanic corrosion, resulting from contact
between two different metals in a saline environment, also
caused the stainless steel set screws to deteriorate. Using
titanium screws would prevent this. To avoid difficulty in
removing the set screws, they were replaced monthly.
A second function of the retaining set screw was to hold in
place a plastic chamber insert containing a square grid of
1.0-mm-spaced holes (Crist Instrument, Hagerstown, MD).
Before a recording session was started, the grid was placed into
the chamber, aligned with a fiduciary mark on the chamber rim,
and locked in place with the set screw. Electrodes were
inserted through grid holes to maintain a Cartesian coordinate
system of recording sites that was consistent from one day to
When recording from surface cortex, it is preferable to
penetrate the dura with a bare electrode to avoid damage from
a metal guide tube. However, the dura inside the recording
chamber becomes abnormally thick with time, making it resis-
tant to puncture. Electrodes are frequently damaged because
the shaft buckles from the compressive force necessary to
penetrate the dura. To overcome this problem, we employed a
system of concentric guide tubes to eliminate electrode buck-
ling. It permitted fragile glass-insulated tetrodes (Thomas Re-
cordings, Geissen, Germany) to penetrate the dura (Fig. 8).
Watertight chamber seal. A principal feature of the cham-
ber’s design was that it resulted in a watertight seal with the
skull surface. This seal prevented the escape of fluid from the
chamber and served as a barrier to infection. To test the seal
formed by the hydroxyapatite gasket, we obtained fluoroscopic
Fig. 8. System to provide axial support to prevent buckling of glass-insulated
electrodes as they penetrate dura. A schematic cross section of the chamber and
grid insert (Crist Instrument, Hagerstown, MD) is shown. The procedure for
electrode penetration is as follows. Remove the lid and place liquid agarose
(Sigma A0169) at 38°C inside the chamber. Immediately insert the grid so that
excess agarose is displaced through the grid holes, and the entire chamber
between the bottom surface of the grid and the dura is filled. Align the grid
with a fiduciary on the chamber rim. Tighten the set screw to hold the grid in
place. Insert the blunt 23-G outer guide tube (yellow) into the selected grid
hole and gently push it down, through the agarose, until it contacts the dural
surface. When gently pressed against the dura, the tube will “bounce back” to
a constant depth. Using a graduated periodontal probe, measure the length of
the tube that protrudes from the grid. Select a spacer (blue) of the same length
(?0.5 mm) from a set of precut 19-G tubes. Remove the 23-G guide tube,
pass it through the spacer, and reinstall it into the grid. The flared end of
the 23-G guide tube (inset 1) will prevent it from passing through the spacer
and advancing any deeper. As a result, the tip of the guide tube will be held
in a stable position against the surface of the dura (inset 2). The electrode
(cyan) is sheathed inside a blunt 31-G tube (red) that is attached to the
micromanipulator. This 31-G tube is the same length as the 23-G guide
tube. Align the 31-G tube using the X/Y stage and insert it into the 23-G
guide tube. Now, the electrode can be driven out of the 31-G tube. Because
it is supported axially, it cannot buckle, and its sharp tip will penetrate
thickened dura. Furthermore, the agarose will prevent lateral movement of
the guide tubes and minimize “tenting” of the dura that could otherwise
result in electrode fracture.
Fig. 7. Cross section of the chamber and a tool for removal of the lid. The tool
resembles an inverted cup with a hole for a freely turning screw in the center
of its base. A Teflon washer (white) is placed under the screw head. A stubborn
lid can be quickly and easily removed as follows: the retaining set screw and
vent screw are removed, and the extraction tool (magenta) is placed over the
chamber (cyan). The tool rests on the chamber’s shoulder. A socket head cap
screw is placed through a clearance hole at the tool’s center and into the
threaded vent hole of the lid (yellow). Rotation of the screw pulls the lid out
of the chamber bore without straining the chamber or headpost attachments to
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images of a chamber that had been implanted for 16 mo. The
chamber was filled with 5 ml of a radiopaque contrast agent.
There was no leakage of the contrast agent into the subdural or
epidural space. More importantly, there was no extravasation
of contrast agent under the scalp surrounding the chamber,
even after 45 min (Fig. 9). This indicated that the seal between
the chamber base and the skull was watertight.
Bacterial colonization. The scalp margin around the cham-
ber was cleaned as needed (usually weekly) by gentle rubbing
with a betadine swab, followed by application of a triple
antibiotic ointment consisting of neomycin, polymyxin, and
bacitracin. A dab of the antibiotic ointment was also placed
inside the chamber after every recording session to maintain
sterility. Surveillance cultures for aerobic, anaerobic, and fun-
gal organisms were performed periodically from the scalp
margins and chamber interior in all three animals. The scalp
cultures always grew Staph aureus, despite weekly application
of antibiotic. The organism was sensitive to bacitracin, oxacil-
lin, cephalothin, vancomycin, sulfisoxazole, timethoprim, tet-
racycline, tobramycin, ciprofloxacin, and moxifloxacin and
resistant to erythromycin. In one animal, cultures were also
positive for corynebacterium, a gram-positive rod found in the
mucosa and normal skin flora of primates.
In two animals, the chamber was not opened for several
weeks, during an interruption in recordings. Cultures were
taken immediately upon reopening the chamber, to see if the
interior remained sterile. In both cases, the cultures were
positive for Staph aureus. Neomycin, polymyxin, and bacitra-
cin antibiotic ointment was placed inside the chamber, and it
was recultured 3 days later, with negative results.
Control of dural proliferation and neovascularizaton. Re-
moval of a disk of skull to allow access for electrode record-
ings induces a proliferative response in the dura. The dura
becomes progressively thickened and vascularized, making it
more difficult to penetrate and prone to bleeding. An anti-
mitotic agent, 5-fluorouracil (5-FU), has been used to inhibit
this process (Spinks et al. 2003). We have tested mitomycin C
and bevacizumab, two other compounds with the potential to
alleviate the problem of dural thickening.
Mitomycin C is a potent DNA cross-linker, giving it anti-
neoplastic and antibiotic properties. It is used intravenously for
chemotherapy and topically in eye surgery to limit scar tissue
formation. In one animal, we tested mitomycin C to prevent
tissue proliferation inside the chamber. To ensure even appli-
cation and to prevent spillage, we placed a disk of gauze on the
chamber floor and soaked it with mitomycin C (1 mg/ml).
After 10 min, the gauze was removed, and the chamber was
irrigated with sterile saline. Mitomycin C was applied weekly
for 3 mo, and then monthly for another 3 mo. The dura
remained thin, avascular, and had a smooth, pale appearance.
During this period, daily recordings were made from single
cells in the operculum of V1 using glass-insulated tetrodes
without any obvious adverse effects from mitomycin C.
An alternative approach to prevent dural proliferation is to
use an agent that suppresses neovascularization. Bevacizumab
(Avastin, Genentech/Roche), an antibody against vascular en-
dothelial growth factor, was tested in one animal. After being
sealed for several months, during a hiatus in recordings, the
dura appeared thickened and vascularized (Fig. 10A). Bevaci-
zumab (5 mg in 0.2 ml) was applied twice. A week later,
granulation tissue could be removed easily with minimal bleed-
ing by wiping the dura with a cotton swab (Fig. 10B). Weekly
treatment with bevacizumab kept the dura soft and thin, so that
Fig. 9. Fluoroscopic images of an acrylic-free chamber taken
to test the seal between chamber and skull. A: side view taken
prior to filling the chamber with contrast agent. B: top view
with 5 ml of contrast agent in the bore. The contrast agent
was left in the chamber for 45 min and then removed.
C: high-sensitivity (shorter exposure) image taken immedi-
ately after removal of the contrast agent. There is no evidence
of leakage. D: side view after contrast agent removal. Seep-
age of the agent between the scalp and the skull would be
visible as a dark line in this image but not in A.
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it could be penetrated daily with glass-insulated tetrodes. There
was no apparent effect on the quality of recordings.
Bone remodeling. Necropsy was performed 16 mo after
implantation in one monkey. New bone growth had partially
covered some of the feet and retaining screws (Fig. 11A). The
base was well opposed to the skull, with no gap (Fig. 11B).
After removal of the chamber, it was evident that the skull had
remodeled to form a perfect impression of the chamber base.
The chamfer cavity and gaps between the chamber feet and
skull were filled completely by bone (Fig. 11C). Even the
imprint of the ?50-?m groves on the titanium surface, result-
ing from the manufacturing process, was visible on the bone
surface (Fig. 11D). Histological examination of the decalcified
specimen showed that the original hydroxyapatite gasket was
replaced by new bone tissue, resulting in intimate contact
between the chamfered base of the chamber and the remodeled
skull surface (Fig. 12). The new growth had the typical char-
acteristics of healthy lamella bone.
A lip of newly formed bone, up to 3 mm wide, had grown
from the edge of the original trephination margin into the
signifies that the bone underneath the implant was healthy. Even-
tually, bone regrowth limits the area of brain available for neural
grow back to close the trephination site entirely.
Survey of recording chambers. To share experience among
different laboratories, we emailed a questionnaire to 65 US
investigators using recording chambers for vision research in
alert monkeys. Replies were received from 37 investigators.
All but one reported using dental acrylic for recording cham-
bers. The majority (33/36) used the traditional approach, em-
bedding both the chamber(s) and headpost in a single acrylic
headcap. Three had adopted an acrylic-free technique to attach
headposts, but used acrylic to anchor the chamber to nearby
screws and to seal gaps between the chamber base and skull.
Most investigators stated that controlling infection and dural
thickening were the most challenging problems associated with
A number of antiseptic agents were applied on a regular
basis to control infection at the skin margin around the implant.
The most common substance was povidone-iodine (Betadine),
used by 24 investigators. Another 13 investigators used chlo-
rhexidine (Nolvasan). Other antiseptic agents used to control
skin margin infection were as follows: silver nitrate, dilute
hydrogen peroxide, Dakin’s solution, benzalkonium chloride,
trypsin (Granulex), 1% iodine in ethylene glycol and propylene
glycol (Xenodine), ichthammol, and isopropyl alcohol. In ad-
dition to antiseptics, 16 investigators regularly applied topical
antibiotics to the skin margins. Neomycin sulfate, polymyxin
sulfate, and bacitracin zinc ointment were used by nine inves-
tigators. Other antibiotics included the following: Neo-Predef
(a combination of neomycin and isoflupredone acetate and
tetracaine), nitrofurazone, and chloramphenicol.
Some investigators expressed doubt about the wisdom of
using antiseptics and antibiotics inside recording chambers
because of potential cerebral toxicity. Nonetheless, 19 inves-
tigators used an antiseptic, sometimes followed by sterile
saline irrigation. Povidone-iodine was used by seven investi-
gators and chlorhexidine by three investigators. Other agents
mentioned were as follows: chlorine dioxide (Clidox), sodium
hypochlorite, and Dakin’s solution. Antibiotics were used
inside the chamber by 17 investigators. Neomycin sulfate,
polymyxin sulfate, and bacitracin zinc ointment were used by
the majority. Other antibiotics mentioned were as follows:
Maxitrol (neomycin-polymyxin-dexamethasone), gentamicin,
To avoid dural thickening, five investigators used 5-FU, and
one used mitomycin-C. There were no reports of adverse
effects from these agents. No investigator reported experience
The use of implanted chambers to allow daily access to the
brain for extracellular recordings in alert monkeys poses many
challenges for the investigator. These devices provide a portal
for introduction of microelectrodes, but they also breach the
brain’s defenses. In essence, an animal with an implanted
chamber has a permanent transcranial fistula, which requires
regular wound care to suppress the proliferation of infectious
agents. The removal of skull by trephination induces a chronic
exudate from the surface of the exposed dura inside the
chamber. This exudate often becomes mixed with cerebrospi-
nal fluid, which drains through perforations created by guide
Fig. 10. Assessment of bevacizumab to reduce vascularization of dura and
granulation tissue in the chamber. A: photograph of chamber interior following
a period of ?3 mo without removal of the lid. B: the same chamber interior 1
wk after applying two 0.5-mg doses of bevacizumab in 0.2 ml on consecutive
days. The dura mater was wiped gently with a sterile Q-tip to remove loose
tissue, causing little bleeding.
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tubes and microelectrodes. A suppurative discharge can leak
between the chamber and skull, providing a conduit for entry
of infectious organisms from the scalp surface. This creates a
risk of meningitis, cerebritis, abscess, and even death.
PMMA was initially used in acute and short-term experi-
mental implants in small quantities as a sealant to close the gap
between the chamber and the skull (Evarts 1966; Evarts 1968;
Sheatz 1961). As the mechanical and technical demands of
implanted devices increased, it was used in greater amounts
and took on an additional structural role. However, its failure
to bond permanently to the skull surface can result in the
ingrowth of soft tissue and the loss of a watertight seal. Fluid
seeps along the interface between the headcap and skull,
providing a moist, sequestered environment for growth of
bacteria and fungi. The common skin flora that colonize this
habitat are potent stimulators of bone resorption (Nair et al.
1995). Weakening and loss of bone tissue surrounding the
metal fasteners that anchor the acrylic cap to the skull can
result eventually in implant detachment.
Recognizing the drawbacks of PMMA, Logothetis (2002)
has pioneered the use of biocompatible acrylic-free implants.
Several physical and chemical properties combine to make
titanium the most suitable material for implantable biomedical
devices. A protective oxide layer renders titanium’s surface
inert, making it compatible with biological tissue. Contact
between titanium and bone promotes the formation of a direct
interface, with no intervening soft tissue (Shi et al. 2008). This
process, known as osseointegration (Brånemark et al. 1969),
allows the apposing bone to proliferate so that the titanium
screws and footplates that anchor the implant become embed-
ded in the skull. The use of a screw-on titanium chamber also
diminishes the size and profile of the implant, making it less
obtrusive and unsightly. Without an acrylic headcap, more
scalp is preserved, leaving the skull mostly covered by normal,
healthy tissue. Bringing the scalp up to the chamber wall
reduces the exposed margin of surrounding tissue, thereby
reducing the burden of chronic wound infection. Colonization
of the scalp margin by infectious organisms can be controlled
by direct application of topical antiseptics and antibiotics. With
acrylic, organisms may proliferate underneath the headcap,
where they are inaccessible to antiseptics and antibiotics.
We used hydroxyapatite, instead of PMMA, to seal the small
gap between the chamber base and skull surface (Fig. 3B).
Hydroxyapatite supports bone proliferation and is replaced
eventually by living bone matrix (Fig. 12). As a result, the
chamber and skull became apposed intimately, forming a
stable seal. Fluoroscopy with a liquid contrast agent proved
that the seal was watertight, preventing leakage of chamber
contents under the scalp.
Despite the tight seal between chamber and skull, S. aureus
grew inside the chamber, unless antibiotics were administered
regularly. On occasions when daily recordings were suspended
and the chamber was left unopened, cultures were positive for
S. aureus. This bacterium is ?0.5 ?m in diameter, small
enough to pass easily through the helical channel between a
screw and its threaded hole. We suspect that bacteria can enter
the chamber via the set-screw hole in the chamber wall.
Addition of a compressible washer or an o-ring may seal this
potential leak to allow a closed chamber to remain sterile
Staphylococci and corynebacteria were cultured repeatedly
from the scalp margins surrounding the recording chambers.
These organisms are normal primate skin flora (Kluytmans et
al. 1997). Their growth can be suppressed by applying anti-
septics and antibiotics to the wound margin, but they cannot be
eliminated entirely (Owens and Stoessel 2008). S. aureus is a
potential pathogen: it is the most common cause of osteomy-
elitis and orthopedic implant infection in humans (Moyad et al.
Fig. 11. Postmortem examination of the titanium/
bone interface in a monkey implanted for 16 mo.
A: high-magnification photograph of one chamber
foot, showing the extent of new bone growth
around and over the metal. B: view inside the bore
of the chamber, focused at its base. A ridge of
new bone (arrow) has covered the circular face of
the trephination to extend inside the chamber
bore. C: the chamber has been removed, and the
outer skull surface photographed. The impression
of the chamber’s foot print is clearly evident
because bone has grown to close the gap between
chamber and skull. D: high-magnification view of
the region outlined with a white box in C. The
bone surface is textured with concentric 50-
?m-wide grooves. These are an imprint of the
machined metal surface. Inset shows the surface
of the chamber’s circular chamfer at the same
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2008). While chamber colonization with S. aureus appeared to
be tolerated in our animals, it would be preferable for the
chamber interior to remain sterile. For this reason, an antibiotic
ointment containing neomycin, polymyxin B, and bacitracin
was placed regularly inside the recording chamber. All staph-
ylococci species are sensitive to this triplet of topical antibiot-
ics (Jones et al. 2006). The survey of fellow investigators
revealed that triple antibiotic ointment was the most widely
used agent both inside and outside recording chambers. How-
ever, different laboratories have developed a variety of suc-
cessful treatment protocols and medication regimens for cham-
ber maintenance. Laboratories should not be required to adhere
to a single treatment regimen, but rather should be free to
devise a system for chamber maintenance that works best in
During penetration of the dura mater, the microelectrode tip
is usually withdrawn inside in a sharp hypodermic tube to
protect it from damage. Once within brain tissue, the electrode
can emerge safely from the protective guide tube. When
recording from surface cortex located immediately under the
dura, it is preferable to insert the microelectrode but not the
guide tube. The dura must be thin enough to allow penetration
of a bare electrode. Treatment with 5-FU arrests normal scar
formation and tissue proliferation, reducing the need for dural
peeling without adverse physiological or anatomical effects on
cortical tissue (Spinks et al. 2003). We tested mitomycin-C as
an alternative to 5-FU. It is used commonly in glaucoma
surgery, where it has a similar efficacy to 5-FU (Abraham et al.
2006; WuDunn et al. 2002). Like 5-FU, mitomycin-C is a
potent antibacterial agent and inhibits vascularization (Tomasz
1995). There is some evidence that mitomycin-C is longer
acting and better suited for topical application than 5-FU
(Anand and Khan 2009; Kim et al. 2008). However, we have
not made any direct comparisons of the two agents.
In 2004, bevacizumab was introduced for the treatment of
colon cancer. This antibody blocks vascular endothelial growth
factor, a signaling molecule responsible for stimulating angio-
genesis. We found that blocking angiogenesis in exposed dura
inhibited the proliferation of granulation tissue, the precursor
to fibrous scar formation. It also reduced bleeding during the
thinning of dural growth. We have not compared bevacizumab
with antimitotics, but a careful study would be useful to
discover if there is any benefit in using one agent over the
other, or even both in combination. While bevacizumab’s
effects on the brain have not been well studied, we have not
observed any adverse effects on neuronal activity. Bevaci-
zumab is regularly injected directly into the human eye to treat
macular degeneration (CATT Research Group 2011). Some
reassurance that it is safe for use in the recording chamber
should be provided by its lack of deleterious effects on human
The evolution of techniques is a critical factor in the ad-
vancement of any scientific field. When methodological refine-
ments carry with them direct implications for animal welfare,
it is important to pursue them. By eliminating PMMA in the
implantation of devices for daily recordings in monkeys, it is
possible to maintain animals in greater comfort for longer
periods, thus improving their quality of life, while increasing
the yield of data. However, PMMA still has advantages when
multiple chambers must be placed on the skull, or when a
chamber must be mounted in a stereotaxic plane to target
Developments in other fields, such as material sciences and
orthopedic medicine, have been valuable in guiding refine-
ments in the field of experimental primate neurophysiology. In
the 50 years since the dawn of neurophysiology in awake,
behaving monkeys, improvements have been made that benefit
both animals and scientific research. It is our hope that tech-
nical advances will continue to be made, aided by open sharing
of collective experience.
We thank colleagues in other laboratories, who have shared generously
their experience with the development of titanium, acrylic-free chambers.
Acknowledgement is made to the veterinarians, technicians, and husbandry
staff at the California National Primate Research Center (CNPRC) and Uni-
versity of California, San Francisco (UCSF) for care of the animals. Vicky
Cevallos at the UCSF Proctor Foundation performed the microbiological
cultures. Yerem Yeghiazarians performed the fluoroscopic study. Robert H.
Wurtz provided comments on the manuscript.
Fig. 12. Histological analysis of remodeled bone. A slice of the skull was cut
from the specimen shown in Fig. 11 (between black lines in C). It was
decalcified, and thin sections were cut and stained for hematoxylin and eosin.
A: transmitted light micrograph of the bone section (pink). The foot plate and
screw cross sections have been drawn in without obscuring the tissue section.
B: dark-field illumination micrograph of the region outlined by the black box
in A. Its histological appearance, with Haversian canals and concentric layers
of lamellae, is characteristic of cortical bone. This location, under the cham-
ber’s chamfer was originally filled with hydroxyapatite paste.
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GRANTS Download full-text
This work was supported by the National Eye Institute (Grant R01
EY10217 and Core Grant EY02162) and Research to Prevent Blindness.
Monkeys were supplied by the CNPRC, supported by National Institutes of
Health Base Grant RR00169.
No conflicts of interest, financial or otherwise, are declared by the author(s).
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