Quantifying anhedonia-like symptoms in marmosets using appetitive Pavlovian conditioning

Abstract

Blunted reward responsivity is associated with anhedonia in humans and is a core feature of depression. This protocol describes how to train the common marmoset, Callithrix jacchus, on an appetitive Pavlovian conditioning paradigm to measure behavioral and cardiovascular correlates of anticipatory and consummatory phases of reward processing. We describe how to use intracerebral infusions to manipulate brain regions whose activity is relevant to impaired reward processing in depression and how the paradigm can be used to test antidepressant efficacy. For complete details on the use and execution of this protocol, please refer to Alexander et al. (2019).

Keywords: Behavior; Model Organisms; Neuroscience.

Graphical abstract

Figure 1

The home cage The home cage housed the marmosets in pairs (A) Entire home cage, dimensions: 280 × 120 × 98 cm. Enrichment items were placed throughout the cage including ropes, logs, ladders, and toys. (B) Clear plastic verandas forming the roof of the home cage. (C) Nest box. The entrance to the nest box included a flap to prevent the cannula implants catching on the doorway. (D) Food tray, where marmosets were fed each day. (E) Plastic tray forming the floor of the home cage.

Figure 2

The Pavlovian discrimination testing chamber and food box apparatus (A) The testing chamber, with a sound attenuating external casing. (B) The doors of the testing chamber opened to visualize the food box apparatus. The houselight (h) consists of two strips of LED lights either side of the main chamber. The cameras (c) record r using three views: top-down, left food box and right food box. The food box (fb) module has a left and right food box and a central clearing for the carry box and marmoset. The telemetry receiver (t) is concealed beneath the floor of the apparatus. A speaker system (s) is used to play auditory cues (CS- and CS+). (C) A close-up view of the internal frame of the food box module. The left food box (fb) is filled with marshmallow, whereas the right food box (fb) is empty. Doors (d) obstruct access to both food boxes – the opaque black door is visible, behind which is a transparent door. The latch (l) is used to attach the carry box into the internal frame of the apparatus. (D) The right opaque door has opened to reveal an empty food box. The food box light is also on. The transparent door still obstructs access to the empty food box chamber. This is equivalent to the US- period.

Figure 4

The PhysioTel Telemetry System and Spike2/video recording software The MX2 Matrix system integrates pressure information from the receiver and the ambient pressure reference monitor, and both the absolute and ambient pressure readings are transferred to the acquisition computer which generates a continuous telemetry readout.

Figure 3

Screenshot of the bespoke software interface used to control the testing chamber Software is available upon request from the lead author. The options for each prompt are shown in brackets ( ) and the default entry for each prompt is shown in square brackets [ ]. This screenshot is an example of a session containing a single CS-/US- trial followed by a single CS+/US+ trial. The first set of prompts concern global task parameters: the monkey name and file name; the name of an SQL database which stores the task parameters (in this case called AversivePavlovianLaith, storing data for both appetitive and aversive Pavlovian tasks we run in the apparatus); the initial offset time at the start of the session (one minute); the inter-trial interval (ITI, set to vary between 70 and 110 s); the houselight (set to comprise of the red, blue, and green LED lights to create a white light); and the number of CS types (two: CS- and CS+). The first CS type describes the CS-/US- trial. The CS duration is 20 s and is set as a ‘wav’ file with the name ‘PhoneVA’ (the sound of a phone ringing). The CS is specified to continue playing during the US, but not to carry on beyond the end of the US. The US is set to ‘food box visible’ (US-) and to last 120 s. The second CS type describes the CS+/US+ trial. The CS parameters are identical except the ‘wav’ file is now ‘DreamharpNB’ (the sound of a harp playing). The US is set to ‘food box open’ (US+) and to last 120 s. Finally, the user is prompted to review and confirm the parameters before beginning the session (not fully shown).

Figure 5

Monitoring equipment for surgical procedures Adapted from Jackson (2016). (A) Capnograph/pulse oximeter, showing clockwise from top left: expired CO₂ partial pressure (EtCO₂), oxygen saturation (SpO₂%), pulse rate, breathing profile, and respiratory rate. The tube with orange connector seen on the top left of the photograph samples gases from inside the intubation tube which is not shown here. (B) Pulse oximetry sensor applied to hand of marmoset during surgery. (C) Close-up view of a capnograph readout. (D) Rectal thermometer with lubricated plastic sheath covering the probe.

Figure 6

The telemetry probe and insertion of the probe during telemetry surgery (A) Telemetry probe. The probe body contains a battery, signal emitter and sensor. The catheter is filled with a non-compressible fluid and bio-compatible gel at the catheter tip. The tip is coated with an anti-thrombogenic film. (B) Implantation procedure. Blood flow in the descending (abdominal) aorta is briefly occluded and a small incision is made in the arterial wall. A catheter introducer with a grooved tip is used to lift a flap at the incision site. The catheter is introduced using forceps and is guided into the vessel through a groove in the catheter introducer. Once the catheter was in place and an adequate signal is detected, the catheter is secured in place with glue and a cellulose patch.

Figure 7

Stereotaxic frame for cannulation surgery The stereotaxic frame was specially modified for the marmoset. The marmoset was secured on the frame using ear bars (e), eye bars (i) and a mouth bar (m).

Figure 8

Output from pre-processing of the raw telemetry trace The tab-separated text file contains seven columns: time of the local maximum of the cardiac cycle used for RR interval/heart rate measurement; RR interval as the time between the local maximum and the previous cardiac cycle maximum (s); heart rate (beats per minute); a second time column corresponding to the local maximum of the cardiac cycle used for systolic blood pressure measurement (identical to the first column); systolic blood pressure (mmHg); a third time column corresponding to local minimum of the cardiac cycle used for diastolic blood pressure measurement; and the diastolic blood pressure (mmHg). Note that the first row of data in the output is ignored during further analysis as the RR interval is calculated using t=0 as the reference point for the previous cardiac cycle maximum (because there is no cardiac cycle preceding the first cycle) generating an erroneous result. Mean arterial pressure (MAP) values for each cycle were calculated later in the analysis.

Figure 9

Intracerebral infusion set-up (A) Infusion set-up. Hamilton syringes are connected to an injector with PTFE infusion lines. The Hamilton syringes are mounted onto a syringe pump with an adjustable rate. (B) Hamilton syringe. (C) Infusion line after an infusion. Prior to an infusion, the syringes and lines are filled with 0.9% sterile saline. A bubble is manually drawn up, and then drug (or saline control) is drawn up so that the fluid after the bubble is the substance to be infused. The bubble is marked with permanent marker. This is used to assess fluid movement – as can be seen, the bubble has moved beyond the initial markings.

Figure 10

Testing the efficacy of ketamine to ameliorate blunted reward processing Adapted from (Alexander et al., 2019). Marmosets receive a single i.m. injection of 0.5 mg/kg racemic ketamine at t = 0, followed by over-activation of sgACC/25 using DHK 4 h, 1 day and 7 days later. Marmosets also received an infusion of DHK 21 days later to determine if the effect of ketamine had abrogated (not shown). 10 min before each timepoint, DHK was infused into sgACC/25 and marmosets were tested on the appetitive Pavlovian discrimination paradigm (CS-/US-, CS+/US+ session which lasts approximately 7 min).

Figure 11

Anticipated outcome of the protocol: successful discrimination performance as evidenced by a rise in blood pressure during the CS+/US+, but not during the CS-/US- (A) Typical performance on the Pavlovian appetitive task, shown for one animal over 30 sessions. The x axis represents the session number, and the y axis represents the CS directed change in mean arterial pressure (MAP). The control (saline) infusion session is indicated by the black arrow. (B) Within-session performance following the saline infusion. The CS+/US+ trial (orange) is plotted on the same axis as the CS-/US- trial (gray) with baseline (BL), CS and US phases shaded. The x axis represents time within the trial (with 0 s being CS onset) and the y axis represents change in MAP at any one point compared to the average mean arterial pressure during the baseline period. The MAP rises during the CS+ period and then rises further during the US+ period.

Figure 12

Anticipated outcome of the protocol: the effect of intracerebral infusions on anticipatory (CS directed) arousal, in this case infusing dihydrokainic acid (DHK) into sgACC/25 Adapted from Alexander et al., 2019. n=5. Symbols represent individual subjects. (A) CS directed change in mean arterial pressure (MAP) values during the CS+ (orange) and CS- (gray) periods comparing control (saline) and over-activation (DHK) infusions into sgACC/25. Over-activation blunted anticipatory blood pressure arousal in a CS-dependent manner (manipulation × CS, F_1,4 = 10.63, p = 0.031), decreasing responding to the CS+, but not the CS− (effect of manipulation: CS+, p = 0.006; CS−, p = 0.301). (B) CS directed change in head-jerk values during the CS+ (orange) and CS- (gray) periods comparing control (saline) and over-activation (DHK) infusions into sgACC/25. Over-activation blunted anticipatory behavioral arousal in a CS-dependent manner (manipulation × CS, F_1,4 = 72.25, p = 0.001), decreasing responding to the CS+, but not the CS− (effect of manipulation: CS+, p < 0.001; CS−, p = 0.407).

Figure 13

Anticipated outcome of the protocol: measuring the efficacy of the antidepressant ketamine Adapted from Alexander et al., 2019. n=4. Symbols represent individual subjects. (A) Ketamine had a time-dependent effect to reverse the blunted blood pressure response induced by sgACC/25 over-activation using DHK (time point × CS, F_2,12 = 14.71, p < 0.001). Compared to control infusions of saline (not shown), sgACC/25 over-activation 4 h after ketamine administration still resulted in significant blunting of blood pressure responses (manipulation × CS, F_1,3 = 60.46, p = 0.004; effect of manipulation on CS+, p = 0.003). Over-activation 1 day following ketamine administration evidenced amelioration of the blood pressure impairment (4 h versus 1 day: CS+, p < 0.0001; CS−, p = 0.863) as did over-activation 7 days following ketamine administration (4 h versus 7 days: CS+, p < 0.001; CS−, p = 0.704). (B) Ketamine had a time-dependent effect to reverse the blunted head-jerk response induced by sgACC/25 over-activation using DHK (time point × CS, F_2,12 = 19.59, p < 0.001). Compared to control infusions of saline (not shown), sgACC/25 over-activation 4 h after ketamine administration still resulted in significant blunting of behavioral arousal (manipulation × CS, F_1,3 = 25.59, p = 0.015; effect of manipulation on CS+, p = 0.012). Over-activation 1 day following ketamine administration evidenced amelioration of the behavioral impairment (4 h versus 1 day: CS+, p < 0.0001; CS−, p = 0.371) as did over-activation 7 days following ketamine administration (4 h versus 7 days: CS+, p < 0.0001; CS−, p = 0.767).

Figure 14

Two-way repeated-measures ANOVA readout from GraphPad Prism using infusion data from the protocol The raw data are shown in Table 1. (A) The results of the two-way ANOVA. There is a significant Drug type × CS type interaction (p=0.0311) indicating a multiple comparisons test should be performed to understand which specific comparisons are significant. (B) The results of Sidak’s multiple comparisons test. The comparison of interest was between saline and drug for both CS- and CS+ arousal responses. There is a significant difference in the response to the CS+ (p=0.0064) but not the CS- (p=0.2995).