Nitrergic neurons of the dorsal raphe nucleus encode information about stress duration

Abstract

Nitrergic neurons of the dorsal raphe nucleus (DRN) may play a role in physiological stress responses. The caudal lateral wings (CLW) are unique compared to other rostral-caudal DRN sub-regions because they contain distinct nitric oxide (NO) synthase (NOS) populations that are independent of tryptophan hydroxylase (TPH). NOS neurons in the CLW are also highly activated during acute restraint stress. However, the effects of acute stress duration on NOS activation in the CLW are unclear. Here NADPH-d, an index of NOS activity, is used to show that sub-regions of the DRN have differential NOS activation in response to 6 hours of restraint stress in rats. We report increased NOS activity through 6 hours of restraint in the caudal lateral wings and ventromedial sub-regions. These data suggest that, NOS neurons may play a dynamic role in the response to stress duration.

Fig 1. Schematic drawing of the rostral and caudal dorsal raphe nucleus.

A. Drawing represents the rostral extension of the dorsal raphe nucleus at Bregma -7.33mm. B. Drawing represents the caudal extension of the dorsal raphe at Bregma -8.33mm. Below the aqueduct is the dorsal region, on each side of the dorsal region are the lateral wings, and below the dorsal region is the ventral region.

Fig 2. 5x objective NADPH-diaphorase staining in the DRN.

Rostral (A, B, C,D) and caudal (E, F, G, H) halves of the DRN in control (A, E), 1 hour (B, F), 3 hour (C, G) and 6 hour (D, H) restraint rats. These photo images are rostral (Bregma -7.64mm) and caudal (-Bregma -8.36) extensions of the DRN through a 6 hour restraint duration. The blue color formed by NADPH-d reaction was adjusted to gray scale for contrast and each image was adjusted against background staining.

Fig 3. Caudal lateral wings and rostral ventromedial have most NOS activation.

These graph shows mean levels of NADPH-d stained neurons in control animals for each DRN sub-region. The CLW and RV areas have the most NADPH-d staining compared to other regions of the DRN. Error bars are representative of standard error. Significance is denoted by an asterisk and these regions are compared to every other region of the DRN.

Fig 4. 40x objective varicosity fibers in the caudal lateral wings.

Caudal extension of the DRN in control (A,), 1 hour (B,), 3 hour (C,) and 6 hour (D,) restraint rats. Arrows point to three varicose structures on axons.

Fig 5. 40x objective varicosity fibers in the ventromedial.

Rostral (A,B,C,D) and caudal (E,F,G,H) halves of the DRN in control (A,E), 1 hour (B,F), 3 hour (C,G) and 6 hour (D,H) restraint rats. Arrows point to varicose structures on axons. Compared to the images in Fig 3, these figures have less abundant varicose axons.

Fig 6. NOS in the DRN caudal lateral wings show differences in activation following restraint stress.

(A) NOS activation increase in the caudal lateral wings region following 3 hours and significantly following 6 hours of restraint. (B) Intervaricosity spacing decreases following 3 hours of restraint but by 6 hours, the intervaricosity spacing is closer to control values. Percentages were calculated by subtracting each animal per experimental group by the mean of the control then dividing by the control mean and multiplying by 100. Significance is denoted by an asterisk and compared to the control. Error bars are representative of standard error.

Fig 7. NOS in the DRN rostral ventromedial region show differences in activation following restraint stress.

(A) NOS activity barely shifts after 1 hour of restraint then increases following 3 hours of restraint and significantly increases following 6 hours of restraint. (B) Intervaricosity spacing decreases following 3 hours of restraint but by 6 hours, the intervaricosity spacing is closer to control values. Percentages were calculated by subtracting each animal per experimental group by the mean of the control then dividing by the control mean and multiplying by 100. Significance is denoted by an asterisk and compared to the control. Error bars are representative of standard error.

Fig 8. NOS in the DRN caudal ventromedial region show differences in activation following restraint stress.

(A) NOS activity in the caudal ventromedial remains stable following 3 hours of restraint but significantly, increase following 6 hours of restraint. (B) Intervaricosity distance decreases following 1 hour of restraint and remains decreased through 6 hours of restraint. Percentages were calculated by subtracting each animal per experimental group by the mean of the control then dividing by the control mean and multiplying by 100. Significance is denoted by an asterisk and compared to the control. Error bars are representative of standard error.

Fig 9. Varicose axon localization in the caudal lateral wing.

The red arrows indicate varicose axons surrounding the aqueduct in the lateral wing of the caudal sub-region. The cluster of neural bodies does not contain many varicose axons.