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Endocrinology, doi:10.1210/en.2004-0931
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Endocrinology Vol. 145, No. 12 5656-5664
Copyright © 2004 by The Endocrine Society

Postnatal Regression of Hypothalamic Dopaminergic Neurons in Prolactin-Deficient Snell Dwarf Mice

C. J. Phelps

Department of Structural and Cellular Biology, Tulane University School of Medicine, New Orleans, Louisiana 70112

Address all correspondence and requests for reprints to: C. J. Phelps, Ph.D., Department of Structural and Cellular Biology, SL-49, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112-2699. E-mail: cjphelps{at}tulane.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Both Snell (Pit-1dw or dwj, dw/dw) and Ames (Prophet of Pit-1df, df/df) dwarf mice fail to produce prolactin (PRL) as well as GH due to deficient transcription factor Pit-1 activity and have reduced numbers of hypothalamic PRL-inhibiting area A12 tuberoinfundibular dopaminergic (TIDA) neurons. It has been reported that the TIDA deficit in Ames dwarf mice develops postnatally as a reduction in number after an initial increase that is comparable to that of normal siblings. The present study was designed to characterize A12 TIDA neuronal development in the Snell dwarf (dw/dw) compared with littermate normal mice. Brains of normal (DW/?) and dwj/dwj mice were examined at 7, 14, 21, 30, and ≥ 60 postnatal days (d) by catecholamine fluorescence and quantification of neuron number after tyrosine hydroxylase immunostaining in dopaminergic (DA) areas A12, A13 (medial zona incerta), and A14 (periventricular nucleus). Fluorescence was less in dw/dw than in DW/? A12 perikarya and median eminence but was not reduced in other DA areas, such as substantia nigra, at all ages; A12 fluorescence was virtually absent in Snell dwarf adults. Numbers of TIDA neurons were comparable in normal and Snell dwarf mice at 7 d. In normal (DW/?) mice, A12 neurons increased in number to adult levels at 14 d and were significantly higher than in Snell dwarf (dw/dw) mice at 14 d (P < 0.05) and at subsequent ages (P < 0.01). In Snell dwarf mice, numbers of A12 neurons did not differ at 7, 14, and 21 d, decreased at 30 d (P < 0.05), and reached, at 60 d, 23% of the population in normal sibling mice (P < 0.01 compared with earlier ages). Neuron numbers in nonhypophysiotropic DA area A13 did not vary with age or phenotype. In A14, cell number was higher in both phenotypes at 14 d (P < 0.05 for DW/?; P < 0.01 for dw/dw); neuron number was lower in dw/dw than in DW/? mice at 30 d (P < 0.05) and 60 d (P < 0.01). Thus, compared with normal mice of the same strain, the A12 deficit is more severe in Snell (dw/dw) than in Ames (df/df) dwarf hypothalamus (48% of DF/?), as previously reported, and develops as a decline from the population present at 7 d rather than first increasing. A reduction in A14 neuron number also occurs in the Snell dwarf. Treatment of DW/dw- and dw/dw-containing litters with ovine PRL (50 µg/d, ip), beginning at 12 or 7 d and continuing until 42 d, resulted in TIDA neuron numbers in Snell dwarfs that were lower than those in normal siblings (P < 0.01 for both) but were higher than in untreated adult dwarfs and comparable to the TIDA population size in dwarfs at 7 d, indicating that PRL maintained this maximal number and prevented TIDA neuron dedifferentiation, which occurs in dwarf postnatal development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROLACTIN (PRL) SECRETION by the anterior pituitary is inhibited tonically (1) by dopamine (DA) produced in neurons in the hypothalamic region known as tuberoinfundibular. These neurons, also known as area A12 according to the nomenclature of Dahlström and Fuxe (2), terminate in the external median eminence (ME) to deliver DA to the pituitary. In turn, PRL exerts a positive feedback on these neurons, elevating DA synthesis and turnover (3) and expression of the rate-limiting enzyme for catecholamine (CA) production, tyrosine hydroxylase (TH) (4). Conversely, hypoprolactinemia leads to decreased DA turnover (5) and TH expression (4). This influence of PRL on tuberoinfundibular dopaminergic (TIDA) neurons extends to effects on developmental differentiation, as shown in animal models of lifelong alteration in PRL production (6).

Ames [Prophet of Pit-1 (Prop-1)df] and Snell (Pit-1dw, dwj) mutations in mice lead to deficient pituitary production of GH, TSH, and PRL and phenotypic dwarfism. In the Snell dwarf, a point mutation (Pit-1dw) or a nucleotide rearrangement (Pit-1dwj) in the gene for the transcription factor Pit-1 (7) results in a protein that is unable to bind to its cognate site in the promoter region of all three pituitary hormones. In the Ames dwarf, a point mutation in the Prop-1 (8) gene results in a protein with greatly reduced ability to bind to the Pit-1 promoter region, reducing Pit-1 transcription. Because Pit-1 is required for transcription of GH, TSH, and PRL (9, 10), both types of mutant mice lack these three pituitary hormones. However, that phenotype is not identical in the two dwarf strains. Gage et al. (11, 12) observed anterior pituitary cells in Ames dwarf mice that were immunoreactive for GH, PRL, or TSH and Pit-1; this laboratory also observed similar clones in Ames dwarf pituitary, although with less frequency (13). These cells have not been observed in pituitaries of Snell dwarf mice (11, 12, 13). Whether the small clusters of hormone-containing cells in the Ames dwarf pituitary produce detectable or significant levels of GH, PRL, or TSH in the circulation has not been determined.

In Ames dwarf mice, TIDA neurons initially develop in a manner comparable to that of normal siblings, with DA levels increasing between 7 and 14 d postnatally (14) and neuronal numbers increasing through age 21 d (15). After 21 d, the TIDA neuron population undergoes a marked reduction, such that, by 60 d of age, cells in the dwarf number less than half of those in normal siblings (15).

The severity of the TIDA neuron deficit in Snell dwarf mice (16) is greater than that in Ames dwarf mice (17). The purpose of this study was to examine the developmental pattern leading to low DA levels (18) and small TIDA population (16). Snell dwarf (Pit-1dwj, dw/dw) and normal (DW/?) littermates were assessed by endogenous CA histofluorescence and TH immunostaining to quantify neuronal numbers at 7, 14, 21, 30, and ≥60 d of age.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue preparation
Snell dwarf (dw/dw) and normal (DW/DW or DW/dw, thus DW/?) mice were reared in a colony whose original breeding pairs were purchased from Jackson Laboratories (Bar Harbor, ME). To increase the numbers of (homozygous recessive) dwarfs in litters, heterozygous (DW/dw) females were mated with dw/dw males brought to fertility by renal capsule grafts of normal mouse pituitaries to provide a source of circulating mouse PRL. Dwarf males were treated with D/L-T4 (2 µg ip, thrice weekly; Sigma Chemical Co., St. Louis, MO) for 4 wk before receiving pituitary renal capsule allografts at 9 wk of age. The pituitary transplant surgery was performed in anesthetized (inhalant isoflurane) animals. Normal donors were male or female and 2–6 months old; they were euthanized with carbon dioxide and decapitated for pituitary gland removal. Whole single glands were placed under the kidney capsules of recipient animals. After surgery, recipients were observed continuously, and body temperature was supported until recovery from anesthesia. Day of birth (by 1000 h) in the colony was considered postnatal d 1. Normal mice (DW/?) were weaned into separate-sex groups at 21–28 d of age and were housed in standard plastic cages of three to five mice per cage. Dwarf mice remained with dams or were housed with another normal female in groups of three to five mice. The colony was maintained under conditions of controlled temperature (22 ± 2 C) and lighting (lights on from 0600–1800 h), with food and water available ad libitum. The procedures for maintenance and euthanasia were approved by the Tulane University Medical Center Institutional Animal Care and Use Committee.

Deeply anesthetized (pentobarbital, 80 mg/kg of body weight) mice were perfused transcardially at 7, 14, 21, 30, or 60 d of age with 0.9% NaCl followed by 4.0% buffered paraformaldehyde and 0.5% glutaraldehyde (Faglu) (19) fixative. After perfusion, brains were removed and postfixed for 24–48 h at 4 C in Faglu containing 30% sucrose. Each brain was sectioned frozen in the coronal plane at 30 µm into six repetitive sets of serial sections in Faglu, such that sections in each set were separated by a rostral-to-caudal distance of 180 µm. Sections to be assessed for CA fluorescence were mounted directly from Faglu. Other sets of sections were transferred to cryoprotectant antifreeze (20) and stored at –20 C.

Hormone treatment
Whole litters comprised of DW/dw normal and dw/dw dwarf (i.e. dwarf-sired) mice were treated beginning at either 12 or 7 d of age with either ovine (o) PRL or oGH (50 µg/d, ip). The mice were euthanized by transcardial perfusion at 42 d of age, and brains were assessed for A12, A13, and A14 cell number using TH immunocytochemistry (ICC).

Endogenous CA fluorescence
For qualitative assessment of steady-state CA content by formaldehyde-induced fluorescence, two sets of every sixth section (i.e. sections taken at 180-µm intervals) from each brain were mounted and examined using blue-violet excitation wavelengths (400–440 nm) and a 455-nm barrier filter on a Nikon Optiphot or E800 microscope (Tokyo, Japan) equipped for epi-illumination.

ICC
TH.
Brain sections retrieved from cryoprotectant were processed free-floating for TH ICC. Sections from several (6, 7, 8, 9, 10, 11, 12) animals were immunostained simultaneously in separate vessels but using aliquots of the same antisera and other reagent solutions. Thus, each ICC set included dw/dw and DW/? sections in a single incubation, when phenotype was discernible (≥21 d of age), or sections from a single DW/dw x dw/dw litter at 7 and 14 d, or animals of several ages, to reduce variance of separate incubations among ages and between types.

The ICC procedure has been described previously (15, 17). One set of sections taken from cryoprotectant were rinsed extensively in 0.1 M PBS and then incubated in nonimmune goat serum to reduce nonspecific-antigen binding to secondary (goat antirabbit IgG) antiserum. Sections were exposed to rabbit anti-TH (Chemicon, Inc., Temecula, CA) at a dilution of 1:5000 for 48 h at room temperature or for 72 h at 4 C. The sections were then incubated with biotinylated goat antirabbit IgG and with avidin-biotinylated peroxidase complex solution (Vectastain ABC kit; Vector Laboratories, Burlingame, CA). The reaction product was developed using a solution of hydrogen peroxide (0.003%) and diaminobenzidine (0.02%). Sections were mounted in rostral-to-caudal order and examined microscopically.

Phenotype identification by pituitary GH ICC.
Dwarf mice retain prepubertal proportions and facial characteristics compared with normal (DW/?) siblings and fail to grow beyond 14–21 d of age, but they are not reliably distinguishable from DW/? littermates at younger ages. Therefore, positive identification of phenotype in mice younger than 21 d was accomplished postmortem by GH ICC in pituitaries. GH immunoreactivity in DW/? but not dw/dw mice is detectable at birth (i.e. d 1), whereas PRL is not detectable until at least 7 d (15). Pituitaries from normal adult mice were used as positive controls. Pituitaries from Faglu-perfused mice were post fixed in Bouin’s acidic picric acid fixative, paraffin-embedded, and sectioned in the horizontal plane at 5 µm. Segments of 25 µm (five adjacent sections) at three levels through the pituitary were sampled and immunostained for GH. The primary antiserum, used at 1:5000 dilution, was an antimouse GH produced in monkey (no. 35-12/1/70 from the National Hormone and Pituitary Program), and the biotinylated secondary antiserum was rabbit antihuman IgG, which recognizes all primate IgG. Reaction product was developed using the avidin-biotinylated peroxidase complex method and diaminobenzidine as chromogen, as described earlier.

Imaging for illustrations.
Endogenous CA fluorescence images were captured using a DX1200 color digital camera on a Nikon E800 microscope. TH immunostaining was photographed using 35-mm color professional film (Kodak EPY 64; Rochester, NY); selected images were scanned using a Nikon CoolScan film scanner at 1350 dpi. Both fluorescence and bright-field images were composited to multiphoto plates using Adobe Photoshop 5.0.2 (Adobe Systems Inc., San Jose, CA).

Neuronal cell counting
Numbers of TH-immunoreactive neuronal cell bodies were counted in areas A12, A13, and A14 in coronal brain sections at 180-µm intervals. This interval included, in adults, approximately six to seven coronal sections in DW/? and four to five sections in dw/dw A12, reflecting a total sagittal length similar to that in Ames dwarf (df/df) of 0.81 ± 0.08 mm and in normal (DF/?) siblings of 1.29 ± 0.06 mm (17). Positive cells were counted in every mouse brain by two or three investigators on coded slides. Cell counts by different persons differed by up to 10%; counts for each mouse represent an average of independent evaluations. In counting, TH-positive cells were not limited to cells with definable nuclei but were only counted if the entire cell outline was visible; criteria were defined initially by comparison counts on single sections, until identical criteria were agreed upon. Cell counts were corrected for sampling frequency (x6, for every sixth section) but not for cells that were double counted or omitted because the product of section thickness (30 µm) and interval (180 µm) far exceeded perikaryal diameter (21). Identification of A12, A13, and A14 was based on the CA classification of Björklund and Nobin (22). A14 neurons were recorded beginning at the level of the union of the anterior commissure and extending to the caudal end of the ME; A12 cells were recorded from the rostral through the caudal appearance of ME; and A13 neurons were recorded in their distinct dorsal distribution in the subthalamus. A stereotaxic atlas of the mouse brain (23) was used for reference. TH-immunopositive neurons in six to 10 animals of each phenotype (dw/dw or DW/?) were recorded in each age group (7, 14, 21, 30, and ≥60 d). Each age group included at least three mice of each sex.

Statistical assessment of cell counts was accomplished using ANOVA for age or treatment effect within each phenotype and phenotype effect in each age or treatment group (one-factor ANOVA) and for effect of age or treatment and phenotype (two-factor ANOVA); significant differences were identified by the Student-Newman-Keuls post hoc multiple range test using a probability of less than 5% as indicative of significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GH immunostaining in pituitaries from 7-d-old mice is shown in Fig. 1Go. In the pituitary of a normal mouse (Fig. 1AGo), GH is widespread and homogeneous. The dw/dw pituitary section (Fig. 1BGo) is devoid of specific signal. Mice designated DW/? had GH-positive pituitaries; animals in which pituitary GH was not detected were designated dw/dw.



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  		FIG. 1. Photomicrographs of GH immunostaining in pituitary of normal (DW/?) and dwarf (dw/dw) mice. Neurointermediate lobe is at the lower left in each photo. GH-containing cells are distributed homogeneously in DW/? anterior pituitary; dw/dw gland is devoid of GH signal. Original objective magnification, x40. The bar at lower right represents 100 µm.

 
CA fluorescence of A12 and ME in normal and Snell dwarf mice during postnatal development is shown in Fig. 2Go. Evaluation of comparative fluorescence was qualitative. At 7 d, fluorescence was faint or undetectable for both normal and dwarf mice in A12 perikarya and ME, so is not shown. At 14 d, fluorescence was detectable in TIDA cell bodies and ME terminals of both normal and dwarf mice. At 21 d, both normal and dwarf mice showed specific perikaryal and ME fluorescence. At 30 d, both perikaryal and ME fluorescence was reduced in dw/dw compared with 14-d-old and DW/?. In adults, perikarya were visible in DW/? but not in dw/dw, and the bright ME CA fluorescence visible in DW/? mice was virtually absent in dwarf. DA fluorescence was not deficient, nor did it decline postnatally, in nonhypophysiotropic brain regions, as illustrated by photomicrographs of perikaryal DA in substantia nigra of dwarf mice in Fig. 3Go, at 14 d (Fig. 3AGo) and 30 d (Fig. 3BGo). DA fluorescence in A13 or A14 was not used for comparison because it was very faint.



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  		FIG. 2. Endogenous CA fluorescence in A12 (TIDA) perikarya and ME in normal (DW/?) and dwarf (dw/dw) mice at 14, 21, 30, and ≥60 d of age. For all panels, the horizontal bar at lower right represents 100 µm. Original objective magnification, x20. Perikaryal and ME fluorescence are shown to decline with age in dwarf, but not normal, A12 cell bodies and ME.

 


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  		FIG. 3. Endogenous DA fluorescence in substantia nigra (CA area A9) in dwarf mice at 14 and 30 d of age. For both micrographs, the horizontal bar at the upper right represents 100 µm. Original objective magnification, x10.

 
TH ICC revealed a striking developmental decline in TIDA neurons in dwarf compared with normal mice. The developmental pattern is shown in composite Fig. 4Go, and the following description of representative photomicrographs in that figure is qualitative. TH-immunostained sections from normal (DW/?) mice are shown in the left column. The right column shows coronally matched TH-immunostained sections from dwarf mice at the same ages. Row 1 of Fig. 4Go shows TH immunostaining at 7 d of age; the subsequent rows show TH ICC at 14, 21, and 30 d of age and in adults (6 months in Fig. 4Go). In 7-d-old mice, A12 perikarya were detectable and were comparable for normal and dwarf mice; ME was rudimentary and showed very little TH in both DW/? and dw/dw mice. At 14 d, TH immunoreactivity was robust in normal mice, and ME TH immunoreactivity was greater than at 7 d. In dw/dw mice, many perikarya were visible, but TH immunoreactivity appeared to be less than that in normal mice of the same age, especially in ME. In 21-d-old mice, the ME began to adopt an adult morphology, and TH immunoreactivity in DW/? mice was robust in terms of both staining intensity and perikaryal numbers. In 21-d dw/dw A12, cell numbers and TH immunostaining intensity were qualitatively less than in age-matched normal mice. At 30 d, DW/? TIDA neurons continued to show strong TH immunostaining in both cell bodies and ME; in the dwarfs, TH immunoreactivity was not only markedly less than in age-matched normal mice but also appeared to have declined from that in dw/dw mice at 21 d. In adult Snell dwarfs, A12 perikarya were faint and very few, and ME TH was minimal, compared with previous ages and with adult DW/? mice.



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  		FIG. 4. TH immunostaining of A12 (TIDA) neurons and ME in normal (DW/?) and dwarf (dw/dw) mice at 7, 14, 21, and 30 d of age and in adult mice (6 months). The horizontal bar at lower right represents 100 µm. Original objective magnification for all the photomicrographs, x10. The comparison of left and right columns shows that TH immunostaining intensity declines over postnatal development in dw/dw, but not DW/?, TIDA neurons and terminals.

 
Figure 5Go shows photomicrographs of TH ICC in diencephalic midcoronal sections from normal (DW/?) and dwarf (dw/dw) mice at 90 d of age. Each section includes areas A13 (dorsal, medial zona incerta), A14 (periventricular DA perikarya), and directly PRL-inhibiting A12. Qualitatively, the deficit is in A12 rather than the other regions.



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  		FIG. 5. Low magnification photomicrographs of TH immunostaining in diencephalic DA regions A13, A14, and A12 in an adult normal (DW/?) and a Snell dwarf mouse (dw/dw). Both mice were of 90 d of age. For both images, the horizontal bar represents 200 µm. Original objective magnification for both images, x4.

 
Figure 6Go illustrates the pattern of TIDA/A12 neuron numbers during postnatal development in Ames dwarf (df/df) and normal sibling (DF/?) mice and was modified from a figure published in Phelps et al. (15). Neuron numbers increased in df/df mice through 21 d, at which age a normal (DF/?) complement of A12 neurons was present. Numbers of A12 neurons were lower in df/df than in DF/? mice at 30 d (P < 0.05) and 60 d (P < 0.01).



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  		FIG. 6. Numbers of A12 TH-immunoreactive neurons during postnatal development in normal (DF/?) and Ames dwarf (df/df) mice. Asterisks represent differences between normal and dwarf phenotype at each age: *, P < 0.05; **, P < 0.01. The figure is modified from a figure previously published in Phelps et al. (15 ).

 
Quantification of TIDA cell number, obtained from immunostained images such as those illustrated in Fig. 4Go, gave the pattern illustrated in Fig. 7Go for development of A12, A13, and A14. Three-way ANOVA indicated no effect of sex or interaction of sex with either phenotype or age in any region, so further analyses compared phenotype or age without regard to sex. In A12, there were significant effects of both type (F(1,65) = 78.856; P = 0.001) and age/type interaction (F(4,65) = 5.904; P = 0.004). Among DW/? mice, there was no effect of age on A12 cell number (F(4,33) = 2.425; P = 0.0676). In dwarf mice, age had a significant effect (F(4,32) = 9.469; P = 0.0001), and cell number was lower at 30 d (P < 0.05) and 60 d (P < 0.01) than at 7–21 d, which did not differ inter alia. Normal and dwarf mice differed in A12 cell number at 14 d (P < 0.05), at 21–30 d (P < 0.01), and at 60 d (P < 0.001), such that A12 cell numbers were comparable for DW/? and dw/dw mice only at 7 d. At 60 d or older, the Snell dwarf A12 cell number was 23% of that in normal DW/? siblings. In A13, there was no effect of age on cell number in either DW/? or dw/dw mice. Perikaryal number did not differ between normal and dwarf mice at any age in A13. For area A14, there was an overall effect of age (F(4,33) = 3.397; P = 0.0197) on cell number in DW/? mice; cell number at 14 d was higher than at 7 d or 30 d (P < 0.05). Among dwarf mice, age affected A14 number (F(4,32) = 10.375; P = 0.001), such that cell numbers were higher at 14 d than at 7, 21, 30, or 60 d (all P < 0.01). Significant differences in A14 cell number between normal and dwarf mice existed at 30 d (P < 0.05) and 60 d (P < 0.01) of age.



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  		FIG. 7. Numbers of TH-immunoreactive neurons during postnatal development in A12, A13, and A14 of normal (DW/?) and Snell dwarf (dw/dw) mice. Asterisks represent differences between normal and dwarf phenotype: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
Figure 8Go shows graphically the effect of neonatal PRL replacement, begun at 12 or 7 d and continuing until 42 d of age, on A12, A13, and A14 neuron numbers in Snell dwarf and normal littermate mice. Three-factor ANOVA showed no effect of sex on cell number in any DA area, so further assessment grouped males and females together. Two-factor ANOVA showed effects of both genotype (F(37,1) = 128.0; P < 0.001) and treatment (F(37,1) = 7.412; P < 0.01) in A12, of genotype (F(38,1) = 64.916; P < 0.001) in A14, but of neither genotype or treatment in A13. Among normal mice, there was no effect of hormone treatment in any DA area. Among Snell dwarf mice, A12 neuron number was higher in mice treated with PRL beginning at either 7 or 12 d than in untreated mice (P < 0.01 for both); cell numbers in A13 and A14 of dwarfs were comparable for untreated and PRL-treated mice. Among mice treated beginning at either 7 or 12 d, dwarf neuron numbers were lower in A12 and A14 than in normal mice (P < 0.01). However, the numbers of A12 neurons in PRL-treated dwarf mice (7 d, 1516 ± 86; 12 d, 1388 ± 61) were comparable to the A12 neuron numbers in untreated 7-d-old Snell dwarfs (1295 ± 138; Fig. 7Go). In litters treated beginning at 12 d with oGH, numbers of A12 neurons in Snell dwarf mice (722 ± 64) were much lower (P < 0.01) than in normal siblings (2088 ± 184) and also lower than in 7-d-old dwarf mice (P < 0.01), but they were higher than in untreated adult dwarfs (521 ± 47; P < 0.05).



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  		FIG. 8. Numbers of TH-immunoreactive neurons in normal (DW/?) and Snell dwarf (dw/dw) mice after daily PRL treatment (50 µg/d) beginning at 12 or 7 d of age compared with numbers in untreated mice. Neuronal numbers were quantified in 42-d-old mice. Asterisks represent differences between normal and dwarf phenotype: **, P < 0.01; ***, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The deficiency in TIDA neuron number in Snell (Pit-1dwj) dwarf mice is severe and is reflected in a postnatal developmental pattern of decline from the population size at 7 d of age, when TIDA numbers are comparable to those in normal littermates. In normal mice (DW/?) of the same strain, TIDA neuron numbers, as identified by TH immunoreactivity, increase, as has been shown for other strains (15), through 14–21 d postnatally. The decrease in Snell dwarf hypothalamus results in a TIDA population that is 23% of normal.

Because neonatal PRL treatment of Ames dwarf mice (24) and of Snell dwarf mice in this report results in adult TIDA neuron populations that are larger than those in untreated dwarfs, it is likely that neuronal decline in development is due to absent PRL feedback stimulation. Similarly, GH deficiency, in hypopituitary dwarf mice or in isolation, is accompanied by reduced hypophysiotropic somatostatin- producing neurons and overexpression of GHRH (13). Collectively, these findings indicate that the pituitary hormone feedback effect on the hypothalamus extends beyond dynamic regulation to influence neuronal developmental differentiation.

Björklund et al. (25) demonstrated that DA fluorescence in A12 neurons and ME is rudimentary in mice at early postnatal ages. In the present study, endogenous CA fluorescence was virtually undetectable in brains of 7-d-old mice. At 14 d, however, A12 perikaryal fluorescence was certainly detectable, including axon terminal fluorescence in ME. In DW/? mice, this pattern persisted, with ME fluorescence that increased with age. In dw/dw mice, ME fluorescence was weak throughout development, possibly reflecting reduced projections by TIDA neuron terminals, and perikaryal DA fluorescence declined after 21 d. In Ames dwarf mice, A12 fluorescence and DA levels fail to rise after 14 d, whereas both increase in normal siblings through 30 d (14).

As opposed to endogenous DA fluorescence at 7 d, TH immunostaining of A12 TIDA neurons was apparent in dwarf as well as normal hypothalamus, although there was very little immunoreactivity in ME, emphasizing the late postnatal development of terminal projections of these neurons. The increased TH immunoreactivity at 14 d in both DW/? and dw/dw mice correlated with increased numbers of neurons in normal but not dwarf mice, suggesting that TH activity in dwarf A12 was increasing in extant neurons but that differentiation of additional neurons was not. The DA fluorescence detectable in dwarfs at ages 14 and 21 d was rather remarkable, considering TIDA cell number, and suggests that these neurons are quite active synthetically in dwarfs as well as normal mice at those ages. Likewise, at 21 d, TH immunoreactivity qualitatively increased further in DW/? but not dw/dw mice, but cell numbers for both mouse types remained comparable to those at 14 d. At 30 d and later, a decline in detectable TIDA perikarya occurred in dw/dw but not in DW/? mice.

Comparison of A12 DA fluorescence and TH immuno-reactivity over development also indicated that TH ICC is more sensitive for cell detection and quantification. This was even more striking for the A13 and A14 DA groups, in which endogenous fluorescence was very faint, and may explain the lower estimates of diencephalic DA neuron populations in rats based on counting fluorescent perikarya using the Falck-Hillarp (26) method, especially in area A14 (27). Subsequent studies using TH ICC in mice (28, 29, 30) have indicated that periventricular DA neurons number in the thousands, which is comparable to findings in this study.

Thus, initial development of TIDA neurons appears to be independent of PRL feedback because PRL is not detectable until 7 d (14, 31) in normal mice and is not detectable at all in dwarfs. It is possible that PRL provided in maternal milk supports this initial development. Transfer of milk PRL to the plasma of neonates has been shown in rats (32) until gap junctions form between intestinal epithelial cells by 2–3 wk of age (33), preventing absorption of such large molecules. In husbandry of the dwarf mice, they are allowed to suckle for a second lactation, and PRL transfer could occur during this period because specific investigation of intestinal epithelial development in dwarf mice has not been performed. However, if milk PRL were transferred to dwarfs at a significant level in plasma, it is likely that TIDA neuron development would not be deficient.

Total numbers of A14 neurons also were lower in dw/dw than in DW/? mice, a difference that manifested at 30 d, following an initial increase in cell number in both mouse phenotypes. Such a difference should not be surprising, if PRL deficit is causative, because of evidence for involvement of periventricular dopaminergic neurons in PRL regulation (34, 35). Therefore, it is curious that A14 cell numbers were unaffected by chronic PRL treatment of Snell dwarfs. Whether A14 neurons express abundant PRL receptors (36), these cells have been shown to respond to PRL feedback (34). An A14 cell reduction was observed in older (12–16 months) male Ames dwarfs (17) but not in young adults (15, 17), which highlights a difference between the two dwarf types on effect of PRL deficiency on A14. The differences between strains may reflect severity of PRL deficiency, such that a very low level of PRL may support A14 differentiation in the Ames dwarf.

Both Ames (Prop-1df) and Snell (Pit-1dw, dwJ) spontaneous mutations in mice lead to dwarfism and deficiency in pituitary production of GH, PRL, and TSH, thus serving as models of multiple pituitary hormone deficiency. Although the overt phenotype of deficiency in GH, PRL, and TSH appears comparable for mutations in both transcription factors, there are specific differences. In humans with Pit-1 or Prop-1 mutations (37), these differences may simply reflect the greater variance in mutation types for each transcription factor, which manifests in a wide range of deficiency in several hormones. The difference between the two dwarf mouse types may stem from the difference in Pit-1 effect on hormone gene expression. The (Ames) Prop-1df mutation leads to a "partial loss of function" (37) in reduced binding of mutant Prop-1 to the promoter region of Pit-1. Ames dwarfs have been shown to exhibit clonal expression of GH, PRL, TSH, and Pit-1 (11, 12, 13); whether this results in circulating hormone levels that are capable of exerting significant feedback is not known. Importantly, Snell dwarfs show no GH, PRL, or TSH expression (11, 12, 13). The difference between pituitary phenotypes may be responsible for the difference in TIDA neuron developmental pattern and adult populations. Because circulating PRL and GH are undetectable in both dwarf types (38, 39), it is possible that small amounts of these hormones reach the hypothalamus from the anterior pituitary in Ames dwarf mice by retrograde flow in hypophysial portal vessels, as originally proposed (40) and only largely refuted by Green and Harris (41) and later supported by Bergland and Page (42).

In Ames dwarf mice, TIDA neuron number rises from 7–21 d of postnatal development (15) and then declines by 60 d to a population that is half that in normal mice. In Snell dwarf mice (present study), decline in TIDA cell number also occurs after 21 d, but there is no increase between 7 and 21 d as occurs in the Ames dwarf. Although PRL treatment initiated at 12 d postnatally can maintain a normal-sized TIDA population in the Ames dwarf (24), treatment beginning at 60 d (43) or at 30 or 21 d (44) cannot, suggesting that cellular phenomena leading to phenotype dedifferentiation or cell death are in progress before the decline in detectable cells. Whether neurons have died or simply no longer express TH in adult dwarfs is not known. A possibility for marking TH-producing neurons permanently is offered by Cre-lox technology (45) and could be used to address this question. However, because TH immunostaining intensity increased in extant neurons without an increase in number among adult Ames dwarf mice (43), the undetectable A12 neurons evidently had lost the capacity to respond to PRL with increased TH production. Timing of such phenomena may be similar in the Snell dwarf because PRL treatment starting at 12 d resulted in maintenance of cells extant at that age.

In the present study, PRL treatment beginning at 7 or 12 d resulted in TIDA neuron populations that were larger than in untreated adult Snell dwarf and that were comparable to the population present at 7–21 d. TIDA neuron numbers in PRL-treated dwarfs were smaller than in normal siblings, but it is important to note that untreated dwarfs never exhibit a normal-sized population during development, as exists in Ames dwarfs at 21 d. Thus, the effect of PRL replacement in both dwarf types is similar, maintaining the maximum population that occurs during development.

Despite these data showing that PRL replacement is sufficient to maintain TIDA neuron number in hypopituitary mice, converse evidence suggests that TIDA neurons may be maintained in the absence of PRL. In PRL knockout mice (46), the population of TIDA neurons is comparable to that of wild-type siblings, although DA and TH levels per neuron appear to be reduced (30). Similar results were found in PRL receptor knockout mice (47) in that the TIDA population was normal (48). These results suggest that GH or TSH or both may maintain TIDA neurons in the absence of PRL. Preliminary data presented in this report suggest that GH treatment is not sufficient to substitute for PRL. However, the GH used was not homologous, and there could be a compensatory mechanism in PRL-deficient transgenic mice, such as increased GH levels; an analogous finding was increased PRL levels in mice with disruption of the GH receptor (49). Perhaps a combination of high GH and TSH is required to replace the PRL effect.


   Acknowledgments
 
Antisera for the detection of mouse-specific pituitary GH, ovine PRL, and ovine GH were provided by the National Hormone and Pituitary Program by Dr. A. F. Parlow. The technical contributions of Martha Romero, Cheryl Malcamp, Shanna Joseph, Irma Estrada, Melina Evdemon Hogan, Jonathan Barnwell, and Sara Clark are gratefully acknowledged.


   Footnotes
 
This work was supported by Public Health Service Grant NS25987.

Abbreviations: CA, Catecholamine; DA, dopamine; ICC, immunocytochemistry; ME, median eminence; PRL, prolactin; Prop-1, Prophet of Pit-1; TH, tyrosine hydroxylase; TIDA, tuberoinfundibular dopaminergic.

Received July 19, 2004.

Accepted for publication August 24, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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