Light- and clock-dependent regulation of ribosomal S6 kinase activity in the suprachiasmatic nucleus

Recent work has revealed that signalling via the p42/44 mitogen-activated protein kinase (MAPK) pathway couples light to entrainment of the circadian clock located in the suprachiasmatic nucleus (SCN). Given that many effects of the MAPK pathway are mediated by intermediate kinases, it was of interest to identify kinase targets of ERK in the SCN. One potential target is the family of 90-kDa ribosomal S6 kinases (RSKs). In this study, we examined light-induced regulation of RSK-1 in the SCN. Immunohistochemical and Western analysis were used to show that photic stimulation during the early and late night triggered the phosphorylation of RSK-1 at two sites that are targeted by ERK. This increase in the phosphorylation state of RSK-1 corresponded with an approximate fourfold increase in kinase activity. Light exposure during the subjective day did not increase the phosphorylated form of RSK-1, indicating that the capacity of light to stimulate RSK-1 activation is phase-restricted. Double immunofluorescent labelling of SCN tissue revealed the colocalized expression of the activated form of ERK with the phosphorylated form of RSK-1 following a light pulse. In vivo pharmacological inhibition of light-induced MAPK pathway activation blocked RSK-1 phosphorylation, indicating that RSK-1 activity is regulated by the MAPK pathway. PDK-1, a coregulator of RSK-1, is also expressed in the SCN and is likely to contribute to RSK-1 activity. RSK-1 phosphorylation was also rhythmically regulated within a subset of phospho-ERK-expressing cells. Together these results identify RSK-1 as a light- and clock-regulated kinase and raise the possibility that it contributes to entrainment and timing of the circadian pacemaker.     

Determining abnormal latencies of multifocal visual evoked potentials: a monocular analysis

Purpose: To describe a methodology for measuring abnormal timing of monocular multifocal visual evoked potentials (mfVEP). Methods: The mfVEPs from 100 individuals with normal visual fields and normal fundus exams were analyzed. The stimulus was a 60 sector, pattern-reversing dartboard display. For each of the 60 locations of the dartboard and each channel and each eye, a template was derived based upon the average of the responses from the 100 normal individuals. In deriving this template, care was taken to exclude those responses reversed in polarity as compared to the average response. The best array of responses for each individual was compared to these templates. The relative latency of each response was measured as the temporal shift producing the best cross-correlation value. Results: The 95% confidence interval (CI) decreased as the signal-to-noise ratio (SNR) of the mfVEP responses increased. For example, the 95% CI decreased from over 17ms to under 9 ms as the SNR increased. Grouping and summing the responses also lead to an increase in SNR and a decrease in CI. Because the number of points exceeding the CI is not randomly distributed among normal individuals, a cluster criterion (e.g. two or more contiguous points within a hemisphere exceeding a given confidence interval) can be helpful. For example, while 18% of the eyes had 5 or more points exceeding the 5% confidence interval, only 6.5% of the eyes had a cluster of 5 of these points. The correlation between relative latency and age was relatively low (r=0.46). Conclusion: For detecting abnormalities in the timing of monocular, mfVEP responses, a template method provides a reasonable approach. In devising a particular test for abnormal timing, the CI should be based upon the SNR of the response. In addition, grouping and summing responses to increase SNR or employing a cluster test may also prove useful.     

Normative ranges and specificity of the multifocal VEP

Purpose: To describe a normative database for the multifocal VEP (mfVEP) and to evaluate specificity for a range of cluster criteria. Methods: One hundred persons (62 females and 38 males) with normal visual fields and ranging in age from 21.6 to 92.4 years participated in this study. Self-reported race in 80 of these 100 persons was `White or Caucasian,' eight were `Black or African-American,' eight were `Asian,' and four were `Hispanic or Latino.' Pattern-reversal mfVEPs were obtained using a dartboard stimulus pattern in VERIS^TM and two 8-min runs per eye were averaged. A bootstrap technique was used to estimate the normal range of mfVEP response signal-to-noise ratio (SNR) and inter-ocular amplitude ratio at each location. Specificity (1 – false alarm rate) was evaluated for a range of cluster criteria, whereby the number and probability level of the points defining a cluster were varied. Results: There was no overall effect of age on SNR (r ²=0.16, p=0.22) nor was the interaction between age and location significant (F=0.83, p=0.82, ANOVA). The location with the largest age effect had an r ² of only 0.13. There was a small but significant effect of sex (t=2.1, p=0.04) such that SNR was slightly (11) larger in females than males, but there was no significant interaction between sex and age (t=0.82, p=0.41). There was a slight trend toward higher SNR in the Asian group and lower SNR in the African-American group, but the overall effect of race was not significant (F=1.99, p=0.12). Specificity depended on the number and probability level of the points defining a cluster. Specificity did not vary by age group in a simple monotonic manner. False positive rates were slightly higher in females than males, and slightly higher in the African-American group as compared with the Asian group. Conclusions: Excellent specificity can be achieved for the mfVEP by using particular cluster criteria for monocular and inter-ocular tests. The effects of age, sex, and race were all very small and only the effect of sex was statistically significant. This normative database can be used for analyses of mfVEP results from individual patients with little risk that demographic factors such as age and sex will confound diagnostic accuracy.     

Selective ganglion cell functional loss in rats with experimental glaucoma

PURPOSE: To characterize retinal functional consequences of elevated intraocular pressure (IOP) in a rat model of experimental glaucoma. METHODS: Unilateral elevation of IOP was produced by hypertonic saline injection into an episcleral vein in 20 adult male Brown-Norway rats. IOP was measured in both eyes of awake animals four to five times per week. After 5 weeks, animals were dark adapted overnight (>12 hours) and full-field electroretinograms (ERGs) were obtained simultaneously from both eyes. Scotopic ERG stimuli were brief white flashes (-6.64-2.72 log cd-s/m(2)). Photopic responses were also obtained (0.97-2.72 log cd-s/m(2)) after 15 minutes of light adaptation (150 cd/m(2)). Eyes were processed the following day for histologic evaluation by light microscopy, including masked determination of optic nerve injury grade (ONIG; 1, normal; 5, severe, diffuse damage). RESULTS: Among experimental eyes, the group average IOP (+/-SD) was 34.5 +/- 4.1 mm Hg, whereas the average for control eyes was 28.1 +/- 0.5 mm Hg (t = 7.1, P < 0.0001). The average ONIG for experimental and control eye groups, respectively, was 3.4 +/- 1.7 and 1.0 +/- 0.02 (t = 6.3, P < 0.0001). The ONIG increased with mean IOP in experimental eyes (r(2) = 0.78, P < 0.0001) and was unrelated to mean IOP in control eyes (r(2) = 0.09, P = 0.18). In experimental eyes with relatively mild IOP elevation (mean IOP < 31 mm Hg) and no structural (histologic) damage to the optic nerve evident by light microscopy (ONIG = 1.1 +/- 0.2, n = 5), there was a selective reduction of the positive scotopic threshold response (pSTR; P < 0.001), whereas other ERG components remained unaltered. In four of the five eyes, pSTR amplitude was reduced by more than 50%, whereas all five had normal scotopic a-wave, b-wave, and OP amplitudes. Eyes with mean IOP of more than 35 mm Hg had reduced a-wave, b-wave, and oscillatory potential (OP) amplitudes. CONCLUSIONS: As demonstrated by prior studies, selective loss of the pSTR is indicative of selective retinal ganglion cell (RGC) injury. In this rat model of experimental glaucoma, selective RGC functional injury occurred before the onset of structural damage, as assessed by light microscopy of optic nerve tissue. The highest IOP levels resulted in nonselective functional loss. Thus, in rodent models of experimental glaucoma, lower levels of chronically elevated IOP may be more relevant to human primary chronic glaucoma.     

Voltage-gated Na+ channels enhance the temporal filtering properties of electrosensory neurons in the torus

Regenerative processes enhance postsynaptic potential (PSP) amplitude and behaviorally relevant temporal filtering in more than one-third of electrosensory neurons in the torus semicircularis of Eigenmannia. Data from in vivo current-clamp intracellular recordings indicate that these "regenerative PSPs" can be divided in two groups based on their half-amplitude durations: constant duration (CD) and variable duration (VD) PSPs. CD PSPs have half-amplitude durations of between 20 and 60 ms that do not vary in relation to stimulus periodicity. In contrast, the half-amplitude durations of VD PSPs vary in relation to stimulus periodicity and range from approximately 10 to 500 ms. Injection of 0.1 nA sinusoidal current through the recording electrode demonstrated that CD PSPs and not VD PSPs can be elicited by voltage fluctuations alone. In addition, CD PSPs were blocked by intracellular application of either QX-314 or QX-222, whereas VD PSPs were not. These in vivo data suggest, therefore, that CD PSPs are mediated by voltage-dependent Na+ conductances.     

Appearance of the frequency doubling stimulus in normal subjects and patients with glaucoma

PURPOSE: To determine whether the spatial structure of the frequency doubling technology (FDT) perimetry stimulus is visible at detection-contrast threshold in normal observers and those with glaucoma and to assess its perceived spatial frequency at threshold and suprathreshold contrast. METHODS: Three subject groups were assessed: 10 young normal observers (aged <40 years), 10 older normal observers (aged >50 years), and 10 subjects with glaucoma. Detection thresholds for centrally and eccentrically presented 10 degrees squares, 0.25-cyc/deg, 25-Hz counterphase flicker sine-wave gratings were obtained by using a yes-no staircase procedure. Eccentric locations were in areas of loss of FDT sensitivity (< or =21 degrees ) in subjects with glaucoma, or at 7 degrees or 21 degrees inferonasally in normal observers. Resolution-contrast thresholds were determined by a two-alternative, forced-choice staircase procedure in which subjects selected the orientation of the grating stimulus tilted at +/-45 degrees. Perceived spatial frequency was determined by having subjects alter the spatial frequency of a temporally interleaved stationary sine-wave grating to match the FDT stimulus. RESULTS: No significant difference was found between detection- and resolution-contrast thresholds, implying that spatial structure was visible at detection threshold. In general, subjects perceived the spatial structure to have a spatial frequency closer to doubled than to veridical, although the young normal subjects reported a lower apparent spatial frequency than older individuals. CONCLUSIONS: When instructed as for clinical testing, subjects respond to the presence of the structure of the grating, and perceive the FDT stimulus to have a spatial frequency greater than its true spatial frequency. These findings were consistent across both normal observers and those with glaucoma, at both central and eccentric test locations.