Sleep responses to chronic sleep restriction (CSR) are fundamentally different from those to short-term total sleep deprivation. Specifically, as CSR continues, animals fail to express compensatory responses in total sleep time and sleep intensity (as measured through NREM EEG delta power). It has been suggested that norepinephrine may play a key role in modulating sleep intensity by promoting cortical long-term potentiation induction via -adrenergic receptors (-AR). Furthermore, c-Fos expression is highly correlated to non-specific neuronal activity. Using a rat model of CSR, we investigated the neuronal activity of cortical neurons expressing -AR in response to CSR in hopes of understanding the mechanism by which -AR mediates sleep time and intensity.

Introduction

Co-expression of β-Adrenergic Receptor and c-Fos in the Rat Brain during Chronic Sleep Restriction

Kaitlyn B. Scalisi1, Youngsoo Kim2, Robert E. Strecker2 and John McCoy1

1) Stonehill College, Easton, MA 2) VA Boston Healthcare System and Harvard Medical School

Subjects• 16 young adult male Sprague-Dawley rats were maintained under a 12:12 light/dark cycle.• Sleep deprivation was performed by placing rats in periodically rotating wheels (4-s on: 12-s off) for 18h/day.• The brain tissues were collected 2h after the end of sleep deprivation on a baseline day and CSR days 1 and 3.

Immunohistochemistry•Fluorescents •NOVARed, TMB, DAB, and DAB-Ni (Vector Laboratory).

Figure 2: Experimental Protocol. A 24-h baseline (BL) sleep recording was collected starting at 6 h after light onset (ZT6). For the 5 days of CSR the rats were sleep deprived (SD) for 18 h/day (ZT6-24) followed by 6-h (ZT0-6) of daily sleep opportunity (SO) in their home cage. Thereafter, animals had 3 days of free recovery sleep (R) opportunity. ‘A’ represents the tissue collection time for histological procedures.

Fluorescents•Using the florescent dye, we identified the coexpression of beta-AR and c-Fos the rat brain on baseline day 1.

Stains•β-AR is best stained using with a high concentration (1:100) using NOVARed.•c-Fos expression detected in the anterior cingulate cortex using DAB-Ni (1:2000).•Best combination of double stains seen using NOVARed and DAB-Ni. •DAB and TMB incompatible with both c-Fos and -AR.

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Results

DiscussionDouble staining with fluorescent dye proved successful. However, this method is not ideal for measuring the number cells expressing both -AR and c-Fos simaltaneously. As a result, non-fluorescent staining was necessary. Single non-fluorescent stains for -AR and c-Fos were successful and double staining for these is a future plan in order to allow the quantification of co-expressed cells. This is the first study showing that cortical neurons which express -AR are activated when animals experience deep sleep. This outcome provides clues about the role that the -AR plays in the sleep-wake cycle and may indicate a neurochemical mechanism that mediates high intensity sleep.

ReferencesGerashchenko, D., Wisor, J.P., Burns, D., Reh, R.K., Shiromani, J., Sakurai, T., de la Iglesia, H. O., Kilduff, T. K. Identification of a population of sleep-active cerebral cortex neurons. PNAS. 2008: 105(29): 10227-10232.Kim, Y., Laposky, A.D., Bergmann, B.M., Turek, F.W. Repeated sleep restriction in rats leads to homeostatic and allostatic responses during recovery sleep. PNAS. 2007: 104(25):10697-10707.

Methods

Figure 1: Activity wheel used for sleep deprivation.

Figure 4: Non-fluorescent staining. Examples of a) -AR in the anterior cingulate cortex with NovaRed (Vector Laboratory) and b) c-Fos in the anterior cingulate cortex with DAB-Ni (Vector Laboratory).

a b

a b c

Figure 3: Fluorescents. Examples of a) β-AR b) double labeling of β-AR and c-Fos, and c) c-Fos in the anterior cingulate cortex.

•C-Fos antibody: various dilutions (SC-52-G, Santa Cruz Biotechnology).-AR antibody: various dilutions (SC-568, Santa Cruz Biotechnology)