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Endocrinology, doi:10.1210/en.2007-1426
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Endocrinology Vol. 149, No. 4 2010-2018
Copyright © 2008 by The Endocrine Society

Long-Term, Homologous Prolactin, Administered through Ectopic Pituitary Grafts, Induces Hypothalamic Dopamine Neuron Differentiation in Adult Snell Dwarf Mice

Christina E. Khodr, Sara M. Clark, David L. Hurley and Carol J. Phelps1

Neuroscience Program, Tulane University School of Medicine, New Orleans, Louisiana 70112

Address all correspondence and requests for reprints to: Christina E. Khodr, Neurobiology Program, Children’s Memorial Research Center, Northwestern University, 2300 Children’s Plaza, Box 209, Chicago, Illinois 60614-3394. E-mail: c-khodr{at}northwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pituitary prolactin (PRL) secretion is inhibited by dopamine (DA) released into the portal circulation from hypothalamic tuberoinfundibular DA (TIDA) neurons. Ames (df/df) and Snell (dw/dw) dwarf mice lack PRL, GH, and TSH, abrogating feedback and resulting in a reduced hypophysiotropic TIDA population. In Ames df/df, ovine PRL administration for 30 d during early postnatal development increases the TIDA neuron number to normal, but 30 d PRL treatment of adult df/df does not. The present study investigated the effects of homologous PRL, administered via renal capsule pituitary graft surgery for 4 or 6 months, on hypothalamic DA neurons in adult Snell dw/dw mice using catecholamine histofluorescence, tyrosine hydroxylase immunocytochemistry, and bromodeoxyuridine immunocytochemistry. PRL treatment did not affect TIDA neuron number in normal mice, but 4- and 6-month PRL-treated dw/dw had significantly increased (P ≤ 0.01) TIDA (area A12) neurons compared with untreated dw/dw. Snell dwarfs treated with PRL for 6 months had more (P ≤ 0.01) TIDA neurons than 4-month PRL-treated dw/dw, but lower (P ≤ 0.01) numbers than normal mice. Periventricular nucleus (area A14) neuron number was lower in dwarfs than in normal mice, regardless of treatment. Zona incerta (area A13) neuron number was unchanged among phenotypes and treatments. Prolactin was unable to induce differentiation of a normal-sized A14 neuron population in dw/dw. Bromodeoxyuridine incorporation was lower (P ≤ 0.01) in 6-month PRL-treated normal mice than in 6-month PRL-treated dwarfs in the subventricular zone of the lateral ventricle and in the dentate gyrus, and lower (P ≤ 0.05) in 4-month untreated dwarfs than in 4-month untreated normal mice in the median eminence and the periventricular area surrounding the third ventricle. Thus, a PRL-sensitive TIDA neuron population exists in adult Snell dwarf mice when replacement uses homologous hormone and/or a longer duration. This finding indicates that there is potential for neuronal differentiation beyond early developmental periods and suggests plasticity within the mature hypothalamus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROLACTIN (PRL) IS AN important regulator of a multitude of biological functions. It is best known for effects on lactation, reproduction, and immune response (1). The majority of serum PRL is secreted by pituitary lactotrophs. Pituitary PRL secretion is inhibited by dopamine (DA) (reviewed in Ref. 2), which is primarily secreted by the hypothalamic tuberoinfundibular DA (TIDA) neurons (3, 4). The TIDA neurons (area A12) are located in the arcuate nucleus (ARC) and extend to the external median eminence (ME), where DA is released into the pituitary portal vasculature (5). PRL feedback to the TIDA neurons increases tyrosine hydroxylase (TH) activity, the rate-limiting enzyme for DA synthesis (6), and thereby DA levels (7, 8).

PRL feedback also has been proven to have a role in TIDA neuron development, as shown in studies of GH and PRL-deficient dwarf mice. Ames (df/df) (9) and Snell (dw/dw) (10) dwarf mice lack GH, PRL, and TSH due to mutation in transcription factor Prop-1 (11) or Pit-1 (12), respectively. Both strains of dwarf mice exhibit a secondary deficiency in TIDA neuron number (13, 14, 15) as well as reduced hypothalamic DA (16, 17, 18). Ovine PRL treatment of Ames dwarf mice for 30 d during early postnatal development maintains a normal TIDA neuron population (19). However, mouse PRL treatment of adult Ames dwarf mice for 30 d does not increase TIDA neuron numbers, although increased TH immunostaining intensity is observed (20). Thus, PRL treatment must be initiated before or during the critical developmental period of this neuron population, which is before 21 d of age in Ames dwarf mice (21). These findings suggest that the TIDA neuron population in Ames dwarf mice undergoes cell death during development, resulting in a dearth of TIDA neurons in the adult or that adult Ames dwarf mice lack PRL-sensitive potential TIDA neurons (20).

These studies in Ames dwarf mice used ovine PRL because it is the most commonly available and only suitable injectable source of PRL at these early developmental ages. However, the effect of long-term treatment with homologous PRL on TIDA neuron number in adult dwarf mice has not been assessed. Renal capsule pituitary grafts, used to induce fertility, provide homologous PRL treatment because the pituitaries continue to secrete PRL once removed from hypothalamic inhibition (22, 23, 24, 25). Preliminary analysis of Snell dwarf mice after pituitary graft surgery suggested that such long-term PRL treatment did induce TIDA neuron differentiation (Khodr, C. E., and C. J. Phelps, personal observation).

Therefore, the purpose of the present study was to investigate the effect of long-term mouse PRL on hypothalamic DA neurons in adult Snell dwarf and normal mice. It was hypothesized that long-term, homologous PRL would induce differentiation of a normal-sized TIDA neuron population in Snell dwarf mice. The TIDA neuron population was assessed using both TH immunohistochemistry and DA histofluorescence in both hypophysiotropic and non-hypophysiotropic neuronal nuclei. Furthermore, the possible recruitment of new TIDA neurons may occur either by the stimulation of differentiation in preexisting neurons or by the stimulation of neurogenesis and differentiation of newly synthesized neurons. Therefore, the mechanism of possible TIDA neuron differentiation was investigated using bromodeoxyuridine (BrdU) treatment of PRL-treated dwarf and normal mice to assess whether PRL induces cell proliferation as a possible mechanism of TIDA neuron recruitment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Snell dwarf (dw/dw) and normal (DW/dw) mice were reared from matings of DW/dw females with dw/dw males. Fertility in dw/dw males was induced by treating with D/L-T4 (2 µg ip; Sigma, St. Louis, MO) three times a week, beginning at 6–8 wk of age, followed by renal capsule pituitary grafts from DW/dw donors at 9 wk of age. Pituitary graft transplant surgeries were performed on isoflurane-anesthetized mice. DW/dw donors included males and females from 2–12 months of age. Donors were anesthetized with sodium pentobarbital and decapitated for pituitary gland removal. Whole single glands then were placed under the kidney capsule of recipient mice as previously described (14). Recipients were observed continuously, and body temperature was maintained until recovery from anesthesia. The breeding colony was maintained under controlled temperature (22 ± 2 C) with lights on from 0600 to 1800 h. Food and water were available ad libitum. Procedures for maintenance (26), recovery from surgery, and euthanasia were approved by the Tulane University School of Medicine Animal Care and Use Committee.

Treatments
At 9 wk of age, male and female DW/dw and dw/dw mice received renal capsule pituitary grafts as a source of continuous homologous PRL. Renal capsule pituitary graft surgeries were performed on experimental animals according to the description of induction of dw/dw fertility described above. Pituitary grafted experimental animals received a single whole pituitary gland, inserted underneath the kidney capsule. Control sham animals underwent complete surgical procedures without insertion of a pituitary in the kidney capsule. Mice treated with or without PRL for 4 months were treated with BrdU (50 µg/g ip; Sigma) at 100, 107, 114, and 117 d after surgery; mice treated with or without PRL for 6 months were treated with BrdU at 160, 167, 174, and 177 d after surgery and then euthanized and perfused at 4 (120 d) or 6 months (180 d) after surgery. Numbers of animals for each experimental group are as follows: sham 4 months (four DW/dw male, four DW/dw female, four dw/dw male, and four dw/dw female), graft 4 months (four DW/dw male, four DW/dw female, four dw/dw male, and four dw/dw female), sham 6 months (three DW/dw male, two DW/dw female, four dw/dw male, and four dw/dw female), graft 6 months (four DW/dw male, three DW/dw female, four dw/dw male, and four dw/dw female).

Tissue preparation
Mice were weighed, anesthetized with sodium pentobarbital (120 mg/kg body weight), and then perfused transcardially with 0.9% NaCl, followed by 4% paraformaldehyde-0.5% glutaraldehyde (Faglu) fixative (27). Cycle phase of animals was not assessed at the time the mice were euthanized. Brains were removed and postfixed overnight at 4 C, after which they were transferred to 30% sucrose-Faglu solution to prevent ice crystal formation. Once saturated with sucrose-Faglu, the brains were sectioned frozen using a sliding microtome (Leica Microsystems, Chicago, IL) at 30 µm thickness in the coronal plane. Brain sections were divided at sectioning into six serial sets, each representative of the entire brain, and postfixed overnight. Sections not analyzed immediately were stored in cryoprotectant (28) at –20 C for subsequent use.

Induction of catecholamine fluorescence
Induction of catecholamine fluorescence was performed using Faglu fixation as previously described (14). Every sixth section was mounted out of Faglu and examined for fixative-induced catecholamine fluorescence using narrow-band excitation wavelengths (395–415 nm) and a violet barrier filter (460 nm) on a Nikon E800 microscope equipped for fluorescence epi-illumination.

Immunocytochemistry (ICC) for BrdU and TH
Tissue sections were pretreated with 0.1% hydrogen peroxide (Sigma) to inhibit endogenous peroxidase activity and 1% aqueous sodium borohydride (Sigma) to reduce glutaraldehyde-fixed linkages and allow antibody penetration. For BrdU ICC, sections were also incubated in 6 N HCl for 15 min at room temperature to denature double-stranded DNA and to expose BrdU epitopes. After extensive rinsing with PBS, sections were incubated with 1.5% normal rabbit serum for 1 h to reduce nonspecific staining. Sections were then incubated with sheep {alpha}-TH (0.025 µg/ml; Pel-Freeze, Rogers, AR) or sheep {alpha}-BrdU (0.05 µg/ml; Abcam Inc., Cambridge, MA) for 48 h and processed further using biotinylated rabbit {alpha}-sheep IgG (1:200; Vector Laboratories, Burlingame, CA) and avidin-biotin complex solutions (Vectastain kit; Vector Laboratories). Immunoreactivity was visualized through development using 0.02% diaminobenzidine tetrahydrochloride (Sigma) and 0.003% H2O2 in Tris buffer. Processing for BrdU immunoreactivity included visualization through development in a diaminobenzidine solution containing 0.5% NiSO4.

Sections were then mounted, dried, and coverslipped using DPX mountant (Fisher Chemical Co., Houston, TX). Sections from all treatment groups were processed for ICC simultaneously using identical antiserum aliquots and reagents. Images of endogenous catecholamine histofluorescence, TH immunostaining, and BrdU immunostaining were taken using a DX1200 color digital camera on a Nikon E800 microscope. Adobe Photoshop 7.0 was used to composite images into multiphoto plates.

TH cell counts
Neurons immunoreactive for TH were manually quantified in hypothalamic dopaminergic areas A12 (TIDA), A13 (zona incerta), and A14 (periventricular nucleus) at 180-µm intervals, according to the classification of Björklund and Nobin (29). Cell counts were corrected for periodicity (X6 for every sixth section) but not for recounted or missed cells because the section thickness (30 µm) and interval (180 µm) exceeded perikaryal diameter (30).

BrdU cell counts
Neurons immunoreactive for BrdU incorporation were manually quantified in the subventricular zone (SVZ) of the lateral ventricle (LV), the dentate gyrus (DG), the periventricular region surrounding the third ventricle (3V), the ME, and the ARC at 180-µm intervals throughout entire brains. Examination was performed at x40 magnification on a Nikon E800 microscope. BrdU-immunoreactive cells were quantified in the entire SVZ of the LV, beginning at the wall of the ventricle and extending approximately 25 µm into the surrounding tissue. For the periventricular region surrounding the 3V, BrdU-immunoreactive cells were quantified in the area encompassing the wall of the 3V to approximately 60 µm into the surrounding tissue, excluding the ARC. Counts were then corrected for periodicity (X6 for every sixth section) for comparison.

Statistical analysis
Data were statistically analyzed using ANOVA (SuperANOVA; Abacus Concepts, Berkeley, CA) followed by Student Newman-Keuls post hoc tests. Differences were considered significant at or below the 5% probability level.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Body weight
Body weights of normal (DW/dw) and Snell dwarf (dw/dw) mice treated for 4 or 6 months with or without PRL (graft or sham, respectively) are graphed in Fig. 1Go. There was an interaction between sex and phenotype on body weight (F1,44 = 14.865; P = 0.0004). Sex affected body weight in DW/dw (F7,20 = 5.995; P = 0.0007). Although male DW/dw (40.7 ± 0.7 g, n = 15) weighed more (P ≤ 0.05) than female DW/dw (32.5 ± 1.1 g, n = 13), DW/dw genders were grouped because treatment did not affect body weight in male or female DW/dw. Gender differences also were observed in dw/dw treated with PRL for 6 months (F3,11 = 22.59; P < 0.0001), but not in other dw/dw treatments. Among dw/dw that received pituitary grafts for 6 months, males (24.6 ± 3.0 g) weighed more (P ≤ 0.01) than females (17.5 ± 1.2 g). ANOVA showed that the effect of phenotype on body weight was significant regardless of treatment (F1,58 = 277.6; P = 0.0001). As expected, dwarf mice (16.4 ± 0.7 g, n = 32) as a group weighed less (P ≤ 0.01) than DW/dw (36.9 ± 1.0 g, n = 28). However, PRL treatment for 6 months significantly increased body weight in male dw/dw (F3,12 = 8.969; P = 0.0022), but not in female dw/dw. Male dwarfs treated with PRL for 6 months (24.6 ± 3.0 g, n = 4) weighed more (P ≤ 0.01) than all other dw/dw treatment groups (15.3 ± 0.4 g, n = 28).


Figure 1
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  		FIG. 1. Average body weight of female Snell dwarf (female dw/dw; black bars), male Snell dwarf (male dw/dw; white bars), or normal (DW/dw; gray bars) mice with (graft) or without (sham) treatment with homologous PRL for 4 or 6 months as indicated on the x-axis. Sample sizes (n) are as follows: 6-month sham DW/dw (n = 5), 6-month sham dw/dw (females, n = 4; males, n = 4), 6-month graft DW/dw (n = 7), 6-month graft dw/dw (females, n = 4; males, n = 4), 4-month sham DW/dw (n = 8), 4-month sham dw/dw (females, n = 4; males, n = 4), 4-month graft DW/dw (n = 8), and 4-month graft dw/dw (females, n = 4; males, n = 4). Asterisks represent differences between dwarf and normal, except in the 6-month graft group as indicated: **, P < 0.01. Letters represent differences between treatments; bars labeled with different letters differ by P < 0.01.

 
Catecholamine fluorescence
Figure 2Go shows photomicrographs of DA histofluorescence in ME and TIDA perikarya of normal and Snell dwarf mice after 4 or 6 months treatment with (graft) or without (sham) homologous PRL. Qualitative evaluation of these photomicrographs indicates that normal (DW/dw) mice treated with PRL for either 4 or 6 months had greater TIDA fluorescence than untreated normal mice, but ME fluorescence was similar between all normal groups. Fluorescence in ME and TIDA perikarya of untreated dwarf mice was minimal, and much reduced compared with that in either normal group. Fluorescence in ME and TIDA increased in intensity in Snell dwarf (dw/dw) mice treated with PRL for either 4 or 6 months. Qualitatively, PRL-treated Snell dwarf mice had similar DA fluorescence to that in normal mice. Intensity in ME in PRL-treated dwarfs was similar to that in all normal groups, and perikaryal DA intensity in PRL-treated dwarfs was similar to that in untreated normal mice but did not appear to be as intense as in PRL-treated normal mice. DA fluorescence was similar in non-hypophysiotropic control regions, such as the substantia nigra, between phenotypes and treatments (data not shown).


Figure 2
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  		FIG. 2. Endogenous DA fluorescence in ARC cell bodies and ME terminals of Snell dwarf (dw/dw; lower row) and normal (DW/dw; upper row) mice with (graft) or without (sham) treatment with homologous PRL for 4 (left two columns) or 6 (right two columns) months. Sample sizes (n) are as follows: 6-month sham DW/dw (n = 5), 6-month sham dw/dw (n = 8), 6-month graft DW/dw (n = 7), 6-month graft dw/dw (n = 8), 4-month sham DW/dw (n = 8), 4-month sham dw/dw (n = 8), 4-month graft DW/dw (n = 8), and 4-month graft dw/dw (n = 8). Coronal sections, original objective magnification, x20. The horizontal bar at the lower right represents 50 µm for all panels.

 
TH immunoreactivity
Photomicrographs of TH immunostaining in ARC and ME of normal and Snell dwarf mice treated with or without PRL for 4 or 6 months is shown in Fig. 3Go. Qualitatively, ARC and ME TH immunoreactivity in all normal treatment groups was of similar intensity. TH immunoreactivity was minimal in sham-treated dwarf mice at 4 or 6 months, but TH staining increased in intensity in dwarf mice treated with PRL for either time. TH immunostaining in Snell dwarfs treated with PRL for 6 months appeared greater in both ARC and ME than that in dwarf mice treated with PRL for 4 months. TH immunoreactivity in the non-hypophysiotropic substantia nigra was similar among all phenotypes and treatments (data not shown).


Figure 3
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  		FIG. 3. TH immunoreactivity in ARC neurons and ME of Snell dwarf (dw/dw; lower row) and normal (DW/dw; upper row) mice treated with (graft) or without (sham) homologous PRL for 4 (left two columns) or 6 (right two columns) months. Sample sizes (n) are as follows: 6-month sham DW/dw (n = 5), 6-month sham dw/dw (n = 8), 6-month graft DW/dw (n = 7), 6-month graft dw/dw (n = 8), 4-month sham DW/dw (n = 8), 4-month sham dw/dw (n = 8), 4-month graft DW/dw (n = 8), and 4-month graft dw/dw (n = 8). Coronal sections, original objective magnification, x10. The horizontal bar at the lower left represents 300 µm for all panels.

 
Quantitative comparison of this immunostaining was performed by counting TH-positive perikarya. Figure 4Go illustrates counts of TH-immunoreactive neurons in areas A12, A13, and A14 in normal (DW/dw) and Snell dwarf (dw/dw) mice treated with or without PRL for 4 or 6 months. Initial analysis by three-way ANOVA indicated that sex did not affect area A12, A13, or A14, so genders were pooled. Table 1Go lists TIDA neuron numbers in male and female DW/dw and dw/dw treated with or without PRL for 4 or 6 months. TH-immunoreactive neuron number in normal mice was similar between treatment groups for each anatomical area. There was a significant effect of phenotype on TIDA neuron number (F1,58 = 84.9; P = 0.0001) regardless of treatment. All dw/dw had lower TIDA neuron numbers (1296 ± 103, n = 32) than DW/dw (2409 ± 53, n = 28). Treatment significantly affected TIDA neuron number in dw/dw (F3,28 = 74.4; P = 0.0001) but not in DW/dw. TIDA neuron number was lowest in control dw/dw (776 ± 35, n = 16) and increased (P ≤ 0.01) with PRL treatment. The 6-month PRL-treated dw/dw had higher (P ≤ 0.01) TIDA neuron numbers (2024 ± 98, n = 8) than dw/dw treated with PRL for 4 months (1607 ± 80, n = 8). PRL-treated dwarfs had higher TIDA neuron numbers (P < 0.05 for 4 months, n = 8; P < 0.01 for 6 months duration, n = 8) than the highest neuronal numbers observed previously in developing (7–21 d postnatal) Snell dwarf mice (denoted by the horizontal line in the A12 graph in Fig. 4Go; 1294 ± 138, n = 6; see Ref. 14). Neuron number in area A13 was similar between all treatments and phenotypes. Phenotype significantly affected A14 neuron number (F1,58 = 46.7; P = 0.0001) regardless of treatment. Neuron numbers in area A14 were lower (P ≤ 0.05) in dwarfs (3092 ± 104, n = 32) than in normal mice (4514 ± 108, n = 28).


Figure 4
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  		FIG. 4. TH-immunoreactive neuron number in areas A12 (top), A13 (middle), and A14 (bottom) of Snell dwarf (dw/dw; black bars) and normal (DW/dw; gray bars) mice treated with (graft) or without (sham) homologous PRL for 4 (left bars) or 6 (right bars) months. Sample sizes (n) are as follows: 6-month sham DW/dw (n = 5), 6-month sham dw/dw (n = 8), 6-month graft DW/dw (n = 7), 6-month graft dw/dw (n = 8), 4-month sham DW/dw (n = 8), 4-month sham dw/dw (n = 8), 4-month graft DW/dw (n = 8), and 4-month graft dw/dw (n = 8). Bars represent average, and error bars denote SEM. Differences between dwarf and normal are indicated by asterisks: *, P < 0.05; **, P < 0.01. Letters represent differences between treatments; bars labeled with different letters differ by P < 0.01. The horizontal line indicates the maximum number of TIDA neurons observed during untreated Snell dwarf development 7–21 d postnatally as reported elsewhere (14 ).

 

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  		TABLE 1. TIDA neuron numbers in male and female Snell dwarf (dw/dw) and normal (DW/dw) mice treated with (graft) or without (sham) PRL

 
BrdU immunoreactivity
BrdU immunoreactivity in normal and Snell dwarf mice treated with or without PRL is illustrated in Fig. 5Go in the SVZ of the LV, DG, and the periventricular region surrounding the 3V. BrdU immunoreactivity in the SVZ of the LV was qualitatively greater in dw/dw than that in DW/dw, regardless of treatment. BrdU immunoreactivity in the DG and the periventricular area surrounding the 3V was qualitatively similar between phenotypes and treatments. In ARC and ME, dwarf mice treated with PRL for 4 months had qualitatively similar BrdU immunostaining among all treatment groups (not shown).


Figure 5
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  		FIG. 5. BrdU immunoreactivity in the SVZ (left panels), the DG (middle panels) and the periventricular region surrounding the 3V (right panels) of Snell dwarf (dw/dw; bottom row) and normal (DW/dw; top row) mice treated with (graft) or without (sham) homologous PRL for 4 or 6 months as indicated on each panel. Sample sizes (n) are as follows: 6-month graft DW/dw (n = 4), 6-month graft dw/dw (n = 4), 4-month sham DW/dw (n = 4), and 4-month sham dw/dw (n = 4). Original objective magnification, x40. The horizontal bar at the lower right represents 50 µm for all panels.

 
Figure 6Go graphs manual counts of BrdU-immunoreactive neuron numbers in the SVZ of the LV, DG, Arc, and the periventricular region surrounding the 3V and ME in normal and Snell dwarf mice with and without 4 or 6 months of PRL treatment. Analysis by three-way ANOVA showed no effect of sex on BrdU incorporation in any area, so gender data were pooled. PRL treatment did not affect BrdU incorporation in DW/dw or dw/dw in any area. ANOVA indicated certain differences in BrdU-positive neuron number in the SVZ of the LV, DG, and ME and the periventricular region surrounding the 3V. BrdU-positive neuron number in both the SVZ of the LV and DG was lower (P ≤ 0.01) in 6-month PRL-treated normal mice (LV, 795 ± 207, n = 4; DG, 276 ± 44, n = 4) than in 6-month PRL-treated dwarf mice (LV, 2364 ± 329, n = 4; DG, 528 ± 40, n = 4). Neurons containing BrdU staining in ME and the periventricular region surrounding the 3V was lower (P ≤ 0.05) in 4-month control dw/dw (48 ± 5; n = 4) than in 4-month control DW/dw (102 ± 21; n = 4). No other significant differences were noted.


Figure 6
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  		FIG. 6. Counts of BrdU-labeled neuron numbers in the SVZ of the LV, the DG, the periventricular region surrounding the 3V, and the ARC of Snell dwarf (dw/dw; black bars) and normal (DW/dw; gray bars) mice treated with (graft) or without (sham) homologous PRL for 4 (left bars) or 6 (right bars) months. Sample sizes (n) are as follows: 6-month sham DW/dw (n = 4), 6-month sham dw/dw (n = 4), 6-month graft DW/dw (n = 4), 6-month graft dw/dw (n = 4), 4-month sham DW/dw (n = 4), 4-month sham dw/dw (n = 4), and 4-month graft dw/dw (n = 4). Bars represent average, and error bars denote SEM. Asterisks indicate differences between phenotypes: *, P < 0.05; **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Heterologous PRL has been shown to affect the development of normal-sized TIDA neuron numbers during early postnatal development in Ames dwarf mice (21). The present results show that homologous PRL treatment for longer duration increases TIDA neuron differentiation after the developmental period where PRL has previously been shown to be required for TIDA neuron development in adult Snell dwarf mice. Thus, the increased TIDA neuron numbers observed in the current investigation appear to result from differentiation of new DA neurons from existing arcuate neurons or from neurogenesis. The definition of TIDA neuron differentiation used in this study, as in previous work, was an increased number of neurons compared with the greatest neuron numbers seen during untreated dwarf development. Long-term, homologous PRL was shown to increase TIDA neuron differentiation in adult Snell dwarf animals if administered for 4 or 6 months as shown by an increase in TH-positive neuronal perikarya (Fig. 4Go). Although ME and ARC DA levels were not directly measured, endogenous DA fluorescence qualitatively indicated that ME and ARC DA stores in adult dwarfs treated with PRL were not deficient compared with those in control normal animals (Fig. 2Go). These results suggest the development of new, appropriately functioning TIDA neurons in response to PRL. The present data did not indicate any effect of sex on TIDA neuron number, although previous reports have shown gender differences in the activity of this cell population (8, 31). The absence of these gender differences could be due to the lack of cycle phase assessment at the time the rats were euthanized. Also, qualitative data were not assessed for sexual differences and TH immunoreactivity and CA fluorescence intensity measurements were not taken.

The current results show that long-term, homologous PRL induces sexually dimorphic weight gain in dwarfs when given for 6 months. Adult male, but not female, dwarf animals experienced significant weight gain after 6 months of PRL treatment. Bone length measurements were not taken, so this weight gain cannot be specifically attributed to long bone growth. PRL has been demonstrated to induce food intake in various species (32, 33), which may be a contributing factor to the increased weight observed in this study. Hormone secretion from these transplanted pituitaries would initially perhaps include some GH, which might account for a portion of the weight gain. However, it would not be possible to perform repetitive serum sampling in these mice after implantation in the kidney capsule to determine GH and PRL levels. Previous analysis of renal capsule pituitary grafts indicate that essentially only PRL is secreted, particularly over a period of 6 months (22, 23, 24, 25). Blood samples taken at euthanasia for hormone analysis were lost in the destruction of Hurricane Katrina, so assay was not possible. However, previous studies in this laboratory have addressed PRL levels in Ames dwarf and normal mice treated with pituitary grafts for 30 d. Pituitary-grafted Ames dwarf mice exhibited PRL levels that were about 2.9 times that in control normal animals, and pituitary-grafted normal animals exhibited PRL levels that were about 4.3 times that in control normal animals (20). Thus, PRL levels in the present experimental animals would be expected to be elevated above basal levels. Pituitary grafts provide a source of PRL that is unregulated by the hypothalamus, thereby allowing continuous PRL secretion, likely leading to PRL levels that are elevated above basal levels and lack of rhythmic release. It is possible that administration of PRL in a pulsatile manner would affect TIDA neuron development differently.

The present data show that homologous PRL can induce TIDA neuron differentiation if given for at least 4 months. Thus, TIDA neuron differentiation could result from treatments of shorter duration than 4 months, but longer than the 30 d that was not successful in adult Ames dwarf mice (20). Although increased TIDA neuron numbers were observed at both 4- and 6-month treatment durations, the numbers of TIDA neurons did increase with duration of PRL treatment. This suggests that the TIDA neurons are increasing due to the process of proliferation and differentiation, rather than recovery from quiescence. However, neither treatment duration induced the differentiation of a completely normal number of TIDA neurons. Therefore, it is possible that homologous PRL replacement lasting longer than 6 months would be capable of inducing the differentiation of a normal-sized TIDA neuron population. The effect of long-term, homologous PRL on TIDA neuron differentiation shown in this study indicates that a PRL-sensitive population of potential TIDA neurons exists in the adult Snell dwarf mouse.

The present results refine the original conclusions of Romero and Phelps (20). In that study, PRL treatment of adult Ames dwarf mice did not induce TIDA neuron differentiation but did increase TH immunostaining intensity. It was suggested that dwarf TIDA cells die throughout development or that potential TIDA neurons become insensitive to PRL throughout development. The most probable explanation for the differences observed between these results is that Romero and Phelps (20) treated Ames dwarf mice with PRL for 30 d, whereas the present study used much longer duration (i.e. 4 or 6 months). Thus, an important implication of this finding is that treatments used to ameliorate adult neuronal deficits should be performed over a long time frame.

The Snell dwarf mice used in the current study also differed from the Ames dwarf mice used previously (20). However, Snell and Ames dwarf mice exhibit very similar phenotypes because each has a mutation that leads to deficiency of Pit-1 (34, 35). Ames dwarf (df/df) mice carry a mutation in transcription factor Prop-1 (11), whereas Snell dwarf (dw/dw) mice carry a mutation in transcription factor Pit-1 (12). However, because Prop-1 is required for Pit-1 expression (11, 36), both mutations lead to similar hypopituitary phenotypes due to deficient Pit-1-regulated transcription of GH, PRL, and TSH-{alpha} (34). Both these dwarf mutants exhibit reduced TIDA neuron development without PRL treatment. Snell dwarf mice display an earlier, more severe TIDA neuron deficit than Ames dwarf mice. Ames dwarf mice establish a normal-sized TIDA neuron population by 21 d of age, after which cell numbers decline to 48% of that in normal animals (15). Snell dwarf mice never establish a normal-sized TIDA neuron population during development and exhibit a deficit of 23% of that in normal animals (14). The effect of 30 d PRL treatment on TIDA neuron number in adult Snell dwarf mice has not been investigated, but findings similar to that in Ames dwarf mice would be expected due to the similarities in the mutations and the more severe TIDA neuron deficit seen in Snell dwarf mice.

Snell dwarf mice also had a reduction in the A14 TH-positive neuron number compared with normal mice, regardless of treatment. The occurrence of this deficit confirms previous indications that PRL induces A14 neuron differentiation (37). However, the lack of PRL effect on the A14 neurons in adult Snell dwarf mice suggests that PRL alone is not sufficient to reverse this deficit. The current findings thus suggest the involvement of factors other than PRL, possibly in conjunction with PRL, in A14 neuron development. Because Snell dwarf mice also lack GH and TSH, either of these hormones, or other secondary targets of their action such as IGF-I, may be the factor affecting A14 neuron number.

According to the present findings, PRL may be inducing the differentiation of quiescent TIDA neurons, or it may be inducing both the proliferation and differentiation of progenitor cells. The latter explanation is more likely due to the different PRL effects observed if given for 30 d vs. 4 or 6 months, assuming that adult Snell dwarf mice respond to 30 d PRL treatment in a similar manner to adult Ames dwarf mice. The difference in outcomes between 30 d and 4 months of treatment could be due to the time needed to recruit new neurons via neurogenesis, which would obviously be longer than stimulating the differentiation of quiescent neurons. Therefore, to examine the mechanism of the observed TIDA neuron recruitment, the present study investigated the effects of long-term, homologous PRL on cell proliferation using BrdU incorporation as a marker of cell proliferation. PRL has previously been shown to induce neurogenesis in the SVZ of the LV in vivo in the adult mouse (38). The present BrdU results indicate that long-term, homologous PRL treatment did not induce cell proliferation, although a few differences were observed between phenotypes in the DG and the SVZ of the LV of 6-month PRL-treated mice and in the periventricular area surrounding the 3V and ME of 4-month control mice. These results are less than those reported by Shingo et al. (38), who found that PRL induces neurogenesis in the SVZ of the LV of adult mice, producing new olfactory neurons. The present study investigated the effect of longer-term PRL (4 or 6 months vs. 6 d in the Shingo et al. study) and did not quantify BrdU-immunoreactive neurons in the olfactory lobe. Animals were injected with BrdU at four intervals in the last month of PRL treatment. Initiation of BrdU treatment at the beginning of PRL treatment might be used to determine whether labeling intensity can be increased.

BrdU-positive cells were observed in each area analyzed in the dwarf mice, including ARC. Cell proliferation in the adult is most clearly defined in the SVZ of the LV and the DG (39). However, some reports show that neurogenesis in the adult can occur in brain regions other than the DG and the SVZ of the LV in response to different factors. These other brain regions include the hypothalamus, which is an area that is not conventionally known to experience postnatal neurogenesis. Markakis et al. (40) have isolated postnatal hypothalamic progenitor cells from 7-wk-old rats and demonstrated differentiation into neuroendocrine phenotypes in vitro. These findings suggest the possibility of postnatal hypothalamic neurogenesis if the quiescent progenitors are appropriately stimulated. In vivo postnatal hypothalamic BrdU incorporation has been reported in cells immunopositive for both mature and immature neuron markers in response to brain-derived neurotrophic factor (41) and ciliary neurotrophic factor (42).

The present study has demonstrated the presence of a PRL-responsive population of potential TIDA neurons in the adult dwarf mouse. These neurons differentiate in response to PRL if given for 4 months, although a duration-dependent response is observed. A neurogenic effect of PRL was not observed, but earlier examination of the PRL effect on cell proliferation will provide a clearer concept of the mechanism of TIDA neuron recruitment by PRL. Thus, the lack of neurogenic effect observed in this study is not enough to assume that PRL is solely inducing differentiation of quiescent neurons to the TIDA phenotype. The present study also demonstrated the inability of PRL, alone, to induce area A14 neuron differentiation in adult Snell dwarf mice.


   Acknowledgments
 
This manuscript is lovingly dedicated to the memory of Dr. Carol J. Phelps, Professor of Structural and Cellular Biology at Tulane University Medical School, and wife of David Hurley. Carol started the dwarf mouse project over 20 yr ago and contributed zeal and drive until the day of her death. We all greatly miss her intellect and humor.


   Footnotes
 
The study was supported by Louisiana Board of Regents Support Fund Grant (to C.E.K.), National Science Foundation Grant IBN-0350537 (to D.L.H.) and U.S. Public Health Service Grant NS25987 (to C.J.P.).

Current address for D.L.H.: Department of Pharmaceutical Sciences, College of Pharmacy, East Tennessee State University, Johnson City, Tennessee 37614-1708. E-mail: hurleyd{at}etsu.edu.

Disclosure Summary: The authors of this manuscript have nothing to disclose.

First Published Online December 20, 2007

1 Carol Phelps died on Dec. 7, 2005. Back

Abbreviations: ARC, Arcuate nucleus; BrdU, bromodeoxyuridine; DA, dopamine; DG, dentate gyrus; Faglu, 4% paraformaldehyde-0.5% glutaraldehyde; ICC, immunocytochemistry; LV, lateral ventricle; ME, median eminence; PRL, prolactin; SVZ, subventricular zone; TH, tyrosine hydroxylase; TIDA, tuberoinfundibular DA; 3V, third ventricle.

Received October 18, 2007.

Accepted for publication December 7, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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