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. Author manuscript; available in PMC: 2012 Mar 7.
Published in final edited form as: J Neurosci. 2011 Sep 7;31(36):12708–12715. doi: 10.1523/JNEUROSCI.2172-11.2011

Figure 3. Neurotrophin (NGF and NT3) expression in the pineal is progressively decreased following exposure to constant light.

Figure 3

(A) A representative Western blot analysis of pineal lysates from animals reared in either LD conditions or following 2 weeks of LL shows decrease in expression of proNGF in animals reared in LL. (B) Time-course of changes in proNGF expression following exposure to constant light represented as percentage proNGF relative to LD control: 50% after 0.25 wks LL (n=6), 23% after 2 wks LL (n=3), 8% at 4 wks LL (n=3) and 14% after 8 wks LL (n=3). (C) Expression of proNGF in the pineal. Daytime pineal lysates from two independent rats (lanes 1 and 2) were resolved by SDS-PAGE and analyzed by immunoblot with the polyclonal anti-NGF antibody (panel a), or with the same antibody pre-incubated with blocking peptide (panel b). Two positive controls were used: lysate of male mouse submandibular gland (lane 3) and 7ng of pure NGF (Sigma, N6009) (lane 4). Note that the 34 kDa band is the only detectable NGF species in the pineal lysates. Pre-incubation of the antibody with the NGF blocking peptide results in no NGF signal (panel b), confirming specificity of the pineal 34kDa band. (D) Time-course of ngf mRNA, analyzed by quantitative real-time PCR (QPCR), in pineal glands of animals maintained in 12:12h LD conditions (n=9), or following exposure to LL, shows decrease in expression of ngf: 13% after 0.25 wks LL (n=4), 21.5% after 2 wks LL (n=3) and 16% after 8 wks LL (n=2). (E) Time-course of nt3 mRNA analyzed by QPCR in pineal glands of animals maintained in LD (n=9) or following extended periods of LL, shows decrease in expression of nt3 following exposure to constant light: 42% after 0.25 wks LL (n=4), 48 % after 2 wks LL (n=3) and 47% after 8 wks LL (n=2). All animals in panels A-D were sacrificed at ZT18. (F) β-adrenergic stimulation with isoproterenol induces expression of ngf and nt3 mRNA. Expression of ngf and nt3 in the pineal, analyzed by QPCR 4 hours after a single intraperitoneal injection of isoproterenol (ISO) administered at ZT6, increased 5.5 fold for ngf (n=4) and 2.3 fold for nt3 compared to vehicle-injected (PBS) controls (n=3). Two-tailed t-test was performed for statistical analysis, p<0.05*, p<0.01**, p<0.0001****. Error bars represent SEM. Animals were sacrificed at ZT18. Silencing of circuit activity was confirmed by the absence of AANAT protein following light exposure in panels A and B and by the suppression of aanat mRNA in panels D and E following light exposure (data not shown).