Research ArticleNEUROSCIENCE

Serotonin rebalances cortical tuning and behavior linked to autism symptoms in 15q11-13 CNV mice

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Science Advances  21 Jun 2017:
Vol. 3, no. 6, e1603001
DOI: 10.1126/sciadv.1603001
  • Fig. 1 15q dup mice have reduced excitatory synaptic transmission onto 5-HT neurons and low glucose metabolism in the DRN.

    (A) Representative action potential recordings of non–5-HT (Tph-immunoreactive–negative, −Tph) and 5-HT (Tph-immunoreactive–positive, +Tph) neurons in DRN in response to depolarizing current injections of 100 and 140 pA, respectively (left). Individual spikes are aligned at the starting point of spike and displayed in an expanded time scale (right). (B) The resting membrane potentials of 5-HT neurons in the DRN were hyperpolarized in 15q dup mice. **P < 0.01, Wilcoxon rank sum test. (C) Representative sEPSC traces recorded from 5-HT neurons in DRN. (D and E) Comparisons between WT and 15q dup mice for the sEPSC amplitude and frequency in 5-HT neurons obtained from the vmDRN and lwDRN subregions. The amplitude but not the frequency of sEPSCs was decreased in 15q dup mice at vmDRN (*P < 0.05, Brunner-Munzel test) and lwDRN (*P < 0.05, Wilcoxon rank sum test). n = cells per mice. (F) Summed PET images from 30 to 60 min after the [18F]FDG injection were made by averaging the images in each group (n = 7 mice, triplicate in each genotype). The heat map indicates the standardized uptake value (SUV) of glucose. (G) The graphs show the mean time–radioactivity curves of [18F]FDG. Regions of interest were manually drawn on the [18F]FDG-PET images based on a morphologically normalized coronal magnetic resonance T1-weighted image. (H) Maps of the t scores obtained by voxel-based statistical comparisons of the [18F]FDG uptake between WT and 15q dup mice. An unpaired two-sample t test was performed. Pseudocolor maps of the t scores are fused on a mouse brain magnetic resonance T1-weighted image. (I to L) The coronal sections correspond to AP1 and AP2 dashed lines in (H) [(I) and (J)], and the horizontal sections correspond to VD1 and VD2 dashed lines in (H) [(K) and (L)]. Low glucose metabolism in the DRN (white arrowheads) of 15q dup mice was observed compared with that in WT littermates. The 15q dup mice also showed high glucose metabolism in the anterior cingulate cortex (Cg; yellow arrows) and hippocampal region (Hip; yellow arrowheads). Statistical significance was defined at a threshold of P < 0.005 (uncorrected), t > 3.05, F = 12. (M) Differences in [18F]FDG uptake ratios (calculated as regional uptake per whole brain uptake of [18F]FDG) between the two groups (n = 7 mice in each group; ***P < 0.001, two-tailed Student’s t test). Box plots represent the median and the 25th and 75th percentiles. Each dot represents individual sample data. The mean is represented by a plus sign. Whiskers represent the minimum and maximum values except for outliers. All other values are means ± SEM.

  • Fig. 2 Sensory-evoked somatosensory receptive field tuning responses are impaired in 15q dup mice.

    (A) The right barrel field (yellow open square) was analyzed in the transcranial calcium imaging. The left C2 whisker is stimulated by a piezo driver at 10 Hz of 10 pulses and at 0.25 Hz of 20 pulses. Scale bar, 2 mm. (B) Representative images of barrel area responses at 10-Hz stimulation in each genotype. The color represents the ΔF/F of averaged image. The area within the bold line indicates the response in principal barrel, and the area within the fine line indicates the response in surrounding barrels of the C2 whisker barrel. Scale bar, 2 mm. (C) The area size of the principal barrel was comparable between genotypes, whereas the area size of the surrounding barrel was larger in 15q dup mice. **P < 0.01, Wilcoxon rank sum test. n.s., not significant. (D) The traces indicate the averaged ΔF/F of the principal barrel (black, WT; red, 15q dup) at 0.25-Hz stimulation. The faint color indicates SEMs in each genotype. (E) The peak amplitude of the principal barrel was smaller in 15q dup mice. (F) The decay slope of the principal barrel was smaller in 15q dup mice. *P < 0.05, two-tailed Student’s t test. n = 9 mice in both genotypes. (G and H) Top: Superimposed traces show the averaged responses to single whisker deflections of the PW, which has the strongest amplitude and fastest onset latency to the whisker stimuli (red, B1), and the S1Ws, which have weaker amplitudes and slower onset latencies (blue, B2; green, β), in a representative case of WT mice and those to the PW (red, B2) and the S1Ws (blue, B1; green, B3) in a representative case of 15q dup mice. Insets: Schema indicates the whisker pattern and deflection points. Bottom: Three-dimensional bar graphs show receptive field maps of averaged postsynaptic potential (PSP) amplitudes, centered to the PW. The PSPs of S1Ws in 15q dup mice showed higher responses than those in WT mice. (I) The PSP amplitude of the PW was normal, but that of S1W was increased in 15q dup mice. (J) The ratio of S1W- to PW-PSP amplitudes was increased in 15q dup mice. WT, n = 12 mice; 15q dup, n = 16 mice. *P < 0.05; **P < 0.01, two-tailed Student’s t test. Box plots represent the median and the 25th and 75th percentiles. Each dot represents individual sample data. The mean is represented by a plus sign. Whiskers represent the minimum and maximum values except for outliers. All other values are means ± SEM.

  • Fig. 3 15q dup mice have fewer inhibitory synapses and a higher excitability of pyramidal neurons in L2/3 of somatosensory cortex.

    (A to D) Representative images show the S1BF with anti–microtubule-associated protein 2 (MAP2) and anti-VGAT immunostaining [A1 to D1 (left column), WT; A2 to D2 (right column), 15q dup]. At low magnification, VGAT signals (white) are comparable between WT (A1) and 15q dup (A2) mice. Scale bars, 1 mm. At high magnification, inhibitory terminals (red, VGAT) on neural dendrites (green, MAP2) in L2/3, L4, and L6 of the S1BF show that only in L2/3 were the VGAT signals weaker in 15q dup mice (B2) than in WT mice (B1). The VGAT signals in L4 (C1 and C2) and L6 (D1 and D2) were comparable. Scale bars, 20 μm. (E) The density of VGAT-positive puncta is reduced in L2/3 of 15q dup mice (*P < 0.05, two-tailed Student’s t test; n = 15 images from five mice per genotype). (F) The density of VGluT1-positve puncta was not changed in L2/3 of 15q dup mice. (G) Representative traces of mIPSCs from L2/3 pyramidal neurons in the S1BF of WT and 15q dup mice. (H) The graphs show box plots and cumulative probabilities for mIPSC frequency and basic synaptic responses (amplitude, 10 to 90% rise time, and decay time constant). In 15q dup mice, the mIPSC frequency was decreased (*P < 0.05, two-tailed Student’s t test), and the cumulative probability of the inter-event interval (IEI) was shifted to the right compared to WT mice. The amplitude and rise time were not changed, but the decay time was increased in 15q dup mice (**P < 0.01, two-tailed Welch’s t test). (I) Representative traces of mEPSCs from L2/3 pyramidal neurons in the S1BF of WT and 15q dup mice. (J) The graphs show box plots and cumulative probabilities for mEPSCs frequencies and basic synaptic responses. There was no difference between genotypes. (K to M) The graphs show the intrinsic properties of L2/3 pyramidal neurons. The input resistance calculated from the voltage trace in response to the hyperpolarizing current of −50 pA was higher in 15q dup neurons (K), whereas the cellular capacitance (L) and the resting membrane potential (M) were unchanged. *P < 0.05, two-tailed Welch’s t test. (N) Representative traces of the Ih current recorded by the hyperpolarizing voltage steps from −50 to −120 mV. The Ih currents were calculated by dividing the instantaneous current measured at the beginning of the negative voltage step by the steady-state current at the end of the step. (O) The Ih currents were smaller in 15q dup neurons [genotype: F1,26 = 9.495, **P < 0.01, two-way repeated-measures analysis of variance (ANOVA)]. (P) The representative voltage traces of L2/3 pyramidal neurons of WT and 15q dup S1BF in response to current injections with amplitudes from 0 to 400 pA with 100-pA steps. (Q) The action potential frequencies are plotted against amplitudes of depolarizing currents. Inset: The area under the curve (AUC) in the line graph was larger in 15q dup mice (*P < 0.05, two-tailed Welch’s t test). n = cells per mice. Box plots represent the median and the 25th and 75th percentiles. Each dot represents individual sample data. The mean is represented by a plus sign. Whiskers represent the minimum and maximum values except for outliers. All other values are means ± SEM.

  • Fig. 4 Chronic FLX treatment during developmental stages restores local synaptic transmission in 15q dup mice

    (A) The administration of FLX via the mother’s milk was started at P3 and finished at P21 (bottom rectangle). The upper rectangles indicate each experimental period. E-phys, electrophysiology. (B) The representative traces of mIPSCs from L2/3 pyramidal neurons in the S1BF of vehicle-treated WT (Veh-WT), vehicle-treated 15q dup (Veh–15q dup), FLX-treated WT (FLX-WT), and FLX-treated 15q dup (FLX–15q dup) mice. (C) The graphs show box plots and cumulative probabilities for mIPSC frequency and basic synaptic responses from the treated mice. The mIPSC frequency of Veh–15q dup was significantly lower, but the mIPSC frequency of FLX–15q dup was comparable with that of Veh-WT. The amplitude and rise time of mIPSCs were not changed among these groups. The decay time of FLX–15q dup was not significantly different from that of Veh-WT but still longer than that of FLX-WT mice. P values were obtained by Steel-Dwass test. (D) The hyperpolarized resting membrane potential of DRN 5-HT neurons in 15q dup mice was not changed by the FLX treatment. P value was obtained by two-tailed Student’s t test. (E and F) The FLX treatment ameliorated reduction in sEPSC amplitude of 15q dup mice. The sEPSC amplitudes in 5-HT neurons at vmDRN and lwDRN of FLX–15q dup were comparable with FLX-WT mice. There was no difference in sEPSC frequency between groups at both vmDRN and lwDRN. n = cells per mice. Box plots represent the median and the 25th and 75th percentiles. Each dot represents individual sample data. The mean is represented by a plus sign. Whiskers represent the minimum and maximum values except for outliers.

  • Fig. 5 Restoration of 5-HT level improves social behaviors of 15q dup mice.

    (A) The tissue levels of 5-HT and 5-HIAA in the midbrain at 9 weeks of age were examined. Veh–15q dup showed a reduction in 5-HT levels (left) and 5-HIAA levels (right), whereas FLX–15q dup showed no difference in both levels compared with Veh-WT. P values were obtained using the Games-Howell test. (B) Approaching time to the stranger cage (S) and the empty cage (C) in the 3-CSI test was compared in each group. Only in Veh–15q dup was there no difference in approaching time to both cages, whereas others showed increased approaching time to the stranger cage. *P < 0.05; **P < 0.01, exact Wilcoxon signed-rank test. (C) The graph shows the performance in training sessions in MWM. S1 to S6, 6 days of learning sessions; RS1 to RS4, 4 days of reversal learning sessions. (D) Probe test performance in the MWM test. Configuration of the four quadrants in the probe test after the original training is shown on the left (TA, target quadrant; OP, opposite quadrant; AR, adjacent right quadrant; AL, adjacent left quadrant). The white bar graphs show the times spent in each quadrant. Veh-WT, Veh–15q dup, and FLX-WT groups spent more time in the TA compared with the other quadrants, whereas FLX–15q dup did not show significance in both OP and AR quadrants. (E) Configuration of the four quadrants in the reversal probe test is shown on the left. Only the Veh-WT group showed a significant difference between TA and OP. *P < 0.05; **P < 0.01; ***P < 0.001, comparison with TA in Steel test. (F) Left: The graph shows total distance in open-field test. Exploratory activity in FLX-treated groups was decreased compared with Veh-WT. Middle: The graph shows time spent in the center area in open field (OF). Only FLX–15q dup mice showed reduction of the time in center. Right: FLX–15q dup mice also showed reduction in the anxiety index, in which the distance traveled in the center area is divided by total distance traveled, for clarification of reduction of the time in center. P values compared with Veh-WT were obtained by Steel test. (G) The number of USV calls in 15q dup pups was larger than that in WT mice in the vehicle group, whereas there was no difference in USV calls between genotypes among FLX-treated groups. The USV calls of 15q dup pups were gradually decreased by FLX dosage (0.5 and 2.5 mg/kg). P values were obtained using the Games-Howell test. (H) The diagram of this study. n = number of mice. Box plots represent the median and the 25th and 75th percentiles. Each dot represents individual sample data. The mean is represented by a plus sign. Whiskers represent the minimum and maximum values except for outliers. All other values are means ± SEM.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/6/e1603001/DC1

    fig. S1. Dendritic morphology of lwDRN 5-HT neurons shows no difference between WT and 15q dup mice.

    fig. S2. Cell densities of inhibitory neurons are unchanged in 15q dup mice.

    fig. S3. The number of symmetry synapses is decreased in the S1BF of 15q dup mice.

    fig. S4. The paired-pulse ratio of inhibitory transmissions in S1BF L2/3 pyramidal neurons is not changed in 15q dup mice.

    fig. S5. 15q dup S1BF have differential expression of GABAA receptor subunits.

    fig. S6. 15q dup mice have decreased dendritic length of S1BF L2/3 pyramidal neurons.

    fig. S7. Profiling of 5-HT receptor expression in 15q dup brain.

    fig. S8. Acute 5-HT application enhances inhibitory transmission and suppresses excitability of S1BF L2/3 pyramidal neurons in 15q dup mice.

    table S1. Properties of action potentials of 5-HT neurons in DRN.

    table S2. Firing properties of L2/3 regular spiking neurons in vivo.

    table S3. Statistical results.

    Supplementary Methods

    References (44, 45)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Dendritic morphology of lwDRN 5-HT neurons shows no difference between WT and 15q dup mice.
    • fig. S2. Cell densities of inhibitory neurons are unchanged in 15q dup mice.
    • fig. S3. The number of symmetry synapses is decreased in the S1BF of 15q dup mice.
    • fig. S4. The paired-pulse ratio of inhibitory transmissions in S1BF L2/3 pyramidal neurons is not changed in 15q dup mice.
    • fig. S5. 15q dup S1BF have differential expression of GABAA receptor subunits.
    • fig. S6. 15q dup mice have decreased dendritic length of S1BF L2/3 pyramidal neurons.
    • fig. S7. Profiling of 5-HT receptor expression in 15q dup brain.
    • fig. S8. Acute 5-HT application enhances inhibitory transmission and suppresses excitability of S1BF L2/3 pyramidal neurons in 15q dup mice.
    • table S1. Properties of action potentials of 5-HT neurons in DRN.
    • table S2. Firing properties of L2/3 regular spiking neurons in vivo.
    • table S3. Statistical results.
    • Supplementary Methods
    • References (44, 45)

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