Supplementary Material for

Transcription

1 Supplementary Material for Neurotransmitter Switching in the Adult Brain Regulates Behavior Davide Dulcis,* Pouya Jamshidi, Stefan Leutgeb, Nicholas C. Spitzer This PDF file includes: *Corresponding author. Published 26 April 2013, Science 340, 449 (2013) DOI: /science Materials and Methods Figs. S1 to S11 Tables S1 and S2 Other Supplementary Material for this manuscript includes the following: (available at Movies S1 to S4

2 Dulcis et al. Supplementary Materials: Materials and Methods Animals. Male Long Evans rats ( g) were housed three per cage with food and water ad libitum on a reversed 12 h light/12 h dark schedule for acclimation for one week prior to experiments. Animals were then maintained in photoperiod chambers on long day (19L:5D), short day (5L:19D) or control (12L:12D) light/dark cycles for different periods of time. Light for each 24 h photoperiod was administered either in continuum (most experiments) or distributed across the 24 h in 72 min cycles (experiments in ig S ). Light intensity was standardized to 130 lumens. After surgeries, rats were housed individually with free access to food and water. Behavioral training and experimental sessions were conducted in the rats dark phase, unless otherwise specified, while cage cleaning was performed during the light phase. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of California, San Diego. Immunocytochemistry. Animals were sacrificed at 3 pm unless otherwise indicated and perfused with 4% PFA fixative. Brains were removed and post-fixed for 24 hr in 4% PFA fixative, then kept at 4 C in 30% sucrose until they sank (usually 4 days). 30 µm sections of the hypothalamus were washed in phosphate-buffered saline (PBS; ph ) for 30 min and blocked for 30 min in 5% horse serum and 0.3% Triton in PBS. For stereology, sections were incubated in tyrosine hydroxylase (TH) (mouse; Millipore, 1:200) or somatostatin (SST) (rabbit; Chemicon, 1:300) primary antibody in blocking solution overnight at 4 C. After two 15-minute washes in PBS, tissue was treated with a biotinylated anti-mouse or anti-rabbit secondary antibody (1:100; Vector) in 0.3% Triton- PBS solution for 1 h at 21 C, then washed 2x for 15 min in PBS and incubated in avidin biotin complex (ABC) solution freshly prepared in PBS with 0.3% Triton and 2% NaCl for 1 h at 21 C. After two 15-minute washes in PBS, tissue was incubated in fresh 3,3 diaminobenzidine tetrahydrochloride (DAB) solution for 5 min, immediately rinsed twice in PBS for 1 min and for 20 min in a rotator at a speed of 100 rpm, and transferred to PBS in 24-well plates. Sections were mounted in gelatin solution and dried overnight at 21 C, processed for Giemsa staining, dehydrated and coverslipped with Cytoseal. Stereological analysis was performed using a Leica DMRE microscope with a motorized 1

3 Dulcis et al. Z and XY stage encoders linked to a computer-assisted stereological system (Stereo Investigator). For fluorescent labeling, sections were incubated with primary antibodies to TH (mouse; Millipore), dopamine (rabbit; Abcam), DAT (rabbit; Santa Cruz), VMAT2 (goat; Santa Cruz), somatostatin (rabbit; Chemicon), BrdU (rabbit, Abbiotec; rat, AbD Serotec), CRF (mouse, Dr. U. Kumar, Univ. British Columbia), D2R (goat; Santa Cruz) or SSTR2/4 (rabbit; Dr. U. Kumar) and secondary antibodies raised against the primary antibody hosts tagged with 488 nm, 594 nm, or 647 nm excitation fluorophores. Confocal imaging was performed with slide scanners. Single molecule fluorescence in situ hybridization. TH and SST mrna were visualized by locked nucleic acid-based probe hybridization optimized for cryostat sections with the tyramide-fluorescein amplification system. Sections were subsequently processed for TH and SST immunocytochemistry and confocally imaged. Dopamine release. Fluorescent False Neurotransmitter FFN511 was used to assess dopamine release. FFN511 makes nerve terminals fluorescent because it is taken up by VMAT2; it prevents serotonin uptake by VMAT2 so that only the equivalent of dopamine uptake is visualized. When FFN is released, it is no longer fluorescent. 250 µm hypothalamic slices were vibratome sectioned, incubated with oxygenated 10 µm FFN511 for 30 min, and washed for 30 min with oxygenated 100 µm ADVASEP-7 to avoid nonspecific background staining of FFN511. Slices were left to rest for 10 min and 90 µm confocal z-stacks of sections were imaged before and after 4 min application of 100 mm KCl ACSF. Fluorescence in collapsed z-stacks was quantified by scoring pixel intensity of horizontal sections of 50 μm x 50 μm areas immediately adjacent to the PeVN, thresholding, and expressing the result as percentage of the black area (Image J, threshold set to 85). BrdU and TUNEL labeling. Intraperitoneal injections of 0.5 ml of BrdU (50 mg/kg) in saline were made twice a day at 12 h intervals at 7:00 am and 7:00 pm on days 2-4 of week-long photoperiod exposure to detect cell proliferation. Animals were sacrificed and brains were sectioned and stained with antibodies to BrdU. Positive BrdU controls were obtained from the dentate gyrus. In situ cell death detection kit fluorescein TUNEL staining was performed on hypothalamic sections of rats exposed to long/short 2

4 Dulcis et al. photoperiods for one week and compared to staining of sections of control rats. Positive TUNEL controls were obtained by 10 min incubation with DNaseI (3 U ml21 in 50 mm Tris-HCl) at 25 o C to induce DNA breaks before labeling procedures. Surgical techniques. Cerebrospinal fluid (CSF) collection and CRF measurement: Anesthetized animals (3-3.5% isofluorane in O 2 ) were stereotaxically secured, the fur on the neck was removed using Oster Clippers and the position of the head was maintained downward at approximately 45. An 18G gauge needle connected to a syringe via 10 in of polyethylene tubing (0.4 mm ID x 1.3mm OD) was inserted vertically and centrally into the cisterna magna. Gentle aspiration drew CSF through the tubing, yielding µl of colorless CSF. Upon the appearance of blood contamination, the tubing was pinched off above the contamination site and cut at this point. The uncontaminated sample was collected and analyzed with a fluorescent immunoassay kit. Blood collection and corticosterone measurement: Blood samples were drawn from the tail vein of anesthetized animals (3-3.5% isofluorane in O 2 ). Animals were made comfortable in a restrainer. A 23G needle was inserted into the vessel and blood was collected using a syringe. Samples were immediately mixed with anticoagulant K2 EDTA and kept at 4 C while they were centrifuged for 10 min at 1.7 RCF to separate the plasma fraction for immunoassay. 6-hydroxydopamine (6-OHDA) infusion: Animals underwent stereotaxic bilateral infusion of 6-OHDA adjacent to the LPO and PaVN nuclei of the hypothalamus (Suppl. Fig. a b) under isoflurane anesthesia (2-2.5% in O 2 ). Burr holes were drilled (AP, -1.1 mm; ML, ±0.6, ±0.8 mm). 6-OHDA was made up in sterile artificial cerebrospinal fluid (ACSF) at a concentration of 128 mm. A 1 µl Hamilton syringe (25G gauge needle) was lowered (DV, -7.4, -7.6 mm from dura) through each of the holes and 0.25 µl (PaVN) and 0.35 µl (LPO) were infused in each hemisphere at a flow rate of 0.1 µl/min via polyethylene tubing from a microinfusion pump. The syringe was left in place for 5 min after each injection to allow completion of the infusion process. The burr holes were sealed with sterile bone wax and the sites of incision were closed and surgically sutured. Cannula implantation and receptor antagonist infusion: Guide cannulas (24G gauge) 3

5 Dulcis et al. were bilaterally implanted stereotaxically adjacent to the midline (AP, -1.1 mm; ML; ±0.7 mm; DV, -7.5 mm) under isofluorane anesthesia (2-2.5% in O 2 ) to provide access to both the LPO and PaVN nuclei for acute infusion of SCH23390 and sulpiride, following established procedures. Cannulas were secured to the skull using stainless steel screws and dental cement. Dummy needles were inserted into the guide cannulas to prevent their obstruction while the animals were in their cages. To minimize potential distress, animals were handled gently for 5 consecutive days before infusions to habituate them to the environment and the investigator. On the day of bilateral infusion, rats were gently held while the dummy needles were removed and two injection needles (32G gauge outer diameter) 1 mm longer than the guide cannulas were inserted. The needles were connected with polyethylene tubing to two 10 µl Hamilton syringes mounted on the microinfusion pump. Approximately 0.2 µl of SCH23390 (608 mm) plus sulpiride (6 µm) per hemisphere were infused over 1.5 min (flow rate: 0.15 µl/min). At the end of infusion, needles were left in place for an additional minute to enable diffusion of the drugs. The needles were then removed and sterile dummy needles were inserted into the cannulas. Sites of infusion were verified histologically before behavioral results were included in the data set for analysis. Behavioral assays Elevated plus maze: The maze was made of medium-density fiberboard with a matte black acrylic surface and consisted of four arms 50 cm long and 10 cm wide (two open without walls and two enclosed by 30 cm high walls). Addition of a 3 5 mm high railing on the open arms of the plus maze encouraged open arm exploration. Each arm of the maze was attached to sturdy plastic legs, elevated 50 cm off a rotating base that allowed pseudorandom orientation within the room. Prior to the test day animals were introduced to the experimental room for 15 minutes daily for five days and handled by the same investigator. The maze was cleaned with 70% alcohol and dried with paper towels before use. On the day of the test each animal was transferred to the experimental room in its home cage and allowed 30 minutes for acclimation. It was then taken out of its cage and placed at the junction of the open and closed arms in dim light, facing the open arm opposite the investigator. All animals were handled in the same manner and placed in the elevated plus maze in the same position at 3 pm unless 4

6 Dulcis et al. otherwise indicated. During the 10-min test the investigator stood in a separate room, observing through a glass window, and recorded the time of exploration in the open arm. An open arm entry was counted only when all four paws of the animal were on the open arm. At the end of the 10-min test, the animal was placed back in its home cage. Forced swimming test: Animals were placed in individual plastic cylinders (height 90 cm; diameter 25 cm) containing 50 cm of water maintained at 25 C at 3 pm. One session of 15-min pretest swimming was performed 24 h prior to the test day. The test entailed a 7-min swimming trial. Trials were video recorded and the duration of immobility was measured for the 7-minute period. After the test, animals were removed and allowed to dry for 15 min before returning to their individual home cages. Statistics: Data are presented as mean ± s.e.m. Significance was assessed with Student s t-test. * P 0.05; ** P 0.01; *** P

7 PaVN PaVN 3rd ventricle PaVN PaVN CRF PeVN PeVN LPO SCN SCN LPO optic chiasm fig. S1. Wiring diagram of the retino-hypothalamic projection. Dopaminergic nuclei of interest are outlined with black dashed lines. Shown are glutamatergic retinal ganglion cell projections (blue), suprachiasmatic nuclei (SCN, green dashed circles), GABAergic SNC interneurons (green), dopaminergic interneurons (red), and corticotropin (CRF) neurons (purple). Scale bar, 300 µm.

8 A 19L:5D PeVN LPO 1 2 L/ 1 2 D 12L:12D PeVN LPO 5L:19D PeVN LPO B 12L:12D A13 fig. S2. Photoperiods regulate numbers of TH-immunoreactive neurons in the PeVN and LPO but not A13 (quantification in Figs. 1A,E). (A) TH neurons in the LPO and PeVN following exposure to 19L:5D, 12L:12D, and 5L:19D for 1 week. Scale bars, 200 and 60 µm. (B) TH neurons in the A13 nucleus. Scale bars, 200 and 60 µm. N=3 animals for each photoperiod. Boxed regions on left are shown at higher magnification at right.

9 A 12L:12D TH BrdU BrdU 5L:19D TH BrdU BrdU PaVN TH BrdU BrdU TH BrdU BrdU PeVN TH BrdU BrdU TH BrdU BrdU LPO Optic nerve Optic nerve Optic nerve Optic nerve BrdU Dentate gyrus B 19L:5D 19L:5D (DNase1) 1500 TUNEL DRAQ5 C DRAQ5 TUNEL TH cells / nucleus (x1000) L:19D 19L:5D DRAQ5 TUNEL TUNEL, DRAQ5 cells / ROI fig. S LPO PaVN PeVN 19L:5D 5L:19D ** ** ** 0 19L: 5D 12L: 12D ** 5L: 19L:5D 19D (DNase1) ** ** 19L:5D 12L:12D 5L:19D **

10 fig. S3. Photoperiods do not regulate neurogenesis or apoptosis. (A) BrdU incorporation is not detected in the PaVN or LPO of rats exposed to 12L:12D and 5L:19D photoperiods but is observed in the dentate gyrus of 12L:12D animals. BrdU incorporation is present in the PeVN, largely in the ependymal cell region, and does not change with change in photoperiod exposure. Scale bars, 150, 80, 150, and 50 µm. (B) TUNEL staining is absent from the PaVN and PeVN nuclei of rats exposed to the 19L:5D photoperiod but is present following DNase1 treatment (left). Scale bar, 100 µ m. Quantification of TUNEL and DRAQ5 immunostaining after exposure to 19L:5D, 12L:12D, and 5L:19D photoperiods (right). (C) TH respecification is reversible. Quantification of TH-IR neurons following successive 1-week exposures to 19L:5D followed by 5L:19D or 5L:19D followed by 19L:5D, compared to 2-week exposure to 19L:5D, 12L:12D, and 5L:19D photoperiods. A-C, N=3 animals for each photoperiod. **, P 0.01 compared to 12L:12D control.

11 19L:5D 12L:12D 5L:19D TH SST TH SST TH SST PeVN fig. S4. The numbers of SST-IR and TH-IR neurons in the PeVN change following exposure to each of the photoperiods. Scale bar, 80 µ m. N=6 animals for each photoperiod.

12 SCN input 19L:5D 12L:12D 5L:19D PaVN interneurons SST upregulation SST downregulation DA downregulation DA upregulation Terminals in the PeVN CRF target cells 3rd ventricle SST2/4R D2R SST storage vesicles DA GABA CRF fig. S5. Model of transmitter and receptor switching. A long-day (19L:5D) photoperiod shifts transmitter expression in PaVN neurons to somatostatin (SST) and a short-day (5L:19D) photoperiod shifts transmitter expression to dopamine (DA). D2 receptor expression on CRF neurons changes in parallel with changes in DA expression while SST2/4 receptor expression remains constant.

13 A TH VMAT2 VMAT2 TH PaVN B TH VMAT2 VMAT2 TH PeVN C TH VMAT2 VMAT2 TH LPO Optic nerve fig. S6. VMAT2/TH colocalization in the hypothalamus. VMAT2 is colocalized with TH in all dopaminergic neurons in the PaVN (A), PeVN (B) and LPO (C) following 6-OHDA ablation and short day photoperiod neurotransmitter respecification. Scale bar, 80 μm. N=3 animals.

14 A TH DAT DAT TH PaVN B TH DAT DAT TH PeVN C TH DAT DAT TH LPO Optic nerve fig. S7. DAT/TH colocalization in the hypothalamus. DAT is colocalized with TH in all dopaminergic neurons in the PaVN (A), PeVN (B) and LPO (C) following 6-OHDA ablation and short day photoperiod neurotransmitter respecification. Scale bar, 80 μm. N=3 animals.

15 hr cycle (dark phase test) ** ** EPM open arm time (sec) hr cycle (light phase test) 72 min cycle (dark phase test) * 0 ** ** *** 19L:5D 12L:12D 5L:19D fig. S8. Behavioral assessment under different dark/light conditions. Elevated plus maze behavior during the dark phase and light phase of the 24 h cycle following one week exposure to each photoperiod and following distributed presentation of dark/ light exposure in 72 min cycles for one week. N=3 animals for each condition for each photoperiod. *, P<0.05. **, P<0.01. ***, P<0.001 compared to 12L:12D control.

16 A Stereotaxic coordinates: ML: ±600 μm; ±800 μm DV: -7.4mm; -7.6 mm AP: -1.1 mm B ML 800 μm 600 μm 7.4 mm 7.6 mm C 80 PaVN GIEMSA cells (x 1000) ** Optic Chiasm 0 Sham 6-OHDA fig. S9. Ablation of neurons with 6-OHDA injections in animals exposed to 12L:12D photoperiod. (A and B) Stereotaxic coordinates and targeted injection sites on representative images of the hypothalamus. Scale bars, 600 and 200 µm. (C) Stereological counts of the total number of PaVN neurons (Giemsa) following 6-OHDA ablation of neurons compared to sham. N=3 animals for each condition. **, P<0.05.

17 EPM/FST /+ D1,2R blockers (loss of rescue) 6-OHDA infusion Short-day treatment Ablation Transmitter switch EPM/FST (loss of function) EPM/FST (behavioral rescue) SST neuron DA neuron fig. S10. Design of rescue experiments. 6-OHDA is infused into the hypothalamus of rats exposed to 12L:12D, adjacent to the PaVN, PeVN and LPO, to ablate DA neurons. Performance on the elevated plus maze (EPM) and forced swim test (FST) determines loss of function. Animals are then maintained for a week on the 5L:19D photoperiod to induce the appearance of newly DA neurons. Performance on the EPM and FST determines the extent of behavioral rescue. Critically, infusion of DA receptor blockers acutely and locally in the region of these nuclei identifies their contribution to behavioral rescue.

18 A 19L:5D 5L:19D FFN mm KCl FFN mm KCl B 3.4±0.8 FFN511 area fraction (%) ±0.7 ACSF 38.9± ±1.3 6-OHDA (19L:5D) ACSF (12L:12D) 6-OHDA (5L:19D) ** ** ** KCl fig. S11. Functional status of neurons after 6-OHDA ablation and photoperiod exposure. (A) Uptake and KCl-induced release of FFN511 in the PeVN after ablation and one week long or short photoperiod exposure. Scale bar, 80 µm. (B) Fluorescence after 6-OHDA injection (left and right bars) measured in boxed regions compared to fluorescence (central bar) in ACSF-injected controls exposed for one week on the balanced photoperiod. N=3 animals for each condition. **, P 0.01 compared to ACSF.

19 Table S1. Comparison of values at different time points to the mean value within each photoperiod photoperiod Long-day (19L:5D) Control (12L:12D) Short-day (5L:19D) Time 6:00PM 12:00AM 6:00AM 12:00PM 6:00PM 12:00AM 6:00AM 12:00PM 6:00PM 12:00AM 6:00AM 12:00PM LPO * * 0.02 * * 0.02 * PaVN * * PeVN 0.02 * ** * 0.02 * 0.09 t-test ns p>0.05 * p 0.05 ** p 0.01 *** p Table S1. Statistical analysis of TH neuron stereological quantification scored at 6 hr intervals after exposure to different photoperiods. Comparison of values at different time points to the mean value within each photoperiod. N=5 animals for each time point. Table S2. Comparison of values at different long- and short-day time points to control values across photoperiods photoperiod ng-day (19L:5D) Control (12L:12D) Short-day (5L:19D) Time 6:00PM 12:00AM 6:00AM 12:00PM 6:00PM 12:00AM 6:00AM 12:00PM 6:00PM 12:00AM 6:00AM 12:00PM LPO *** *** *** *** *** *** 0.001*** ** PaVN *** *** *** *** 0.006** *** *** 0.019** PeVN *** 0.001*** 0.002** *** 0.02* 0.001*** 0.05* ** t-test ns p>0.05 * p 0.05 ** p 0.01 *** p Table S2. Statistical analysis of TH neuron stereological quantification scored at 6 hr intervals after exposure to different photoperiods. Comparison of values at different long- and short-day time points to control values across photoperiods. N=5 animals for each condition.

20 Movies S1 and S2 Elevated plus maze (EPM) behavior (36 sec) following exposure to 12L:12D (movie 1) or 5L:19D (movie 2) photoperiod for one week. Movies S3 and S4 Forced swimming test (FST) behavior (10 sec). Swimming phase (movie 3) and immobility phase (movie 4) displayed by rats exposed to the control photoperiod.