Article Figures & Data
Figures
- Figure 1.
Multisite electrophysiological recordings in WT and Clock-Δ19 mice. A, Raster plot of LFPs and 35 neurons simultaneously recorded from NAC, PrL, and VTA in an awake behaving animal. B, Diagram of mesolimbic dopamine reward circuit (note that projections from NAC to VTA are not direct). C, LFP spectral power in WT and Clock-Δ19 mice during recording session.
- Figure 2.
Cross-frequency phase coupling across mesolimbic brain areas in WT and Clock-Δ19 mice. A, Two-second 1–4 Hz NAC oscillation trace (green) overlaid on simultaneously recorded 30–55 Hz oscillations (red). Red arrows show bouts of increased gamma oscillation amplitudes that were phase coupled to 1–4 Hz oscillation peaks. B, Sample of NAC LFP channel from Clock-Δ19 mouse. Notice peaks in gamma oscillatory power are not consistent with a 1–4 Hz oscillation phase. C, The modulation index (M) was determined for all mesolimbic brain areas examined. The images depict group averages of stepwise modulation values for amplitude oscillations occurring between 15 and 100 Hz and phase oscillations occurring between 0.5 and 19.5 Hz. Clock-Δ19 mice displayed diminished coupling between the phase of 1–4 Hz oscillations and the amplitude of low-gamma (30–55 Hz) oscillations. The blue line indicates threshold for significant phase coupling; *Corrected P < 0.05. D, Modulation index of NAC low-gamma oscillations versus total distance traveled. E, Clock-Δ19 mice failed to display CFPC during periods when they were moving <4 cm/s.
- Figure 3.
Neuronal phase locking across mesolimbic brain areas in WT and Clock-Δ19 mice. A, Example of four neurons isolated from NAC. From left to right: depiction of the four extracellularly recorded waveforms of the unit (x-axis 1400 μs; y-axis 188 μV); projection of the clusters correspondent to the units and the noise based on analysis of the first two principal components of the waveforms recorded (x-axis, PC1; y-axis, PC2); and interspike interval histogram. B, Example of single-neuron phase locking, and the phase distribution of NAC neuron shown above. The activity of the depicted neuron was phase locked to the 1–4 Hz oscillation peak. C, Raleigh statistic for all NAC, PrL, and VTA neurons in WT and Clock-Δ19 mice. D, NAC, PrL, and VTA neurons in WT and Clock-Δ19 mice optimally phase locked to the rising phase of delta oscillations. E, Clock-Δ19 mice displayed a significantly lower proportion of NAC neurons that were phase locked to delta oscillatory activity than did WT mice. Two-tailed paired z test with FDR correction, *Corrected P < 0.05.
- Figure 4.
Chronic lithium treatment reverses NAC phase timing deficits seen in Clock-Δ19 mice. A, B, Treatment with lithium significantly increased coupling between the phase of 1–4 Hz oscillations and the amplitude of 30–55 Hz oscillations in NAC (compare with Fig. 2C). Mann–Whitney U test; *P < 0.05. The blue line indicates threshold for significant phase coupling. C, Treatment with lithium significantly increased NAC neuronal phase locking to delta oscillations in Clock-Δ19 mice, but had no significant effect on phase locking in WT mice. D, Treatment with lithium significantly decreased novelty-induced hyperactivity in Clock-Δ19 mice; n = 5–11 per group; Student's t test.
- Figure 5.
Analysis of NAC glutamatergic function in WT and Clock-Δ19 mice. Levels of AMPA and NMDA receptor subunits were quantified by Western blotting in Clock Δ19 mutants and WT controls treated with vehicle or lithium. Representative blots are shown below the corresponding graph. There was a significant decrease in GluR1 (A) and phospho-ser845 GluR1 (B) in ClockΔ19 mutant mice relative to WT. Lithium decreased protein levels of GluR1 and P-GluR1 in WT mice only. No changes in GluR2 (C), NR1 (D), NR2A (E), or NR2B (F) levels were observed. All values are expressed as ratios relative to levels of GAPDH. n = 5–6/group; comparisons made by two-way ANOVA followed by post hoc analysis, *p < 0.05, **p < 0.01, for across genotype comparisons. #p < 0.05, ##p < 0.01, for lithium treatment comparisons.
- Figure 6.
Analysis of the structural plasticity of NAC neurons in WT and Clock-Δ19 mice. Brains from Clock-Δ19 and WT animals with or without 10 d of lithium treatment (600 mg/L) were subjected to Golgi staining and then sliced into 150 μm sections. Twenty-five to twenty-nine cells from the NAC were analyzed and averaged from five mice per group. A, Example of a Golgi-stained medium spiny neuron from NAC. B, Dendritic spine density was measured. There were no differences in spine density between groups. C, Dendritic length across Sholl circles was measured. Clock-Δ19 mice have an increase in overall dendritic length across all Sholl radii compared to WT mice (P < 0.01). Lithium treatment tended to reduce overall dendritic length in the Clock-Δ19 mice, though this trend did not reach statistical significant (P = 0.095). Lithium treatment increased the length of dendrites in WT mice (P < 0.01). D, The number of dendritic intersections of Sholl circles was calculated. Dendrites from Clock-Δ19 mice intersect Sholl circles more often than those of WT mice (P < 0.01). Lithium treatment significantly decreases these intersections in Clock-Δ19 mice (P < 0.05). Lithium treatment increases the number of dendritic intersections of Sholl circles in WT mice (P < 0.01). E, The number of dendritic nodes was calculated across Sholl circles. No significant differences were found between Clock-Δ19 and WT mice; however, lithium treatment increased the number of nodes in WT animals (P < 0.05). Insets for C–E show totals measured across all lengths; *P < 0.05 for across genotype comparison, #P < 0.05 for within genotype comparison of lithium effect.
Tables
Area AP (mm) ML (mm) DV (mm) NAC +1.25 +1.15 −3.9 PrL +2.5 or +1.7 ±0.25 −0.75 or −1.8 VTA −3.2 +0.3 −4.25 All coordinates are measured from bregma.
NAC PrL VTA WT mice 5.1 6.7 16.8 Clock-Δ19 mice 6.4 4.6 21.4
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