Figure 2. Chronic chemogenetic activation of CamKIIα-positive forebrain excitatory neurons during the early postnatal window results in a long-lasting increase in anxiety- and despair-like behavior in adult male mice.

(A) Shown is a schematic of the experimental paradigm to induce chronic CNO-mediated hM3Dq DREADD activation in CamKIIα-positive forebrain excitatory neurons using bigenic CamKIIα-tTA::TetO-hM3Dq mouse pups that were fed CNO (PNCNO; 1 mg/kg) or vehicle from P2 to P14 and then left undisturbed for 3 months prior to behavioral analysis performed in adulthood on male mice. (B) Shown are representative tracks of vehicle or PNCNO-treated CamKIIα-tTA::TetO-hM3Dq bigenic adult male mice in the open field test (OFT). A history of chronic postnatal hM3Dq DREADD activation of CamKIIα-positive forebrain excitatory neurons resulted in increased anxiety-like behavior on the OFT in adulthood, as noted by a significant decrease in the percent distance traveled in center (C), number of entries to the center (E), and the total distance traveled in the OFT arena (F) in PNCNO-treated mice as compared to vehicle-treated controls (n = 15 per group). The percent time spent in the center was not significantly altered (D) in PNCNO-treated mice as compared to vehicle-treated controls. (G) Shown are representative tracks of vehicle or PNCNO-treated CamKIIα-tTA::TetO-hM3Dq bigenic adult mice on the elevated plus maze (EPM). Adult mice with chronic postnatal hM3Dq DREADD activation of CamKIIα-positive forebrain excitatory neurons exhibited increased anxiety-like behavior on the EPM as revealed by a significant decrease in the number of entries to the open arms (J), and a trend toward a decrease in percent time spent in the open arms (I) in PNCNO-treated mice as compared to vehicle-treated controls (n = 15 per group). The percent distance traveled in the open arms (H) and the total distance traveled in the EPM arena (K) was not altered in PNCNO-treated mice as compared to vehicle-treated controls. (L) Shown is a schematic of the light-dark box used to assess anxiety-like behavior. Chronic postnatal hM3Dq DREADD activation of CamKIIα-positive forebrain excitatory neurons resulted in an increased anxiety-like behavior in the LD box test in adulthood, as revealed by a significant decline in the number of entries to the light box (M), and a trend toward decline in the time spent in the light box (N) in PNCNO-treated mice as compared to vehicle-treated controls (n = 15 per group). (O) Shown is a schematic representation of the forced swim test (FST) apparatus used to assess despair-like behavior. Chronic postnatal hM3Dq DREADD activation of CamKIIα-positive forebrain excitatory neurons resulted in an increased despair-like behavior on the FST in adulthood, as revealed by a significant increase in time spent immobile (P) in PNCNO-treated mice as compared to vehicle-treated controls (n = 13 per group). Results are expressed as the mean ± S.E.M. *p<0.05, ^$p=0.08, ^#p=0.07; as compared to vehicle-treated controls using the two-tailed, unpaired Student’s t-test.

Figure 2—figure supplement 1. Chronic chemogenetic activation of CamKIIα-positive forebrain excitatory neurons during the early postnatal window does not alter weight during CNO administration and in adulthood.

(A) Shown is a schematic of the experimental paradigm used to determine the influence of chronic CNO-mediated hM3Dq DREADD activation of CamKIIα-positive forebrain excitatory neurons in CamKIIα-tTA::TetO-hM3Dq bigenic mouse pups from P2 to P14 on gross weight during the time of treatment and in adulthood. (B) CamKIIα-tTA::TetO-hM3Dq bigenic mouse pups fed with CNO (1 mg/kg) once daily from P2 to 14 did not show any significant change in gross weight across the duration of treatment as compared to their vehicle-treated controls (n = 9 for vehicle; n = 11 for PNCNO). (C) Adult CamKIIα-tTA::TetO-hM3Dq bigenic mice with a history of postnatal CNO treatment from P2 to 14 did not show any significant change in gross adult weight as compared to vehicle-treated controls (n = 9 for vehicle; n = 11 for PNCNO). Results are expressed as the mean ± S.E.M.

Figure 2—figure supplement 2. Chronic chemogenetic activation of CamKIIα-positive forebrain excitatory neurons during the early postnatal window does not alter the developmental emergence of reflex behaviors.

(A) Shown is a schematic of the experimental paradigm used to determine the influence of chronic CNO-mediated hM3Dq DREADD activation of CamKIIα-positive forebrain excitatory neurons in CamKIIα-tTA::TetO-hM3Dq bigenic mouse pups from P2 to P14 on the postnatal emergence of reflex behaviors. (B) Surface righting was not significantly altered at P9 (n = 10 for vehicle; n = 11 for PNCNO) and P12 (n = 12 for vehicle; n = 16 for PNCNO) in PNCNO-treated mice as compared to vehicle-treated controls. (C) Negative geotaxis was not significantly altered at P9 (n = 12 for vehicle; n = 19 for PNCNO) and P12 (n = 12 for vehicle; n = 16 for PNCNO) in PNCNO-treated mice as compared to vehicle-treated controls. Results are expressed as the mean ± S.E.M.

Figure 2—figure supplement 3. Chronic chemogenetic activation of CamKIIα-positive forebrain excitatory neurons during the early postnatal window results in a long-lasting increase in anxiety-like behavior in adult female mice.

(A) Shown is a schematic of the experimental paradigm to induce chronic CNO-mediated hM3Dq DREADD activation in CamKIIα-positive forebrain excitatory neurons using bigenic CamKIIα-tTA::TetO-hM3Dq mouse pups that were fed CNO (PNCNO; 1 mg/kg) or vehicle from P2 to P14, and then left undisturbed for 3 months prior to behavioral analysis performed in adulthood on female mice. (B) Shown are representative tracks of vehicle or PNCNO-treated CamKIIα-tTA::TetO-hM3Dq bigenic adult female mice in the open field test (OFT). A history of chronic postnatal hM3Dq DREADD activation of CamKIIα-positive forebrain excitatory neurons resulted in increased anxiety-like behavior on the OFT in adulthood, as noted by a significant decrease in the percent distance traveled in center (C), and the number of entries to the center (E) in PNCNO-treated mice as compared to vehicle-treated controls. The percent time spent in the center (D), and the total distance traveled in the OFT arena (F) were not significantly altered across treatment groups (n = 11 per group). (G) Shown are representative tracks of vehicle or PNCNO-treated CamKIIα-tTA::TetO-hM3Dq bigenic adult mice on the elevated plus maze (EPM). Adult female mice with chronic postnatal hM3Dq DREADD activation of CamKIIα-positive forebrain excitatory neurons exhibited increased anxiety-like behavior on the EPM as revealed by a significant decrease in the percent distance traveled in the open arms (H), and a decrease in percent time spent in the open arms (I) in PNCNO-treated mice as compared to vehicle-treated controls. The number of entries to the open arms (J) and the total distance traveled in the EPM arena (K) was not altered in PNCNO-treated mice as compared to vehicle-treated controls (n = 11 per group). (L–N) Shown is a schematic of the light-dark box used to assess anxiety-like behavior. Chronic postnatal hM3Dq DREADD activation of CamKIIα-positive forebrain excitatory neurons did not alter anxiety-like behavior in the LD box test in adulthood in PNCNO-treated mice as compared to vehicle-treated controls (n = 11 per group). (O) Shown is a schematic representation of the forced swim test (FST) apparatus used to assess despair-like behavior. Chronic postnatal hM3Dq DREADD activation of CamKIIα-positive forebrain excitatory neurons did not influence despair-like behavior on the FST (P) in adulthood in PNCNO-treated female mice as compared to vehicle-treated controls (n = 14 for vehicle; n = 11 for PNCNO). Results are expressed as the mean ± S.E.M. *p<0.05 as compared to vehicle-treated controls using the two-tailed, unpaired Student’s t-test.

Figure 2—figure supplement 4. Chronic chemogenetic activation of CamKIIα-positive forebrain excitatory neurons during the early postnatal window does not alter repetitive behavior in adult male mice.

(A) Shown is a schematic of the experimental paradigm to induce chronic CNO-mediated hM3Dq DREADD activation in CamKIIα-positive forebrain excitatory neurons using bigenic CamKIIα-tTA::TetO-hM3Dq mouse pups that were fed CNO (PNCNO; 1 mg/kg) or vehicle from P2 to P14 and then left undisturbed for 3 months prior to behavioral analysis to assess repetitive behavior on the marble burial test performed in adult male mice. (B) Shown is a representative image of marble burial test. PNCNO-treated mice did not show any significant difference in the number of marbles buried (C) as compared to vehicle-treated controls (n = 12 per group). Results are expressed as the mean ± S.E.M.

Figure 2—figure supplement 5. Chronic CNO administration during the early postnatal window does not influence anxiety- and despair-like behavior in genotype-control, adult male mice.

(A) Shown is a schematic of the experimental paradigm to assess the influence of chronic CNO administration in genotype-control mice. Mouse pups, single-positive for either CamKIIα-tTA or TetO-hM3Dq and referred to as genotype-controls, were fed CNO (PNCNO; 1 mg/kg) or vehicle from P2 to P14 and then left undisturbed for 3 months prior to behavioral analysis performed in adulthood on male mice. (B) Shown are representative tracks of vehicle or PNCNO-treated genotype-control adult male mice in the open field test (OFT). A history of chronic postnatal CNO treatment did not influence anxiety-like behavior on the OFT in adulthood, with no change observed in the percent distance traveled in center (C), percent time spent in the center (D) and the number of entries to the center (E) of the OFT arena in PNCNO-treated genotype-control mice as compared to vehicle-treated genotype-control group (n = 13 for vehicle; n = 11 for PNCNO). We noted a small, but significant decline in the total distance traversed in the OFT arena (F) in the PNCNO-treated genotype-control mice as compared to vehicle-treated controls. (G) Shown are representative tracks of vehicle or PNCNO-treated genotype-control male mice on the elevated plus maze (EPM). Chronic postnatal CNO treatment did not alter anxiety-like behavior on the EPM, with no change observed in percent distance traveled (H), percent time spent (I), or number of entries (J) in the open arms of the EPM between PNCNO-treated genotype-control mice as compared to vehicle-treated controls (n = 13 for vehicle; n = 14 for PNCNO). The total distance traveled in the EPM arena (K) was also not different between the two groups. (L) Shown is a schematic of the light-dark box used to assess anxiety-like behavior. Chronic postnatal CNO administration did not influence anxiety-like behavior in the LD box test in adulthood, with no change noted for either the number of entries to the light box (M) or the time spent in the light box (N) in PNCNO-treated genotype-control mice as compared to vehicle-treated controls (n = 13 for vehicle; n = 14 for PNCNO). (O) Shown is a schematic representation of the forced swim test (FST) apparatus used to assess despair-like behavior. Chronic postnatal CNO administration did not influence despair-like behavior on the FST in adulthood, with no change in time spent immobile (P) in PNCNO-treated genotype-control mice as compared to vehicle-treated controls (n = 12 per group). Results are expressed as the mean ± S.E.M. *p<0.05 as compared to vehicle-treated genotype-controls using the two-tailed, unpaired Student’s t-test.

Figure 2—figure supplement 6. Chronic CNO administration during the early postnatal window does not influence anxiety- and despair-like behavior in C57BL/6J adult male mice.

(A) Shown is a schematic of the experimental paradigm to assess the influence of chronic CNO administration in the background strain for the bigenic CamKIIα-tTA::TetO-hM3Dq, namely the C57BL/6J mouse strain. C57BL/6J mouse pups were fed CNO (PNCNO; 1 mg/kg) or vehicle from P2 to P14 and then left undisturbed for 3 months prior to behavioral analysis performed in adulthood on male mice. (B) Shown are representative tracks of vehicle or PNCNO-treated background strain, adult male mice in the open field test (OFT). A history of chronic postnatal CNO treatment did not influence anxiety-like behavior on the OFT in adulthood, with no change observed in the percent distance traveled in center (C), percent time spent in the center (D), the number of entries to the center (E), or the total distance traversed in the OFT arena (F) in PNCNO-treated background strain mice as compared to the vehicle-treated background strain control group (n = 12 per group). (G) Shown are representative tracks of vehicle or PNCNO-treated background strain male mice on the elevated plus maze (EPM). Chronic postnatal CNO treatment did not alter anxiety-like behavior on the EPM, with no change observed in percent distance traveled (H), percent time spent (I), number of entries (J) in the open arms of the EPM, or the total distance traveled in the EPM arena (K) between PNCNO-treated background strain mice as compared to vehicle-treated background strain controls (n = 12 per group). (L) Shown is a schematic of the light-dark box used to assess anxiety-like behavior. Chronic postnatal CNO administration did not influence anxiety-like behavior in the LD box test in adulthood, with no change noted for either the number of entries to the light box (M) or the time spent in the light box (N) in PNCNO-treated background strain mice as compared to vehicle-treated controls (n = 12 per group). (O) Shown is a schematic representation of the forced swim test (FST) apparatus used to assess despair-like behavior. Chronic postnatal CNO administration did not influence despair-like behavior on the FST in adulthood, with no change in time spent immobile (P) in PNCNO-treated background strain mice as compared to vehicle-treated controls (n = 12 per group). Results are expressed as the mean ± S.E.M.

Figure 2—figure supplement 7. Chronic chemogenetic activation of CamKIIα-positive forebrain excitatory neurons during the early postnatal window using the DREADD agonist Compound 21 (C21) results in a long-lasting increase in anxiety-like behavior in adult male mice.

(A) Shown is a schematic of the experimental paradigm to induce chronic C21-mediated hM3Dq DREADD activation in CamKIIα-positive forebrain excitatory neurons using bigenic CamKIIα-tTA::TetO-hM3Dq mouse pups that were fed C21 (PNC21; 1 mg/kg) or vehicle from P2 to P14 and then left undisturbed for 3 months prior to behavioral analysis performed in adulthood on male mice. (B) Shown are representative tracks of vehicle or PNC21-treated CamKIIα-tTA::TetO-hM3Dq bigenic adult male mice in the open field test (OFT). A history of chronic postnatal hM3Dq DREADD activation of CamKIIα-positive forebrain excitatory neurons resulted in increased anxiety-like behavior on the OFT in adulthood, as noted by a significant decrease in the percent time spent in center (D) (n = 15 per group). The percent distance traveled in center (C), number of entries to the center (E), and total distance traveled (F) was not altered in PNC21-treated mice as compared to vehicle-treated controls. (G) Shown are representative tracks of vehicle or PNC21-treated CamKIIα-tTA::TetO-hM3Dq bigenic adult mice on the elevated plus maze (EPM). Adult mice with chronic postnatal hM3Dq DREADD activation of CamKIIα-positive forebrain excitatory neurons exhibited increased anxiety-like behavior on the EPM as revealed by a significant decrease in percent distance traveled in the open arms (H), as well as percent time spent in the open arms (I) (n = 15 per group). The number of entries to open arms (J) and the total distance traveled in the EPM arena (K) was not altered in PNC21-treated mice as compared to vehicle-treated controls. Results are expressed as the mean ± S.E.M. *p<0.05 as compared to vehicle-treated controls using the two-tailed, unpaired Student’s t-test.