SUPPLEMENTARTY INFORMATION “Disinhibition of somatostatin positive interneurons results in an anxiolytic and antidepressant-like brain state” Thomas Fuchs1*, Sarah J. Jefferson1*, Andrew Hooper4, Pei-Hsuan Patricia Yee1, Jamie Maguire5 and Bernhard Luscher1-3 1
Department of Biology, Pennsylvania State University, University Park, PA 16802.
2
Department of Biochemistry & Molecular Biology, Pennsylvania State University, University Park, PA 16802. 3
Center for Molecular Investigation of Neurological Disorders (CMIND), Pennsylvania State University, University Park, PA 16802. 4
Graduate Program in Neuroscience, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, MA, United States 5
Department of Neuroscience, Tufts University School of Medicine, Boston, MA 02111
* These authors contributed equally to this work Correspondence: Bernhard Luscher, Ph.D., Department of Biology, Penn State University 301 Life Sciences Building, University Park, PA 16802, E-mail:
[email protected], Phone office: 814-865 5549 MATERIALS AND METHODS Animals All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Pennsylvania State University and performed in accordance with all relevant guidelines and regulations of the National Institute of Health (NIH). SSTCre mice 1, also known as Ssttm2.1(cre)Zjh/J, were obtained from JAX mice (Stock #013044, Jackson Laboratory, Bar Harbor, ME). The γ2f/f mice carrying a Gabrg2 locus with exon 8 flanked by lox P sites (JAX Stock# 013044016830, Gabrg2tm2Lusc/J) were produced in house as previously described 2 3-5. The LSL-YFP Cre reporter strain also known as ROSA26-EYFP 6 was obtained from JAX mice
(Stock #006148, Gt(ROSA)26Sortm1(EYFP)Cos/J). All mice were backcrossed to the 129X1/SvJ genetic background for at least six generations. The genotypes compared in experiments were
1
produced as litter-mates by mating γ2f/f mice with SSTCre:γ2f/f mice or by mating γ2f/+:LSL-YFP with SSTCre:γ2f/f mice and identified by PCR analyses of tail DNA isolated at the time of weaning, using PCR conditions previously described 5. The mice were maintained on a 12:12 h light-dark cycle with food and water available ad libitum. The mice compared were produced as littermates in our own breeding facility and matched for sex and age, except for behavioral testing where the sexes were analyzed separately.
Immunohistochemistry Mice were deeply anesthetized with Avertin and perfused transcardially with ice cold, oxygenated ACSF containing 125 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 2 mM MgCl2, 26 mM NaHCO3, 1.25 mM NaH2PO4, 25 mM glucose, pH 7.4, at a flow rate of 5 mL/min. The brains were rapidly removed, cut in halves and emersion fixed in 4% paraformaldehyde in phosphate buffered saline (PBS), pH 7.4, for 60 min. The tissue was rinsed in PBS, cryoprotected overnight in 30% sucrose in PBS (4 °C) and then frozen with powdered dry ice and stored at −80 °C until sectioning. Free floating sections (30 μm) were cut from frozen blocks with a cryostat and collected in PBS, blocked in 0.2% TritonX-100, 2% normal serum and 2% BSA for one hour and then immunostained with chicken anti GFP (1:1000, #GFP-1020, Aves Labs, Tigard, OR), guinea pig anti GABAA receptor γ2 (1:500, #224 004, Synaptic Systems, Göttingen, Germany) and mAb7a anti gephyrin (1:500, #147011, Synaptic Systems) at 4 °C for 48 h. Sections were rinsed four times and then overnight in PBS followed by incubation in secondary antibodies goat anti chicken Alexa 647 (1:800, Jackson Immuno Research, West Grove, PA), goat anti guinea pig Alexa 488 and goat anti mouse Cy3 (Molecular Probes, Eugene, OR) for one hour at room temperature before washing and mounting onto slides. +
Confocal image stacks (3 μm) of SST/YFP neural somata were sampled from CA1 strata pyramidale and radiatum, using an Olympus FV-1000 confocal microscope with a 100X/NA1.4 oil objective, a 3x zoom and 0.28 μm Z-axis steps. Image stacks were collapsed and quantitated
2
using Image J and hand tracing of GFP-positive somata. Measures of area and number of puncta were used to determine puncta densities.
Electrophysiological Recordings The mice were anesthetized with isoflurane, decapitated, and the brains removed and placed in ice cold, oxygenated normal artificial cerebral spinal fluid (nACSF) containing 126 mM NaCl, 26 mM NaHCO3, 1.25 mM NaH2PO4, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, and 10 mM dextrose (300-310 mOsm) supplemented with 3 mM kynurenic acid. Coronal sections (350 μm) were cut with the tissue submerged in the same solution using a Leica vibratome. The slices were stored at 34°C in nACSF with adequate O2 tension at physiological pH (~7.4) for at least 1 h before recording. Hemislices were placed into a recording chamber also maintained at 34°C and perfused with nACSF continually bubbled with a gas mixture of 95% O2/5% CO2 at a minimum of 4 ml/min. Whole cell voltage clamp recordings were performed on visually-identified SST+ interneurons and principal neurons in the CA1 subregion of the hippocampus and in L2/3 of the cingulate cortex as previously described 7 8. Intracellular recording solution containing 140 mM cesium-methylsulfonate, 10 mM Hepes, 0.2 mM EGTA, 5 mM NaCl, 2 mM MgATP, 0.2 mM NaGTP (~280 mOsm, pH ~7.25) and electrodes with DC resistance of 5-8 MΩ were used for recording spontaneous excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs) at VH = -70 mV and VH = 0 mV, respectively. The frequency, peak amplitude, and weighted decay (τw) of EPSCs and IPSCs were measured during a 5 min period at each holding potential. Miniature IPSCs (mIPSCs) and miniature EPSCs (mEPSCs) were recorded at each respective holding potential in the presence of 50 µM CdCl2. Tonic GABAergic currents were measured in the whole cell voltage clamp configuration using an high Cl- intracellular solution containing 140 mM CsCl, 1 mM MgCl2, 10 mM HEPES, and 4 mM Na-ATP (~280 mOsm, pH ~7.25) as previously described 7, 9-11. The mean holding current was measured over 10 ms epochs collected every 100 ms throughout the duration of the experiment. A Gaussian
3
function was fit to these points in the over a min recording period in the presence or absence of SR95531 (>200 μM, Sigma, St. Louis, MO). The tonic current was measured as the difference in the peaks of the Gaussian curves. Current clamp recordings were performed on visuallyidentified SST+ interneurons and principal neurons in the CA1 subregion of the hippocampus and in L2/3 of the cingulate cortex using an intracellular recording solution containing: 130 mM K-gluconate, 10 mM KCl, 4 mM NaCl, 10 mM HEPES, 0.1 mM EGTA, 2 mM Mg-ATP, 0.3 mM Na-GTP (pH = 7.25, 280–290 mosm) to determine the number of action potentials generated in response to a series of 500 ms current injections from 20–300 pA in 20 pA steps. Input resistance was calculated using Ohm’s law in response to a -100 pA current injection. Inputoutput curves were fit with a Boltzmann equation: f(W) = (MAX/(1 + exp((I - I50)/k)) + MAX), where I is current injected, MAX is the maximum response, k is a slope factor, and I50 is the current injection amplitude that elicits 50% of MAX. In these set of experiments, the MAX was considered the maximum number of spikes fired in response to the maximum (300 pA) current injection. Data acquisition was carried out using an Axon Instruments Axopatch 200B and Powerlab hardware and software (ADInstruments, Colorado Springs, CO) and data analysis was performed using either LabChart Pro (ADInstruments) or MiniAnalysis software (Synaptosoft, Decatur, GA). Series resistance and whole-cell capacitance were continually monitored and compensated throughout the course of the experiment. Recordings were eliminated from data analysis if series resistance increased by > 20%.
Behavioral Testing The minimal sample size for behavioral studies was estimated based on effect sizes of comparable previous analyses of GABAAR γ2+/- mice 4, 12. For a 2-way ANOVA and an α level of 0.05, the minimal effect size of 0.85 SD and a power of 0.8 suggested that a minimal group size of 12 is sufficient for detection of main sex and genotype effects (G*Power, http://www.gpower.hhu.de). Animals produced as litter mates were weaned into same sex
4
groups (up to 10 per group), genotyped, equipped with ear tags, and sorted by genotype into same sex groups (maximally six) in gang cages, in one-sex-only-rooms, under a reversed lightdark cycle (lights on at 7 PM). Each cage was coded with a random number displayed on a cage card. No genotype information was displayed. Animals were tested two at a time using two identical test setups and returned to a fresh empty cage with the same cage number. This was repeated for each cage, cycling through cages until all animals were removed from the old cages. The order of cages and animal testing was changed randomly between behavioral tests and scoring was conducted blind to genotype. Behavioral testing started at nine weeks of age, with one test per week and starting with the Open Field test (OFT), followed by Elevated Plus Maze (EPM), Novelty Suppressed Feeding Test (NSFT), Forced Swimming Test (FST). The Learned Helplessness Test (LHT) was conducted with a separate cohort of mice. All testing was performed under red light, between two and six h after the beginning of the dark phase and at least 48 h after the last cage change. All experiments were carried out and scored by investigators blind to genotype. OFT and EPM were conducted in an “odor saturated environment”, i.e. the testing equipment was saturated with mouse odor prior to testing. After each trial feces were removed and the equipment was wiped clean with paper towels. Only same sex groups were tested on any given day.
The OFT was used to assess locomotion in a novel environment, under red light. Mice were allowed to freely explore an opaque Plexiglas arena (50 x 50 x 20 cm). The behavior was video recorded and motor activity (path length) was analyzed in 5 minute bins using the EthoVision XT video tracking system (Noldus Information Technologies, Leesburg, VA).
The EPM 13 apparatus consisted of an elevated (40 cm) crossbar with two open and two closed arms (30 cm X 5 cm). Closed arms were surrounded by 20 cm walls of clear Plexiglas. The edges of open arms were raised by 2 mm to minimize falling. Mice were placed into the center
5
square of the maze facing a closed arm. Behavior was video recorded for five min using the EthoVision XT video tracking system.
For the NSFT 14, mice were food deprived for 18 h and then placed into a corner of a Plexiglass arena (50 x 50 x 20 cm) containing 3 cm of saw dust bedding and a pellet of rodent chow placed in the center on a white cotton nesting square (6 x 6 x .5 cm). The latency to feed was scored when the mouse was chewing on the pellet while sitting on its hind paws. Trials were stopped after 10 min when no feeding occurred.
The FST 15 assessed escape behavior in a plastic beaker measuring 19 cm in diameter and 27 cm in height, containing 25 ±1°C water to a height of 18 cm. Mice were placed into the beaker and their activity was video recorded for 6 min. Latency to the first episode of passive floating (Time to first immobility) was recorded manually. The total time immobile during the last 4 min and average swim speed during the first minute of each trial were analyzed with the Ethovision XT video tracking system.
The LHT was adapted from reference16. The mice were exposed to 120 inescapable foot shocks (.3 mA, 15s) at an average interval of 45 s. Sessions took place two mice at a time in adjacent compartments of a two compartment shuttle box with the connecting gate closed (SanDiego Instruments, San Diego, CA). Twenty-four h later mice were tested individually in the same shuttle box in a 30 trial active avoidance task (escapable foot shock). The foot shock (.3 mA, 15 s max) was signaled 3 s prior to onset by a signal light and the opening of the connecting gate. Escape failures were recorded automatically.
Sucrose Preference Test. The mice received a 12 h training session during which their drinking water was replaced with 25 ml pipette of a 2 % sucrose solution. Thirty-six h later the mice were
6
water deprived for 12 h followed by a choice between two 25 ml pipettes, containing either water or a 2% sucrose solution for the next 24 h. The positions of water and sucrose pipettes were counter-balanced. Amounts of sucrose and water consumed were monitored at two time points (4 and 24 hours).
Morris Water Maze 17. Mice were trained for five days with four trials per day to locate a submerged platform in a 140 diameter pool of water clouded with black acrylic paint. Latencies to reach the platform were recoded with Ethovision software and averaged for each day of acquisition. Seven days after the final acquisition trial the mice were tested in the absence of a goal platform in a 60 s test trial. Time to reach the target area and number of area entries were recorded with Ethovision software.
Tissue extract preparation and western blotting Hippocampus and medial prefrontal cortex from adult mice were dissected on ice and sonicated in lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X100, 1 mM NaVO3, 5 mM NaF and 1X protease inhibitor cocktail (Roche, Switzerland)]. Tissue extracts were cleared from debris by centrifuging at 14,000 rpm for 10 min at 4°C. The protein concentration of supernatants was determined using DC protein assay (Bio-Rad, Hercules, CA). For western blotting, 40 μg protein/lane was loaded onto 4-12% SDS-PAGE gels for electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with Odyssey blocking buffer (LI-COR, Lincoln, NE) and probed overnight at 4°C with mouse anti β-tubulin (1:10,000, T8328, Sigma-Aldrich, St. Louis, MO) or the following antibodies (all from Cell Signaling Technology, Danvers, MA): rabbit anti-phospho-eEF2 (Thr56) (1:500, #2331), rabbit anti-eEF2 (1:500, cat# 2332), rabbit anti-phosphor-mTOR (Ser2448) (1:500, #5536), mouse anti-mTOR (1:500, #4517), rabbit anti-phosphor-p70S6K (Thr389) (1:500, #9205), rabbit anti-p70S6K (1:500, #9202). Immunoreactive bands were developed
7
and quantitated using IRDye secondary antibodies and an Odyssey® CLx infrared imager (LICOR). Western blots were done with brain extracts of 5-6 age- and sex-matched animals per genotype. Initial trends were followed up by a second experiment and the data combined for statistical analyses.
ELISA Hippocampus and medial prefrontal cortex from adult mice were dissected on ice and immediately frozen in liquid N2. Tissue extracts were prepared by sonicating in lysis buffer [20 mM Tris-HCl, 137 mM NaCl, 0.2% Triton X-100, 10% glycerol, and 1X protease inhibitor cocktail (Roche)], centrifuging at 13,000 rpm for 30 min at 4°C and saving supernatant. Somatostatin concentration was measured using a mouse somatostatin ELISA kit (F12622, LifeSpan Biosciences, Seattle, WA).
Real-time qPCR Hippocampus and medial prefrontal cortex from adult mice were dissected on ice and immediately frozen in liquid N2. Total RNA was extracted using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich). Reverse transcription was performed using the qScript cDNA Supermix (Quanta Biosciences, Gaithersburg, MD). Each PCR reaction was performed in triplicate and compared to a β-actin internal control. Primers were designed using Primer Express software (Thermo Fisher Scientific, Waltham, MA). Quantification was performed using the comparative threshold cycle (Ct) measurement with SYBR green fluorescence signal (Quanta Biosciences, Gaithersburg, MD).
8
SUPPLEMENTARY FIGURES, FUCHS ET AL
Supplementary Figure S1. Recordings from SST+ neurons. (A, B) mEPSC recordings from SST+ neurons of SSTCre:γ2f/f mutants and SSTCre:γ2f/+ control mice in hippocampus CA1 (A) and L2/3 cingulate cortex (B). Representative traces shown on top with summary quantifications of frequency and amplitude below. No significant genotype differences were found in either brain area (p, n.s., all comparisons, n = 6 and 8 neurons and slices, 3 mice/genotype, t-tests). (C–E) Current injection data from SST+ neurons of mutant vs. control mice recorded in the presence and absence of 25 µM BIC. (C) Note that BIC increased the excitability of control cells to mutant levels and did not alter the excitability of mutant cells, consistent with drastically reduced expression of functional GABAARs in the mutants (p, n.s., Bolzman fit, W50, t-test). (D) The average number of action potentials observed in response to the maximum current (300 pA) showed no difference between mutants, mutants + BIC and controls + BIC. The average number of action potentials elicited in controls was reduced compared to all other groups (ANOVA, t-tests). (E) The Input resistance recorded from slices of control mice was lower than from mutant, mutant + BIC, and control + BIC slices. The input resistance of mutant cells was not altered by BIC. However, BIC increased the input resistance of control cells to mutant levels (ANOVA, t-tests, n = 10 and 12 cells and slices, 3 mice per genotype). *, p < .05.
9
Supplementary Figure S2. The excitability of pyramidal cells is unaffected in SSTCre:γ2f/f mice. (A–H) Current injection data recorded from SST+ neurons in CA1 and L2/3 cingulate cortex, with representative traces for CA1 (A) and L2/3 cingulate (E) and summary statistics for CA1 (B–D) and L2/3 (F–H). Excitability of pyramidal cells did not differ between SSTCre:γ2f/f mutants and controls in both brain areas (B, F), except for a trend toward fewer action potentials elicited in CA1 of SSTCre:γ2f/f mice [(B), CA1: p = .09, (F), L2/3: p, n.s., Bolzman fit, W50]. The maximal number of action potentials (APs) and the resting membrane potentials (RMP) were unaffected by genotype [(C, D, G, H) p, n.s. all comparisons, n = 9].
10
Supplementary Figure S3. SSTCre (A-C) and SSTCre:γ2f/+ mice (D-F) are behaviorally similar to WT and γ2f/f controls. (A) Motor activity in female SSTCre mice was significantly decreased compared to γ2f/f controls during the first 5 min of a 10 min OFT (F1/13 = 6.32, p < .05, min 0-5: p < .05, min 5-10: p, n.s., n = 7 and 8). Male SSTCre mice did not differ from controls (F1/13 = .54, p, n.s., n = 9). (B) FST immobility was comparable between SSTCre mice and controls of both sexes (p, n.s.). (C) Male SSTCre mice examined in the EPM showed reductions in both open arm entry- and open arm time percentages (entries: p < .05, time: p < .05, n = 9). Female SSTCre mice did not differ from controls in the EPM (p, n.s. n = 7 and 8). (D) Motor activity of SSTCre:γ2f/+ male mice during the first or second 5 min episode of an OFT did not differ from γ2f/f controls (F1/18 = .23, p, n.s.). (E) The time spent immobile in a FST was indistinguishable between SSTCre:γ2f/+ and γ2f/f mice (p, n.s.). (F) Percentage of open arm entries and open arm time in the EPM did not differ between SSTCre:γ2f/+ and γ2f/f mice (p, n.s., n = 10 for all comparisons). * p < .05, t-tests.
11
Supplementary Figure S4: SSTCre:γ2f/f mice show normal body weight combined with moderate novelty-induced hyperlocomotion mainly in female mice (A) The body weight of SSTCre:γ2f/f mice of both sexes did not differ from γ2f/f controls (p, n.s., n = 15 and 19). (B) Motor activity of SSTCre:γ2f/f mice in a 10 min OFT was significantly increased compared to γ2f/f controls. Female activity levels remained high during the entire 10 min period (p < .001 for both comparisons, n = 16). Male activity levels were significantly elevated during the first 5 minutes of the test (min 0–5: p < .05, min 6-10: p >.05, n.s., n = 15 and 19). The home-cage activity of female mice measured over two days was not different between SSTCre:γ2f/f mice and γ2f/f controls (p, n.s., n = 8). *, p < .05, ***, p < .001; t-tests.
12
Supplementary Figure S5: SSTCre:γ2f/f mice show unaltered sucrose preference and normal learning and memory in the Morris water maze. (A) Sucrose preference measured over 24 h was comparable between genotypes, independent of sex (females: p, n.s., n = 16, males: F2/32 = .1, p, n.s. n = 10). However, note the high sucrose preference even in γ2f/f and SSTCre:γ2f/+ control mice, suggesting ceiling effects. ***, p < .001, t-tests. (B) SSTCre:γ2f/f mice and γ2f/f controls show similar learning curves in the Morris Water Maze (males plus females, days: F4/184 = 16.6, p < .001, genotype: F1/46 = .1, p, n.s., n = 24). A 60 s test trial seven days after the final training session showed no genotype differences in “latency to reach the goal area” and “number of goal area entries” (p, n.s., both comparisons).
13
REFERENCES (Supplementary Information, Fuchs et al.)
1. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11.
Taniguchi H, He M, Wu P, Kim S, Paik R, Sugino K et al. A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex. Neuron 2011; 71(6): 9951013. Schweizer C, Balsiger S, Bluethmann H, Mansuy IM, Fritschy JM, Mohler H et al. The gamma 2 subunit of GABA(A) receptors is required for maintenance of receptors at mature synapses. Mol Cell Neurosci 2003; 24(2): 442-450. Earnheart JC, Schweizer C, Crestani F, Iwasato T, Itohara S, Mohler H et al. GABAergic control of adult hippocampal neurogenesis in relation to behavior indicative of trait anxiety and depression states. J Neurosci 2007; 27(14): 3845-3854. Shen Q, Lal R, Luellen BA, Earnheart JC, Andrews AM, Luscher B. gamma-Aminobutyric acid-type A receptor deficits cause hypothalamic-pituitary-adrenal axis hyperactivity and antidepressant drug sensitivity reminiscent of melancholic forms of depression. Biol Psychiatry 2010; 68(6): 512-520. Shen Q, Fuchs T, Sahir N, Luscher B. GABAergic control of critical developmental periods for anxiety- and depression-related behavior in mice. PLoS One 2012; 7(10): e47441. Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, Jessell TM et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev Biol 2001; 1: 4. Sarkar J, Wakefield S, MacKenzie G, Moss SJ, Maguire J. Neurosteroidogenesis is required for the physiological response to stress: role of neurosteroid-sensitive GABAA receptors. J Neurosci 2011; 31(50): 18198-18210. O'Toole KK, Hooper A, Wakefield S, Maguire J. Seizure-induced disinhibition of the HPA axis increases seizure susceptibility. Epilepsy Res 2014; 108(1): 29-43. Maguire JL, Stell BM, Rafizadeh M, Mody I. Ovarian cycle-linked changes in GABA(A) receptors mediating tonic inhibition alter seizure susceptibility and anxiety. Nat Neurosci 2005; 8(6): 797-804. Maguire J, Mody I. Neurosteroid synthesis-mediated regulation of GABA(A) receptors: relevance to the ovarian cycle and stress. J Neurosci 2007; 27(9): 2155-2162. Maguire J, Mody I. GABA(A)R plasticity during pregnancy: relevance to postpartum depression. Neuron 2008; 59(2): 207-213.
14
12.
13. 14. 15. 16. 17.
Ren Z, Pribiag H, Jefferson SJ, Shorey M, Fuchs T, Stellwagen D et al. Bidirectional homeostatic regulation of a depression-related brain state by gamma-aminobutyric acidergic deficits and ketamine treatment. Biol Psychiatry 2016; 80(6): (in press, available online). Lister RG. The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology 1987; 92(2): 180-185. Santarelli L, Saxe M, Gross C, Surget A, Battaglia F, Dulawa S et al. Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants. Science 2003; 301(5634): 805-809. Lucki I. The forced swimming test as a model for core and component behavioral effects of antidepressant drugs. Behav Pharmacol 1997; 8(6): 523-532. Newton SS, Thome J, Wallace TL, Shirayama Y, Schlesinger L, Sakai N et al. Inhibition of cAMP response element-binding protein or dynorphin in the nucleus accumbens produces an antidepressant-like effect. J Neurosci 2002; 22(24): 10883-10890. D'Hooge R, De Deyn PP. Applications of the Morris water maze in the study of learning and memory. Brain Res Brain Res Rev 2001; 36(1): 60-90.
15
