Animals

Male Wistar rats (Charles River, Denmark), weighing 220–350 g at beginning of the experiment, were housed together with two or three animals in each cage. The animals had ad libitum access to food and water, fixed 12 h day/light cycle and the cages were enriched by a cardboard tunnel and nesting materials. After arrival to the facilities, the rats were left in the stables for at least 1 week before they were subjected to any operations in order to enable acclimatization to the new environment. All experiments were approved by the Malmö/Lund Ethical Committee for Experimental Animals (Permit number M49-15) and performed according to international guidelines for the use of research animals.

A total number of 77 rats were used in the KA induced seizure test and randomly divided into 9 treatment groups by an experimenter not aware of the treatment conditions. The resulting group size was 8 in all groups, except for increased numbers of rats administered AAV8-NPY/Y2 (n = 12) and AAV8-Y2/NPY (n = 9), since 5 rats were added in these groups due to lost data at video recordings that were later recovered. 12 rats were used in the electrophysiology test (n = 4/group). The group sizes were by experience considered appropriate for statistical comparison.

Viral vectors

Viral vectors were manufactured by GeneDetect (New Zealand) and provided by CombiGene AB (Sweden). A set of cDNA constructs, with a size of 4.3kbp, carrying the sequence for human NPY and the human NPY Y2-receptor, separated by an internal ribosome entry site (IRES), with either NPY first (NPY/Y2) or the Y2-receptor first (Y2/NPY), were contained in three different AAV-serotypes, AAV1, AAV2 and AAV8. The vectors all contained the Woodchuck post-transcriptional regulatory element (WPRE) and bovine growth hormone polyadenylation sequence (BGHpA). Also, three empty control vectors (EMPTY) were used, one for each serotype, resulting in a total number of nine vectors for evaluation. The synthetic CAG (chicken-beta-acting promoter hybridized with the cytomegalovirus [CMV] immediate-early enhancer sequence) were driving the constructs. All viral vectors were titrated together using qPCR targeting the WPRE and had a titer of 1.1*1012 viral genomes (vg)/ml.

Viral vector delivery

The viral vectors were delivered bilaterally to the hippocampus via stereotaxic surgery. Rats were anesthetized with 4% isoflurane (Isoba Vet, Intervet, Netherlands) air mixture lowered to 2% when paw withdrawal response was absent. The rat’s heads were shaved and placed in a stereotaxic frame (Kopf Instruments, Tujuga, CA, USA). Local anaesthesia (Marcain, AstraZeneca, Denmark) was administered before an incision was made exposing the scull bone. Drops of hydrogen peroxide (4%) were applied to stop bleeding after scalpel scrapes and to expose bregma. Holes were drilled, prior to each injection, using predefined coordinates (see below). The viral vector, kept on ice at all time before the injection, was loaded into a glass capillary attached to a microliter syringe (Hamilton Company, Switzerland) and delivered into the hippocampus at a rate of 0.2 µl/min. Dorsal hippocampus (AP −3.3 mm. ML ± 1.8 mm, DV −2.6 mm) was injected with 1.0 µl vector and ventral hippocampus (AP −4.8 mm, ML ± 5.2 mm) was injected with 1.0 µl at two different depths (DV −6.4 and −3.8) [30]. Thus, a total of 6 µl viral vector at a concentration of 1.1*1012 vg/ml were delivered to each brain, resulting in a dose of 6.6*109 vg/brain. Reference points were bregma for anterior/posterior coordinate, midline for medial/lateral and dura for dorsal/ventral. The tooth bar was set to -3.3 mm. After vector delivery, the incision was closed using staples, and the animals were allowed to recover in quarantine for 48 h before being relocated to their stables.

Controlling for orexigenic effects of NPY and Y2 overexpression

As NPY is an established orexigenic factor [31,32,33], we were interested to study if the different vectors had any effect on animal weight development. No difference in weight gain per day was detected between the groups (Supplementary Fig. 1).

KA preparation and induction of SE

KA was prepared freshly the same day as SE induction. 50 mg of KA (Abcam, ab120100) was dissolved in 4 ml of saline and adjusted with 1 M NaOH to a pH of 7.4, in order to reduce a pain response in connection to the injection. The concentration was set to 10 mg/ml by adding saline to a total volume of 5 ml. Continuous measurement of pH during NaOH titration and comparing to previous titration curves facilitated reproducibility. The rats were injected with a dose of 10 mg/kg in the neck region subcutaneously (s.c.) and immediately placed in a transparent Plexiglas cage with the dimension 30 x 19 x 29 cm. Ten animals per time were treated with KA and recorded for 2 h with a high-definition camera.

Assessment of KA-induced seizures

The animals were assessed for behavioral manifestation of seizures for two hours following KA injection. Behavioral scoring of seizures was performed according to a modified Racine scale [24, 34], focusing exclusively on grade 3–5 seizures. Grade 3 seizures included a minimum of 15 s of clonic activity in the forelimbs, in a grade 4 seizure the rat was rearing while the clonus in the forelimb continued and a grade 5 seizure caused the animal to rear and fall on side or back. The analysis of the video recordings was performed by an experienced experimenter blinded to the treatment conditions. Three parameters were assessed. First, latency to first motor seizure was measured as the time that passed from the injection until the 1st grade 3 seizure. Second, the time spent in motor seizures (grade 3 or above) was measured. Again, a seizure had to be at least 15 s to add to the cumulative seizure duration. Finally, the time elapsed from injection until SE, defined as continuous clonic activity of at least 10 min, was measured. The monitoring was terminated after 2 h by deeply anesthetizing the animals with isoflurane and decapitation.

Immunohistochemistry and assessment of NPY expression levels

The brains were quickly removed from the skull, directly frozen on crushed dry ice and stored at −80 °C until being cut in 16 µm thick coronal slices utilizing a cryostat. The resulting 16 series of slices, covering the dorsal and ventral injection points, were also stored in −80 °C on glass slides until further processing.

Before immunohistochemical staining, the slices were fixed by soaking the glass slides with slices in 4% paraformaldehyde (PFA) for 20 min. After washing in 0.02 M KPBS, the tissue was pre-incubated in 10% donkey serum in 0.25% T-KPBS for 1 h followed by primary antibody (Rabbit anti-NPY, N9528, Sigma Aldrich, 1:500) incubation overnight at 4 °C in 5% donkey serum and 0.25% T-KPBS. Next day the slices were washed and then incubated with secondary antibody (Cy3 conjugated Donkey anti Rabbit, Jackson Immunoresearch, 1:200) in 1% donkey serum, 0.25% T-KPBS solution for 2 h at room temperature. After washing a final time in 0.2 M KPBS the slices were cover-slipped in DABCO (Sigma Aldrich). A negative control (primary antibody omitted) was used to control for unspecific binding of the secondary antibody.

For analysis, 6 fluorescent microscopy pictures (Olympus BX61) were taken from each animal. Two of the pictures covered the dorsal injection points and four the ventral hippocampus. Settings for image acquisition were the same for all images (ISO800, 1500 ms exposure, 4x objective). The mean gray value of the fluorescence in the hippocampus was measured by ImageJ and was corrected for by the background level in a cortical region were no NPY overexpression was observed (see representative images of NPY immunohistochemistry in the dorsal hippocampus in Supplementary Fig. 2).

Y2 receptor functional binding

Visualization of Y2 functional binding has been described earlier [24, 35, 36]. The tissue sections were defrosted in room temperature for 30 min before rehydration in assay buffer A (50 mM Tris-HCl, 3 mM MgCl, 0.2 mM ethylene glycol tetraacetic acid [EGTA], 100 mM NaCl, pH 7.4) for 10 min at room temperature. Thereafter, a pre-incubation assay buffer B (assay buffer A + 0.2 mM dithiothreitol, 1 µM 1,3-dipropyl-8-cyclopentylxanthine [DPCPX; #C-101, Sigma Aldrich, DK], 0.5% w/v bovine serum albumin, 2 mM guanocine-5’-diphosphate [GDP; #G7127, Sigma Aldrich]) was applied for 20 min at room temperature. Subsequently, the sections were incubated in assay buffer C (assay buffer B + 40 pM [35S]-GTPγS [1250 Ci/mmol; NEG030H250UC; PerkinElmer, DK]) together with 1 µM NPY (rat synthetic, Schafer-N, Copenhagen, DK) and the Y1 (1 µM BIBP3226, #E3620, Bachem AG, Switzerland) and Y5 (10 µM L-152,804, #1382, Tocris Cookson, UK) antagonists for 1 h at 25 °C. The assay was terminated by rinsing the sections 2 × 5 min in ice-cold 50 mM Tris-HCl buffer (pH 7.4) and subsequently dried. Exposure to Kodak BioMax MR films was done together with 14C micro scales (Amersham Life Sciences, UK) during 5 days in −20 °C and the films were thereafter developed in a Kodak GBX developer.

The NPY stimulated binding was analysed by measuring the mean grey value of the hippocampus. The value from the hippocampus was corrected for by the background grey value in a cortical region were no Y2 overexpression was observed (see representative image of Y2 receptor functional binding in Supplementary Fig. 3).

Preparation of acute hippocampal slices

Preparation of acute slices was performed as previously described [37]. Animals were sacrificed by decapitation under isoflurane anaesthesia and the brain was quickly removed, with the rAAV-injected hemisphere cut transversely on a vibratome in sucrose-containing artificial cerebrospinal fluid (sucrose-aCSF), containing in (mM) 75 sucrose, 67 NaCl, 26 NaHCO3, 25 glucose, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, and 7 MgCl2. Hippocampal slices (400 µm) were collected and placed in a holding chamber with aCSF containing (in mM): 119 NaCl, 2.5 KCl, 1.3 MgSO4, 26.2 NaHCO3, 1 NaH2PO4, 11 glucose and 2.5 CaCl2, oxygenated at room temperature (RT), and left to rest for approximately 1 h. For recordings, slices were transferred to the recording chamber perfused with oxygenated aCSF (RT; 2 ml/min).

Electrophysiology

Field recordings of excitatory postsynaptic potentials (fEPSPs) were conducted in the stratum radiatum of hippocampal CA1 region. Schaffer collaterals were stimulated with square constant current pulses (0.1 µs) through silver-chloride electrodes in glass capillaries filled with aCSF. Field EPSPs were recorded from the same layer with a recording capillary filled with aCSF (with a pipette resistance of 1–3 MΩ). Each individual slice was considered as one n. Field EPSP recordings were started by determining the input-output relationship between the amplitude of the presynaptic fiber volley (PSFV) and the amplitude of the fEPSP. Only slices capable of generating fEPSP amplitudes of more than 1 mV were included in the study. Test stimuli inducing 30–50% of the maximal fEPSP responses were used throughout all experiments. Short-term plasticity of fEPSPs was assessed by paired-pulse (PP) stimulations at different interstimulus intervals (ISI; 25, 50, 100, 200 ms; at 0.033 Hz). Paired-pulse ratio (PPR) of fEPSPs was calculated as change in the initial slope of the second fEPSP as compared to the first.

The effect of transgene NPY expression on excitatory neurotransmission during repetitive synaptic activation was examined by applying high-frequency stimulation (HFS) trains of 100 Hz with 10 stimulations of one synaptic pathway (SP.1) preceded and followed with 500 ms interval by PP-stimulation in a neighboring, convergent but independent synaptic pathway (SP.2). Synaptic independence was determined by evoking a fEPSP in SP2, with or without a fEPSP 50 ms prior in SP.1. The lack of paired-pulse facilitation supports the absence of shared synapses between the inputs. The stimulation was repeated eight times and the responses were averaged. To avoid interference with post-tetanic potentiation (PTP), each train was delivered at 5 min intervals. To prevent long-term potentiation (LTP) induction in stimulated synapses, the specific N-methyl-D-aspartate (NMDA) antagonist, D(2)-2-amino-5-phosphonopentanoic acid (D-AP5, 50 µM) was applied together with aCSF. The specific Y2-receptor antagonist BIIE0246 (0.6 μM, Sigma-Aldrich) was applied to the aCSF and allowed to wash in during paired-pulse stimulation for 10 min. After recordings, slices were fixed in PFA (12 h at 4 °C), rinsed 3 × 20 min in KPBS and stored in Walters antifreeze solution (−20 °C).

Immunohistochemistry in acute hippocampal slices

Sections were washed three times in KPBS for 10 min and imbedded in a solution containing 300 g/L egg-albumin (Sigma-Aldrich) and 30 g/L gelatin (Sigma-Aldrich), frozen at −80 °C and sub-sliced on a cryotome at −20 °C (Cellab Nordia AB) to 16 μm thick slices. Slices were mounted on positively charged glasses (+Menzel glass, Thermo Scientific) and stored at −20 °C. These slices were then preincubated in 10% Donkey serum, 0.25% Triton X-100 in KPBS for 1 h. After adding rabbit anti-NPY antibody (1:500, #N9528, Sigma-Aldrich, DK) in 5% Donkey serum, 0.25% Triton X-100 in KPBS, sections were incubated overnight at 4 °C. Sections were washed 3 × 10 min in tKPBS, incubated for 2 h at RT with Cy3-conjugated Donkey anti-rabbit antibody (1:200, Jackson Immunoresearch USA) in 1% Donkey serum, 0.25% Triton X-100 in KPBS and washed 1 × 10 min in tKPBS and 2 × 10 min in KPBS. The slides were mounted with DABCO (Sigma-Aldrich) and digitized images obtained using Olympus BX61 microscope and CellSens software.

Human organotypic slice preparation and AAV-vector mediated NPY and Y2 receptor overexpression

Human temporal lobe (hippocampus) tissue was obtained by surgical resections from three patients treated for intractable epilepsy at Rigshospitalet in Copenhagen, Denmark, as previously described [38, 39]. Prior to each surgery a written informed consent was obtained from all subjects. The use of resected human brain tissue and following procedures was approved by the local Ethical Committee in Copenhagen (H-2-2011-104) in accordance with the Declaration of Helsinki. The resected tissue was immediately submerged in ice-cold sucrose solution containing in mM: 200 sucrose, 21 NaHCO3, 10 glucose, 3 KCl, 1.25 NaH2PO4, 1.6 CaCl2, 2 MgCl2, 2 MgSO4 (all from Sigma-Aldrich, Sweden), bubbled with carbogen (O2, 95% and CO2, 5%) adjusted to 300–310 mOsm and 7.4 pH, and transferred from the surgical theatre to the laboratory. Time of transport of tissue from Copenhagen Hospital operating theatre to Lund laboratory was approximately 1 h. Coronal slices (250 μm thick) were cut on a Leica VT1200 vibratome in the same sucrose-solution as mentioned above. The slices were then transferred to ice-cold rinsing medium containing Hank’s balanced saline solution (HBSS) with 20 mM HEPES, 17.5 mM glucose and 0.5% penicillin/streptomycin solution (all from Life Technologies, Thermo Fisher Scientific Inc, Sweden) before placing them on cell culture inserts (Millipore, Sweden, #PICM03050) in 6-well plates with 960 μl equilibrated culturing medium. This medium contained 50% minimum essential media (MEM), 25% horse serum, 18% HBSS, and 2% B27 supplemented with 0.5% penicillin/streptomycin solution (all from Life Technologies, Thermo Fisher Scientific Inc, Sweden), glutamine 2 mM, glucose 11.8 mM, and sucrose 20 mM (all from Sigma-Aldrich, Sweden). Slices were cultured as interface cultures at 37 °C, 5% CO2, and ambient O2 in 90% humidity.

The slices were allowed to settle for 12 h before addition of the AAV1-NPY/Y2 vector (titre of 1012 vg/ml), with approximately 0.035 µl/mm2 added (3.5 * 107 vg/mm2). After 10 days of culturing, slices for immunohistochemistry were fixed in 4% paraformaldehyde in phosphate buffer (PB) for 12–24 h, rinsed in PB and then stored until further processing in anti-freeze solution (ethylene glycol and glycerol in PB) at −20 °C.

NPY immunohistochemistry in organotypic human hippocampal slices

Sections were fixed in 4% paraformaldehyde for 20 min. Sections were washed three times in KPBS for 10 min and preincubated in 10% Donkey serum, 1% Triton X-100 in KPBS for 1 h. After adding rabbit anti-NPY antibody (1:500, N9528, Sigma-Aldrich, DK) in 5% Donkey serum, 0.25% Triton X-100 in KPBS, sections were incubated overnight at 4 °C. Sections were washed 3 × 10 min in tKPBS, incubated for 2 h at RT with Cy3-conjugated Donkey anti-rabbit antibody (1:200, Jackson Immunoresearch USA) in 1% Donkey serum, 0.25% Triton X-100 in KPBS and washed 1 × 10 min in tKPBS and 2 × 10 min in KPBS. The slides were then mounted with DABCO (Sigma-Aldrich) and digitized images were obtained using Olympus BX61 microscope and CellSens software. Average fluorescence intensity of NPY immunoreactivity in hippocampus was evaluated using ImageJ. The experimenter performing the evaluation was unaware of the specific treatment given to individual slices.

Quantitative PCR measurements of NPY-related genes in organotypic human hippocampal slices

Sections of organotypic slices treated with AAV1-NPY/Y2 and corresponding empty control vector were kept in −80 °C freezer until processing. The tissue was scraped from the glass sections and homogenized with QIAzol lysis reagent (QIAgen) and QIAshredder spin column (QIAgen, Denmark). Total RNA was extracted using RNeasy Lipid Tissue Kit (QIAgen) according to manufacturer’s protocol. The concentration of total RNA was measured on Nanodrop (Thermo Fisher Scientific), and the quality was evaluated by using the 260/280 nm ratio parameter. RNA (0.5 μg) was reverse transcribed to cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) following the manufacturer’s recommendations. Quantitative PCR (qPCR) was performed using the following NPY gene primers with GAPDH as the reference gene [40].

hNPY: forward: 5’-GGA GGA CAT GGC CAG ATA CT -3’, reverse: 5’-ATC TCT GCC TGG TGA TGA GG -3’; hY2: forward: 5’-GGC CAT CTT CCG GGA GTA TT -3’, reverse: 5’-GCC AGG CCA CTT TTC AGT AC -3’; hY1: forward: 5’-AAT ACC AGC GGA TCT TCC C -3’, reverse: 5’-TTC CCT TGA ACT GAA CAA TCC -3’; hY5: forward: 5’-GCC CCG AGG TCT GCT CAT TGT -3’, reverse: 5’-TGT GGC AGG TCA GTT GTT ACC GA -3’; GAPDH: forward: 5’-TCA CCA CCA TGG AGA AGG C -3’, reverse: 5’-GCT AAG CAG TTG GTG GTG CA -3’.

The real-time qPCR was run on LightCycler480 (Roche). The cycling conditions were 1 cycle of pre-incubation at 95 °C for 5 min, followed by 45 three-segment cycles of amplification (95 °C for 10 s; 60 °C for 15 s, and 72 °C for 10 s) where fluorescence was automatically measured during PCR, and 1 cycle three-segment cycle of product melting (5 s at 95 °C, 1 min at 65 °C and then continuous acquisition mode at 97 °C for 5 acquisitions per °C). The last step was one cooling cycle for 10 s with ramp rate 2 °C/s.

Statistics

Data analysis was done in Prism (GraphPad) and in Igor Pro (Wavemetrics). One-way ANOVA tests followed by Bonferroni’s multiple post-hoc comparison test or two-tailed Student’s t-test (one comparison) was used in the case of normally distributed data. Electrophysiology data was analysed in Igor (Wavemetrics) and Prism (GraphPad) using two-tailed Student’s paired t-test and one-way ANOVA followed by Tukey’s post hoc test. All data is presented as mean ± SEM. A difference was considered significant if P < 0.05.