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Vertebrate Animals
1. The objective of the Shenoy lab is to understand how neural processing in the cerebral cortex of non-human
primates contributes to the preparation and execution of arm movements, and to then go on to design neural
prosthetic systems for translation into human patients suffering from a range of neurological diseases and
injuries. To achieve these goals it is essential to conduct neurophysiological experiments in awake, behaving
animals. Cognitive functions such as perception, attention, decision-making and motor planning occur only
in intact, functioning nervous systems. We therefore conduct simultaneous behavioral and
electrophysiological experiments in alert monkeys that are trained to perform tasks such as delayed reaches
to visual targets. By recording electrophysiologically the activity from single neurons during performance of
such tasks, we gain initial insights into the relationship of neuronal activity to the animal's behavioral
capacities. Hypotheses concerning this relationship are then tested by modifying neural activity within local
cortical circuits, using electrical (or soon optical) microstimulation or drug injections, to determine whether
behavior is affected in a predictable manner. Computer modeling techniques are then used to develop more
refined hypotheses concerning the relationship of brain to behavior that are both rigorous and testable.
All experiments will be performed on macaque monkeys (Macaca mulatta or Macaca fascicularis), male or
female, ages 4-12 years. The proposed experiments will require the use of ten (10) monkeys over the five
(5) year project period. All of our surgical procedures are standard for behavioral and electrophysiological
work with macaque monkeys. Each animal will undergo surgery for implantation of a head holding device
and a recording cylinder, that permits microelectrode access to relevant brain structures, or a chronic
electrode array. Chronic electrode arrays will be the main recording technique for the proposed experiments.
Other surgeries may include chronically-implanted EMG electrodes and simulating electrodes and / or
optical fibers (for use with optogenetic experiments). “In-rig” behavioral training is accomplished by
operant conditioning methods, using fluids as a reward for desired behavior. “In-cage” behaviors, when they
are actually trained, will proceed similarly. During experimental study, the animals are maintained on a
controlled water intake regimen with fluid levels that are adequate for full health while assuring that the
animals are motivated for behavioral experiments. Our procedures are not intrinsically harmful to the
animals. Most of our animals remain in experimental study for several years and are euthanized ultimately
only because of our need for histology.
2. Of the experimental preparations currently available for studying cognitive, preparatory and motor function
in alert animals, the most powerful and versatile is the awake, behaving monkey. Macaque monkeys can be
trained with operant conditioning techniques to perform a wide variety of simple cognitive tasks such as
short-term memory, sequential motor planning, and many others. We will use rhesus monkeys (Macaca
mulatta) for most of our experiments, but may occasionally use cynomologous macaques (Macaca
fascicularis) depending on the availability of rhesus. Rhesus is our species of choice for several reasons.
First, previous experiments have shown that the visual-motor capabilities and pathways of this species
closely resembles those of humans. Similarities make it highly probably that new discoveries in this species
will be directly applicable to our understanding of visual-motor function in humans. Secondly, rhesus
monkeys are ideal for experiments in which the animal's behavioral cooperation is required. They can be
trained on a wide variety of visual-motor tasks and will perform these tasks reliably in a laboratory
environment. Moreover, their natural behavior involves reaches, dexterous manipulations, locomotion, and
postures rather similar to humans. Finally, decades of research have given us a broad range of knowledge
concerning the organization of the visual-motor system. We are therefore able to ask relatively sophisticated
questions with this animal that would be impossible in a species for which more basic aspects of visualmotor function are poorly understood. Lower animals such as rats and mice can be employed to study
certain restricted functions at which they excel, such as spatial navigation (mazes) and olfactory function.
But it is exceedingly unlikely that rodents possess the full range of cognitive and visual-motor abilities that
we wish to study. For all of these reasons behaving monkeys are our ideal candidate for developing An
Animal Model of Freely Moving Human.
Ten (10) monkeys are required for the proposed research. All of the proposed experiments are highly
innovative and will therefore likely to yield very novel results, in our opinion. Such novel results must be
demonstrated in multiple animals to ensure that the data are sound and do not result from idiosyncratic
aspects of individual animals. Multiple animals to support each scientific discovery is also the standard in
the field, and for publishing results in top tier journals.
3. Our animals receive outstanding veterinary care and husbandry provided by the Veterinary Service Center
(VSC) at Stanford. All of Stanford's animal facilities are AAALAC accredited. The veterinarians and their
technical staff are experienced, well trained, and maintain the highest professional standards of their
profession. VSC employs several veterinarians, most of whom are also faculty members of the Department
of Comparative Medicine. At least one veterinarian is on-call at all times, and one of the veterinarians has
been designated as a specialist for the non-human primate labs (our neighboring monkey labs at Stanford
include Professors William Newsome, Tirin Moore, Jennifer Raymond, and Corinna Darian-Smith). The
veterinary staff are very responsive and are willing to work with us in great detail to solve any problem that
threatens the health of an animal. A veterinarian is typically present in our lab once or twice each week for
general monitoring or to address a specific clinical problem with an individual animal. Daily husbandry
(feeding, cage cleaning, general monitoring) is performed by trained VSC technical personnel. In addition,
my laboratory technician oversees all special husbandry that is performed by laboratory personnel,
consisting mainly of daily cleansing and care of cranial implants and recording cylinders. All animals are
tested for TB four times per year, and for herpes virus B once a year. Post-operative care is performed in the
VSC under our care and VSC staff care.
4. On arrival in the laboratory, monkeys typically exhibit varying degrees of fear of humans. We begin
immediately to tame and acclimatize them by spending a few tens of minutes with each monkey daily, and
giving them fruit or other treats such as peanuts or raisins. After a monkey has adjusted to living in the cage,
he is fitted with a collar. Collars are inspected at regular intervals to ensure that they fit correctly. The first
step in training is to condition the monkey to leave his cage and go voluntarily to a primate chair. A metal
pole is attached to the collar, the trainer holds the pole and moves the monkey from the cage to the floor,
and finally on into the primate chair where he receives reinforcement rewards. Eventually monkeys
typically learn to jump directly from their cage to the primate chair. For behavioral training, monkeys are
placed in the primate chair and brought to the laboratory.
“In rig” training occurs daily in sessions lasting up to 6 hours. After each training session, the monkey is
returned immediately to the home cage. Our studies require that monkeys fixate and/or reach to spots of
light projected on a screen. Monkeys receive juice or water reinforcements for correctly performing each
trial of such tasks. When we first begin training monkeys to reach to targets displayed on a screen they do
not reach in a consistent manner (an infrared camera system tracks the location of their hand in real time).
They do not consistently reach with their left or the right arm. Our neuro-physiological studies require that
monkeys consistently reach with one arm or the other. Several laboratories around the country lightly
restrain one of the arms, by loosely strapping (e.g., nylon ribbon) it to an arm rest or by placing the arm in a
loosely fitting pipe (e.g., plastic) that is secured to an arm rest. We adjust these restraints to assure a
comfortable fit. Both methods result in a comfortable resting position for the lightly-restrained arm and
monkeys soon learn to relax this arm and not attempt to reach with it. The restraint is left in place
throughout the duration of the training or recording session. Monkeys routinely reach to the visual targets
thousands of times during each training or experimental session, which is quite similar to the total number
of reaches that they would perform if they could reach with either arm. This suggests that consistently
reaching with one arm does not introduce significant fatigue.
These “in rig” experiments are conducted with awake, behaving monkeys sitting in primate chairs located
inside the laboratory. Experiments are typically conducted 5 days per week, but sometimes 7 days a week.
Each experimental session typically lasts 4-6 hours after which the monkey is returned to his cage.
Monkeys' heads are restrained by attaching the previously implanted head-holder receptacle to the top of the
primate chair. Monkeys then begin to perform behavioral tasks (e.g., fixating a spot of light, reaching to the
spot of light) and are rewarded for correct performance. The maximum time per day that a monkey will be
seated in a primate chair, whether or not his head is fixed and whether or not he is performing behavioral
tasks, is eight (8) hours. To record the activity of single neurons during the behavioral tasks, a metal
microelectrode (or microelectrodes) is (are) introduced under control of a micromanipulator. The electrode
is passed through the dura and advanced through the cerebral cortex to the desired recording site in cortex.
One to three electrode penetrations are made daily. On some penetrations microstimulation currents (e.g.,
20-80 A) are briefly (e.g., 100-400 ms) passed to stimulate eye or arm movements in order to assess the
anatomical location of the microelectrode. Stimulation is immediately discontinued if any discomfort is
observed. At the end of the day's experiment, the electrodes are removed. The inside of the stainless steel
recording cylinder is cleaned, partially filled with antibiotic solution and securely capped. Alternatively, to
record the activity of a population of neurons which is what we aim to do in the majority of the proposed
experiments, we neurosurgically implant silicon-based, 100-electrode arrays into the superficial layers of
cerebral cortex using standard neurosurgical techniques, practices, and materials. A connector is also
embedded in the head cap, allowing electrical connection to be made from our recording instrumentation to
the electrode array. When our “Hermes” miniature, head-mounted recording system is to be used a small
aluminum enclosure is added around this connector and recording electronics reside within this water-tight
enclosure.
Our “in cage” experiments will proceed similarly, but intentionally will not involved the elaborate training
necessary for “in rig” experiments. Animals will have neural and EMG (muscle) data wirelessly transmitted
to near-by receivers in the housing room. Cameras will monitor the animals’ every move, day and night.
Together these measurements will allow us to determine (e.g., massive “reverse correlation” problem) the
relationship between neural activity and behavior over a very broad range of behavioral contexts. Ultimately
it will be possible to automatically compute the neural-behavioral relationship day and night. Moreover, we
will also wirelessly activate either electrical microstimulation or optical stimulation (opsins are virally
delivered to specific neurons in cerebral cortex rendering them light excitable or light suppressed; in
collaboration with our neighbor, Karl Deisseroth, and his lab here at Stanford). This will allow us to
causally perturb neural activity, at the time of our choosing relative to ongoing neural or behavioral events
which we will monitor in real time, and will facilitate the direct testing of our hypotheses.
Mackenzie Risch, is our laboratory animal care technician. Mackenzie administers all analgesics,
anesthetics and tranquilizers to our animals in conjunction with medical procedures. The following plan for
alleviating pain and distress associated with our occasional medical procedures has been reviewed and
approved by our attending veterinarians and by the Stanford University Institutional Animal Care and Use
Committee (IACUC) referred to as the Administrative Panel on Laboratory Animal Care (A-PLAC). An
attending veterinarian, from the Stanford University Veterinary Service Center (VSC), is on call and is
typically in the Research Animal Facility (RAF) building. While a veterinarian is not present at all times
during procedures, a veterinarian is always available for consultation. Pre-Anesthetics: agent: Ketamine
hydrochloride, dose range: 5-15 mg/kg, route: IM; agent: Diazepam, dose range: 0.5 mg/kg; route: IV,
agent: atropine; dose range: 0.04 mg/kg, route: IM; agent: suprax, dose range: 1.5 mg/kg, route: PO;
Anesthetics: agent: Isoflurane, dose range: 1-5\%, route: IM; Postoperative analgesic: agent: torbugesic,
dose range: 0.02 mg/kg, route: IM, frequency: immediately after surgery; agent: Buprenorphine, dose range:
0.01-0.03 mg/kg, route: IM, frequency: q 8-12 h prn.
5. Monkeys typically participate in several studies over many years. This is possible because the initial
implantation surgery can often allow monkeys to participate in a few studies and, by simply shifting the
location of the recording cylinder or electrode array, these highly trained monkeys can participate in
subsequent investigations of other areas of the brain. We and the veterinary staff monitor each animal's
overall health, the condition of the implants and the total number of survival surgeries. When any of these
fall out of range, and we are not able to effectively treat the condition, euthanasia is deemed necessary.
Also, if scientific results require histological evaluation of brain slices then we again deem euthanasia
necessary. While retiring rhesus monkeys to large caged runs is sometimes possible, because we can remove
all implants, it is not the policy of Stanford University to allow such retirement. In practice such retirement
is often difficult and impractical as well. Thus all of our laboratory animals will ultimately be euthanized.
The standard procedure for euthanasia is consistent with the recommendations of the Panel on Euthanasia of
the American Veterinary Medical Association. Ketamine is administered as a pre-anesthetic then a
Nembutal injection, 40 mg/kg IV catheter in saphenous vein, is administered.