All experiments were conducted with adult (2–3 months of age), sexually naïve, female musk shrews (Suncus murinus) weighing between 20 and 28 g. All animals were born in the breeding colony at the University of Virginia (Charlottesville, VA). After weaning at 21 days of age, the animals were housed individually in cages (28 × 17 × 12 cm) with food (Purina Cat Chow, Nestle Purina PetCare, St. Louis, MO) and water available ad libitum (except in Experiment 3 in which food restriction was required). The room, containing only females, was maintained on a 14:10 light:dark photoperiod at temperature of 23 ± 2 °C. All experiments were performed in compliance with regulations of the Animal Care and Use Committee of the University of Virginia.
Experiment 1: Food Intake and NPY
In Experiment 1 the impact of NPY on acute feeding was examined. Adult females were subjected to stereotaxic surgery and a cannula was placed in the lateral ventricle. After 7–10 days of recovery from the surgery, basal food intake was measured for 4 days. Animals were briefly anesthetized and received an intracerebroventricular (icv
) infusion with one of four doses of NPY (20, 2, 0.2 and 0.02 nmol; n=8–10 per dose group). Control animals received an infusion of vehicle, artificial cerebral spinal fluid (aCSF; n=8). Next, food intake was measured one, three, and 24 hours after the infusion. All shrews were infused at the same time of the day about 30 minutes before lights in the animal room go off. This time was selected because the majority of feeding occurs in the dark portion of the day (Kauffman and Rissman, 2004
). All feeding groups were initially matched for average body weight and baseline food intake.
Experiment 2: Sexual behavior and NPY
In Experiment 2 the effect of NPY on female receptivity was examined. Adult females were subjected to stereotaxic surgery and a cannula was placed in the lateral ventricle. After 7–10 days of recovery from the surgery, animals were briefly anesthetized and received an icv infusion with one of two doses of NPY (2 nmol; n=12 and 0.2 nmol; n=10). Control animals received an infusion of vehicle, aCSF (n=12). Fifteen minutes after infusion females were paired with a stud male that was habituated to a neutral test box. All shrews were infused and tested at the same time of the day, during the last 4 hours prior to when the room lights went off; musk shrews can display mating behavior at any time in the day (Rissman, unpublished observation). All groups were initially matched for average body weight.
Experiment 3: Immunocytochemistry for NPY
Adult female musk shrews were assigned to one of four feeding groups (n=6 per group); AL, receiving unlimited access to food at all times, FR, food restricted to 60% of baseline intake for 48 hours prior to sacrifice, RF-90, or RF-180, receiving ad libitum
access to food for 90 or 180 min, respectively, after 48 hours of food restriction. These time points were selected based on our past work in which we found that this schedule of food restriction suppressed mating behavior which was reversed in the majority of females by 90 minutes of ad libitum
access to food (Temple and Rissman, 2000b
). Upon sacrifice brains were removed, fixed, and the tissue was processed for immunocytochemistry with a primary antibody against NPY peptide. Pattern of NPY immunoreactivity and differences in the staining between groups was determined. All musk shrews were sacrificed at the same time of the day.
Experiment 4: Immmunocytochemistry for NPY and GnRH II
To assess the colocalization of NPY and GnRH II we conducted a study with brains from 4 females that were food restricted for 12 hours prior to perfusion. The tissue was processed for dual-labeled immunocytochemistry in sequence with first a primary monoclonal antibody against NPY peptide and then a primary rabbit polyclonal antibody made against GnRH II. Presence of NPY- immunoreactive (ir) fibers near GnRH II-ir perikarya was assessed qualitatively. All musk shrews were sacrificed at the same time of the day.
In Experiments 1 and 2, the animals underwent stereotaxic implantation of cannula into the lateral ventricle as previously described (Temple et al., 2003
). Briefly, animals were anesthetized with sodium pentobarbital (4.5mg/ml/kg body weight) and/or isoflurane inhalant. Shrews received a midline incision along the top of the head and bupivacaine (0.25% in 0.1 ml) was injected into the muscles above the skull. Shrews were fitted into a modified mouse stereotaxic apparatus and a guide cannula (26-guage from Plastics One) containing an internal dummy cannula was centered on bregma and positioned −4.5mm rostral-caudal, −1.0mm medial-lateral. A hole was drilled in the skull and the cannula was lowered to a depth of 2.2mm aimed at the lateral ventricle. The cannula was fixed to the skull with glue and dental acrylic and the tip of cannula was secured with dummy cannula (33-guage). After the surgery the animal received an injection of saline (sc) and analgesic (Ketoprofen, 2 mg/kg body weight).
In Experiments 1 and 2, on the day of infusion females were briefly anesthetized with isoflurane inhalant (Burns Veterinary Supplies, Inc. Paul, MN, USA) and infused with 2 μl of either artificial cerebral spinal fluid (aCSF, control group) or different doses of NPY. Using an internal cannula (33 gauge) with a 0.5 mm projection attached to a syringe and delivered slowly with an infusion pump over the course of 1 minute in Experiment 1 and 2 minutes in Experiment 2. The internal cannula was left in place for an additional 30–45 secs after the infusion in order to prevent backflow. Then the dummy cannula was replaced and the shrew returned to its cage. At the end of the study to confirm the placement of cannula shrews were anesthetized with an overdose of sodium pentobarbital and 10 μl of India ink was injected into the cannulas. The brains were removed and sectioned on a cryostat. Cannulas were considered placed correctly if ink was present in the ventricular linings.
Food intake measurement and food restriction
In Experiments 1 and 3 animals received pre-weighed food in excess of their normal 24-h intake. The uneaten food was weighed at 24 hours intervals for 4 days and the difference used to calculate the daily average baseline food intake for each individual. In Experiment 1, after the icv infusion, individual food intake was recorded one, three and 24 hours later. Animals received pre-weighed food and at the specific time points the remaining food was weighed and the difference used to calculate the food intake. In Experiment 3, once the baseline food intake was determined, animals in groups FR, RF-90 and RF-180 were food restricted for 48 hours (they received 60% of their baseline food intake). After the food restriction period, animals in groups RF-90 and RF-180 received food available ad libitum for 90 and 180 min, respectively.
Sexual behavior testing
Female musk shrews do not have estrous cycles and their follicular development and ovulation are both induced by mating (Clendenon and Rissman, 1990
; Rissman, 1992
; Rissman, Silveira, and Bronson, 1988
). Receptivity is initiated by contact with a male. During a typical mating event, sexually naïve females are initially aggressive toward males. The onset of sexual receptivity is marked by a significant reduction in aggression accompanied by tail wagging at which time the male begins to mount and intromit. Usually after a series of mounts that include a single missed, placed or deep intromissions the male ejaculates.
Each female in Experiment 2 was tested once for sexual receptivity 7–10 days after surgery. Tests were conducted between 0900–1300 EST (in the second half of the light portion of the day). Directly after icv infusions females were placed in their home cage for 14 minutes and then introduced into a Plexiglas (dimensions: 39 cm × 18 cm × 10 cm high) testing cage containing a sexually experienced male which had habituated to the test box for at least ten minutes. Each test lasted for 60 minutes or until the male attained an ejaculation. If at the end of 60 minutes the animals were engaged in mounting the test was extended until 2 minutes elapsed without any contact between the two shrews. Latencies for the female to begin tail wagging, receive the first mount, first missed and placed intromissions and the latency to the male’s ejaculation were recorded. Frequencies of male behaviors including the numbers of missed, placed, and deep intromissions were recorded. Numbers of female vocalizations (“yelling”) after the onset of receptive tail-wagging were also recorded.
Brain tissue collection and processing
In Experiment 3 after the appropriate period of food restriction or re-feeding shrews were deeply anesthetized with isofluorane inhalant (Burns Veterinary Supplies, Inc. Paul, MN, USA) and sacrificed by cervical dislocation. The brains were quickly removed, placed into 5% acrolein and incubated on a shaker (160 rpm). After two hours, acrolein was replaced by fresh solution and the brains were fixed overnight at 4°C on the shaker. The next day, brains were placed into 30% sucrose for cryoprotection at 4°C. In Experiment 4 shrews were deeply anesthetized with an overdose of sodium pentobarbital and rapidly perfused first with heparinized saline followed by Zamboni’s fixative (15% picric acid in 4% paraformaldehyde). Brains were removed and placed into 30% sucrose.
The fixed brains were cut into 30 μm thin coronal sections in a cryostat into 2 or 3 series. All rinses and solutions were made in 0.02 M Tris-buffered saline (TBS, pH 7.8). The sections were pretreated in 0.3 % hydrogen peroxide in TBS buffer and then incubated in 1 % sodium borohydride to remove residual aldehydes. The sections were then transferred into avidin and subsequently into biotin blocking solution (Avidin Biotin Blocking Kit, Vector Laboratories, Burlingame, CA) to block endogenous biotin. Next, the tissue was incubated in the primary NPY antiserum (1:10,000; IHC 7180, Peninsula Lab., Belmont, CA) overnight at room temperature. After rinses, tissue was incubated in secondary biotinylated anti-rabbit IgG antiserum made in goat (1:500; Vector Laboratories) and then treated with avidin-biotin complex (1:1,000; Vectastain ABC Kit, Vector Laboratories). Immunoreactivity was visualized with nickel intensified diaminobenzidine solution (0.25 % nickel ammonium sulfate and 0.05 % diaminobenzidine) and activated by 0.001% hydrogen peroxide. In order to minimize the differences in staining all the sections were processed in a single run.
In Experiment 4, to visualize both NPY and GnRH-II in the musk shrew brain sections were cut as described and sequential dual labeling was conducted. We used a monoclonal NPY antibody (1:500; Abnova Corp. Taipei, Taiwan) that was validated by western blot (data not shown) and biotinylated horse anti-mouse (1:200; Vector Laboratories) followed by ABC (1:500; Vectastain ABC Kit, Vector Laboratories) and developed with nickel DAB to yield a black appearance. After rinses in TBS, the tissues were incubated overnight in a GnRH II specific polyclonal antibody (#741 generously provided by Dr. Robert Millar; 1:500) which we have validated for use in musk shrew brain previously (Rissman and Li, 1998
). Next tissues were incubated in biotinylated goat anti-rabbit followed by ABC at the same concentrations listed above, and developed in DAB to give a brown appearance.
For the NPY polyclonal antibody negative controls from three series of brain sections were processed: the first without the primary antibody, the second control was run without the secondary antibody and the third one was run following the regular procedure except the primary antibody was pre-incubated with 50 μg of NPY peptide prior to staining the brain tissue. After the staining, the sections were mounted on gel-coated glass slides (Superfrost Microscope Slides, Fisher), dehydrated, coverslipped with mounting medium (Cytoseal XYL, Apogent, Kalamazoo, MI) and dried. The staining was analyzed with Olympus BX60 light microscope and the fibers density was measured with MetaMorph Series Software (Molecular Devices Corp. Downingtown, PA).
The sections with brain regions of interest were captured and the images were analyzed with MetaMorph Series Software (Molecular Devices, West Chester, PA). Preoptic area (POA), periaqueductal central grey (PAG) and the region in the musk shrew brain containing the GnRH II neuronal cell bodies were examined with a 10x magnification, while bed nucleus of stria terminalis (BNST), medial habenula (mHB), paraventricular hypothalamic nucleus (PVN), arcuate nucleus (ARC), median eminence (ME), and paraventricular thalamic nucleus (PVT) were captured with a 20x magnification. The landmarks used to define brain regions were based on the mouse brain atlas (Franklin and Paxinos, 1997
) as follows: BNST and POA (bregma 0.38 mm to −0.10 mm), mHB and PVT (bregma −0.82 mm to −1.82 mm), PVN (bregma −0.58 mm to −1.22 mm), ARC (bregma −1.34 mm to −2.46 mm), and PAG (bregma −2.70 mm to −4.16 mm). The region that contains the GnRH II cell bodies is defined by the following landmarks; it is dorsal to the PAG, runs medial to the fasciculus retroflexus in the anterior portion of its extent into a region similar in location to the rostral linear nucleus of the raphe in the mouse (bregma −2.92 to −3.88 mm; Dellovade, King, Millar, and Rissman, 1993
For each brain region fiber densities of immunoreactivity were analyzed by computerized gray-level thresholding using MetaMorph image analysis. The light intensity and camera settings were kept constant across all sections and areas to standardize the measurements. Immunoreactivity was expressed as the amount of NPY-ir staining (μm2). A series of sections was used and the data are based on average area covered with NPY-ir per section per animal. The size of the area varied by region but not between subjects.
In Experiments 1 and 3 differences between groups were assessed using analysis of variance (ANOVA) and planned comparisons were conducted with Fisher’s LSD multiple-comparison test. For latency measurements in Experiment 2, only animals that engaged in the behaviors were included in the analysis. Kruskal-Wallis One way ANOVA on ranks were used followed by Z-value tests.