Neuroscience Letters, 130 (1991) 255-258
255
1991 Elsevier Scientific Publishers Ireland Ltd. ADONIS 030439409100525K NSL 08042
Circadian changes in arginine vasopressin level in the suprachiasmatic nuclei in the rat Keitaro Yamase I, Sumio Takahashi 1, Kouji N o m u r a l, K a z u h i k o H a r u t a 1 and Seiichiro K a w a s h i m a 2 IZoological Institute, Faculty of Science, Hiroshima University, Hiroshima (Japan) and 2Zoological Institute, Faculty of Science, University of Tokyo, Tokyo (Japan) (Received 8 May 1991; Revised version received 7 June 1991; Accepted 11 June 1991)
Key words: Arginine vasopressin; Suprachiasmatic nuleus; Circadian rhythm; Rat Arginine vasopressin (AVP) neurons were preferentially localized in the dorsomedial part of the suprachiasmatic nucleus (SCN). To know the role of AVP neurons in the SCN, male rats were kept under a normal light-dark cycle (L-D), or under constant darkness (D-D) for 20 days. In L-D condition, AVP levels in the SCN showed the circadian change. In D-D condition, the patterns in AVP levels showed a free-running rhythm, and an about 12-h shift per 20 days. This result suggests that the activity of AVP neurons may be closely associated with the endogenous circadian rhythm of the SCN.
The suprachiasmatic nucleus (SCN) of the hypothalamus is involved in the generation of various circadian rhythms and their entrainment to environmental light and dark cycles [8, 19]. The arginine vasopressin (AVP) neurons were located in the SCN as well as in the supraoptic nucleus (SON), paraventricular nucleus (PVN), and retrochiasmatic nucleus in the rat [2, 3, 17, 23]. The AVP contents in the SCN, SON and PVN showed diurnal changes [11]. The AVP levels in cerebrospinal fluid (CSF) also showed a diurnal change and the AVP in the CSF is mainly secreted from AVP neurons in the SCN [13, 15]. However, the physiological roles of AVP neurons in the SCN are not known. As far as we know, there is no report about effect of constant darkness on the change in AVP levels in the SCN. Therefore, in order to clarify the nature of the diurnal change in AVP levels in the SCN we studied the changes in AVP contents in the SCN and SON of rats kept under the normal light and dark cycle or constant darkness. Also, we studied the distribution of AVP neurons in the SCN. As circadian change in corticosteroid secretion is controlled by the SCN [9], we studied plasma corticosterone levels to know the relationship between the AVP in the SCN and the corticosterone secretion. Male Wistar rats were obtained from CLEA Japan Inc. (Osaka) at I month of age, and were housed in a temperature-controlled room at a 12-h light (06.00Correspondence: S. Takahashi, Zoological Institute, Faculty of Science, Hiroshima University, Naka-ku, Hiroshima 730, Japan.
18.00 h) and 12-h dark cycle with free access to laboratory chow and tap water. Rats were used at 3-4 months of age in each experiment. Eighty-six rats were used in the study of circadian changes in AVP levels in the SCN and SON, and corticosterone levels. All rats had been individually kept under normal light--dark cycle (L-D) conditions in a plastic cage. Thereafter, half of them were kept for 20 days in constant darkness (D-D) condition. At 03.00, 05.00, 09.00, 11.00, 15.00, 19.00 and 23.00 h, rats were killed by decapitation in a surgery room next to the animal room within 20 s after being removed from the home cage under the red safety light. The blood was collected from the trunk, and plasma samples were stored at -20°C. The brain was quickly removed, frozen on dry ice and kept at -80°C. For the AVP assay, the frozen brain was cut frontally with a cryostat in 250/tm thickness according to the brain atlas [6]. The SCN and SON were microdissected with stainless-steel tubes (i.d. 1,100 and 500/tm, respectively) [12]. The microdissected nuclei were homogenized in 750/tl of 0.1 N HCI with an ultrasonicator, neutralized with 0.3 N NaOH. After centrifugation, the supernatant was frozen and stored at -20°C. AVP levels were measured as described previously [24]. For the corticosterone assay, plasma was extracted with dichloromethane, and plasma levels were measured by the radioimmunoassay using the antiserum raised against corticosterone-21-hemisuccinyl thyroglobulin (BioMakor, Rehovot). Specificity of the antiserum written in
256
the attached data sheet was that at 50% displacement the cross-reactivity with other steroids was 17% with progesterone, 27% with 1l-deoxycorticosterone and less than 8% with the other steroids. For the morphological study, brains were fixed with cardiac perfusion of Zamboni's fixative, and cut coronally into serial frozen sections of 10 pm and divided into two series. One series was immunohistochemically stained for AVP by the avidin-biotin-peroxidase complex method using anti-AVP serum (RV-1K, [5]). The other was stained with Cresyl violet. The boundary of SCN was traced on a profile of brain sections and the localization of AVP neurons was plotted with the aid of camera lucida. The SCN area was demarcated into 4 equal parts: the dorsomedial part (dm), dorsolateral part (dl), ventromedial part (vm) and ventrolateral part (vl), as shown in Van den Pol [21]. The number of AVP neurons was counted in 4 different divisions of the SCN (Fig. 4). The results were expressed as mean + S.E. Duncan's multiple range test was used for statistical analysis. A probability of less than 0.05 was assumed to denote a significant difference. Fig. 1 shows AVP levels in the SCN. L-D condition: during the light period, the AVP levels were generally higher than during the dark period. The AVP level at 23.00 h was significantly lower than the levels at 15.00 or 19.00 h. The AVP level at 05.00 h markedly increased from the level at 03.00 h. D-D condition: at 11.00 and 15.00 h the AVP levels were significantly lower than the level at 09.00 h. The AVP level at 19.00 h was signifi-
0.6
I"----.,
L-D
o--o D-D
cantly higher than the levels at 11.00 and 15.00 h. The pattern of AVP levels in D-D rats indicated a shift for longer circadian periods of about 12 h after 20-day residence in D-D. Fig. 2 shows AVP levels in the SON. L-D condition: during the light period, the AVP level remained higher than during the dark period. The AVP level at 23.00 h was significantly lower than the levels at 15.00 and 19.00 h. At 03.00 h the AVP level remained low. The AVP level at 05.00 h was significantly higher than the levels at 23.00 and 03.00 h. D-D condition: the AVP levels did not show any significant circadian fluctuations and remained high. We found that after 20 days in D-D condition the circadian change of AVP contents in the SCN still persisted but showed a free-running pattern, indicating that the AVP rhythm in the SCN represents its nature of intrinsic circadian rhythmicity. The disappearance of AVP rhythm in the SON in D-D condition indicates that the circadian change in AVP levels in the SON may not be regarded as an intrinsic rhythm. The circadian change in AVP levels in the SON may be involved in the circadian changes in drinking or locomotor behaviors under the normal lighting condition. We observed the circadian change in plasma corticosterone level (Fig. 3) and confirmed that it was the endogenous circadian rhythm [9]. L-D condition: during the morning period the corticosterone levels were low and increased at 15.00 h, and then it remained high until 23.00 h. The corticosterone levels at 15.00, 19.00 and 23.00 h were significantly higher than those at 05.00 or 07.00 h when the corticosterone levels were the lowest. "--" L-D o--o D-D
/ I, k /~'X,x~
1.6
0 1.4
,~ 0.4 ~ 0.3 E ~Q. 0 . 2
._~
~ 0.8 ~ 0.6 0.4
0
' 7
' 9
11
' 13
~ 15
J 17
I 19
I 21
I 23
I 1
I 3
I 5
Time of day (hours) Fig. 1. Changes in AMP levels in the SCN in the normal light and dark cycle (L-D) condition (solid line) or in the constant darkness (D-D) condition (dotted line). At each sampling time 5 or 6 rats were used. Vertical bars depict the S.E.M. L-D condition: the AVP levels at 15.00 or 19.00 h vs. the level at 23.00 h, P < 0 . 0 1 in both comparisons; the AVP level at 03.00 h vs. the level at 05.00 h, P < 0 . 0 1 . D - D condition: the AVP level at 09.00 h vs. the levels at 11.00 or 15.00 h. P < 0 . 0 1 in both comparisons; the AVP levels at 11.00 or 15.00 h vs. the level at 19.00 h, P < 0 . 0 1 in both comparisons.
0.2 0
i
|
,
|
i
7
9
11
13
15
i 17
rime of
19
day
21
23
1
3
5
(hours)
Fig. 2. Changes in AVP levels in the SON in L-D condition (solid line) or in D - D condition (dotted line). At each sampling time 5 or 6 rats were used. Vertical bars depict the S.E.M. L-D condition: the A V P level at 15.00 or 19.00 h vs. the level at 23.00 h, P < 0.01, P < 0.05, respectively; the AVP levels at 23.00 or 03.00 h vs. the level at 05.00 h, P < 0.01 in both comparisons. D-D condition: the AVP levels did not show any significant changes.
257 30
~--~ L-D _~
2S
"0
20 C 0
(..)
7
e
11
13
is Time
17
10
of day
21
=3
1
3
s
(hours)
Fig. 3. Changes in plasma corticosterone levels in L-D condition (solid line) or in D-D condition (dotted line). At each sampling time 5 or 6 rats were used. Vertical bars depict the S.E.M. L-D condition: the corticosterone levels at 05.00 or 07.00 h vs. the levels at 15.00, 19.00 or 23.00 h, P < 0.01, respectively. D-D condition: the corticosterone level at 7.00 h vs. the level at 09.00 h, P
255
1991 Elsevier Scientific Publishers Ireland Ltd. ADONIS 030439409100525K NSL 08042
Circadian changes in arginine vasopressin level in the suprachiasmatic nuclei in the rat Keitaro Yamase I, Sumio Takahashi 1, Kouji N o m u r a l, K a z u h i k o H a r u t a 1 and Seiichiro K a w a s h i m a 2 IZoological Institute, Faculty of Science, Hiroshima University, Hiroshima (Japan) and 2Zoological Institute, Faculty of Science, University of Tokyo, Tokyo (Japan) (Received 8 May 1991; Revised version received 7 June 1991; Accepted 11 June 1991)
Key words: Arginine vasopressin; Suprachiasmatic nuleus; Circadian rhythm; Rat Arginine vasopressin (AVP) neurons were preferentially localized in the dorsomedial part of the suprachiasmatic nucleus (SCN). To know the role of AVP neurons in the SCN, male rats were kept under a normal light-dark cycle (L-D), or under constant darkness (D-D) for 20 days. In L-D condition, AVP levels in the SCN showed the circadian change. In D-D condition, the patterns in AVP levels showed a free-running rhythm, and an about 12-h shift per 20 days. This result suggests that the activity of AVP neurons may be closely associated with the endogenous circadian rhythm of the SCN.
The suprachiasmatic nucleus (SCN) of the hypothalamus is involved in the generation of various circadian rhythms and their entrainment to environmental light and dark cycles [8, 19]. The arginine vasopressin (AVP) neurons were located in the SCN as well as in the supraoptic nucleus (SON), paraventricular nucleus (PVN), and retrochiasmatic nucleus in the rat [2, 3, 17, 23]. The AVP contents in the SCN, SON and PVN showed diurnal changes [11]. The AVP levels in cerebrospinal fluid (CSF) also showed a diurnal change and the AVP in the CSF is mainly secreted from AVP neurons in the SCN [13, 15]. However, the physiological roles of AVP neurons in the SCN are not known. As far as we know, there is no report about effect of constant darkness on the change in AVP levels in the SCN. Therefore, in order to clarify the nature of the diurnal change in AVP levels in the SCN we studied the changes in AVP contents in the SCN and SON of rats kept under the normal light and dark cycle or constant darkness. Also, we studied the distribution of AVP neurons in the SCN. As circadian change in corticosteroid secretion is controlled by the SCN [9], we studied plasma corticosterone levels to know the relationship between the AVP in the SCN and the corticosterone secretion. Male Wistar rats were obtained from CLEA Japan Inc. (Osaka) at I month of age, and were housed in a temperature-controlled room at a 12-h light (06.00Correspondence: S. Takahashi, Zoological Institute, Faculty of Science, Hiroshima University, Naka-ku, Hiroshima 730, Japan.
18.00 h) and 12-h dark cycle with free access to laboratory chow and tap water. Rats were used at 3-4 months of age in each experiment. Eighty-six rats were used in the study of circadian changes in AVP levels in the SCN and SON, and corticosterone levels. All rats had been individually kept under normal light--dark cycle (L-D) conditions in a plastic cage. Thereafter, half of them were kept for 20 days in constant darkness (D-D) condition. At 03.00, 05.00, 09.00, 11.00, 15.00, 19.00 and 23.00 h, rats were killed by decapitation in a surgery room next to the animal room within 20 s after being removed from the home cage under the red safety light. The blood was collected from the trunk, and plasma samples were stored at -20°C. The brain was quickly removed, frozen on dry ice and kept at -80°C. For the AVP assay, the frozen brain was cut frontally with a cryostat in 250/tm thickness according to the brain atlas [6]. The SCN and SON were microdissected with stainless-steel tubes (i.d. 1,100 and 500/tm, respectively) [12]. The microdissected nuclei were homogenized in 750/tl of 0.1 N HCI with an ultrasonicator, neutralized with 0.3 N NaOH. After centrifugation, the supernatant was frozen and stored at -20°C. AVP levels were measured as described previously [24]. For the corticosterone assay, plasma was extracted with dichloromethane, and plasma levels were measured by the radioimmunoassay using the antiserum raised against corticosterone-21-hemisuccinyl thyroglobulin (BioMakor, Rehovot). Specificity of the antiserum written in
256
the attached data sheet was that at 50% displacement the cross-reactivity with other steroids was 17% with progesterone, 27% with 1l-deoxycorticosterone and less than 8% with the other steroids. For the morphological study, brains were fixed with cardiac perfusion of Zamboni's fixative, and cut coronally into serial frozen sections of 10 pm and divided into two series. One series was immunohistochemically stained for AVP by the avidin-biotin-peroxidase complex method using anti-AVP serum (RV-1K, [5]). The other was stained with Cresyl violet. The boundary of SCN was traced on a profile of brain sections and the localization of AVP neurons was plotted with the aid of camera lucida. The SCN area was demarcated into 4 equal parts: the dorsomedial part (dm), dorsolateral part (dl), ventromedial part (vm) and ventrolateral part (vl), as shown in Van den Pol [21]. The number of AVP neurons was counted in 4 different divisions of the SCN (Fig. 4). The results were expressed as mean + S.E. Duncan's multiple range test was used for statistical analysis. A probability of less than 0.05 was assumed to denote a significant difference. Fig. 1 shows AVP levels in the SCN. L-D condition: during the light period, the AVP levels were generally higher than during the dark period. The AVP level at 23.00 h was significantly lower than the levels at 15.00 or 19.00 h. The AVP level at 05.00 h markedly increased from the level at 03.00 h. D-D condition: at 11.00 and 15.00 h the AVP levels were significantly lower than the level at 09.00 h. The AVP level at 19.00 h was signifi-
0.6
I"----.,
L-D
o--o D-D
cantly higher than the levels at 11.00 and 15.00 h. The pattern of AVP levels in D-D rats indicated a shift for longer circadian periods of about 12 h after 20-day residence in D-D. Fig. 2 shows AVP levels in the SON. L-D condition: during the light period, the AVP level remained higher than during the dark period. The AVP level at 23.00 h was significantly lower than the levels at 15.00 and 19.00 h. At 03.00 h the AVP level remained low. The AVP level at 05.00 h was significantly higher than the levels at 23.00 and 03.00 h. D-D condition: the AVP levels did not show any significant circadian fluctuations and remained high. We found that after 20 days in D-D condition the circadian change of AVP contents in the SCN still persisted but showed a free-running pattern, indicating that the AVP rhythm in the SCN represents its nature of intrinsic circadian rhythmicity. The disappearance of AVP rhythm in the SON in D-D condition indicates that the circadian change in AVP levels in the SON may not be regarded as an intrinsic rhythm. The circadian change in AVP levels in the SON may be involved in the circadian changes in drinking or locomotor behaviors under the normal lighting condition. We observed the circadian change in plasma corticosterone level (Fig. 3) and confirmed that it was the endogenous circadian rhythm [9]. L-D condition: during the morning period the corticosterone levels were low and increased at 15.00 h, and then it remained high until 23.00 h. The corticosterone levels at 15.00, 19.00 and 23.00 h were significantly higher than those at 05.00 or 07.00 h when the corticosterone levels were the lowest. "--" L-D o--o D-D
/ I, k /~'X,x~
1.6
0 1.4
,~ 0.4 ~ 0.3 E ~Q. 0 . 2
._~
~ 0.8 ~ 0.6 0.4
0
' 7
' 9
11
' 13
~ 15
J 17
I 19
I 21
I 23
I 1
I 3
I 5
Time of day (hours) Fig. 1. Changes in AMP levels in the SCN in the normal light and dark cycle (L-D) condition (solid line) or in the constant darkness (D-D) condition (dotted line). At each sampling time 5 or 6 rats were used. Vertical bars depict the S.E.M. L-D condition: the AVP levels at 15.00 or 19.00 h vs. the level at 23.00 h, P < 0 . 0 1 in both comparisons; the AVP level at 03.00 h vs. the level at 05.00 h, P < 0 . 0 1 . D - D condition: the AVP level at 09.00 h vs. the levels at 11.00 or 15.00 h. P < 0 . 0 1 in both comparisons; the AVP levels at 11.00 or 15.00 h vs. the level at 19.00 h, P < 0 . 0 1 in both comparisons.
0.2 0
i
|
,
|
i
7
9
11
13
15
i 17
rime of
19
day
21
23
1
3
5
(hours)
Fig. 2. Changes in AVP levels in the SON in L-D condition (solid line) or in D - D condition (dotted line). At each sampling time 5 or 6 rats were used. Vertical bars depict the S.E.M. L-D condition: the A V P level at 15.00 or 19.00 h vs. the level at 23.00 h, P < 0.01, P < 0.05, respectively; the AVP levels at 23.00 or 03.00 h vs. the level at 05.00 h, P < 0.01 in both comparisons. D-D condition: the AVP levels did not show any significant changes.
257 30
~--~ L-D _~
2S
"0
20 C 0
(..)
7
e
11
13
is Time
17
10
of day
21
=3
1
3
s
(hours)
Fig. 3. Changes in plasma corticosterone levels in L-D condition (solid line) or in D-D condition (dotted line). At each sampling time 5 or 6 rats were used. Vertical bars depict the S.E.M. L-D condition: the corticosterone levels at 05.00 or 07.00 h vs. the levels at 15.00, 19.00 or 23.00 h, P < 0.01, respectively. D-D condition: the corticosterone level at 7.00 h vs. the level at 09.00 h, P
