In-vivo voltammetry has successfully been used to detect dopamine release in rodent brains, but its application to monkeys has been limited. We have previously detected dopamine release in the caudate of behaving Japanese monkeys using diamond microelectrodes (Yoshimi 2011); however it is not known whether the release pattern is the same in various areas of the forebrain. Recent studies have suggested variations in the dopaminergic projections to forebrain areas. In the present study, we attempted simultaneous recording at two locations in the striatum, using fast-scan cyclic voltammetry (FSCV) on carbon fibers, which has been widely used in rodents. Responses to unpredicted food and liquid rewards were detected repeatedly. The response to the liquid reward after conditioned stimuli was enhanced after switching the prediction cue. These characteristics were generally similar between the ventral striatum and the putamen. Overall, the technical application of FSCV recording in multiple locations was successful in behaving primates, and further voltammetric recordings in multiple locations will expand our knowledge of dopamine reward responses.
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In this article, we share our recent technical challenges and successes in monkey voltammetry. Because our initial attempts failed to detect relevant phasic changes in our first animal, several modifications in electrodes, behavioral procedures, and voltammetric techniques were tried to find the best experimental condition to detect reliable reward responses. While our report represents primarily technical advancement, it now permits the detection of statistically significant event-related phasic dopamine changes in more than half of the recordings. We believe that this technique is now sufficiently advanced to undertake an analysis of actual dopamine release by behavioral events at multiple locations in the monkey brain.
Three Japanese monkeys (monkeys U, C and S; Macaca fuscata, female, 5 to 7 kg) participated in the voltammetry experiments. Monkey U and S were used for MFB stimulation, and S and C were for behavioral tasks (experiments S1-8 and C1-7 in Tables 1 and 2). The brain from one additional male monkey, which was euthanized after our similar experiment, was used for immunohistochemical staining of dopamine neurons. One monkey was provided by Kawahara Bird-animal Trading (Tokyo, Japan), and two were provided by NBRP "Japanese Monkeys" through the National BioResource Project (NBR) of the MEXT, Japan. K.Y. and M.I. are approved for certificate of NBR primate care instruction course (#380 and #358 respectively). The procedures were conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments established by the Science Council of Japan, and all experiments were approved by the Ethics Review Committee for Animal Experimentation of Juntendo University School of Medicine (protocol number 935). All possible efforts were made to minimize the number of animals used and their suffering. Throughout the study, animals were monitored in consultation with the institution's clinical veterinarian and also medical doctors of Department of Neurology of Juntendo University School of Medicine. No animals were euthanized during this study.
Animals were housed individually in 70x70x80cm cages and were on 12 hour light/dark cycle. Animals followed a primate diet (PS-A Oriental Yeast CO.LTD) and received daily sliced sweet potato. Animals also had access to primate toys and biting wood (S1 Fig), which were rotated on a weekly basis. Our monkey facility had wide windows, and natural sunlight lit the room beside 12 hr light-dark illumination 0700 to 1900. Animals had partial access to the outside view from the 11th floor window. Animal rooms are set to a constant temperature of 24 deg Celsius and the moisture was set 40 to 60%.
On weekday mornings, general behavioral responses of the animals were observed while offering small pieces of soft biscuit. At least three times a week, the animal was moved to the monkey chair to inspect and clean the head chamber with saline and treated with antibiotic ointment (gentacin or ofloxacin). The ointment was also spread on the border of the skin and dental cement, and the body weight was recorded. Water restriction was minimized for just two days immediately before behavioral voltammetric recordings (150ml/day). On the other days, a 500ml water bottle was given at meal-time or after the training. Water and monkey food chow (PS-A Oriental Yeast CO., LTD. Tokyo Japan) was given around noon, unless no other treatments or trainings were made. Thus, animals are monitored at least twice daily by lab personnel on weekdays.
All surgical procedures were performed under general anesthesia using aseptic techniques. A chronic recording chamber and a head holder were implanted in the monkey skull (Fig 1E). The chamber was placed in either the right (monkey U and S) or left (monkey C) striatum. Before surgery, the animals were pretreated with 0.015mg/kg of atropine and anesthesia was induced with ketamine-HCl (4 mg/kg, i.m.) and xylazine (1 mg/kg, i.m.). Antibiotics (cefazolin 50mg/body, i.m.) and diclofenac sodium suppository were given. Heart rate and SPO2 monitors were attached immediately before placement of the animal in the stereotaxic frame. An intravenous catheterization was made and pentobarbital sodium (10 mg/kg, i.v.) and butorphanol (0.03mg/kg, i.v.) were injected slowly to maintain anesthesia during the surgery. A recording chamber (25 x 30 mm) and a head-post holder were attached with dental cement and titanium anchor screws. Craniotomy was made under the recording chamber. Following surgical procedures, animals were monitored and given antibiotics (cefazolin 50 mg/body, i.m.) for 5 days. No scleral search coil to monitor eye position was implanted in this study.
Coronal MRI images of the head and brain were taken prior to the experiment with a 0.3-T magnetic resonance scanner (AIRIS2, Hitachi Medical Co., Tokyo, Japan) to determine the position of implantation for each monkey. For MRI scanning, animals were anesthetized with ketamine-HCl (4mg/kg, i.m.) and medetomidine (0.15mg/kg, i.m.). The chamber was visualized on MRI image by filling either with 2% agar or ointment with paraffin liquid. The relative position of the chamber was determined from the MRI images. Unit recordings were also used to verify the depth from the chamber.
Two locations were selected for the voltammetric recordings in each monkey. The positions of the ventral striatum and putamen were determined using MRI images. The anterior positions of coronal slices were adjusted to a brain atlas [21]. The recording positions were anterior to interaural center +25.0 to 26.5, right 6.0, and 17 to 19 mm below the dural surface in the ventral striatum of monkey S; +22.5 anterior, left 2.5 to 4.0, and 18 to 26 mm below the dural surface in the ventral striatum of monkey C; +22.0 anterior, right 12.0, and 18 to 20 mm below the dural surface in the putamen of monkey S; and +18.0 to 19.5, left 10.0, and 20 to 22 mm below the dural surface in the putamen of monkey C.
(A) Pavlovian cue-reward task. A timing cue (white dot, -2.5 to -1.5 s) and CS+ (red circle, -1.5 to 0 s to liquid reward delivery) were presented on the screen in front of the monkeys. The blue square was not followed by a juice reward in ordinary sessions. Free juice was delivered without these visual cues. (B) Recording positions indicated on the MRI image. (C) Licking movement associated with the task. A representative example of experiment S8. Infra-red (IR) motion sensor output placed beside the mouth of the monkey. Only calm trials (97 out from 111 trials) without movements during the baseline (-5.4 to -2.5s) are averaged in this figure. Free (no CS): black dots, CS-: blue, CS+: red with circle. The abscissa indicates the time from juice onset/CS offset. Average of 25 to 49 trials in experiment S8.
Small 0.5 g biscuits (Tamago-boro, Iwamoto Seika Co., Ltd., Aichi, Japan) were given to the animals restricted in the monkey-chair. The examiner (K.Y.) sat in front of the monkey face to face and took the pellets one by one from the backside table, approximately every 20 s. The delivery motion (look back, pick one biscuit, put it in front of the mouth of the monkey and activate a foot pedal timing-switch) occurred within 2 seconds. The body of the examiner was electrically connected to the common ground of the potentiostat. The food reward trials were effective to detect significant responses in our first animal (monkey S), instead of the typical Pavlovian liquid reward task.
A formaldehyde-fixed Japanese monkey brain was sliced into 40 μm coronal sections in a cryostat, and the sections were immunostained with an anti-TH rabbit antibody (Calbiochem) and visualized using the elite-ABC kit (Vector) and DAB as described previously [23].
We started with a typical Pavlovian task of liquid reward, following the previous studies [11, 14, 17]. As is the nature of this type of trial and error for technical development, the task schedule was modified continuously and was thus partially inconsistent between experimental days, particularly for the first monkey, S. The licking responses, detected by an infra-red motion sensor beside the juice spout revealed clearly differentiated licking responses between CS+ and CS- trials in monkey C, but not in monkey S (Table 1). We confirmed differentiated behavior of Monkeys S at the beginning of this study, but the differentiated behavior was lost after experiment S2. The 50% probability CS trials (green cross), which was not shown in the daily training but introduced only in the recording sessions of monkey S, potentially confused monkey S so these trials were omitted from S7 and from all sessions with monkey C. The food trial was introduced from experiment S5. CS reversal was also tried in some of the experiments, but the technique was tried only four times to avoid the establishment of new reversed learning. 2ff7e9595c
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