The Journal of Neuroscience, November 30, 2005, ():
Gastrin-Releasing Peptide Promotes Suprachiasmatic Nuclei Cellular Rhythmicity in the Absence of Vasoactive Intestinal Polypeptide-VPAC2 Receptor Signaling
J. Neurosci. Brown et al.
25: 11155
Supplemental data
Files in this Data Supplement:
- supplemental material -
Fig S1. Analysis of single and multiunit activity.
Individual spikes corresponding to the activity of putative single units were discriminated from multiunit recordings (a). Firing rate histograms for three putative single units extracted from the raw multiunit data (c,e,g) show high amplitude rhythms in action potential discharge. The average waveforms of spikes contributing to the putative single unit data on days 1 and 2 were identical (c,e,g inset traces & b; overlay) and plots of the amplitude of the primary positive (Amp. P1) and negative (Amp. P2) components of each individual spike revealed well separated clusters (j). Single unit activity was validated as the contribution of an individual neuron by the presence of an epoch where there were no spikes at the beginning of an interspike interval histogram, corresponding to the refractory period (d,f,h). On the basis of these criteria, g was rejected as a true single unit due to the absence of a clear refactory period (h). The sum of all neural activity detected in the raw data (i; MUA: multiunit activity) also shows a clear circadian rhythm. Overlayed curve in i was fitted to the equation y=AsinBx by the Newton-Raphson iterative method (see materials and methods for details). Data in c-i represents the mean firing rate (Hz) each min, shaded areas correspond to periods of darkness extrapolated from previous LD cycle.
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Fig S2. GRP-BB2 signaling is involved in the expression of neural rhythms in the Vipr2-/- SCN. Phasic application of GRP (6 h on/18 h off) to SCN slices prepared from Vipr2-/- mice that lacked behavioral rhythms in constant darkness (a) induced weak rhythmicity in 2/4 MUA recordings (b) and 8/11 single units observed so far (c). Chronic Application of The BB2 receptor antagonist DPDMB to SCN slices from behaviorally rhythmic Vipr2-/- mice (f) reversibly suppressed cellular activity and prevented the expression of MUA (e) and single unit activity rhythms (f). Bars in b,c,e & f indicate timing and duration of drug application.
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Fig S3. WT SCN sustains circadian rhythmicity for more than 3 days in vitro. SCN slices from WT mice housed under LD sustain MUA (a) and single unit firing rate rhythms (b) for > 80 hrs in vitro. In the presence of the VPAC2 receptor antagonist PG 99-465, GRP promotes short period rhythmicity in WT MUA (c) and single unit discharge (d). Despite displaying decreased cellular activity, PG 99-645/GRP co-treated slices respond to NMDA application after ~73 h in vitro, indicating long-term drug treatment does not seriously compromise slice viability. Shaded areas in a-d correspond to periods of darkness extrapolated from previous LD cycle, bars in c & d indicate timing and duration of drug vapplication.
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Table 1
