**2. Materials and methods**

#### **2.1 General**

98 Prolactin

to dopamine; and interact with lactotrophs in the peripheral region of the gland (Boockfor & Frawley, 1987; Boockfor et al., 1986; Frawley & Boockfor, 1991; Nagy et al., 1991; Nagy & Frawley 1990; Diaz et al., 2002). In these studies, it is possible that the release of PRL variants

In previous reports (Huerta-Ocampo et al., 2007; Mena et al., 2010) we showed that conditioned media (CM) and PRL variants i.e., from 7-14 and 70-97 kDa, from lactating rat APs, characterized by Western blotting and eluted from SDS-PAGE, promoted the *in vitro* vesicular release of the hormone from preformed, mature PRL granules of male rat APs, and that such release was independent of PRL synthesis (Huerta-Ocampo et al., 2007). Autocrine and paracrine types of actions have also been shown to occur within the AP (Denef, 2008; Freeman et al., 2000; Lorenson and Walker, 2001; Schwartz & Cherny, 1992; Schwartz, 2000; Welsch et al., 1968; Diaz, et al., 2002; Huerta-Ocampo et al., 2007; Mena et al., 2010), and were demonstrated when the central and peripheral AP regions of lactating rats were incubated *in vitro* with CM from pituitaries of lactating, pregnant and steroid-treated castrated males or females, but not from untreated castrated rats, intact male rats or by a PRL Standard (Huerta-Ocampo et al., 2007; Mena et al., 2010). Also, more potent effects occurred with CM from APs of early- than from mid- or late- lactating rats and from rats non-suckled for 8 or 16 h than from those non-suckled for 32 h (Mena et al., 2010). These results suggest that, under certain conditions, PRL variants released from lactating and non-

lactating rat APs may regulate the release of PRL variants from the lactotrophs.

In the present study, CM proteins, i.e., PRL variants, that were released *in vitro* from the AP regions of lactating rats were separated and electroeluted from SDS-PAGE and tested using *in vitro* incubation techniques. We sought to determine first, whether PRL variants, which are known to occur within the AP (Schwartz & Cherny, 1992; Schwartz, 2000; Wang & Walker, 1993; Sinha, 1992; Bollengier et al., 1989; Huerta-Ocampo et al., 2007; Mena et al., 2010; Mena & Grosvenor, 1972; Asawaroengchai et al., 1978; Nicoll et al., 1969; Mansur & Hymer, 1985), and are released *in vitro* (Mena et al, 1984; Mena et al., 1992; Huerta-Ocampo et al., 2007; Mena et al., 2010; Mena & Grosvenor, 1972; Grosvenor et al., 1967; Grosvenor et al., 1979; Mena et al., 1989; Mena et al., 1993) after the suckling-induced PRL transformation i.e., the transfer of the hormone from a pre-releasable to a releasable state (Mena & Grosvenor, 1972; Grosvenor et al., 1967; Mena et al., 1993) would influence the release of PRL variants from lactating rat lactotrophs; and second, whether the effects of dopamine (DA), thyrotropin-releasing hormone (TRH) and oxytocin (OT) upon PRL release would manifest their effects upon PRL secretion by regulating the release of PRL variants from

Several reports indicate that PRL has some neuro and gliatrophic properties, and that it mediates the development and maturation of dopaminergic neurons in the hypothalamopituitary system (Möderscheim et al., 2007). We showed previously (Morales et al., 2001) that intrathecal injection of PRL in the spinal cord promoted the sympathetic inhibition of milk ejection in lactating rats, and that prolactin variants in CM from the central and peripheral regions of the anterior pituitary from lactating, but not from male rats promoted

may have influenced the regulation of PRL release.

lactating rat lactotrophs (Mena et al., 2011).

Animal studies were performed under a protocol similar to the USPHS Guide for the Care and Use of Laboratory Animals and the Official Mexican Guide from Secretary of Agriculture (SAGARPA NOM-062-Z00-1999) published in 2001. Wistar primiparous lactating rats (8-10 pups per litter) were housed individually in a room with a reversed lightdark cycle (14 h light, 10 h darkness) and constant temperature (23-25°C) and were fed *ad libitum* (Purina Chow, Ralston Purina Co., Chicago IL, USA). On postpartum days 10-12 (7 am, local time) groups of mothers had their pups removed, and 6 h later their pups were or were not returned to the mothers and suckled for 15 min. At the end of the suckling or nonsuckling periods, the mothers were killed by decapitation after light ether anesthesia. From all animals employed (see below), the pituitary was removed under a dissecting microscope, the posterior lobe was discarded and, using fine forceps as originally described by Papka et al. 1986, and by Bookfor & Frawley, 1987, the central region around the neurointermediate lobe and the peripheral region i.e., the rest of the AP tissue (Boockfor & Frawley, 1987; Diaz et al., 2002) were dissected independently, and incubated in Earle's medium as described below.

#### **2.2. Preparation of concentrated conditioned media**

In individual flasks containing 300 μl of Earle's medium, media were conditioned by incubating tissue fragments corresponding to the central (CR) and peripheral (PR) pituitary regions from lactating rats. The pituitary fragments were incubated immediately after removal to prevent disruption of hormone storage dynamics (Mena et al., 1992; Diaz et al., 2002). Flasks containing the pituitary fragments were gassed with 95% O2, 5% CO2, sealed with rubber stoppers and incubated at 37°C in a water bath shaker (American Optical, Buffalo NY, USA) for 1h. CM from pituitary fragments of each group of rats employed was concentrated and desalted in a Centricon micro-concentrator (Centripep, Millipore, Bredford MA, USA) and stored frozen until assayed, along with the corresponding primary cultures of pituitary cells or with cultures of sympathetic neurons.

The dose-response effects of DA (0.5, 1.0, 1.5 μM), TRH (0.1, 1.0, 10 μM) and OT (0.1-10 μM), upon the in vitro release of PRL variants previously exposed to the electroeluted PRL variants from NS and S lactating rat APs were determined by Enzyme-linked immunoabsorbant assay (ELISA).

Autocrine and Paracrine Regulation of Prolactin Secretion

Cultures of sympathetic neurons were obtained as previously reported by Fiordelisio & Hernandez-Cruz, 2002. Briefly, ganglia from 10-day-old rats were removed under aseptic conditions after ether anaesthesia and cervical dislocation. After cleaning and chopping, the neurons were incubated in Ca2+/Mg2+-free Hanks solution with 1 mg/ml trypsin (Worthington Biochem Co.) and 2 mg/ml DNAse I for 30 min at 37ºC. After digestion, trypsin was inactivated by dilution in DMEM containing 10% fetal bovine serum (FBS) and 1 mg/ml trypsin inhibitor (Sigma) and the tissue was incubated in Hanks solution with 2 mg/ml collagenase and 2 mg/ml DNAse I for 30 min at 37ºC. After trituration with a Pasteur pipette, the cell suspension was centrifuged, washed twice in Hanks solution, and resuspended in fresh control culture medium. Cells were seeded on poly L-lysine-treated #1 round glass coverslips (1r105 cells per well), and maintained in control culture medium supplemented with 30 ng/ml of 7S NGF (Sigma) at 37ºC in a humidified atmosphere of 95% air and 5% CO2. Culture medium was changed three times per week. All experiments were carried out with

Astrocyte ion currents were recorded using the whole-cell voltage-clamp technique, as shown previously by Hernández-Morales and García-Colunga, 2009. Primary astrocytes between 4-7 days of culture were placed in a recording chamber and continuously superfused with control solution containing (mM): 136 NaCl, 2.5 KCl, 10 HEPES, 4 CaCl2, 0.5 MgCl2, 10 glucose, pH 7.2. Pipettes were filled with a solution containing (mM): 130 Kgluconate, 10 NaCl, 10 EGTA, 10 HEPES, 2 ATP, 0.2 GTP, pH 7.2, having a resistance of 3-5 M. Astrocytes were held at a potential of -60 mV. The data were analyzed with pClamp 8.2 software and Origin 7. We determined changes in electrical activity (EA) in response to CM's from lactating and from male rats in male lactotrophs, as well as in neurons from the

Primary astrocytes from hippocampus, medial preoptic area, sympathetic neurons or male rat lactotrophs, between 4-7 days of culture, were incubated for 30 min with the Ca2+ indicator Fluo 4-AM. Following this, cells were washed out three times for 10 min with solution containing (mM): 136 NaCl, 2.5 KCl, 10 HEPES, 4 CaCl2, 0.5 MgCl2, 10 glucose, pH 7.2. Cells were recorded on a Confocal Microscope LSM-510. Local application of CM was carried on with U-tube system placed at 250 μm from the recorded cell. Cells were excited at 488 nm-Ar laser. Fluorescent intensity represents the change in intracellular calcium concentration and is measured in the whole soma. Data are expressed as a change from basal intensity (F0) to maximum intensity reached (%ΔF), %ΔF/F0. Then, we determined fluctuations in intracellular Ca2+ concentration, [Ca2+]i, in male lactotrophs in response to CM

**2.6. Cultures of sympathetic neurons** 

cultures less than 5 days old.

**2.7. Electrophysiology** 

central nervous system.

from lactating and from male rats.

**2.8. Measurements of intracellular Ca2+ concentration** 

by Prolactin Variants and by Hypothalamic Hormones 101

#### **2.3. SDS-PAGE**

The PRL released into the media was determined by non-denaturing SDS-PAGE (12.5% gels) and Western blotting. The gels of SDS-PAGE were divided into 6 fractions which encompassed PRL variants from 6 to 97 kDa. The proteins in each fraction were electrophoretically eluted, dialyzed, lyophilized, and then assayed by ELISA for PRL content, as well as for their effects upon PRL secretion on primary culture of pituitary cells from the lactating rats, different concentrations of hypothalamic hormones, and in central and peripheral cultures of sympathetic neurons and astrocytes from hippocampus.

#### **2.4. Primary cultures of pituitary cells**

Pituitary fragments from male APs, or from NS and S lactating rats (n=5) corresponding to the central and peripheral regions of the anterior pituitary were dissected and processed separately. Primary cultures (lactotrophs) were prepared as described by Fiordelisio & Hernandez-Cruz, 2002. The tissue fragments were gently triturated with a Pasteur pipette; the cells were collected by centrifuging for 10 min at 185 x g, and washed twice with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% BSA. The pellet was resuspended in DMEM medium, supplemented with 10% horse serum, 2% Fetal Bovine Serum, 10,000 U penicillin, 10 mg/ml streptomycin, all from Gibco BRL, Grand Island NY, USA. The cultures were maintained for 24 h at 37°C in a humidified atmosphere (95% air and 5% CO2). The primary cultures were placed in the bottom of 24-multiwell culture plates (Costar, Cambridge, MA, USA) at a density of 2 x104 cells per well and three replicates were used in each experiment.

#### **2.5. Cultures of astrocytes from the hippocampal and medial preoptic areas**

Astrocyte cultures were obtained as shown previously by Hernández-Morales and García-Colunga, 2009. Two newborn Wistar rats were killed by decapitation and their brains removed. Slices from hippocampal CA1 region, or medial preoptic area were dissected and then the tissue was dissociated (5000 cells/mL). The suspended cells were placed on a glass coverslip in a 35-mm Petri dish coated with poly-L-ornithine with DMEM supplemented with 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 11 μg/mL piruvate was added. After 24 h, the medium was changed by Neurobasal medium supplemented with G5 (specific for growing astrocytes) and 100 U/mL penicillin, and 0.1 mg/mL streptomycin and 2 mM of L-glutamine. Cultures were kept under controlled air and temperature.

#### **2.6. Cultures of sympathetic neurons**

100 Prolactin

(ELISA).

**2.3. SDS-PAGE** 

**2.4. Primary cultures of pituitary cells** 

used in each experiment.

The dose-response effects of DA (0.5, 1.0, 1.5 μM), TRH (0.1, 1.0, 10 μM) and OT (0.1-10 μM), upon the in vitro release of PRL variants previously exposed to the electroeluted PRL variants from NS and S lactating rat APs were determined by Enzyme-linked immunoabsorbant assay

The PRL released into the media was determined by non-denaturing SDS-PAGE (12.5% gels) and Western blotting. The gels of SDS-PAGE were divided into 6 fractions which encompassed PRL variants from 6 to 97 kDa. The proteins in each fraction were electrophoretically eluted, dialyzed, lyophilized, and then assayed by ELISA for PRL content, as well as for their effects upon PRL secretion on primary culture of pituitary cells from the lactating rats, different concentrations of hypothalamic hormones, and in central

Pituitary fragments from male APs, or from NS and S lactating rats (n=5) corresponding to the central and peripheral regions of the anterior pituitary were dissected and processed separately. Primary cultures (lactotrophs) were prepared as described by Fiordelisio & Hernandez-Cruz, 2002. The tissue fragments were gently triturated with a Pasteur pipette; the cells were collected by centrifuging for 10 min at 185 x g, and washed twice with Dulbecco's Modified Eagle's Medium (DMEM) containing 10% BSA. The pellet was resuspended in DMEM medium, supplemented with 10% horse serum, 2% Fetal Bovine Serum, 10,000 U penicillin, 10 mg/ml streptomycin, all from Gibco BRL, Grand Island NY, USA. The cultures were maintained for 24 h at 37°C in a humidified atmosphere (95% air and 5% CO2). The primary cultures were placed in the bottom of 24-multiwell culture plates (Costar, Cambridge, MA, USA) at a density of 2 x104 cells per well and three replicates were

**2.5. Cultures of astrocytes from the hippocampal and medial preoptic areas** 

2 mM of L-glutamine. Cultures were kept under controlled air and temperature.

Astrocyte cultures were obtained as shown previously by Hernández-Morales and García-Colunga, 2009. Two newborn Wistar rats were killed by decapitation and their brains removed. Slices from hippocampal CA1 region, or medial preoptic area were dissected and then the tissue was dissociated (5000 cells/mL). The suspended cells were placed on a glass coverslip in a 35-mm Petri dish coated with poly-L-ornithine with DMEM supplemented with 10% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin and 11 μg/mL piruvate was added. After 24 h, the medium was changed by Neurobasal medium supplemented with G5 (specific for growing astrocytes) and 100 U/mL penicillin, and 0.1 mg/mL streptomycin and

and peripheral cultures of sympathetic neurons and astrocytes from hippocampus.

Cultures of sympathetic neurons were obtained as previously reported by Fiordelisio & Hernandez-Cruz, 2002. Briefly, ganglia from 10-day-old rats were removed under aseptic conditions after ether anaesthesia and cervical dislocation. After cleaning and chopping, the neurons were incubated in Ca2+/Mg2+-free Hanks solution with 1 mg/ml trypsin (Worthington Biochem Co.) and 2 mg/ml DNAse I for 30 min at 37ºC. After digestion, trypsin was inactivated by dilution in DMEM containing 10% fetal bovine serum (FBS) and 1 mg/ml trypsin inhibitor (Sigma) and the tissue was incubated in Hanks solution with 2 mg/ml collagenase and 2 mg/ml DNAse I for 30 min at 37ºC. After trituration with a Pasteur pipette, the cell suspension was centrifuged, washed twice in Hanks solution, and resuspended in fresh control culture medium. Cells were seeded on poly L-lysine-treated #1 round glass coverslips (1r105 cells per well), and maintained in control culture medium supplemented with 30 ng/ml of 7S NGF (Sigma) at 37ºC in a humidified atmosphere of 95% air and 5% CO2. Culture medium was changed three times per week. All experiments were carried out with cultures less than 5 days old.

#### **2.7. Electrophysiology**

Astrocyte ion currents were recorded using the whole-cell voltage-clamp technique, as shown previously by Hernández-Morales and García-Colunga, 2009. Primary astrocytes between 4-7 days of culture were placed in a recording chamber and continuously superfused with control solution containing (mM): 136 NaCl, 2.5 KCl, 10 HEPES, 4 CaCl2, 0.5 MgCl2, 10 glucose, pH 7.2. Pipettes were filled with a solution containing (mM): 130 Kgluconate, 10 NaCl, 10 EGTA, 10 HEPES, 2 ATP, 0.2 GTP, pH 7.2, having a resistance of 3-5 M. Astrocytes were held at a potential of -60 mV. The data were analyzed with pClamp 8.2 software and Origin 7. We determined changes in electrical activity (EA) in response to CM's from lactating and from male rats in male lactotrophs, as well as in neurons from the central nervous system.

#### **2.8. Measurements of intracellular Ca2+ concentration**

Primary astrocytes from hippocampus, medial preoptic area, sympathetic neurons or male rat lactotrophs, between 4-7 days of culture, were incubated for 30 min with the Ca2+ indicator Fluo 4-AM. Following this, cells were washed out three times for 10 min with solution containing (mM): 136 NaCl, 2.5 KCl, 10 HEPES, 4 CaCl2, 0.5 MgCl2, 10 glucose, pH 7.2. Cells were recorded on a Confocal Microscope LSM-510. Local application of CM was carried on with U-tube system placed at 250 μm from the recorded cell. Cells were excited at 488 nm-Ar laser. Fluorescent intensity represents the change in intracellular calcium concentration and is measured in the whole soma. Data are expressed as a change from basal intensity (F0) to maximum intensity reached (%ΔF), %ΔF/F0. Then, we determined fluctuations in intracellular Ca2+ concentration, [Ca2+]i, in male lactotrophs in response to CM from lactating and from male rats.
