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Am J Physiol Regul Integr Comp Physiol 280: R1169-R1176, 2001;
0363-6119/01 $5.00
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Vol. 280, Issue 4, R1169-R1176, April 2001

Lactation decreases angiotensinogen mRNA expression in the midcaudal arcuate nucleus of the rat brain

Robert C. Speth, M. Susan Smith, and Kevin L. Grove

Division of Neuroscience, Oregon Regional Primate Research Center, Oregon Health Sciences University, Beaverton, Oregon 97006


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In lactating rats, ANG II receptor binding in the arcuate nucleus (ARH) and median eminence is decreased. To further evaluate brain angiotensinergic activity during lactation, we assessed angiotensinogen (AON) mRNA by in situ hybridization in forebrains of day 10 or 11 postpartum lactating and diestrous rats. AON mRNA was abundantly expressed in the ARH, preoptic, suprachiasmatic, supraoptic, paraventricular, and dorsomedial hypothalamic nuclei, and other regions, similar to that reported in male rat brains. AON mRNA levels were decreased 27% in the midcaudal ARH of lactating rats but did not differ between lactating or diestrous rats in any of the other brain areas examined. Immunofluorescence for AON and glial fibrillary acidic protein or tyrosine hydroxylase confirmed that the AON immunoreactivity in the ARH was limited to astrocytes. Confocal microscopy revealed close appositions of AON-positive astrocytes to dopaminergic neurons in the ARH. The decrease in AON mRNA in the midcaudal ARH during lactation coupled with decreased ARH ANG II receptor binding suggests that lactating rats are less subject to ANG II-mediated inhibition of prolactin secretion.

tuberoinfundibular dopamine neurons; astrocytes; prolactin secretion; hypothalamus; in situ hybridization histochemistry


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ONE OF THE MANY FUNCTIONS of the brain renin-angiotensin system (RAS) is the regulation of prolactin release (41). The AT1 subtype of ANG II receptors is present on dopaminergic neurons in the arcuate nucleus (ARH) of the rat and stimulates dopamine release into the pituitary portal vasculature in response to ANG II (20). Dopamine directly inhibits prolactin secretion from the anterior pituitary (2). Previous studies from this laboratory have demonstrated that ANG II receptor binding in the ARH and median eminence of lactating rats is decreased compared with nonlactating diestrous rats (40). This suggests that the ability of brain ANG II to inhibit prolactin release is diminished in lactating rats.

Another way in which the inhibitory effects of the brain RAS on prolactin secretion in lactating rats could be diminished is through decreased ANG II production, particularly in the ARH. Angiotensinogen (AON) is a critical component of the RAS. It is the only known precursor of ANG II (see review Ref. 29). Transgenic rats expressing antisense to AON mRNA that have more than a 90% reduction in brain AON exhibit reduced blood pressure and diabetes insipidus, confirming the pivotal role of AON in the function of the brain RAS (37).

The ARH has been reported to contain the highest amount of AON immunoreactivity (ir) in the rat brain (13). AON mRNA displays a distinct topographical distribution in the male rat brain and is expressed in high abundance in the ARH (3). The high abundance of this ANG II precursor in the ARH is consistent with the brain RAS having an important role in the stimulation of dopamine release from the ARH, and consequently having major effects on prolactin secretion.

The lactating rat has extremely low estrogen levels, concurrent with elevated glucocorticoids (46) and progesterone (39). This is of potential importance for the regulation of brain AON expression. AON and AON mRNA expression in the liver are enhanced by glucocorticoids, estrogen, and a number of other hormones (27, 29). Astrocytes, in which AON mRNA is primarily expressed in the brain (3, 37, 42), have been shown to contain receptors for glucocorticoids (9), estrogen (22, 34), and progesterone (21). In the brain, glucocorticoids also increase AON mRNA (35, 36). However, this effect appears to be region specific, as AON mRNA in the ARH and the subfornical organ (SFO) is not increased by glucocorticoids (4). The effect of estrogen on brain AON mRNA is also region specific (16). There is no information on the effect of progesterone on brain AON mRNA; however, estrogen-plus-progesterone treatment of ovariectomized rats causes a large increase in ANG II in the brain (32). Thus it is difficult to predict how brain AON mRNA, specifically that in the ARH, would be altered during lactation.

To test the hypothesis that formation of ANG II in the ARH may be diminished in lactating rats, this study examined the expression of AON mRNA in the ARH of lactating and cycling female rats at diestrus by in situ hybridization. In addition, AON mRNA expression in a number of other forebrain regions was examined to determine whether there are other region-specific differences in AON mRNA expression between lactating and diestrous rat brains and to compare the distribution of AON mRNA in the female rat brain with that previously reported in male rat brains (3, 5, 35, 36).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal procedures. Five pregnant female rats and five intact cycling female rats (B & K Universal, Kent, WA) were housed individually and maintained on rat chow and water ad libitum. Lights were on from 0700 to 1900. Animals were checked daily for the presence of pups; the day of delivery was considered postnatal day 0 (P0). The litters were culled to eight pups on P2 to allow for an equivalent suckling stimulus for all lactating rats. The lactating rats were killed by decapitation on P9 or P10. Cycling rats were monitored daily for stages of the estrous cycle by vaginal smear. Rats that had completed at least two complete cycles were killed by decapitation on the day of diestrus. The brains were removed, frozen, and stored at -80°C.

Two female rats (250-300 g) ovariectomized 2 wk before being killed were used for the immunohistochemical analyses. They were perfused intracardially with ice-cold 0.9% saline after deep anesthetization with pentobarbital sodium. The brains were fixed by intracardial perfusion with ice-cold 4% paraformaldehyde, postfixed overnight in 4% paraformaldehyde at 4°C, saturated in 20% sucrose, frozen, and stored at -80°C.

All animal procedures were approved by the Oregon Regional Primate Research Center Institutional Animal Care and Use Committee.

In situ hybridization histochemistry. The brains from lactating and diestrous rats were sectioned coronally at 20 µm using a cryostat starting at 1 mm rostral to bregma to ~6 mm caudal to bregma. The tissue sections were collected in repeating sets of three and thaw-mounted onto slides (Superfrost/Plus, Fisher Scientific, Pittsburgh, PA). The sections were dried and stored at -80°C until further processing.

In situ hybridization was performed as previously described (26, 38). The AON cRNA probe was transcribed from a 329-bp cDNA subcloned into a pCRII plasmid (kindly provided by Dr. Terry Elton) using a T7 promoter with 25% of the UTP being 35S labeled (NEG-039H, DuPont/New England Nuclear, Boston, MA). The specific activity of the riboprobes averaged 5.42 × 108 dpm/µg and varied by less than 1%. A saturating concentration of the probe (0.3 µg · ml-1 · Kb-1, ~5 million dpm per slide) was used to label the mRNA in the brain sections. Briefly, brain sections were fixed in 4% paraformaldehyde and treated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). The sections were rinsed in 2× sodium chloride-sodium citrate (SSC), dehydrated through a graded series of alcohols, delipidated in chloroform, and rehydrated through a second series of alcohols, after which they were air-dried. All of these steps were carried out at 22-24°C.

The sections were hybridized overnight with the AON cRNA probe in a humidified chamber at 55°C. After the incubation, the slides were washed in 4× SSC, RNase A treated at 37°C, and further rinsed in 0.1× SSC at 60°C. Slides were then dehydrated through graded series of alcohols and dried. Slides were exposed to film (BioMax, MR-1, Kodak, Rochester, NY) at 4°C for 24-48 h. A corresponding set of sections from a rat brain was exposed to 35S-labeled AON sense probe.

Immunohistochemistry. The immunohistochemical protocols are similar to those previously described (25). Brains were sectioned coronally from ~2 mm caudal to bregma to 4.5 mm caudal to bregma at a thickness of 25 µm with a freezing microtome in repeating sets of 12, placed in cryoprotectant medium (30% sucrose, 30% polyethylene glycol, buffered with NaPO4, pH 7.2), and stored at -20°C until used for immunohistochemical labeling studies.

On removal from the cryoprotectant solution, brain sections were extensively rinsed in potassium phosphate-buffered saline (KPBS, 50 mM potassium phosphate, 100 mM NaCl at pH 7.4) at 22-24°C. The sections were then placed in a KPBS-blocking solution containing 2% nonfat dry milk and 0.4% Triton X-100 detergent for a minimum of 60 min at 22-24°C. The sections were subsequently incubated with a cocktail of polyclonal rabbit antibody to rat AON (1:50,000, kindly provided by Drs. Christian Deschepper and Pierre Corvol) and a monoclonal antibody to rat tyrosine hydroxylase (TH, 1:150,000, catalog no. MAB318, Chemicon, Temecula, CA) or a polyclonal antibody to glial fibrillary acidic protein (goat anti-GFAP C-19, 1:1,000, catalog no. SC-6170, Lot no. L159, Santa Cruz Biotechnology, Santa Cruz, CA). The sections were incubated with the primary antibodies for 1 h at 22-24°C and for 36-48 h at 4°C. After incubation with the primary antibodies, the sections were rinsed in KPBS as before.

Primary antibody binding was tagged by incubation with Alexa Fluor fluorophor-labeled secondary antibodies (Molecular Probes, Eugene, OR) or FITC- and tetramethyl rhodamine isothiocyanate (TRITC)-labeled secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) at a dilution of 1:200 in KPBS, 0.4% Triton X-100 for 1 h at 22-24°C in the dark. AON was visualized with goat anti-rabbit Alexa Fluor 488 (AON × TH) or donkey anti-rabbit FITC (AON × GFAP), TH was visualized with goat anti-mouse Alexa Fluor 546, and GFAP was visualized with donkey anti-goat TRITC.

The sections were again rinsed extensively in KPBS. The sections were then mounted onto subbed slides and placed under a coverslip with buffered glycerin, aqueous mounting medium.

Image analysis. The films were analyzed for the amount of 35S-labeled riboprobe binding by densitometry using a computer program (MCID, Imaging Research, St. Catherine's, ON, Canada). The observer (R. C. Speth) taking the measurements was blinded to the treatment groups to which the brains belonged to assure objective quantification of the signal.

A sampling area was established empirically for each brain region surveyed. Once established, the sampling area was kept constant for all brains. The sampling area generally bordered on or exceeded the boundaries of the region of interest. To limit the area within the sampling area to the confines of the brain region of interest, we empirically established a threshold optometric density (OD) reading for each region of interest. OD values below this threshold value were not recorded. This allowed for a more accurate measurement of the irregular shapes that describe the brain regions sampled. Once established, this threshold was kept constant for the measurement of all brains.

For each section for each brain region surveyed, an OD value, as well as the area sampled, was obtained. At least four sections were measured for each brain region. The number of sections assayed for each brain region was similar for each brain. A total of 18 brain regions was assayed based on an initial survey of the distribution of AON mRNA and the potential relevance of the brain region to lactation-induced alterations. Brain sections from each rat were anatomically matched based on several landmarks, e.g., the crossing of the anterior commissure at 0.3 mm caudal to bregma, the joining of the lateral and third ventricles and the beginning of the suprachiasmatic nucleus at 0.9 mm caudal to bregma, the presence of the compact zone of the dorsomedial hypothalamus at 3.3 mm caudal to bregma, and the crossing of the posterior commissure at 4.5 mm caudal to bregma. There was no difference in the areas measured for each nucleus between the two groups. Therefore, only values for the density of 35S-labeled AON mRNA probe are reported. The data were analyzed using an unpaired Student's t-test and linear regression analysis using PRISM (Graphpad Software, San Diego, CA). Values are expressed as means ± SE.

For histological analysis of the distribution of AON mRNA, slides were dipped in Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY) diluted 1:1 in 600 mM ammonium acetate and then placed in light-tight boxes containing desiccant and stored at 4°C for 10-21 days. The slides were then developed and stained with cresyl violet. The distribution of silver grains was analyzed by dark-field microscopy with an indirect light source illuminator (Meridian Instruments, Seattle, WA).

The immunohistochemical staining of the sections was analyzed by confocal microscopy (Leica, TCS SP confocal system) as described previously (25). Sections were scanned through a Leica Corp IRB/E inverted microscope using an argon laser emitting light at 488 nm for visualization of the Alexa Fluor 488 or FITC fluorophore and a krypton laser emitting light at 568 nm for visualization of the Alexa Fluor 546 or TRITC fluorophore. The confocal microscopic images were obtained using ×25 numerical aperture (NA) 0.75 and ×40/NA 1.25 objectives. For each experiment, fluorophore signals were checked individually for bleed-through to the apposing detector. All bleed-through was eliminated by adjusting laser intensity and detector window width. A series of optical sections with a resolution of 0.5 µm was taken at 1-µm intervals along the z-axis of the brain section for each fluorophore and saved as a series of 512 × 512-pixel images. These images were processed with the MetaMorph Imaging System (Universal Imaging, West Chester, PA) for presentation as a stack of optical images. The brightness and contrast of the images were adjusted using Photoshop software (Adobe Systems, San Jose, CA) to optimize their representation.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Regional distribution of AON mRNA in the forebrain of the lactating rat. The OD of film exposure corresponding to the 18 forebrain regions surveyed for AON mRNA with the 35S-labeled AON antisense probe is shown in Table 1. Incubation of brain sections with 35S-labeled AON sense probe yielded a negligible signal with no specific pattern of labeling of the brain sections.

                              
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  		Table 1.   Distribution of angiotensinogen mRNA in the forebrains of lactating and diestrous rat brains

Overall there was a high density of AON mRNA in the lactating and diestrous rat brains throughout the hypothalamus including the median preoptic (Fig. 1B), suprachiasmatic, supraoptic (Fig. 1C), paraventricular (Fig. 1D), dorsomedial, and ARH nuclei (Fig. 1, E and F). A moderate expression of AON mRNA was present in the lateral hypothalamic nucleus (Fig. 1F). There was a high expression of AON mRNA in a band of cells surrounding the perimeter of the mammillary complex (not quantitated). There was also moderate AON mRNA expression in the dorsomedial aspect of the thalamus adjacent to the third ventricle, the subthalamic nucleus, and the medial and lateral geniculate nuclei (not quantitated). In addition, there was a band of high AON mRNA expression along the cerebroventricular and basal margins of the diencephalon that did not correspond to any defined nuclei. In the midbrain there was considerable AON mRNA expression in the habenula (Fig. 1G) medial terminal nucleus of the accessory optic tract (Fig. 1H), a nucleus not previously reported to express AON mRNA, and the superior colliculus (Fig. 1H). There was also moderately high AON mRNA in the olivary pretectal nucleus, and a lower expression of AON mRNA in the interfascicular nucleus and central gray (not quantitated).


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  		Fig. 1.   Distribution of angiotensinogen (AON) mRNA in the forebrain of a lactating rat. Images are from film apposed to coronal sections of rat brain incubated with 35S-labeled AON cRNA. Numbers at top right of each panel indicate the anteroposterior coordinates of the brain section (31). A: OVLT, organum vasculosum of the lamina terminalis; MSept, medial septum. B: MnPO, median preoptic nucleus; GP, globus pallidus; SCN, suprachiasmatic nucleus. C: SFO, subfornical organ; SON, supraoptic nucleus. D: PVH, paraventricular hypothalamic nucleus; Amyg, amygdaloid complex. E: PVH, paraventricular hypothalamic nucleus; r-ARH, rostral arcuate nucleus; ME, median eminence. F: LH, lateral hypothalamus; mc-ARH, midcaudal ARH. G: c-ARH, caudal arcuate nucleus; HAB, habenula. H: SC, superior colliculus; MT, medial terminal nucleus of the accessory optic tract. Calibration bar = 1 mm.

In the telencephalon, there was moderate AON mRNA expression in the medial septum, diagonal band of Broca (Fig. 1A), globus pallidus (Fig. 1B), and amygdaloid complex (Fig. 1D). There was a low to moderate expression of AON mRNA in the pyramidal cell layer of the hippocampus (not quantitated).

Two circumventricular organs (CVO), the organum vasculosum of the lamina terminalis (OVLT) (Fig. 1A) and SFO (Fig. 1C), displayed high and moderate levels of AON mRNA, respectively. The SFO displayed a marked rostrocaudal gradient of AON mRNA. The OD of the most rostral section of SFO sampled (~0.9 mm caudal to bregma) was 0.346 ± 0.010, whereas that of the section 0.3 mm caudal to it was 0.256 ± 0.004. In contrast to the OVLT and the SFO, a third forebrain CVO, the median eminence (Fig. 1, E and F), had barely detectable levels of AON mRNA either by film or grain density analysis. The subcommissural organ, another forebrain CVO but one that has not been reported to have apparent angiotensinergic activity or responsivity, had only background levels of silver grains.

Comparison of brain AON mRNA in lactating and diestrous rats. There was very little difference in the expression of AON mRNA between the diestrous and lactating rat brains (Table 1). When combined, the average ODs for all the brain regions sampled differed by less than 0.1% between the lactating and diestrous rats. However, in the midcaudal portion of the ARH there was a significant 27% reduction in AON mRNA (P = 0.023) in lactating, compared with diestrous, rats (Fig. 2). There were no significant differences in AON mRNA expression between the lactating and diestrous rats in the rostral and caudal portions of the ARH. There was a significant gradient of AON mRNA expression in the ARH (P < 0.0001) for both groups of rats. The OD for AON mRNA decreased from the rostral to caudal extent of the ARH by 0.078 and 0.064 OD/mm, (r2 = 0.81 and 0.78) for lactating and diestrous brains, respectively.


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  		Fig. 2.   AON mRNA expression along the rostrocaudal extent of the ARH. Sections of brains separated by intervals of ~60 µ from each rat were matched for anteroposterior (A-P) coordinates based on landmarks such as the crossing of the anterior and posterior commissures (see METHODS). A shows the average ± SE of optometric density (OD) readings for the ARH of lactating and diestrous rat brains from A-P coordinates of 1.88-4.29 mm caudal to bregma. The solid rectangle from 3.16 to 3.65 mm caudal to bregma represents the area defined as the midcaudal ARH. Arrows indicate the divisions of the ARH that were compared. Vertical dashed lines denote the boundaries of the 4 subdivisions of the ARH [arcuate (Arc)-A, Arc-B, Arc-C, and Arc-D] delineated in our laboratory's previous studies (38, 47). B compares the average ± SE of the OD values for the rostral, midcaudal, and caudal ARH from 1.88 to 3.16 mm, 3.16 to 3.65, and 3.65 to 4.29 caudal to bregma, respectively.

To further characterize the distribution of AON mRNA within the ARH, we qualitatively evaluated silver grain distribution in the ARH by dark-field microscopy (Fig. 3). In the rostral ARH (Fig. 3, A and C), AON mRNA expression was similar in the lactating and diestrous rats. Again, in the midcaudal ARH, the AON mRNA expression was considerably reduced in lactating rats compared with diestrous rats (Fig. 3, B and D). The decrease in AON mRNA appeared to be greatest in the ventral aspect of the midcaudal ARH.


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  		Fig. 3.   Distribution of silver grains indicative of AON mRNA localization in the rostral and midcaudal ARH of lactating and diestrous rats (Di). A and C show rostral ARH sections corresponding to an A-P plane of 2.3 mm caudal to bregma. B and D represent midcaudal ARH sections corresponding to an A-P plane of 3.4 mm caudal to bregma. L8, lactating rat with 8 pups. Calibration bar = 100 µm.

Immunocytochemical localization of AON, GFAP, and TH. Examination of the characteristics of AON-expressing cells in the brain revealed that all of the AON-ir-positive cells in the ARH (Fig. 4, A and C) were also positive for GFAP, a glial cell marker (Fig. 4, B and C). However, not all GFAP-ir-positive cells (Fig. 4B) expressed AON (Fig. 4C).


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  		Fig. 4.   Confocal microscopic representation of immunofluorescent labeling of AON-, tyrosine hydroxylase (TH)-, and glial fibrillary acidic protein (GFAP)-like immunoreactivities (ir) in the ARH of the rat. A: localization of AON-ir (green) in the ARH of a Di rat. B: localization of GFAP-ir (red) in the same region of the ARH as in A. C: colocalization of AON-ir (green) and GFAP (red) in the same region of the ARH as in A and B. Yellow areas, colocalization of AON-ir and GFAP-ir in the same cells. D: diagrammatic representation of a hemicoronal section of the rat brain corresponding to the plane represented in A, B, C, and E. Red box, the size of the area depicted in A, B, C, and E. E: AON-ir (green) and TH (red)-ir cells in the ARH. Yellow areas, very close apposition between the AON-positive cells and the TH-positive cells. Dashed line, the medial border of the ARH abutting the third ventricle (3V). F: higher resolution image of area contained within white box from E. The white arrow points to an AON-positive cell in close apposition to a tyrosine hydroxylase-positive cell. F-1, F-2, and F-3 represent single, 0.5-µm-thick planes, separated by 1-µm intervals, of the confocal microscopic image shown in F. The white arrow points to the same AON-positive cell as in F. Note that this cell can be seen in the absence of the TH-positive cell, indicating that the close apposition seen in F is not due to colocalization of the AON-ir in the TH-positive cell.

Because ANG II, the active hormonal product of AON, is known to stimulate dopaminergic neurons in the ARH, the distribution of AON-ir cells was compared with TH-ir cells in the ARH nucleus. TH antibody was used to identify ARH dopaminergic neurons as they are the only TH-containing cells in the ARH (43). AON-ir was not colocalized with TH-ir; however, there was close apposition of AON-ir-positive cells with TH-ir-positive cells (Fig. 4, E and F). AON-ir-positive cells were more widely distributed in the ARH than the TH-ir-positive cells with no apparent enrichment of AON-ir-positive cells in the immediate vicinity of TH-ir-positive cells.

The distribution of AON-ir-positive cells in other areas of the brains (in the same coronal sections that contained the ARH) of ovariectomized rats (not shown) was similar to the distribution of AON mRNA. There was no apparent increase in the number of AON-ir-positive cells in other nearby TH-ir-positive regions of the brain (A11, A13 nuclei).

The overwhelming majority of AON-containing cells appeared to be GFAP positive and had morphology suggestive of astrocytes. However, some AON-ir-containing neurons were present in the CA1 region of the hippocampus in addition to a considerably more abundant population of astrocyte-like AON-ir-positive cells in the pyramidal layer of the hippocampus (not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The distribution of AON mRNA in the forebrain of both lactating and diestrous female rats is similar to that reported for the male rat brain (3, 36). Overall, the highest amount of AON mRNA is in the hypothalamus, with dense concentrations in the suprachiasmatic, dorsomedial, paraventricular, preoptic, and ARH of the hypothalamus. Therefore, AON mRNA expression in the rat forebrain, with the possible exception of the medial terminal nucleus of the accessory optic tract (Fig. 1H), does not appear to be sexually dimorphic. There is a lesser but reasonable agreement between the AON mRNA distribution and that of AON protein (8) and immunohistochemically identified AON (14, 45).

Among the brain regions surveyed for AON mRNA, only the ARH showed a reduction in AON mRNA in the lactating rat brain compared with that of the diestrous rat. However, this decrease was limited to the midcaudal portion of this nucleus (Fig. 2). The smaller reductions in AON mRNA in the rostral and caudal ARH of lactating rats were not statistically significant. The localization of this decrease in AON mRNA to the midcaudal aspect of the ARH is of interest because this overlaps the arcuate (Arc)-C portion of the ARH in which lactating rats show an increased neuropeptide Y (NPY) mRNA expression (38).

The moderate decrease in AON mRNA expression from the rostral to the caudal aspect of the ARH is of interest because it parallels the rostrocaudal decrease in TH mRNA expression in this nucleus (47). This may indicate a functionally significant relationship between AON producing cells and dopaminergic neurons in the ARH. TH mRNA in the ARH is also reduced in lactating rats; however, the reductions in TH activity in the ARH of lactating rats occur in the more rostral Arc-A and Arc-B regions of the ARH as well as in the Arc-C region (47). Thus the extent to which the brain ANG II system influences tuberoinfundibular dopaminergic (TIDA) neuron activity relative to other factors may be small.

Brain AON appears to be produced predominantly, if not exclusively, in astrocytes (3, 42). This study did not attempt to colocalize AON mRNA with astrocytic markers, e.g., GFAP. However, double-label immunohistochemical staining for AON, GFAP, and TH indicated that AON-ir-containing cells in the ARH also contained GFAP-ir but did not contain TH-ir. Moreover, the fact that some of the AON-positive cells in the ARH were found in close apposition to TH-positive neurons in the ARH (Fig. 4) is again suggestive of a functional interaction between AON-producing astrocytes and the TIDA neurons of the ARH.

The mechanism whereby lactation is associated with a reduction in AON mRNA in the midcaudal ARH is of interest. In addition to the influence of gonadal and adrenal steroids on AON mRNA expression, changes in thyroid hormone (6, 15, 23), dietary salt intake (30), dehydration (1), and plasma glucose (48) are additional factors that are potential regulators of AON mRNA in the lactational state.

Many of these factors also have region-specific effects on AON mRNA expression in the brain. In addition to the region-specific alterations in brain AON mRNA expression caused by glucocorticoids and estrogen (4, 16), 1% sodium chloride in drinking water decreases AON mRNA expression in the brain stem but not in the hypothalamus (30). Dehydration increases AON mRNA expression more in the rostral than in the caudal SFO (1).

A more likely route by which AON mRNA expression may be regulated in the ARH is by neurotransmitters. The suckling stimulus alters the activity of a number of neurotransmitter systems that impinge on the ARH (24), and astrocytes contain a variety of neurotransmitter receptors that can affect their function (18, 33). Of considerable interest is the presence of NPY receptors on astrocytes (17). NPY is increased in the ARH of the lactating rat (38), and NPY-positive nerve terminals are present in close apposition to, but do not make synaptic contact with, dopaminergic neurons in the ARH (12). Thus it is possible that NPY could interact with astrocytes in the ARH and inhibit AON mRNA synthesis or stability. Further studies to determine the functional significance of NPY and other suckling-activated neurotransmitter systems on astrocyte AON synthesis in the ARH will be needed to resolve this issue.

Under normal conditions, prolactin provides an excitatory stimulus to TIDA neurons in the ARH (28). However, during lactation, TIDA neurons are suppressed despite high circulating levels of prolactin (11, 47). The mechanism by which the stimulatory effect of prolactin on TIDA neurons is interrupted by lactation is not known. If the pathway by which prolactin stimulates TIDA neurons requires angiotensinergic activity, the reduction in ARH AON mRNA seen in this study, coupled with the previous observation of a decrease in ANG II receptor binding in the ARH (40), may explain the loss of prolactin stimulation of TIDA neuron activity.

AON is synthesized and excreted constitutively from astrocytes (44) leading to a high local concentration of this ANG II precursor. As shown by Schinke et al. (37), inhibition of brain AON mRNA leads to a 90% reduction in AON and profound inhibition of brain ANG II-mediated events. In addition, the correlation between the distributions of AON mRNA and AON-ir in different brain regions infers that changes in AON mRNA closely parallel changes in AON secretion. Renin, the enzyme that cleaves ANG I from AON, is contained in nerve terminals in the rat brain (19) and is likely released on stimulation in a manner reminiscent of the release of renin from the juxtaglomerular cells of the kidney. ANG-converting enzyme, the enzyme that converts ANG I to ANG II, is localized to the outer membrane of cells (10) and is present in the ARH (7). Thus it is likely that under normal, i.e., nonlactating, conditions, high concentrations of AON are present in the immediate vicinity of TIDA neurons and can give rise to high concentrations of ANG II that stimulate TIDA neurons. During lactation, the reduction in AON could reduce ANG II in the immediate vicinity of TIDA neurons leading to a withdrawal of stimulation of TIDA neurons. If the feedback stimulation of TIDA neurons by prolactin requires ANG II, then lactation-induced reduction of angiotensinergic influence on TIDA neurons would interrupt this feedback loop, allowing prolactin secretion to continue unabated.

Perspectives

Given the large number of changes in hormonal status and neurotransmitter function in the lactating rat, the overall lack of change in AON mRNA in forebrain regions other than the ARH is remarkable. It would suggest that for the most part the synthesis of AON in the brain is rather stable and subject only to major perturbations, e.g., complete loss of regulatory hormones or application of supraphysiological/pharmacological concentrations of the hormones.

The reduction in AON mRNA in the caudal ARH, along with the reduction in ANG II receptors, may be of importance in the maintenance of high levels of prolactin secretion during lactation. However, the functional significance of this pathway remains to be tested. The fact that most of the TIDA neurons are located rostral to the area in which there was a significant reduction in AON mRNA would seem to indicate that the overall effect of ANG II on TIDA neuron function may be limited to a small population of TIDA neurons. Future studies involving the effects of chronic infusion of ANG II or AT1 receptor antagonists into the ARH of lactating and nonlactating rats on prolactin release or examination of prolactin secretion in transgenic animals under or overexpressing AON will be needed to answer this question.


    ACKNOWLEDGEMENTS

We thank R. Campbell for comments on the manuscript.


    FOOTNOTES

During this study R. C. Speth was a Visiting Scientist at the Oregon Regional Primate Research Center.

This work was supported by National Institutes of Health Grants HD-14643 and RR-00163.

Address for reprint requests and other correspondence: R. C. Speth, Dept. Vet. Comp. Anat. Pharmacol. Physiol., P.O. Box 646520, Washington St. Univ., Pullman, WA 99164-6520 (E-mail: speth{at}wsu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 28 September 2000; accepted in final form 6 December 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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