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Endocrinology Vol. 138, No. 9 3741-3748
Copyright © 1997 by The Endocrine Society

ARTICLES

Reduced Response of the Hypothalamo-Pituitary-Adrenal Axis to {alpha}1-Agonist Stimulation during Lactation1

R. J. Windle, M. M. Brady, T. Kunanandam, A. P. C. da Costa, B. C. Wilson, M. Harbuz, S. L. Lightman and C. D. Ingram

Neuroendocrine Research Group, Department of Anatomy, School of Medical Sciences (R.J.W., M.M.B., T.K., A.P.C.d.C., C.D.I.), Bristol, United Kingdom BS8 1TD; the Department of Medicine, University of Bristol, Bristol Royal Infirmary (R.J.W., A.P.C.d.C., M.H., S.L.L.), Bristol, United Kingdom BS2 8HW; and the Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph (B.C.W.), Guelph, Ontario, Canada N1G 2W1

Address all correspondence and requests for reprints to: Dr. C. D. Ingram, Neuroendocrine Research Group, Department of Anatomy, School of Medical Sciences, Bristol, United Kingdom BS8 1TD. E-mail: c.ingram{at}bristol.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine whether altered noradrenergic activation of the hypothalamo-pituitary-adrenal (HPA) axis contributes to the attenuated neuroendocrine response to stress observed during lactation, the effect of intracerebroventricular injection of the {alpha}1-agonist methoxamine (100 µg) was compared between virgin and lactating rats. Virgin rats showed significant increases in plasma corticosterone after methoxamine, reaching 317 ± 44 ng/ml at 10 min and remaining significantly elevated for more than 120 min, but lactating rats showed no significant increase in corticosterone levels. Furthermore, methoxamine induced an increase in paraventricular nucleus (PVN) CRF messenger RNA expression in virgin, but not lactating, animals. Both groups of rats exhibited comparable elevations in plasma PRL after methoxamine treatment. Arginine vasopressin messenger RNA expression within the parvocellular PVN was greater in the lactating animals than in the virgin controls, but methoxamine injection was without further effect. Studies performed on ovariectomized virgin rats and ovariectomized rats receiving estradiol or progesterone replacement failed to reproduce the attenuated HPA responses seen after methoxamine treatment, although methoxamine-induced PRL levels were greatly increased by estradiol, probably arising from an effect on hormone synthesis. In vitro electrophysiological recordings of PVN neurons in hypothalamic slices from proestrous virgin and lactating rats showed that 45–52% of neurons in both groups exhibited excitatory responses to 10-4 M methoxamine, but there was a differential response to 10-5 M methoxamine, with PVN neurons from lactating animals failing to show a response. These data show a selective down-regulation of {alpha}1-mediated activation of the HPA axis in lactating animals. This may contribute to the attenuated stress-induced activation of the HPA axis during lactation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN RATS during a period extending from late pregnancy to the end of lactation, the neuroendocrine system is characterized by a marked down-regulation of responses to physical and psychological stresses (1, 2). In particular, there is a decrease in the stress-induced release of ACTH and corticosterone (CORT) by the hypothalamo-pituitary-adrenal (HPA) axis (3, 4, 5, 6, 7) as well as the release of PRL (7, 8, 9, 10) and oxytocin (5, 11, 12). Furthermore, this down-regulation of HPA responses can be seen at the hypothalamic level, in that the induction of CRF messenger RNA (mRNA) transcription within the parvocellular portion of paraventricular nucleus (pPVN), which normally occurs after an acute stress (13, 14), is absent during lactation (5). This alteration in the responsiveness of the HPA axis appears not to be due to an increase in corticosteroid negative feedback (7) arising from the higher morning concentrations of CORT (3, 6, 7, 15, 16), as a similar down-regulation of the ACTH response to stress is seen in adrenalectomized dams (6). Indeed, removal of negative feedback by adrenalectomy has been shown to cause comparable accumulation of CRF mRNA in both virgin and lactating rats (5), suggesting that the down-regulation is specific to the hypothalamic responses to stress and probably involves pathways impinging on the pPVN.

The CRF-containing neurons of the pPVN have been shown to receive noradrenergic inputs (17, 18), which travel via the ventral noradrenergic bundle (VNAB) from catecholaminergic cell groups of the brainstem (19). Although disruption of the VNAB has variable effects on the stress-induced activation of the HPA axis (20, 21, 22), these noradrenergic inputs appear to participate in the activation of the pPVN, as electrical stimulation of the VNAB induces CRF secretion into the portal system that can be blocked by {alpha}1-antagonists (23). Furthermore, icv injection of noradrenaline or {alpha}1-agonists will cause dose-related increases in the secretion of CRF (23) and ACTH (20, 24, 25). This effect appears to be mediated via {alpha}1-adrenoceptors within the PVN itself (26), as microinfusions close to the PVN are capable of stimulating HPA activity (20, 27, 28) and will induce CRF mRNA expression (28).

The following studies examined the possibility that the attenuated response of the HPA axis during lactation arises from the modulation of {alpha}1 activation of CRF neurons of the pPVN. This hypothesis is suggested by the observations that the circulating levels of gonadal steroids show marked changes over the peripartum period (29, 30) when the hyporesponsive state begins, and these steroids have important effects on the density of hypothalamic {alpha}1B-binding sites (31, 32, 33, 34). Furthermore, we have previously shown that the release of oxytocin, another hormone that is not released by stress during lactation (5, 11, 12), may be evoked by central administration of the long acting {alpha}1-agonist methoxamine to virgin, but not lactating, rats (25). In the current studies, intracerebroventricular (icv) injection of methoxamine was performed in virgin and lactating rats, and HPA function was measured by the changes in plasma CORT and expression of CRF mRNA within the pPVN. Comparisons were made with steroid-treated virgin rats to determine whether gonadal steroids could modulate methoxamine-induced responses, and in vitro electrophysiological recordings of PVN neurons were used to investigate whether lactation was associated with a change in postsynaptic {alpha}1-mediated activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Studies were carried out on primiparous lactating rats (253–284 g) between days 7–14 of lactation; these animals were compared with randomly cycling virgin females (212–261 g). However, in the case of the electrophysiological recordings, all virgin rats were proestrus, as determined by taking daily vaginal smears. Animals were individually housed with their litters and maintained under standard environmental conditions (temperature, 21 C; photoperiod, 14 h of light and 10 h of darkness; lights on at 0500 h), with food and water available ad libitum. All procedures were performed in accordance with United Kingdom animal welfare legislation.

Anesthesia and surgical procedures
Animals were anesthetized using im Hypnorm (0.32 mg/kg fentanyl citrate and 10 mg/kg fluanisone; Janssen Pharmaceuticals, Oxford, UK) and ip Diazepam (0.25 mg/kg; Phoenix Pharmaceuticals, Gloucester, UK). The right jugular vein was implanted with a SILASTIC-tipped (Dow Corning, Midland, MI) catheter that passed sc and exteriorized on top of the head. A small area of the parietal bone was exposed, and an icv cannula was stereotaxically positioned in the lateral ventricle. The cannula was composed of a metal guide and approximately 20 cm of fine bore PTFE tubing (id, 0.28 mm; Goodfellow, Cambridge, UK). The dead volume of each cannula was accurately measured (5.3–6.4 µl) before filling with 0.15 M sterile NaCl preceding permanent implantation into the lateral ventricle. Both the icv cannula and iv catheter were then passed through a protective steel spring that was attached to the parietal bones using two stainless steel screws, and the icv guide tube and head of the spring were held in place with a covering of dental acrylic. The upper end of the spring was then attached to a mechanical swivel that allowed the animals freedom of movement. The iv catheters were flushed daily with sterile saline containing 10 U/ml heparin. Four days after surgery at 1500 h, the free end of the icv cannula was attached to a 10-µl microsyringe (Hamilton, Bonaduz, Australia) attached to the mechanical swivel and filled with either 0.15 M NaCl or 0.15 M NaCl containing 25 mg/ml methoxamine hydrochloride (Sigma Chemical Co., St. Louis, MO). An injection equivalent to the dead volume minus 1 µl was made, leaving the remaining 1 µl NaCl as a buffer between the test solution in the cannula and the lateral ventricle. Between 0800–0900 h on the following morning, blood samples (0.1 ml) were collected at -30, 0, 10, 30, and 120 min relative to a 5-µl icv injection containing either 0 or 100 µg methoxamine. This single dose was selected on the basis that it had previously been shown to evoke a differential plasma oxytocin response in virgin and lactating rats (25). The animals were then given an overdose of pentobarbitone, and the brains were collected onto dry ice. An additional group of virgin animals treated with methoxamine had blood samples collected at -30, 0, 10, 30, 120, 180, 240, and 360 min to provide an extended time course of the endocrine response.

Effect of gonadal steroids
Studies of gonadal steroid effects on the response to methoxamine were performed on intact virgin rats or ovariectomized (OVX) rats receiving steroid replacement. Ovariectomies were performed by bilateral laparotomy under Hypnorm/diazepam anesthesia 2 weeks before the experiment. At the time of ovariectomy, animals received 1) one 25-mm SILASTIC implant containing oil (control group); 2) one 25-mm SILASTIC implant containing 150 µg/ml 17ß-estradiol (E2 group); or 3) two 40-mm implants containing 50 mg/ml progesterone (P group). Implants were constructed from SILASTIC medical grade tubing (id, 0.062 in.; od, 0.1252 in.) filled with steroids dissolved in olive oil and sealed with rubber compound. Implants were replaced at the time the cannulations were performed 5 days before sampling. Cannulations, injections, and sampling were carried out as described above, except that all animals received methoxamine.

RIAs
All blood samples were collected into heparinized tubes on ice and centrifuged immediately, and the plasma was stored at -20 C until assay. Total CORT was measured after dilution of the samples in sodium citrate buffer (pH 3.0) using antiserum kindly donated by Prof. G. B. Makara (Institute of Experimental Medicine, Budapest, Hungary). Plasma PRL was determined in unextracted plasma using rabbit antirat PRL serum obtained from NIDDK, and plasma levels of PRL are expressed in terms of NIAMMD rat PRL RP-2.

In situ hybridization histochemistry
Hybridization procedures are essentially the same as those described previously (13, 14). Coronal sections (11.5 µm) were cut from the anterior commissure to the posterior margin of the PVN. The oligonucleotide probe to arginine vasopressin (AVP) mRNA was a 48-mer probe complementary to the region coding for the last 16 amino acids of the glycopeptide region of preprovasopressin. The probe for CRF mRNA was a 48-mer probe complementary to bases 496–543 of the rat CRF gene coding for the C-terminal amino acids 166–181 of the rat prepro-CRF. Both probes were 3'-end labeled using 35S-labeled {alpha}-thio-deoxy-ATP (New England Nuclear, Boston, MA) and deoxynucleotidyl transferase (Pharmacia, Piscataway, NJ), diluted to 1000–3000 cpm/µl, and applied to the sections in hybridization buffer. Hybridization took place overnight at 37 C, followed by stringent washes. The dried slides were then apposed to autoradiographic film (Hyperfilm HP, Amersham, Arlington Heights, IL) for exposure times of 48 h for AVP mRNA and 14 days for CRF mRNA. Hybridization signal was quantified by computer-assisted densitometric analysis (Image 1–22, developed by W. Rasband, NIH, Bethesda, MD). The area of the PVN above the background, and the optical density of this area were multiplied, and data were expressed as a percentage of the respective virgin control group.

Electrophysiological recordings
This study was performed on lactating and proestrous virgin rats using extracellular electrophysiological techniques described previously (35). Recordings were made from 400-µm hypothalamic slices maintained at 37 C in oxygenated artificial cerebrospinal fluid (composition: 124 mM NaCl, 2.4 mM MgSO4, 1.25 mM KH2PO4, 3.25 mM KCl, 1.0 mM CaCl2, 26 mM NaHCO3, and 10 mM D-glucose; equilibrated with 95% O2-5% CO2) at the liquid-gas interface of an incubation chamber. Extracellular recording was carried out using 0.5 M NaCl-filled glass microelectrodes coupled to an AC differential amplifier (x1000), and spike events were stored on computer disc for later analysis. Each neuron was recorded for a period of at least 5 min to determine the basal activity before changing the medium to one containing either 10-5 or 10-4 M methoxamine for a period of 2 min. Neurons were classified as responsive on the basis of changes in firing rate to the higher concentration. Note that the records have not been corrected for the 2-min lag time of the perifusion system. All neurons were recorded from the region of the PVN, and this was confirmed by iontophoretic deposition of Pontamine sky blue after recording. Slices were then fixed overnight in 4% paraformaldehyde and transferred to 30% sucrose for 24 h, before frozen sections (45 µm) were cut and stained with neutral red for confirmation of the recording site.

Data analysis
Values are presented as the mean ± SE. Where appropriate, hormone levels were compared before and after icv injection using paired Student’s t test. Hormone levels and expression of CRF and AVP transcripts were compared between groups and studies using either one- or two-way ANOVA with repeat measures together with post-hoc Dunnett’s multiple comparison tests. For the electrophysiological recordings, firing rates of neurons were integrated into 10-sec bins, and responsive neurons were averaged within groups. A neuron was classified as excited if the maximum firing rate sustained for 60 sec after the application of methoxamine exceeded the 95% confidence interval of the mean basal firing rate calculated from five consecutive 60-sec periods before drug application. The proportion of responsive neurons was compared by {chi}2 test, and the basal firing rate and change induced by methoxamine were compared by independent t tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Methoxamine-induced change in HPA activity and PRL secretion
CORT levels measured immediately before injection were comparable in all treatment groups and did not vary significantly from the values measured at -30 min (Fig. 1AGo). Intracerebroventricular injection of saline had no significant effect on CORT levels in either virgin or lactating animals, whereas icv injection of methoxamine caused a differential response in virgin and lactating rats (P < 0.026; Fig. 1AGo). In the virgin group, methoxamine treatment caused a significant elevation of plasma CORT compared with preinjection values. This peaked at 10 min (P < 0.002; Fig 1AGo), and the levels were still maximally elevated at 120 min. CORT levels were significantly higher in the methoxamine-treated than in the saline-treated virgin animals at all time points after injection. Prolonging the sampling time after methoxamine injection showed that plasma CORT levels had returned to basal concentrations by 240 min (Fig. 1BGo). Although CORT levels also tended to rise in the lactating group, this was not significant compared with the preinjection value at any of the time points studied. CORT levels were significantly higher in the methoxamine-treated lactating rats compared with the saline-treated animals 30 min after injection (194 ± 29 and 79 ± 21 ng/ml, respectively; P < 0.05), but by 120 min, CORT levels were comparable in the two groups (Fig. 1AGo).



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  		Figure 1. Plasma concentrations of CORT (A and B) and PRL (C) in virgin (squares) or lactating (circles) Sprague-Dawley rats before and after icv administration of saline (open symbols) or 100 µg methoxamine (filled symbols) at time zero, as indicated by the arrow. Values are the mean ± SEM. *, P < 0.05 for virgin rats treated with methoxamine; #, P < 0.05 for lactating rats treated with methoxamine (vs. values at 0 min). +, P < 0.05 (virgin vs. lactating rats treated with methoxamine, by ANOVA). n = 9 for virgin, saline; n = 8 for virgin, methoxamine; n = 8 for lactating, saline; n = 8 for lactating, methoxamine.

 
Before icv injection, plasma PRL levels in the virgin animals were consistently low (Fig. 1CGo), whereas those in lactating animals were significantly higher at -30 min, but showed a very marked decline between those at -30 and 0 min, most likely as a result of the interruption to suckling caused by the sampling procedure. These levels continued to fall after icv injection of saline and did not differ from those in the virgin group. By contrast, icv injection of methoxamine caused a transient elevation of plasma PRL concentrations in both groups of animals (Fig. 1CGo). Although levels were higher in the lactating animals, there was no significant group difference in the methoxamine-induced increase (by ANOVA).

Virgin rats had significantly higher levels of CRF mRNA expression than lactating animals (P < 0.001; Fig. 2AGo). Furthermore, methoxamine injection significantly increased CRF mRNA expression in the virgin animals (P < 0.05), but had no effect on CRF mRNA expression in the lactating animals. In contrast to CRF mRNA expression, parvocellular AVP mRNA expression was significantly lower in the virgin animals than that in the lactating rats (P < 0.005), and methoxamine treatment had no significant effect in either virgin or lactating rats (Fig. 2BGo).



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  		Figure 2. Expression of CRF mRNA (A) and AVP mRNA (B) in the PVN of virgin and lactating rats after icv injection of saline (open bars) or 100 µg methoxamine (closed bars). Bars show the mean ± SE for mRNA determinations in arbitrary optical density units expressed as a percentage of the mean value in the virgin saline-infused group. Multiple ANOVA revealed that CRF mRNA expression was significantly lower (P < 0.001) and AVP mRNA expression was significantly higher in the lactating rat (P < 0.005) compared with those in the virgin animals. Methoxamine injection caused a significant increase in CRF mRNA expression in the virgin group only (P < 0.05). n = 16 for virgin, saline; n = 9 for virgin, methoxamine; n = 10 for lactating, saline; n = 10 for lactating, methoxamine.

 
Effect of gonadal steroids on methoxamine-induced responses
Basal CORT levels did not differ between intact and steroid-treated groups, and icv injection of methoxamine induced an increase in all OVX groups (Fig. 3Go). However, the responses of the OVX groups were initially smaller than that of the intact virgin group (Fig. 3AGo; P < 0.05 for all groups compared with virgin animals at 30 min). However, by 120 min, CORT levels were comparably elevated in all four groups (Fig. 3AGo). At this time, PVN CRF mRNA levels were similar among the four groups (Fig. 4Go). Basal PRL levels did not differ between groups, but steroid treatment had a marked effect on the response to methoxamine; ovariectomy alone reduced the response compared with that in intact virgin mice, although E2 treatment significantly potentiated the response to methoxamine (Fig. 3BGo). In contrast, progesterone appeared to further diminish the response, such that there was no significant effect of methoxamine in this group. As in the first study, the response to methoxamine was transient, and levels returned to basal at 30 min.



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  		Figure 3. Plasma concentrations of CORT (A) and PRL (B) after icv administration of 100 µg methoxamine (arrow) to virgin Sprague-Dawley rats (squares) or to OVX rats bearing implants containing oil (triangles), estradiol (diamonds), or progesterone (circles). Values are the mean ± SEM. *, P < 0.05, all groups vs. values at time zero; +, P < 0.05, all steroid-treated groups vs. virgin rats; #, P < 0.05, virgin, OVX, and estradiol-treated animals vs. values at time zero and also vs. each other response at this time point (by ANOVA). Note that the plasma PRL concentration was only determined at 0, 10, and 30 min relative to the injection. n = 9–11/group for CORT; n = 5–6/group for PRL.

 


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  		Figure 4. Expression of CRF mRNA in the PVN of methoxamine-treated virgin Sprague-Dawley rats (open bars) or OVX rats bearing implants containing oil (solid bars), estradiol (stippled bars), or progesterone (hatched bars). Bars show the mean ± SE for the number of replicates shown in parentheses and represent mRNA determinations in arbitrary optical density units expressed as a percentage of the mean value in the virgin group.

 
Electrophysiological recordings of methoxamine-induced excitation
Application of 10-4 M methoxamine induced a rapid and reversible increase in the firing rate in 13 of 29 (45%) neurons recorded in hypothalamic slices from proestrus virgin rats (Fig. 5Go, A and B). This effect was dose dependent, with a significantly greater and more long lasting response of the agonist to 10-4 than to 10-5 M (Fig. 6Go, A and B), indicating that these concentrations were on the slope of the dose-response curve. The higher dose of methoxamine (10-4 M) induced excitation of a similar proportion of neurons in slices from lactating rats (13 of 25, 52%; {chi}2 = 0.298; P = NS; Fig. 6Go and Table 1Go), with the effect again being dose dependent (Fig. 5Go, C and D). However, significantly fewer neurons attained the criterion for excitation in response to 10-5 M methoxamine (4 of 9; {chi}2 = 3.905; P < 0.05; vs. 9 of 10 for virgin rats), and the mean response showed no significant effect at this dose (Fig. 6CGo). Although more neurons from lactating animals were tested first with the higher dose of methoxamine (10 of 11 vs. 4 of 11 for virgin animals), the inability of PVN neurons to respond to 10-5 M methoxamine appears not to be due to tachyphylaxis because 1) neurons from virgin animals showed responses to both doses regardless of the sequence of presentation (Fig. 5Go, A and B); 2) there was no evidence of tachyphylaxis when tested repeatedly with the same dose (Fig. 5Go, A and B); and 3) neurons from lactating animals failed to respond to 10-5 M methoxamine even when the interval between doses exceeded 60 min. Although the mean firing rate in response to 10-4 M methoxamine was higher in the lactating group (cf. Fig. 6Go, B and D), this was principally due to the higher basal firing rate of the responsive neurons in this group, and there was no difference in the methoxamine-induced change of firing (Table 1Go).



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  		Figure 5. Ratemeter records showing examples of electrophysiological responses of hypothalamic PVN neurons in brain slices obtained from proestrous virgin (A and B) and lactating Wistar (C and D) rats. The open squares show the application of 2-min pulses of either 10-4 or 10-5 M methoxamine. Spikes have been integrated into 10-sec bins.

 


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  		Figure 6. Averaged excitatory responses of PVN neurons from either proestrous virgin (A and B) or lactating (C and D) Wistar rats to 2-min applications of 10-5 M (A and C) or 10-4 M (B and D) methoxamine. Symbols show the mean, and the lines show the SE of the firing rates in 10-sec bins aligned to the period of methoxamine administration (horizontal bars). n = 9–11 neurons/group.

 

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  		Table 1. Basal and stimulated firing rates of responsive PVN neurones recorded from virgin and lactating Wistar rats following the application of the {alpha}1 agonist methoxamine

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies show that the {alpha}1-agonist, methoxamine, has a differential effect on the activation of the HPA axis between virgin and lactating rats. Methoxamine-induced CORT secretion and CRF mRNA accumulation were significantly lower in the lactating animals, and this may have arisen from a shift in the sensitivity of PVN neurons to {alpha}1-receptor activation. This supports the hypothesis that the attenuation of stress-induced activation of the axis that occurs during lactation arises from down-regulation of the afferent pathway.

The observed {alpha}1-mediated activation of the HPA axis is in agreement with previous reports of effects of icv injection of methoxamine on the release of ACTH and CORT (19, 24, 25, 36) and on the depletion of CRF immunoreactivity in the median eminence (37) and of neurosecretory vesicles from terminals of the CRF/AVP coexpressing neurons (36). The methoxamine-induced increase in CRF mRNA is similar to that reported for direct microinjection into the PVN of noradrenaline (28), and in parallel to the differential hormonal effect, methoxamine produced a significantly smaller stimulation of CRF mRNA expression in the lactating animal.

Although methoxamine can pass the blood-brain barrier and could conceivably exert a peripheral action on the pituitary (38, 39), we have previously demonstrated that iv administration of methoxamine at the dose used here was without effect on HPA activity (25). Furthermore, unlike methoxamine, peripheral administration of noradrenaline has no effect on lactotroph or corticotroph activity due to its inability to access central adrenoceptors (39), and although direct microinjection of 100 µg methoxamine into the PVN leads to a long lasting increase in plasma CORT, no similar effect is induced by injections into the caudate nucleus (27). It is likely, therefore, that the neuroendocrine effects were mediated via direct activation of the {alpha}1-binding sites that are preferentially located in the medial PVN (26). This was confirmed by electrophysiological recordings from this region.

It is well known that noradrenergic afferents activate tuberoinfundibular PVN neurons in vivo (40), and that {alpha}-adrenergic agonists will increase the activity of these neurons both in vivo (41) and in vitro (42, 43). The reduced ability of PVN neurons from lactating rats to respond to a threshold dose of methoxamine in vitro suggests that one possible explanation of the attenuated HPA response is that the hypophysiotropic (CRF and AVP) neurons have an elevated threshold for postsynaptic {alpha}1 excitation. This is consistent with the shorter duration of the CORT response to icv methoxamine. However, aside from this apparent shift in the dose-response relation, the excitatory responses in both groups of animals were similar to those of previous in vitro electrophysiological recordings of periventricular PVN neurons in the mouse (42) and rat (43), which showed that a high proportion of neurons were excited by {alpha}1 agonists. Furthermore, the dose range of methoxamine required to obtain an excitatory response in the virgin rats was similar to that reported for excitation of neurons in the supraoptic (44) and other hypothalamic nuclei (45, 46), although a lower dose (10-6 M) induced CRF release from rat hypothalami in vitro (47). Interestingly, similar to the present data, Wakerley and Negoro (48) have shown that responses of magnocellular neurons of the supraoptic nucleus to the {alpha}1-agonist phenylephrine (10-5 M) were also smaller in the lactating rat. This suggests that down-regulation of {alpha}-adrenoceptor responses may be a phenomenon that also affects neurohypophyseal neurosecretory neurons and may explain the reduced stress-induced oxytocin release during lactation (5, 11, 12).

Although the neurons recorded in the present study were not identified as parvocellular tuberoinfundibular neurons, the recordings were concentrated on the medial part of the PVN, and no neuron showed clear phasic activity characteristic of magnocellular AVP neurons either before or after methoxamine (44) or demonstrated the high frequency bursts reported for phenylephrine effects in lactating rat magnocellular SON (48).

A possible cause of the hyporesponsive state is the dynamic fluctuations in gonadal steroid levels that occur before parturition (29, 30). Lactation is associated with estrogen levels similar to those found in OVX animals (49). Estrogens elevate the density of {alpha}1B-receptors in certain hypothalamic nuclei (32, 33) and potentiate {alpha}1-induced cAMP production (31). Therefore, it is possible that a lowered level of estrogen has the inverse effects, decreasing the responses seen during {alpha}1 stimulation. As the stimulatory effect of noradrenaline on CRF neurons appears to be through {alpha}1-receptors (20, 24, 28), the magnitude of responses may depend upon the levels of circulating E2. At the doses employed here, the steroid treatments had no effect on methoxamine-induced CRF mRNA expression and no differential effect on plasma CORT levels. This contrasts with the effect of steroids on basal HPA activity (50, 51) and also suggests that gonadal steroid interaction with catecholaminergic transmission may not be the primary cause of the down-regulated response seen during lactation. Interestingly, the blunted CORT response displayed by all OVX animals suggests that the ovary does contribute to maintaining normal HPA responses.

Gene expression for AVP, the other principal regulator of the HPA axis, was also measured in these studies. Consistent with previous observations (15, 52, 53), AVP mRNA expression was higher in the lactating animal than in the virgin, and this may relate to activation of magnocellular AVP neurons due to the fluid demand imposed by milk secretion. Although both parvocellular and magnocellular AVP neurons are known to express {alpha}1-receptors (26) through which methoxamine will cause neuronal excitation (41, 42, 43, 44), methoxamine was without effect on the expression of AVP mRNA in either group of animals. The abundance of AVP mRNA in magnocellular neurons and the presence of ectopic magnocellular neurons within the parvocellular area of the PVN make small changes in parvocellular AVP mRNA difficult to detect. Further studies using intronic probes to measure heteronuclear mRNA may be helpful to determine whether there is a dissociation between activation and gene transcription, or whether any change in AVP gene expression was masked by the high basal magnocellular AVP mRNA content.

PRL release was also induced by methoxamine, although there was no difference between virgin and lactating animals. This suggests that the down-regulation of {alpha}1 responsiveness may be selective for the HPA axis, and that the differential stress-induced PRL response seen during lactation (7, 8, 9, 10) does not arise from altered noradrenergic transmission. The differences in the magnitude of responses after steroid treatment probably reflect effects on pituitary PRL synthesis (54). However, despite these differences and the long acting nature of methoxamine, all responses were transient and did not show the prolonged response nature of the CORT release, suggesting a different mechanism of action. The transient nature of responses is consistent with the rapid half-life of PRL in lactating rats (55) and suggests a rapid switch-off of secretion. No previous studies have examined the effects of icv administration of selective {alpha}1-agonists on PRL, but the currently observed transient responses contrast with the more sustained responses to injection of noradrenaline into the anterior hypothalamus (56) or iv infusions of methoxamine (39). This suggests that the transient nature of the response may have arisen from rapid clearance from the ventricular system rather than from densensitization.

In summary, there is a reduction in CORT and CRF mRNA responses to {alpha}1 activation in lactating rats that is similar to the down-regulation of stress-induced HPA responses that occurs during lactation (3, 4, 5, 6, 7). Given the role of catecholaminergic pathways in the transduction of the stress response (19) and activation of neurosecretory PVN neurons (40, 41, 42, 43, 44), it is possible that this is one of the factors contributing to the attenuated neuroendocrine response to stress during lactation.


   Acknowledgments
 
The authors express their gratitude to Dr. N. Shanks for helpful discussions of this work, to Mrs. Susan Wood for her technical assistance with the RIAs, and to Dr. M. Harbuz for advice concerning the in situ hybridization.


   Footnotes
 
1 This work was supported by the Wellcome Trust and the Canadian Medical Research Council (to B.C.W.). Back

Received November 4, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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