Chemogenetic Inhibition of the Amygdala Modulates Emotional Behavior Expression in Infant Rhesus Monkeys

Jessica Raper; Lauren Murphy; Rebecca Richardson; Zoe Romm; Zsofia Kovacs-Balint; Christa Payne; Adriana Galvan

doi:10.1523/ENEURO.0360-19.2019

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Figure 1.
Schematic of the study design. Two infant rhesus monkeys received bilateral injections of AAV-hM4Di DREADD into the amygdala at 9 months of age, followed by 6 months of testing with either Vehicle (Veh), CNO (10 mg/kg), or clozapine (0.1 mg/kg). Before DREADD transduction, infants were tested on the human intruder paradigm with CNO to rule out any impact of the drug on the behavior in naive animals. MRI was conducted immediately before surgery to aid with amygdala injection localization and again at 4 months postsurgery in relation to another study not reported here. Monkeys were killed at 7 months postsurgery to determine DREADD expression with histology.
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Figure 2.
Time–concentration profiles of CNO and clozapine in infant monkeys. A, B, Plasma concentrations of CNO (yellow line) and its metabolite clozapine (green line) following subcutaneous administration of CNO at 10 mg/kg (A) and clozapine at 0.1 mg/kg (B) in the infant male (blue square) and female (pink circle) monkeys.
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Figure 3.
In vivo neuroimaging in infant rhesus monkeys. A, B, T1-weighted MRI scan presurgery (A) and again at 4 months postsurgery (B) for the female and the male monkeys. In the left hemisphere of the female, a reduction in the amygdala was observed at 4 months postsurgery (B), which was later confirmed in the Nissl section (inset). The green dashed lines approximately outline the amygdala.
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Figure 4.
Expression of DREADDs after virus injections in the amygdala. A, Injection tracks (blue lines, male; red lines, female) identified in Nissl stains; and DREADD expression coverage (gray outline) in the amygdala, as identified with immunoperoxidase. B–D, Examples of HA immunoperoxidase labeling in the amygdala. White rectangles indicate areas shown at higher magnification in subsequent panels. Arrows point to examples of HA(hM4Di)-positive neuronal cell bodies. In B–D, the approximate outline of the amygdala is indicated by green dashed lines. Scale bars and orientation in B apply to C and D. AP indicates the approximate anteroposterior plane from the interaural line, according to coronal drawings from a normalized infant rhesus monkey brain (J. Bachevalier, unpublished observations). AB, Anterior basal amygdala; AC, anterior commissure; B, basal amygdala; CE, central amygdala; Dor., dorsal; GPe, external segment of the globus pallidus: L, lateral amygdala; Lat., lateral; PL, paralaminar amygdala, Put, putamen.
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Figure 5.
Behavioral responses on the human intruder paradigm before DREADD transduction. A, B, Freezing (A) and anxiety (B) behaviors measured across task conditions (A, alone; P, profile; S, stare) in the infant female (pink circle) and male (blue square) monkeys before transduction (striped bar), compared with control monkeys from a previous study (historic controls, gray bars; Raper et al., 2013b). #Indicates a significant effect of condition on the task (p < 0.05).
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Figure 6.
Behavioral responses on the human intruder paradigm with and without DREADD inhibition of the amygdala. A–D, Freezing during the Profile condition (A), anxiety (B), hostility (C), and vocalizations (D) during the Stare condition after CNO or clozapine (CNO/clozapine, yellow bars) or vehicle (Veh, open bars) administration. *Indicates a significant effect of ligand (p < 0.05).
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Figure 7.
Looking behavior during the socioemotional attention task during DREADD-mediated inhibition of the amygdala. A represents examples of the video stimuli type, as well as outlines the specific areas of interest (AOIs) mouth (red), eyes (blue), and body (green) for social stimuli. B and C illustrate the percentage looking to the mouth or eyes of neutral (N), lipsmack (L), or threatening (T) social videos. D illustrates the percentage looking at the neutral (N) or aversive (A) nonsocial object after CNO or clozapine (yellow bars) or vehicle (open bars) administration. *Indicates a significant difference from vehicle (p < 0.05).

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Table 1.

Behavioral ethogram for human intruder

Category and specific behaviors	Measurement	Brief descriptions
Freeze	Duration	Rigid, motionless posture except slight head movement
Hostile	Cumulative frequency
Threat bark	Frequency	Low pitch, high intensity, rasping, guttural
Threat	Frequency	Any of the following: open mouth (no teeth exposed), head bobbing, or ear flapping
Cage aggression	Frequency	Vigorously slaps, shakes, or slams body against cage
Lunge	Frequency	A quick, jerky movement toward the stimulus
Vocalizations	Cumulative frequency
Coo	Frequency	Clear soft pitch and intensity, sounds like “ooooh”
Scream	Frequency	High pitch, high-intensity screech or loud chirp
Anxiety	Cumulative frequency
Scratch	Frequency	Rapid scratch of body with hands or feet
Body shake	Frequency	Shake of the whole body or just head and shoulders region
Tooth grind^a	Frequency	Repetitive, audible rubbing of upper and lower teeth
Yawn	Frequency	Open mouth widely, exposing teeth
Affiliative^b	Cumulative frequency
Lipsmack	Frequency	Rapid movement of pursed lips, accompanied by a smacking sound
Present	Frequency	Rigid posture (knees locked) with tail elevated and rump oriented toward the stimulus object
Fearful^b	Cumulative frequency
Withdrawal	Frequency	Quick, jerky motion away from the stimulus object (jump back)
Grimace	Frequency	Refracted lips, exposed clenched teeth
Stereotypies^b	Cumulative duration
Pacing	Duration	Repetitive motor pattern around the test cage
Motor stereotypy	Duration	Repetitive, abnormal voluntary or involuntary motor patterns (e.g., swinging, twirling, flipping)

List of all behaviors scored, how they are measured and a brief definition.
^aBehavior for which total duration was also measured.
^bBehavior that was rarely or never seen in the current study.

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Table 2.

Individual human intruder paradigm data

Session			Freezing (duration)			Anxiety (frequency)			Hostility (frequency)			Vocalizations (frequency)
order	Ligand	Subject	Alone	Profile	Stare	Alone	Profile	Stare	Alone	Profile	Stare	Alone	Profile	Stare
1	CNO (prior to transduction)	F M	0 0	203.97 313.34	0 0	0 0	0 0	26 28	0 0	0 0	193 151	0 0	0 0	80 41
2	CNO	F M	0 1.63	33.83 170.26	0 0	0 0	0 0	2 15	2 0	0 0	157 158	0 0	0 0	47 45
3	Vehicle	F M	0 0	222.58 342.97	0 0	0 0	0 0	38 71	8 0	0 1	148 112	0 0	0 0	33 19
4	CNO	F M	0 0	40.81 72.9	0 0	0 0	0 0	7 33	4 0	1 0	86 87	0 0	0 0	0 16
5	Clozapine	F M	0 0	53 68.07	0 0	0 0	0 0	0 26	1 0	3 0	147 124	0 3	0 0	116 67
6	Vehicle	F M	0 0	68 250.54	0 0	0 0	0 0	15 32	2 8	1 1	167 62	39 8	162 0	100 48

List of behaviors scored on the human intruder paradigm and the individual values for the female (F) and male (M) infant monkeys across the 6 test sessions and ligand administered during each session.

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Table 3.

Individual data social stimuli

Session			Presentation	Mouth			Eyes			Body
order	Ligand	Subject	order	Neutral	Lipsmack	Threat	Neutral	Lipsmack	Threat	Neutral	Lipsmack	Threat
1	Vehicle	F	1 2	0.00 0.96	0.00 4.37	0.00 1.84	0.31 19.87	33.33 13.85	37.88 19.04	29.60 45.25	20.63 24.84	0.87 47.21
1	Vehicle	M	1 2	0.00 4.99	0.00 7.79	0.00 1.62	3.16 9.11	18.90 13.71	28.67 21.83	24.66 24.55	44.35 19.97	25.72 35.01
2	CNO	F	1 2	7.67 4.35	10.02 10.80	12.90 11.35	32.75 42.77	30.48 22.86	19.42 12.21	48.01 44.36	50.92 57.28	46.71 49.69
2	CNO	M	1 2	0.29 10.94	4.53 9.26	9.94 13.24	1.75 16.86	8.05 8.97	8.27 6.94	28.50 45.63	28.42 35.98	19.80 41.78
3	Vehicle	F	1 2	2.13 7.86	2.42 4.60	2.87 5.88	9.56 6.76	0.14 10.66	21.73 12.19	33.28 43.95	25.11 44.00	17.54 27.12
3	Vehicle	M	1 2	2.24 8.89	1.21 2.28	0.32 9.63	31.28 0.00	20.98 3.33	22.00 0.00	28.70 44.17	35.91 31.07	29.45 28.85
4	Clozapine	F	1 2	15.21 5.57	18.04 35.65	2.18 7.64	30.23 9.14	38.21 6.32	7.74 9.95	45.38 68.35	36.60 36.52	39.91 50.47
4	Clozapine	M	1 2	4.76 10.00	3.78 11.87	11.10 13.98	8.37 7.57	12.71 6.24	10.36 8.11	35.92 33.35	27.37 39.65	24.75 25.07
5	Vehicle	F	1 2	19.31 6.82	5.08 3.78	24.09 9.74	9.24 15.06	15.72 16.62	4.17 3.74	60.13 61.65	63.73 61.95	59.86 65.18
5	Vehicle	M	1 2	17.23 5.47	0.18 2.60	4.52 3.13	20.40 1.29	17.36 9.36	13.18 1.18	18.91 37.53	23.70 49.22	46.12 48.11

Average percentage looking at specific AOIs for the first and second presentation of social stimuli containing neutral, lipsmack, or threatening emotional valences. Individual values for the female (F) and male (M) infant monkeys across the five test sessions and ligand administered during each session of the Socioemotional Attention task.

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Table 4.

Individual data nonsocial stimuli

				Object
Session order	Ligand	Subject	Presentation order	Neutral	Aversive
1	Vehicle	F	1 2	47.60 70.63	38.95 70.22
1	Vehicle	M	1 2	28.21 58.27	40.17 67.16
2	CNO	F	1 2	74.61 87.86	87.72 83.51
2	CNO	M	1 2	58.56 76.47	90.26 69.40
3	Vehicle	F	1 2	54.10 41.48	25.12 27.80
3	Vehicle	M	1 2	58.17 54.98	55.99 36.10
4	Clozapine	F	1 2	65.32 66.00	75.28 64.79
4	Clozapine	M	1 2	34.11 50.30	66.02 73.88
5	Vehicle	F	1 2	49.33 61.40	26.99 37.21
5	Vehicle	M	1 2	68.30 51.33	54.82 46.36

Average percentage looking for the first and second presentation of neutral and aversive nonsocial stimuli. Individual values for the female (F) and male (M) infant monkeys across the 5 test sessions and ligand administered during each session of the Socioemotional Attention task..