BRAIN RESEARCH

E L S E V I E R Brain Research 689 (1995) 122-128

Research report

Cyclic changes in the release of norepinephrine and dopamine in the medial basal hypothalamus: effects of aging

Srinivasan ThyagaRajan, Puliyur S. MohanKumar, S. Kaleem Quadri * Neuroendocrine Research Laboratory, Department of Anatomy and Physiology, Kansas State University, VMS 228, Manhattan, KS 66506, USA

Accepted 18 April 1995

Abstract

Push-pull perfusion and HPLC were used to measure the release of norepinephrine (NE) and dopamine (DA) in the medial basal hypothalamus of young (4-5 months old), middle-aged (8-10 months old), and old (22-24 months old) rats. In the young animals, the afternoon of proestrus was characterized by a gradual increase in NE release and a simultaneous gradual decrease in DA release. The peak in NE release and the nadir in DA release occurred at about the time when the proestrous surges in serum LH and PRL are known to occur. No changes in NE and DA releases occurred in the afternoon of diestrus when serum LH and PRL are known to remain stable. In the middle-aged proestrous animals, the patterns of NE and DA releases were similar to those in the young proestrous animals, but the peak in NE release was attenuated and did not reach statistical significance. This corresponded with the reported attenuation in the LH surge in middle age. In the old persistently diestrous animals, NE and DA were released at constant rates, which correlated with the well-documented constant levels of serum LH and PRL in old age. These data provide an explanation for the simultaneous proestrous surges of LH and PRL and lead us to conclude that NE plays a facilitatory role in the LH surge, while DA, through its inhibitory action, regulates the PRL surge. These studies, by monitoring NE and DA releases from adulthood through middle-age to old age, indicated that cyclicity in catecholamine (CA) activities begins to be dampened in middle-age and eventually completely disappears in the acyclic period of old age which is also characterized by a marked deficiency in CA activities.

Keywords: Norepinephrine; Dopamine; Hypothalamus; Estrous cycle; Aging; Push-pull perfusion

1. Introduct ion

In the late 1940s Sawyer and colleagues reported inhibi- tion of ovulation in rabbits and rats with adrenergic block- ers [8,25-27]. This led to the measurement of a number of catecholamine (CA)-related parameters in the hypothala- mus to determine if activities of the catecholaminergic neurons change during the estrous cycle and if these changes could explain the alterations in the release of luteinizing hormone (LH) during the estrous cycle. Now, almost five decades later, although CAs are established as the primary regulators of LH release, the roles of individ- ual CAs, norepinephrine (NE) and dopamine (DA), in LH release have remained unclear and little is known about the simultaneous changes in NE and DA activities which lead to the surge in serum LH which occurs in the afternoon of proestrus [19].

* Corresponding author. Fax: (1) (913) 532-4557.

0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 5 ) 0 0 5 5 1 - X

The failure, as the massive amount of literature indi- cates, was not due to lack of effort, but resulted from a variety of reasons. The measurements of such parameters as the content and concentration of hypothalamic CAs resulted in accumulation of a considerable amount of highly contradictory and confusing data [6,12,13,24,28]. The confusion arose not only from questions about the reliability of these parameters as indicators of neuronal activities but also due to methodological problems. Often these parameters were measured in the whole hypothala- mus or in gross subdivisions of the hypothalamus, with methods which were insensitive, semiquantitative or lacked the ability to distinguish between NE and DA. The mea- surement of other parameters such as CA turnover rates provided important clues to changes in NE and DA activi- ties in the afternoon of proestrus [9,34], but all the methods used for this purpose can determine only relative nonabso- lute turnover rates and have come under criticism for a variety of reasons including the gross disturbances they produce in brain chemistry [4,5,31]. The experiments in- volving the use of drugs gathered valuable evidence of CA

S. ThyagaRajan et al. /Brain Research 689 (1995) 122-128 123

involvement in LH release but failed to clarify the specific roles of NE and DA in LH release due to inability of the drugs to selectively influence the NE and DA systems [23].

A serious limitation of all the methods used for measur- ing content, concentration, and turnover rates of CAs is that due to the necessity to kill the animals they can measure these parameters only one time in a group of animals. This precludes the possibility of developing pro- files of continuous changes in NE and DA activities which lead to the LH surge. Another serious complication arises from the occurrence of the surge in serum prolactin (PRL) at about the same time as the LH surge on the afternoon of proestrus. Because CAs, especially DA, are also the major regulators of PRL release, the profiles of NE and DA activities in the afternoon of proestrus have to be able to explain not only the LH surge but also the PRL surge. This is not possible on the basis of the currently available information, especially due to the uncertainty regarding the role of NE in PRL release and the highly contradictory information on the role of DA in LH release [19].

Thus, although the various methods used so far have established a definite role for CAs in LH and PRL regula- tion, they have not succeeded in deciphering the individual roles of NE and DA and in providing a basis for the simultaneous surges of LH and PRL in the afternoon of proestrus. It is recognized that to achieve this goal, we have to be able to measure simultaneous releases of NE and DA in discrete hypothalamic areas of conscious, unre- strained animals [23].

In recent years, the availability of high performance liquid chromatography with electrochemical detection (HPLC-EC) and the push-pull perfusion technique has provided a powerful tool for continuous and simultaneous measurements of NE and DA releases in highly specific areas of the brain of conscious, freely-moving animals [17-19]. The purpose of the present study was to use these techniques to examine profiles of NE and DA releases in the afternoon of proestrus and diestrus in young rats and determine if they can explain the known patterns of serum LH and PRL during the estrous cycle. NE and DA releases were monitored simultaneously and continuously during 6-to 7-h periods in the medial basal hypothalamus (MBH) which is critical in the release of LH and PRL. Similar measurements were also made in cycling middle-aged and acyclic old animals to determine whether changes in NE and DA activities in these animals can be correlated with the known age-related changes in hormones and estrous cyclicity.

2. Materials and methods

2.1. Animals

Female Sprague-Dawley rats were obtained form Amitech (Omaha, NE) at the age of 3 months and housed

in air-conditioned animal quarters (23 + 2°C) with lights on from 07:00-19:00 h. They were provided rat chow and water ad libitum. The estrous cycles in young (4 months old) and middle-aged (8-9 months old) rats were moni- tored by examination of daily vaginal smears. The rats which showed two consecutive regular estrous cycles were selected and implanted with push-pull cannulae in the third estrous cycle. After a rest of 10-12 days, the estrous cycles were monitored again. The rats which cycled regu- larly by exhibiting two consecutive regular cycles were used for push-pull perfusion on the day of proestrus or diestrus 2 of the next cycle. The old rats (22-24 months old) were selected for cannula implantation on the basis of continuous diestrous smears (pseudopregnancy) for 10 days. They were used for push-pull perfusion two weeks after cannula implantation.

2.2. Push-pull cannula implantation

Construction of the push-pull cannula has been de- scribed previously [11,17,18]. Briefly, it consisted of a 10 mm-long outer cannula made from a 22 G hypodermic needle. It contained a removable stylet made from a 29 G stainless steel tubing. The stylet protruded 0.5 mm beyond the outer cannula. The rats were anesthetized with sodium pentobarbital (50 m g / k g i.p.) and given atropine sulphate (2.2 m g / k g i.p.). They were placed in a stereotaxic appa- ratus (Kopf, Tujunga, CA) and implanted with the cannula in the MBH. The coordinates used for cannula implanta- tion were 10 mm dorsoventral, 0.2 mm lateral and 3.3 mm anteroposterior [21]. After implantation the cannula was secured in place as described previously [17], and the animals were housed in individual cages.

2.3. Push-pull perfusion (PPP)

On the day of perfusion, the rats were placed in the PPP chamber at least an hour before commencing perfusion. The PPP chamber was made of plexi-glass and resembled an individual rat cage. The rats moved freely in the cage and were provided rat chow and water ad libitum. To initiate PPP, the styler was removed from the outer cannula and replaced with an inner cannula assembly which con- sisted of two 29 G stainless steel tubes of unequal lengths. The longer tube (3.5 cm) which protruded 0.5 mm beyond the outer cannula, was used to introduce (push) the perfu- sion medium at the implantation site, whereas the shorter tube (2.0 cm) was used to collect (pull) perfusate from the implantation site. The two tubes were kept together in 2 mm-long Silastic tubing fixed to a piece of tuberculin syringe cut at the 0.05 ml mark. The inner cannula assem- bly was connected to two identically calibrated and bal- anced peristaltic pumps (Pharmacia, Sweden) through two polyethylene tubings (PE-20, Clay Adams, Parsipanny, N J). The pumps were considered balanced when a drop hanging at the tip of a cannula connected to the pumps

124 S. ThyagaRajan et al. / Brain Research 689 (1995) 122-128

remained unchanged in size for two hours while the pumps were in operation. Artificial cerebrospinal fluid (ACSF) was used as the perfusion medium. It consisted of CaC12 (0.087 g / l ) , NaCI (7.188 g / l ) , KCI (0.358 g / l ) , MgSO 4 (0.296 g / l ) and NazHPO 4 (1.703 g / l ) mixed in pyrogen- free water and adjusted to pH 7.3. The pump speed was maintained at 10 /z l /min.

The perfusate samples were collected at 1-h intervals during a continuous 6-or 7-h period between 13:00 h and 20:00 h. Each animal was used only once for perfusate collection. The peffusate samples were collected on ice in polypropylene microsample vials containing sufficient 0.5 M HCIO 4 to give a v / v perfusate to HC104 ratio of 25:1 [20]. The samples were stored at - 7 0 ° C until their analy- sis for NE and DA by high performance liquid chromatog- raphy with electrochemical detection (HPLC-EC).

2.4. HPLC-EC

The HPLC-EC procedure has been described in detail previously [17,20,30]. Briefly, after thawing the perfusate sample at 60°C for 1 min, 45 /xl of the sample were mixed with 5 /.tl of the internal standard (isoproterenol) and injected onto a C-18, 3 /xm ODS reverse phase, 100 × 3.2 mm column (Bioanalytical Systems, West Lafayette, IN). The column was maintained at a temperature of 37°C in a CTO-6A column oven (Shimadzu, Columbia, MD). The mobile phase consisted of monochloroacetic acid (12.285 g / l ) , octanesulfonic acid disodium salt (0.25 g / l ) , ethy- lenediaminetetraacetic acid (0.25 g / l ) and acetonitrile (2.5%) dissolved in pyrogen-free water and the pH ad- justed to 3.1 with sodium hydroxide. The mobile phase was pumped through the HPLC system at a flow rate of 1.3 m l / m i n with the help of an LC-6A pump (Shimadzu). The sensitivity of the LC-4B amperometric detector (Bio- analytical Systems) was 1 nA full scale and the potentials of the working electrodes were 0.8 V for channel 1 and 0.65 V for channel 2. The data were analyzed using a C-R6A Chromatopac integrator (Shimadzu).

2.5. Histology

After perfusion, the animals were sacrificed and their brains were quickly removed and frozen with dry ice. Coronal sections (60 /xm thick) of the brain were cut using a cryostat ( - 10°C), stained with cresyl violet, and exam- ined under a light microscope to verify the cannula implan- tation site. The numbers of animals used in various groups were as follows: young proestrous 9, young diestrous 9, middle-aged proestrous 8, old persistently diestrous 6.

2.6. Statistical analysis

To determine if catecholamine release rates changed with time, the data in each group were analyzed using one-way A N O V A for repeated measures followed by

P1 P2 P3 P4 P5

\

10

! Young Proestrous (n=9) Young Diestrous (n=9) Lateral 0.4 mm Middle-aged Proestrous (n=8) Old Persistently diestrous (n=6)

Fig. 1. Schematic representation of the push-pull cannulae implantation sites in the medial basal hypothalamus of young (4-5 months old) proestrous and diestrous, middle-aged (8-9 months old) proestrous, and old (22-24 months old) persistently diestrous rats. The numbers P1-P5 represent coronal plates extending rostrocaudally 1 mm (P1) to 5 mm (P5) posterior from the zero plane passing through the bregma. The numbers 8-10 on the margin represent the distance (mm) below the horizontal plane (bregma). LA, lateroanterior hypothalamic nucleus; AH, anterior hypothalamic area; VMH, ventromedial hypothalamus; DMC, dorsomedial nucleus compact; ARC, arcuate nucleus; 3V, third ventricle; SOD, supraoptic decussation. The exact location of the push-pull cannula in each animal was established by examination of stained, serial sections of the brain under a light microscope.

Fisher's LSD test. To determine if the average cate- cholamine release rates among various groups were differ- ent from each other, first the average rates in individual animals within each group were calculated and then ana- lyzed by one-way A N O V A followed by post-hoc Fisher's LSD test.

3. Results

3.1. Location o f the PPP cannulae

The PPP cannulae in young proestrous and diestrous, middle-aged proestrous, and old persistently diestrous rats were located in the arcuate and ventromedial nuclei of the MBH (Fig. 1). The perfusion area around the cannula tip was about 0.5 mm in diameter. After cannula implantation, about 35% of the young and middle-aged rats continued to cycle while the rest exhibited diestrus for about 7 - 1 0 days before resuming regular cycles. Cannula implantation did not affect the state of constant diestrus in the old animals. For a variety of mechanical reasons (twisting of the lines, air bubbles in the lines, etc.) perfusion was not always successful. Only those animals in which perfusion was completely successful (75-85%) are included in this re- port. The pituitaries in about 25% of the old animals were tumorous. These rats are not included in the report.

S. ThyagaRajan et al. / Brain Research 689 (1995) 122-128 125

12

D.

Y o u n g 20 Proestrous

15

10 jz_~

/ . - J \ s

0 13 15 17 19

Middle-Aged] 20 P r o e s t r o u s j

15

5

m= 0 . . . .

"t" 3 15 17 19

~- 20 Old .E P e r s i s t e n t l y =" Diestrous

~5 o z

10

13 15 17 19 0 , , , , , , , , 0

13 15 17 19

TTrne ( X l O 0 hrg)

Fig. 2. Norepinephrine (NE) release in the afternoon in young (4-5 months old) proestrous, and diestrous, middle-aged (8 -9 months old) proestrous, and old (22 -24 months old) persistently diestrous rats. * Dif- ferent from 13:00 h (P < 0.05).

3.2. NE release

The patterns of NE release in the young, middle-aged, and old animals are shown in Fig. 2. In the young proe- strous animals, NE release increased gradually from 13:00 h to reach a peak at 17:00 h ( P < 0.01). NE release was essentially unchanged at 18:00 h but decreased markedly at 19:00 h. There was variability in NE release profiles among individual animals. Of the 9 animals used in this group, 6 showed peaks at 17:00 h, 1 at 16:00 h, 1 at 18:00 h and 1 showed no peak. In the young diestrous animals, no significant change in NE release occurred throughout the period of observation, but the basal release rate in the beginning of the perfusion (13:00 h) was more than twice that in the young proestrous animals. There was consider- ably less variability in individual profiles of NE release on the day of diestrus than on the day of proestrus.

In the middle-aged animals, the NE release pattern, characterized by a gradual increase to peak levels late in the afternoon, was similar to that in the young proestrous animals, except that the peak appeared 2 h late at 19:00 h, and did not reach statistical significance when analyzed by ANOVA for repeated measurements. In the middle-aged proestrous animals, NE release in the beginning of the perfusion (13:00 h) was more than twice that in the young proestrous animals. The peak levels in the middle-aged animals at 19:00 h were also more than twice the peak levels in the young proestrous animals. Of the 9 animals in the middle-aged group, 6 showed peaks at 19:00 h, 1 at 17:00 h, 1 at 18:00 h, and 1 failed to show any peak.

In the old persistently diestrous animals, as in the young diestrous animals, NE release remained essentially stable throughout the afternoon. NE release in these animals also

m

o 10

© 8

r C . _

o 4 Z

~ 2

13 YP YD MP OPD

Fig. 3. The overall average ( + S.E.M.) norepinephrine release rate in the entire afternoon in young proestrous (YP), young diestrous (YD), middle-aged proestrnus (MP), and old persistently diestrous (OPD) ani- mals. * Different from YP and OPD (P < 0.001).

showed much less individual variability than that in the other two age groups.

The overall average NE release rates in the entire afternoon in the three age groups are shown in Fig. 3. In the young rats, on average, more NE was released in the afternoon of diestrus than in the afternoon of proestrus ( P < 0.001). Comparisons among the three age groups indicated that the middle-aged proestrous rats released more NE than young proestrous rats ( P < 0.001), whereas the old persistently diestrous rats released significantly less NE than the young diestrous rats ( P < 0.001).

3.3. DA release

DA release patterns are shown in Fig. 4. In the young proestrous rats, DA release decreased from high levels at

1

"Z 2 E ol 0 ~ , , , , , , 0 O.

v 13 15 17 19 o 10

.E M i d d l e - A g e d 10 E Proestrous ~. a 8 -.\ D

6

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2

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i 6

13 15 17 19

l °'° J Persistently Diestrous

6

4

I

, , , , , , , 0 ,

13 15 17 19 3 15 17 19

T i m o ( X l O 0 hrs)

Fig. 4. Dopamine (DA) release in the afternoon in young proestrous and diestrous, middle-aged proestrous, and old persistently diestrous rats. * Different from 13:00 h ( P < 0.001).

126 S. ThyagaRajan et aL / Brain Research 689 (1995) 122-128

5 i n

4 n , . ,

. h ° i

~ v 2

o 1

I 0 ~'P +D MP OPD '

Fig. 5. The overall average ( + S.E.M.) dopamine release rate in the entire afternoon in young proestrous (YP), young diestrous (YD), middle-aged proestrous (MP), and old persistently diestrous (OPD) animals. * Differ- ent from YP and OPD (P < 0.001).

13:00 h to low levels at 19:00 h (P < 0.05). In contrast, in the young diestrous animals no significant changes in DA release were observed throughout the period of observa- tion. The DA release rate at 13:00 h in the young proe- strous rats was significantly (P < 0.05) less than that in the young diestrous rats. In the middle-aged proestrous rats, as in the young proestrous rats, DA release decreased from high levels at 13:00 h to low levels at 20:00 h (P < 0.05). However, the basal DA release at 13:00 h was significantly greater in the middle-aged proestrous animals than in the young proestrous animals (P < 0.05). As in the young diestrous rats, no changes in DA release were observed in the old persistently diestrous rats, but DA release at 13:00 h in the old group was only about one-half of that in the young group (P < 0.05).

The average DA release rates in the entire afternoon in the three age groups are shown in Fig. 5. In the young animals, significantly more DA was released in the after- noon of diestrus than in the afternoon of proestrus (P < 0.001). Comparisons among the three age groups indicated that the middle-aged proestrous rats released more DA than the young proestrous rats, whereas the old persistently diestrous rats released significantly less DA than the young diestrous rats (P < 0.001).

4. Discuss ion

These results demonstrate that the afternoon of proestrus in young animals is characterized by a gradual increase in the release of NE and a simultaneous gradual decrease in the release of DA in the MBH. The absence of these events on the day of diestrus indicates that they are not part of a daily rhythm but are associated with the estrous cycle. This association was strengthened by the observa- tions in the aging animals. In the middle-aged proestrous animals both NE and DA release patterns were similar to those in the young proestrous animals, except that the NE

peak appeared later and was not significant when analyzed by ANOVA for repeated measures. In the old animals, the absence of cyclicity in NE and DA releases matched the absence of estrous cyclicity. In these persistently diestrous animals, NE and DA releases at constant rates were similar to those in the young diestrous animals, except that in the old animals NE and DA release rates were markedly reduced compared to those in the young animals. These data indicate that cyclicity in NE activity begins to un- dergo modifications in middle-age and completely disap- pears in old age, which is also characterized by absence of cyclicity in DA release and marked reductions in the activities of NE as well as DA.

The MBH and other components of the preoptico- suprachiasmatic tuberoinfundibular system are believed to be the areas where NE acts to regulate LH release. How- ever, the absence of information on NE release in these areas had made it difficult to explain with certainty the nature of NE involvement in the surge in serum LH which occurs in the afternoon of proestrus. In the present study, serum LH was not measured but we have recently reported that in young rats of the same age and strain and main- tained under the same conditions as in the present study, the LH surge occurs at about the same time on the afternoon of proestrus as the NE peak in the present study [19]. The fact that in the present experiments NE release showed no change on the day of diestrus when serum LH is known to remain stable, but increased on the day of proestrus to reach a peak at a time when the LH surge is known to occur indicates that NE plays a facilitatory role in the LH surge. Although turnover studies have several drawbacks, they, nonetheless, support our conclusion that increased LH secretion on the afternoon of proestrous is associated with increased NE activity. Thus, in agreement with the NE release pattern observed in the present study, NE turnover rate in the arcuate nucleus was low between 12:00-14:00 h on the day of proestrus, but increased markedly during the LH surge at 15:00-17:00 h [34]. Additional support comes from turnover studies in estra- diol-treated ovariectomized rats. In these animals also NE turnover rate in the median eminence and other parts of the preopticosuprachiasmatic tuberoinfundibular system showed a marked increase at the time of the LH surge [35].

With the use of the push-pull perfusion technique, a positive relationship between rising levels of NE release and LH peaks has also been demonstrated in two other species [10,29]. In female rabbits, the postcoital LH surges were accompanied by marked increases in NE release in the MBH and the anterior hypothalamus [10]. Similarly, in ovariectomized monkeys NE release in the stalk-median eminence was synchronous with LHRH release which, in turn, was synchronous with LH release [29].

The fact that the basal and overall average release rates of NE in the afternoon of diestrus were higher than those in the afternoon of proestrus, indicates that it is not the level of NE release, but a specific pattern of NE release

s. ThyagaRajan et al. / Brain Research 689 (1995) 122-128 127

characterized by a gradual increase leading to peak, is required for the LH surge. A similar conclusion was drawn with regard to the pattern of NE release in the MPA and its association with the LH surge [19]. Other yet to be defined cyclic changes in other hypothalamic neurochemicals and the known alterations in ovarian steroids are no doubt important in the LH surge.

Our conclusion that the NE release pattern observed in the young proestrous animals is required for the LH surge and that changes in NE release pattern would lead to alterations in the LH surge was reinforced by the data in the aging animals. In the middle-aged animals, the attenua- tion of the NE peak matched the attenuation in the LH surge which has been reported by others [3,33] and also observed by us [19]. In the old animals, NE release at a constant rate corresponded with the well-documented con- stant release of LH in old age [15,16].

It is noteworthy that although the peak in NE release in the middle-aged proestrous animals was attenuated, the basal NE release was significantly elevated compared to that in the young proestrous animals. The significance of this increase is not clear, but it is in agreement with the reported increases in NE concentrations, turnover rates and release in several hypothalamic areas of middle-aged rats [7,19,32]. This increase in the basal release was, however, transitory and was replaced by a marked decrease in NE release rate in the old animals.

Unlike NE, the role of DA in LH release is not clear [14,23]. In the present study, DA release in the young animals declined in the afternoon of proestrus and reached its lowest levels at a time in the afternoon when LH is known to reach a peak [19]. This discounts the possibility that DA has a stimulatory role in the LH surge and suggests that DA is either not involved or has an inhibitory role in LH release. Contrary to the uncertainty about the role of DA in LH regulation, the inhibitory effect of DA on PRL release is well established [1,2]. The gradual decline and the nadir in DA release observed in this study closely parallels the well-documented rise and the eventual peak in serum PRL in the afternoon of proestrus, as reported by several investigators [33] and also recently observed by us in the rats of the same age and strain [19]. This leaves little doubt that the decline in DA release as observed in the present study is a critical event which permits the occurrence of the proestrous surge of PRL. This conclusion was reinforced by the fact that there was no difference in the patterns of decline in DA release in the young and middle-aged proestrous animals which matched with the well-documented absence of any differ- ence in the profiles of PRL surges in these two age groups, as documented by others [33] and also by us [19]. In the old animals, the lack of changes in DA release correlated with the absence of alterations in serum PRL levels. In these persistently diestrous animals DA release was similar to that in the young diestrous animals except that the average release rate in the old animals was less than half

of that in the young diestrous animals. This marked defi- ciency is no doubt the cause for the marked increase in serum PRL in old age [15,16]. CA deficiency is believed to be the main reason for the onset of reproductive acyclicity in old age and its removal has been shown to reinitiate estrous cyclicity in old acyclic animals [22].

It is noteworthy that the profiles of NE and DA releases in the MBH during the estrous cycle and during aging as described in this study are very similar to those observed during the estrous cycle and aging in the MPA [19]. This increases the confidence in these data and indicates that these changes constitute a generalized phenomenon in the preoptico-suprachiasmatic tuberoinfundibular system which is known to be critical in the regulation of LH as well as PRL.

In summary, by combining the techniques of PPP and HPLC-EC we have demonstrated that the afternoon of proestrus in young animals is marked by a gradual increase in NE activity and a gradual decrease in DA activity in the MBH. These simultaneous but diametrically opposite events provide the explanation for the simultaneous occur- rence of LH and PRL surges in the afternoon of proestrus. The absence of changes in NE and DA releases on the day of diestrus corresponded with the absence of changes in LH and PRL release in this stage of the estrous cycle. In the middle-aged proestrous animals, the attenuation in the peak of NE release matched the well-documented attenua- tion in LH peak in these animals, whereas the absence of changes in DA release was consistent with the reported absence of changes in the proestrous PRL surge in middle age. In the old animals, marked reductions and absence of fluctuations in NE and DA releases corresponded with the well-established constant levels of serum LH and PRL and absence of estrous cyclicity during this final phase of life.

Acknowledgements

Preliminary results of this study were presented in the 3rd IBRO World Congress of Neuroscience, Montreal, Canada in 1991. This work was supported by NIH Grant AG 05980.

References

[1] Chiocchio, S.R., Cannata, M.A., Fesnes, J.R.C. and Tramezzani, J.H., Involvement of dopamine in the regulation of prolactin release during suckling, Endocrinology, 105 (1979) 544-547.

[2] Chiocchio, S.R., Chafuen, S. and Tramezzani, J.H., Changes in adenohypophysial dopamine related to prolactin release, Endocrinol- ogy, 106 (1980) 1682-1685.

[3] Cooper, R.L., Conn, P.M. and Walker, R.F., Characterization of the LH surge in middle-aged female rats, Biol. Reprod., 23 (1980) 611-615.

[4] Costa, E., Simple neuronal models to estimate turnover rate of noradrenergic transmitters in vivo. In Costa, E. and Galcobine, E.

128 S. ThyagaRajan et al. /Brain Research 689 (1995) 122-128

(Eds.), Biochemistry of simple neuronal models, Advances in Bio- chemical Psychopharmacology, Raven Press, New York, 1970, pp. 169-196.

[5] Costa, E. and Neff, N.H., Estimation of turnover rates to study the metabolic regulation of the steady state level of neural amines. In Lajtha, A. (Ed.), Handbook of Neurochemistry, Control Mechanisms in the Nervous System, Plenum Press, New York, 1977, vol 4, pp. 45.

[6] Donoso, A.O. and De Gutierrez Moyano, N.B., Adrenergic activity in hypothalamus and ovulation, Proc. Soc. Exp. Biol. Med., 135 (1970) 633-635.

[7] Estes, K.S. and Simpkins, J.W., Age-related alteration in cate- cholamine activity within microdissected brain regions of ovariec- tomized Fischer 344 rats, ./. Neurosci. Res., 11 (1984) 405-417.

[8] Everett, J.W., Markee, J.E. and Hollinshead, W.H., A neurogenic timing factor in control of ovulatory discharge of luteinizing hor- mone in the cycling rat, Endocrinology, 44 (1949) 234-243.

[9] Honma, K. and Wuttke, W., Norepinephrine and dopamine turnover rates in the medial preoptic area and the mediobasal hypothalamus of the rat brain after various endocrinological manipulations, En- docrinology, 106 (1980) 1848-1853.

[10] Kaynard, A.H., Pau, K.Y., Hess, D.L. and Spies, H.G., Go- nadotropin-releasing hormone and norepinephrine release from the rabbit mediobasal and anterior hypothalamus during the mating-in- duced luteinizing hormone surge, Neuroendocrinology, 127 (1990) 1176-1185.

[11] Levine, J.E. and Ramirez, V.D., In vivo release of luteinizing hormone-releasing hormone estimated with push-pull cannulae from the mediobasal hypothalami of ovariectomized steroid primed rats, Endocrinology, 107 (1980) 1782-1790.

[12] Lichtensteiger, W., Cyclic variations of catecholamine content in hypothalamic nerve cells during the estrous cycle of the rat, with a concomitant study of the substantia nigra, J. Pharmacol. Exp. Ther., 165 (1969) 204-215.

[13] Lofstr6m, A., Catecholamine turnover alterations in discrete areas of the median eminence of the 4-and 5-day cyclic rat, Brain Res., 120 (1977) 113-131.

[14] McCann, S.M., Monoaminergic and peptidergic control of anterior pituitary secretion. In Farner, D.S. and Lederis, K. (Eds.), Neurose- cretion: Molecules, Cells, Systems, Plenum Press, New York, 1980, pp. 139-151.

[15] Meites, J., Changes in neuroendocrine control of anterior pituitary functions during aging, Neuroendocrinology, 34 (1982) 151-156.

[16] Meites, J., Role of hypothalamic catecholamines in aging process, Acta Endocrinol. (Copenh), 125 (1991) 98-103.

[17] MohanKumar, P.S., Thyagarajan, S. and Quadri, S.K., Interleukin-1 stimulates the release of dopamine and dihydroxyphenylacetic acid from the hypothalamus in vivo, Life Sci., 48 (1991) 925-930.

[18] MohanKumar, P.S. and Quadri, S.K., Systemic administration of interleukin-1/3 stimulates norepinephrine release in the paraventricu- lar nucleus, Life Sci., 52 (1993) 1961-1967.

[19] MohanKumar, P.S., Thyagarajan, S. and Quadri, S.K., Correlations of catecholamine release in the medial preoptic area with proestrous surges of luteinizing hormone and prolactin: Effects of aging, En- docrinology, 135 (1994) 119-126.

[20] Palazzolo, D.L. and Quadri, S.K., Interleukin-1 stimulates cate- cholamine release from the hypothalamus, Life Sci., 47 (1990) 2105-2109.

[21] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordi- nates, Second Edition, Academic Press, New York, 1986.

[22] Quadri, S.K., Kledzik, G.S. and Meites, J., Reinitiation of estrous cycles in old constant estrus by central acting drugs, Neuroen- docrinology, 11 (1973) 248-255.

[23] Ramirez, V.D., Feder, H.H. and Sawyer, C.H., The role of brain catecholamines in the regulation of LH secretion: A Critical inquiry. In: Martini, L. and Ganong, W.F. (Eds.), Frontiers in Neuroen- docrinology, Raven Press, New York, 1984, vol 8, pp. 27-84.

[24] Sandler, R., Concentration of norepinephrine in the hypothalamus of the rat in relation to the estrous cycle, Endocrinology, 83 (1968) 1383-1386.

[25] Sawyer, C.H., Everett, J.W. and Markee, J.E., A neural factor in the mechanisms by which estrogen induces the release of luteinizing hormone in the rat, Endocrinology, 44 (1949) 218-223.

[26] Sawyer, C.H., Markee, J.E. and Everett, J.W., Further experiments on blocking pituitary activation in the rabbit and the rat, J. Exp. Zool., 113 (1950) 659-663.

[27] Sawyer, C.H., Markee, J.E. and Hollinshead, W.H., Inhibition of ovulation in the rabbit by adrenergic blocking agent dibenamine, Endocrinology, 41 (1947) 395-399.

[28] Selmanoff, M.K., Pramid-Holdaway, M.J. and Weiner, R.I., Concen- trations of dopamine and norepinephrine in discrete hypothalamic nuclei during the rat estrous cycle, Endocrinology, 99 (1976) 326- 329.

[29] Terasawa, E., Krook, C., Hei, D.L., Gearing, M., Schultz, N.J. and Davis, G.A., Norepinephrine is a possible neurotransmitter stimulat- ing pulsatile release of luteinizing hormone-releasing hormone in the rhesus monkey, Endocrinology, 123 (1988) 1808-1816.

[30] ThyagaRajan, S., Meites, J. and Quadri, S.K., Underfeeding-induced suppression of mammary tumors: counteraction by estrogen and haloperidol, Proc. Soc. Exp. Biol. Med., 203 (1993) 236-242.

[31] Weiner, N., A critical assessment of methods for the determination of monoamine synthesis and turnover rates in vivo. In Usdin (Ed.), Neuropharmacology of Monoamines and Their Regulatory enzymes, Raven Press, New York, 1974, pp. 143.

[32] Wilkes, M.M., Lu, K.H., Hopper, B.R. and Yen, S.S.C., Altered Neuroendocrine status of middle-aged rats prior to the onset of senescent anovulation, Neuroendocrinology, 29 (1979) 255-261.

[33] Wise, P.M., Alterations in proestrous LH, FSH and prolactin surges in middle-aged rats, Proc. Soc. Exp. Biol. Med., 169 (1982) 348-354.

[34] Wise, P.M., Norepinephrine and dopamine activity in microdissected brain areas of the middle-aged and young rats on proestrus, Biol. Reprod., 27 (1982) 562-574.

[35] Wise, P.M. Estradiol-induced daily luteinizing hormone and pro- lactin surges in young and middle-aged rats: Correlations with age-related changes in pituitary responsiveness and catecholamine turnover rates in microdissected brain areas, Endocrinology, 115 (1984) 801-809.