Fig 1.
Bbs1M390R/M390R mice have impaired long-term context fear conditioning.
A.) Schematic diagram of the delay fear conditioning procedure. On the first day, a mouse is placed in a chamber, and a sound is paired with a shock multiple times. On the second day, a mouse is placed in an altered chamber. The chamber is triangle shaped (represented by the blue triangle) with a smooth floor, and a sound is given to test long-term cue fear conditioning. On the third day, the mouse is placed back in the same chamber (context) as day 1, without a sound cue. This set up is used to test long-term context fear conditioning. B.) Day 1 acquisition between the control mice (n = 16) and the Bbs1M390R/M390R mice (n = 16) for long-term fear conditioning differed significantly (2-way ANOVA, time X genotype, F (13, 420) = 0.4488, P = 0.950, time, F (13, 420) = 179.8, P<0.0001, genotype, F (1, 420) = 11.46, P = 0.0008). The thick lines above the curve indicate when the sound cue was given, and the thick lines below the curve indicate when the shock was given. C.) The immediate fear conditioning indicates training to the day 1 fear conditioning. The immediate fear conditioning is measured as the difference of the freezing time (%) just before conditioning (first three minutes) and just after conditioning (last minute). The immediate fear conditioning did not differ significantly between the control mice (n = 16) and Bbs1M390/M390R mice (n = 16) used for long-term fear conditioning (Welch’s t-test, P = 0.2107). The post 24 hr fear conditioning represents cue fear conditioning, and is portrayed as Day 2 on the schematic diagram. The 24 hr fear conditioning (cue) is measured as the difference of the freezing time (%) before the tone (cue) on day 2 and during the tone (cue) on day 2. The 24 hr fear conditioning (cue) did not differ significantly between the control mice (n = 16) and the Bbs1M390R/M390R mice (n = 16) (Welch’s t-test, P = 0.2414). The post 48 hr fear conditioning represents context fear conditioning, and is portrayed as Day 3 on the schematic diagram. The 48 hr fear conditioning is measured as the difference of the freezing time (%) just before conditioning (first three minutes of day 1) and during the context on day 3. The 48 hr fear conditioning (context) between the control mice (n = 16) and the Bbs1M390R/M390R mice (n = 16) differed significantly (Welch’s t-test, P = 0.0240). control mice = Bbs1M390R/+ mice, M390R = Bbs1M390R/M390R mice, hr = hour, ns = not significant, * P< 0.05.
Fig 2.
Bbs1M390R/M390R mice have normal short-term context fear conditioning.
A.) Schematic diagram of the one day delay fear conditioning procedure. On the first day, a mouse is placed in a chamber, and a sound is paired with a shock multiple times. One hour later, the mouse is placed back in the chamber, and freezing is measured for short-term context fear conditioning. B.) Day 1 acquisition between the control mice (n = 9) and the Bbs1M390R/M390R mice (n = 9) used for the short-term fear conditioning differed significantly (2-way ANOVA, time X genotype, F (13, 224) = 0.5574, P = 0.8858, time, F (13, 224) = 56.19, P<0.0001, genotype, F (1, 224) = 9.369, P = 0.0025). The thick lines above the curve indicate when the sound cue was given, and the thick lines below the curve indicate when the shock was given. C.) The immediate fear conditioning indicates training to the day 1 fear conditioning. The immediate fear conditioning is measured as the difference of the freezing time (%) just before conditioning (first three minutes) and just after conditioning (last minute). The immediate fear conditioning did not differ significantly between control mice (n = 9) and Bbs1M390/M390R mice (n = 9) used for the short-term fear conditioning (Welch’s t-test, P = 0.8004). The 1 hr fear conditioning represents short-term context fear conditioning. The 1 hr fear conditioning was measured as the difference of the freezing time (%) just before conditioning and 1 hour after conditioning. The day 1 fear conditioning for context between the control mice (n = 9) and the Bbs1M390R/M390R mice (n = 9) did not reveal a significant difference (Welch’s t-test, P = 0.3436). control mice = Bbs1M390R/+ mice, M390R = Bbs1M390R/M390R mice, hr = hour, ns = not significant.
Fig 3.
Bbs8 is involved in long-term context fear conditioning postnatally.
A.) Timeline of the tamoxifen I.P injections of the experimental mice, BBS8 CKO (Bbs8flox/- and Bbs8flox/flox; UBC-CreERT2) and littermate control mice, Control (Bbs8flox/- and Bbs8flox/flox; UBC-CreERT2-). To induce Bbs8 deletion in BBS8 CKO mice, Tamoxifen was injected at P9, P12, and P15 (denoted by the syringe image). At 2 months of age, the mice were tested for long-term fear conditioning. The first day was the acquisition phase for fear conditioning, the second day was cue fear conditioning, and the third day was context fear conditioning. B.) Day 1 acquisition curve between the Control mice (n = 9) and BBS8 CKO mice (n = 9) differed significantly (2-way ANOVA, time X genotype, F (13, 224) = 1.721, p = 0.0579, time, F (13, 224) = 31.71, P<0.0001, genotype, F (1, 224) = 39.16, P<0.0001). The thick lines above the curve indicate when the sound cue was given, and the thick lines below the curve indicate when the shock was given. C.) The immediate fear conditioning indicates training to the day 1 fear conditioning. The immediate fear conditioning was measured as the difference of the freezing time (%) just before conditioning (first three minutes) and just after conditioning (last minute). The immediate fear conditioning did not differ significantly between the Control mice (n = 9) and BBS8 CKO mice (n = 9) used for long-term fear conditioning (Welch’s t-test, P = 0.3717). The post 24 hr fear conditioning represents cue fear conditioning, and is portrayed as Day 2 on the schematic diagram. The 24 hr fear conditioning (cue) was measured as the difference of the freezing time (%) before the tone (cue) on day 2 and during the tone (cue) on day 2. The 24 hr fear conditioning (cue) did not differ significantly between the Control mice (n = 9) and BBS8 CKO mice (n = 9) (Welch’s t-test, P = 0.4325). The post 48 hr fear conditioning represents context fear conditioning, and is portrayed as Day 3 on the schematic diagram. The 48 hr fear conditioning was measured as the difference of the freezing time (%) just before conditioning (first three minutes of day 1) and during the context on day 3. The 48 hr fear conditioning (context) between the Control mice (n = 9) and BBS8 CKO mice (n = 9) differed significantly (Welch’s t-test, P = 0.0099). Control = Bbs8flox/- and Bbs8flox/flox; UBC-CreERT2- mice, BBS8 CKO = Bbs8flox/- and Bbs8flox/flox; UBC-CreERT2 + mice, hr = hour, del = deletion, flx = flox, hr = hour, ns = not significant, ** P< 0.01.
Fig 4.
Bbs1 in the forebrain is involved in long-term fear conditioning.
A.) Schematic diagram of the three day delay fear conditioning procedure. On the first day, a mouse is placed in a chamber, and a sound is paired with a shock multiple time. On the second day, a mouse is placed in an altered chamber that is triangle shaped (represented by the blue triangle) with a smooth floor, and a sound is given to measure cue fear conditioning. On the third day, the mouse is placed back in the same chamber, and measured for freezing without sound. This gives the context fear conditioning. B.) Day 1 acquisition between the Emx1-Cre mice (control, mixed strain of C57BL/6 and 129/SVeV, n = 12) and the Bbs1flox/-; Emx1-Cre (BBS1 CKO, mixed strain of C57BL/6 and 129/SVeV, n = 13) differed significantly (2-way ANOVA, time X genotype, F (13, 322) = 3.483, P<0.0001, time, F (13, 322) = 93.95, P<0.0001, genotype, F (1, 322) = 137.5, P<0.0001). The thick lines above the curve indicate when the sound cue was given, and the thick lines below the curve indicate when the shock was given. C.) The immediate fear conditioning indicates training to the day 1 fear conditioning. The immediate fear conditioning is measured as the difference of the freezing time (%) just before conditioning (first three minutes) and just after conditioning (last minute). The immediate fear conditioning did not differ significantly between the control Emx1-Cre mice (n = 12) and Bbs1flox/-; Emx1-Cre (n = 13) used for the long-term fear conditioning (Welch’s t-test, P = 0.8999). The post 24 hr fear conditioning represents cue fear conditioning, and is portrayed as Day 2 on the schematic diagram. The 24 hr fear conditioning (cue) is measured as the difference of the freezing time (%) before the tone (cue) on day 2 and during the tone (cue) on day 2. The 24 hr fear conditioning (cue) did not differ significantly between the control Emx1-Cre mice (n = 12) and Bbs1flox/-; Emx1-Cre (n = 13) (Welch’s t-test, P = 0.7005). The post 48 hr fear conditioning represents context fear conditioning, and is portrayed as Day 3 on the schematic diagram. The 48 hr fear conditioning is measured as the difference of the freezing time (%) just before conditioning (first three minutes of day 1) and during the context on day 3. Day 3 fear conditioning for context between the control Emx1-Cre mice (n = 12) and Bbs1flox/-; Emx1-Cre (n = 13) differed significantly (Welch’s t-test, P = 0.0438, Mann-Whitney-Wilcoxon Test, P = 0.0398). control = Emx1-Cre mice, BBS1 CKO = Bbs1flox/-; Emx1-Cre mice, hr = hour, ns = not significant, * P< 0.05.
Fig 5.
Bbs1M390R/M390R mice have decreased hippocampal proliferation.
A.) Normalized initial slope (%) recordings of field excitatory post synaptic potentials (fEPSP) in the hippocampal CA1 Schaffer-collateral pathway between 2 month male control mice (n = 17, 4 mice) and Bbs1M390R/M390R mice (n = 16, 4 mice). LTP was induced by 12 theta burst stimulation (TBS). B.) The Long-Term Potentiation (average of last five minutes of normalized initial slope of fEPSP) in the hippocampal CA1 Schaffer-collateral pathway between 2 month male control mice (n = 17, 4 mice) and Bbs1M390R/M390R mice (n = 16, 4 mice) did not differ significantly (Welch’s t-test, P = 0.8407). C.) Schematic diagram of the BrdU injections of postnatal day 3 (P3) mice. P3 mice were IP injected with 300mg/kg BrdU, and taken down four hours later. Sac = Sacrifice. D.) Inverted fluorescent microscope images of the P3 Dentate Gyrus. The sections were stained with Bromodeoxyruidine (BrdU) and counterstained with the nuclear marker, DAPI. BrdU immunostaining (red) and DAPI nuclear staining (blue). The yellow dotted line outlines the dentate gyrus. The Red Bar line represents 50μm.E.) Schematic diagram of the BrdU procedures for postnatal day 44 (P44) mice. At P30, mice were started on BrdU injections (2x50mg/kg) for five days. At P44, mice were taken down. Sac = Sacrifice. F.) Inverted fluorescent microscope images of the P44 Dentate Gyrus. The sections were stained with Bromodeoxyruidine (BrdU) and counterstained with the nuclear marker, DAPI. BrdU immunostaining (red) and DAPI nuclear staining (blue). The yellow dotted line outlines the dentate gyrus. The Red Bar line represents 50μm. G.) Decreased proliferation in the dentate gyrus of P3 Bbs1M390R/M390R mice. The natural logarithm of BrdU+cell/mm3 in the dentate gyrus between the control mice (n = 4) and the Bbs1M390R/M390R mice (n = 4) differed significantly (Welch’s t-test, P = 0.0038). H.) Decreased proliferation in the dentate gyrus of P44 Bbs1M390R/M390R mice. The natural logarithm of BrdU+cell/mm3 in the dentate gyrus between the control mice (n = 6) and the Bbs1M390R/M390R mice (n = 4) differed significantly (Welch’s t-test, P = 0.0018). control = Bbs1+/+, Bbs1M390R/+ mice, M390R = Bbs1M390R/M390R mice, hr = hour, ns = not significant, ** P< 0.01.
Fig 6.
Chronic Lithium treatment rescued long-term context fear conditioning in Bbs1M390R/M390R mice.
A.) Schematic diagram of the Bromodeoxyuridine (BrdU) procedures for postnatal day 44 (P44) mice. At P30, mice were started on Lithium water (45mM) or continued with water (vehicle). Mice were also started on BrdU injections (2x50mg/kg) for five days. At P44, mice were taken down. Sac = Sacrifice. B.) Images of immunohistochemistry for neurogenesis of vehicle and lithium treated Bbs1M390R/M390R mice. Z-stack, 20X, images of Dentate Gyrus at postnatal day 44 (P44). The tissue sections were stained with BrdU, Doublecortin (DCX), and counterstained with the nuclear marker DAPI. BrdU immunostaining (green), Doublecortin immunostaining (red) and DAPI nuclear staining (blue). The yellow dotted line outlines the dentate gyrus. The White Bar line represents 100 micrometers. C.) Proliferation in the dentate gyrus of P44 control and Bbs1M390R/M390R mice. We assessed two factors, and found a significant interaction for genotype and treatment, and a difference in treatment and genotype (2-way ANOVA, treatment X genotype, F (1, 20) = 6.428, P = 0.0026, treatment, F (1, 20) = 1.569, P = 0.0177, genotype, F (1, 20) = 46.89, P<0.0001). A Sidak’s multiple comparisons test showed a significant difference in treatment for Bbs1M390R/M390R mice (n = 5 Vehicle, n = 6 Lithium, P = 0.7875) but not for control mice (n = 8 Vehicle, n = 5 Lithium, P = 0.0010). D.) Neurogenesis in the dentate gyrus of P44 control and Bbs1M390R/M390R mice. We assessed two factors, and found a significant interaction between genotype and treatment, and a difference in treatment and genotype (2-way ANOVA, treatment X genotype, F (1, 20) = 11.37, P = 0.0005, treatment, F (1, 20) = 0.0368, P = 0.5779, genotype, F (1, 20) = 29.54, P<0.0001). A Sidak’s multiple comparisons test showed a significant difference in treatment for Bbs1M390R/M390R mice (n = 5 Vehicle, n = 6 Lithium, P = 0.0006) but not for control mice (n = 8 Vehicle, n = 5 Lithium, P = 0.3301). E.) Timeline of LiCl treatment. At 4–5 weeks of age, mice were treated with LiCl (45 mM) water or continued on water (vehicle). After two weeks of treatment, mice were tested on a 3 day fear conditioning set up. Day 1 is the training (acquisition phase) for fear conditioning. The second day is testing for cue fear conditioning. The third day is testing for context fear conditioning. F.) Day 1 fear conditioning acquisition between the vehicle treated control mice (n = 13) and Bbs1M390R/M390R (45 mM) treated mice (n = 10), and lithium treated control mice (n = 9) and Bbs1M390R/M390R (45 mM) treated mice (n = 11). Graph was presented without standard error. We found a significant difference in Treatment, Genotype, and Time (3-way ANOVA, Time x Genotype x Treatment, F (13, 560) = 0.2572, P = 0.9963; Genotype x Treatment, F (1, 560) = 0.4214, P = 0.5165; Time x Treatment, F (13, 560) = 1.338, P = 0.1860; Time x Genotype, F (13, 560) = 0.5960, P = 0.8582; Treatment, F (1, 560) = 5.475, P = 0.0196; Genotype, F (1, 560) = 39.48, P<0.0001; Time, F (13, 560) = 157.3, P<0.0001). The thick lines above the curve indicate when the sound cue was given, and the thick lines below the curve indicate when the shock was given. G.) The immediate fear conditioning indicates training to the day 1 fear conditioning. The immediate fear conditioning was measured as the freezing time (%) increase of the freezing time (%) just after conditioning (last minute) to the freezing time (%) just before conditioning (first three minutes). The immediate fear conditioning was not significantly different between the control mice given vehicle (n = 13) and control mice given LiCl water (n = 10) (Welch’s t-test, P = 0.0689) and did not significantly differ between the Bbs1M390R/M390R mice given vehicle (n = 10) and Bbs1M390R/M390R mice given LiCl water (45 mM) (n = 11) (Welch’s t-test, P = 0.2834). The post 24 hr fear conditioning represents the cue fear conditioning. The 24 hr fear conditioning (cue) was measured as the freezing time (%) increase of the freezing time (%) during the tone (cue, day 2) to the freezing time (%) before the tone (cue, day 2). The 24 hr fear conditioning (cue) was not significantly different between the control mice given vehicle (n = 13) and control mice given LiCl water (n = 10) (Welch’s t-test, P = 0.7483) and was not significantly different between the Bbs1M390R/M390R mice given vehicle (n = 10) and Bbs1M390R/M390R mice given LiCl water (45mM) (n = 11) (Welch’s t-test, P = 0.3625). The post 48 hr fear conditioning represents the context fear conditioning. The 48 hr fear conditioning was measured as the freezing time (%) increase of the freezing time (%) during the context on day 3 to the freezing time (%) just before conditioning (first three minutes of day 1). Day 3 fear conditioning for context was not significantly different between the control mice given vehicle (n = 13) and control mice given LiCl water (n = 10) (Welch’s t-test, P = 0.9285) but was significantly different between the Bbs1M390R/M390R mice given vehicle (n = 10) and Bbs1M390R/M390R mice given LiCl water (45mM) (n = 11) (Welch’s t-test, P = 0.0235). control = Bbs1+/+, Bbs1M390R/+ mice, M390R = Bbs1M390R/M390R mice, hr = hour, ns = not significant, * P< 0.05, ** P< 0.01, ***P<0.001, ****P<0.0001.