1
Supplementary information
Data presentated in Figs. S1, S2, S3, S4 are obtained from experimental groups different from those
described for main text figures. These analysis were done in rats injected with the rAAV-NPY or
rAAV-Empty vectors but not exposed to electrical stimulation.
Supplementary methods
Light and Electron microscopy (see Fig. S1 online). Four weeks after vector injection, Emptyvector and rAAV-NPY injected rats (n=3 each experimental group) were deeply anaesthetized with
Equithesin (1% pentobarbital + 4% chloral hydrate; 3.5 ml/kg, i.p.) and transcardially perfused with
saline, followed for 15 min by a fixative composed of 4% paraformaldehyde, ~0.2% picric acid,
0.05% glutaraldehyde made up in 0.1M phosphate-buffer (PB, pH 7.2-7.4). Brains were quickly
removed, extensively rinsed in PB and sectioned in the coronal plane (40 µm for light microscopy
and 70 µm thickness for electron microscopy), using a vibratome.
The immunocytochemical procedures were identical to those described earlier (Schwarzer et al.,
1996). Free-floating sections were first incubated for 1 h in 20% normal goat serum diluted in TBS
(0.6% TRIS-base, 0.9% NaCl). Triton-X 100 (0.3%) was added to incubation solutions only when
the sections were prepared for light microscopy. Immunoperoxidase reactions were carried out
using the avidin-biotin-HRP complex method (ABC, Elite kit, Vector). Sections were then routinely
processed for either light or electron microscopic examination. For electron microscopy, after
osmium treatment (2%) and contrasting with uranyl acetate (1%), the sections were embedded in
Durcupan ACM (Fluka). Ultra-thin sections (70 nm) were collected on pioloform-coated copper
slot grids and analyzed in a Philips CM120 electron microscope. At least two different blocks of
each animal were cut for electron microscopy.
2
NPY release experiments and HPLC identification (see Fig. S2 online). In this experiment, rats
were injected bilaterally in the sepal hippocampus only using the same procedure described in
Methods. Four weeks after vector injection, rAAV-Empty (n=3) and rAAV-NPY (n=4) injected rats
were killed by decapitation. Brains were removed and the dorsal hippocampi of both hemispheres
were dissected out and transferred into oxygenated Krebs-bicarbonate buffer (OKB) (Rizzi et al,
1993). Coronal slices (250 µm) were obtained using a McIllwain tissue chopper. Slices were
washed 3 times in OKB (37°C) containing 0.2% bovine serum albumin and 0.03% bacitracin. In
each release experiment, samples were run in triplicates. Four slices were put into each basketshaped sieves and after 40 min wash-out in OKB, the sieves were transferred every 5 min into
subsequent wells (48 well tissue culture clusters, Costar, Cambridge, MA, USA) containing 500 µl
OKB (37°C). After 20 min (basal release) slices were exposed to OKB containing 50 mM KCl and
proportionally lowered NaCl. A different group of rAAV-NPY injected rats (n=3) was used to
assess the Ca2+-dependence of 50 mM KCl-induced efflux of NPY by adding 2 mM EGTA into
OKB. At the end of the experiments, medium was removed, 25 µl 10 M acetic acid was added and
fractions were frozen. Slices were transferred into 2 M acetic acid containing 0.2 % bovine serum
albumin and 0.03% bacitracin and NPY was extracted by sonication and centrifugation. Medium
and slice-derived samples were lyophilized and NPY immunoreactivity was determined by
radioimmunoassay. An aliquot of the slice-derived sample was analyzed by HPLC (Waters, high
pore RP8 reversed phase column). Running phase consisted of 27% acetonitrile in 0.1 trifluor acetic
acid for 5 min followed by a linear gradient in which the acetonitrile concentration was increased to
50% for the subsequent 45 min (flow rate was 1.0 ml/min). Fractions (1.0 ml) were collected,
lyophilized and NPY immunoreactivity was determined in each fraction by radioimmunoassay.
NPY Y1 and Y2 receptor binding (see Fig S3 online). Four weeks after vector injection, rAAVEmpty and rAAV-NPY injected rats (n=5 each experimental group) were killed by decapitation.
Brain coronal sections (20 µm thick, plate 21 of the Paxinos and Watson,1986 rat brain atlas) were
3
cut and mounted on gelatine-coated slides. Binding assays were carried out by receptor
autoradiography as previously described with slight modifications (Roder et al, 1996). Briefly,
slides were preincubated for 30 min at 25°C in Krebs-Ringer phosphate buffer (KRB, 138 mM
NaCl, 11 mM NaHCO3, 1 mM NaH2PO4, 5 mM KCl, 1 mM MgSO4, 11 mM glucose, 1 mM CaCl2,
10 mM Tris HCl, pH 7.4). For measuring NPY-Y1 receptor binding, slices were transferred to
fresh KRB supplemented with 0.1% BSA, 0.05% bacitracin and 15 pM [125I]Pro34PYY (specific
activity 2200 Ci/mmol, iodinated by G.S.) and incubated for 120 min at 25°C, in the absence (total
binding) or in presence of 300 nM BIBO-3304, a selective Y1 receptor antagonist (Dr. KarlThomae GmbH, Germany). For measuring NPY-Y2 receptor binding, slices were transferred to
fresh KRB supplemented with 0.1% BSA, 0.05% bacitracin and 15 pM [ 125I]PYY3-36 (specific
activity 2200 Ci/mmol, iodinated by G.S.) and incubated for 180 min at 25°C in the absence (total
binding) or in presence of 300 nM BIIE-0246, a selective Y2 receptor antagonist (Dr. Karl-Thomae
GmbH). Non-specific binding was determined by including 100 nM unlabelled NPY in the
incubation medium. Two adjacent sections were used for each of these experimental conditions. At
the end of the incubations, sections underwent 2 min washes (x3) in ice-cold buffer, then rapidly
dipped in ice-cold distilled water and dried under a stream of cold air.
subsequently apposed, together with Amersham
125
The sections were
I plastic standard scales, to Hyperfilm 3H and
stored at -70°C for about 15 days. Computer-assisted autoradiographic image analysis was done
using the AIS 3.0 software (Imaging Research Inc., Canada). Optical densities were measured in
selected brain regions from both ipsi- and contralateral sides in two adjacent sections. Absolute
values were calculated from optical densities using
tissue weight.
125
I-microscales and expressed as fmol/g wet
4
Hippocampal learning and memory test (see Fig. S4 on line). Fourteen naïve rats were
stereotaxically injected either with rAAV-Empty or rAAV-NPY as previously described in the
method section. Four weeks after vector injection, these rats were used for behavioral assessment of
hippocampal learning and memory.
Spatial discrimination abilities of rAAV-Empty and rAAV-NPY injected rats were assessed by a
two-discrimination platform test sensitive to changes in hippocampus-associated learning processes
(Morris et al., 1986; Carli et al., 2001). A circular ‘swimming pool’ (150x50 cm), filled to a depth
of 0.29 m with water at 26 ± 1°C, was used. The pool was placed in the middle of a large room and
was surrounded by various visual cues. Two grey visible square (11x11 cm) platforms protruding
1.2-2.0 cm above the water were used. One platform was rigid and provided support and the other
sank when rats tried to climb onto it. Rats were trained to swim to the rigid escape platform while
avoiding the floating one. For all rats, the fixed escape platform (correct) was always in the same
place at the center of one of the eight sectors. The floating platform (incorrect) was positioned over
successive trials in a quasi-random sequence of eight locations around the pool. The rats were
trained with 10 trials a day for seven days. A trial began with the rat being placed in the pool and
ended when the rat chose one of the two platforms. In the case of the rigid one it was allowed to sit
on the platform for 15 sec. Inter-trial intervals were approximately 2-4 min so each rat’s daily
testing lasted approximately 30 min. A correct trial was one in which the rat climbed onto the rigid
platform without touching the floating platform with its forepaws or snout. If the rat did not choose
to escape onto either platform (correct or incorrect) within 60 sec it was taken to the rigid platform
and left on it for 15 sec. We measured (1) the first choice in each trial (correct/ incorrect), (2) the
latency to escape (s) and (3) the number of omissions.
5
Supplementary figures
Fig. S1 Light and electron microscopy.
(a) Digital brightfield photomicrograph of a coronal section of the dentate gyrus from a
representative rAAV-NPY injected rat taken 4 weeks after vector injection. NPY immunoreactivity
is present in the somata of hilar interneurones (white arrow), fiber tracts and in the neuropil of the
inner third of the dentate molecular layer (black arrowheads). (b, c) Electron micrographs of
immunoperoxidase labeling for NPY in presynaptic axon boutons (t) making type I synaptic
contacts (indicated by arrowheads) with dendritic spines (s) in the inner third of the dentate
molecular layer. (d) Immunoperoxidase labeling of NPY in sporadic dendritic shafts within the
dentate molecular layer. Note one dendrite (d2) with peroxidase immunoreaction diffused
throughout its cross-section and near an immunolabeled presynaptic bouton (t) forming a type II
synapse (indicated by an arrow) with an unlabelled dendrite (d1). Panel (e) depicts
immunoperoxidase labeling of NPY in presynaptic axon boutons (t) making type II synaptic
contacts (arrows) with a dendritic shaft (d) in the dentate molecular layer of a rat injected with the
rAAV-NPY vector. A representative control (Empty-vector injected) rat is depicted in (f). These
data indicate that rAAV-mediated overexpression of NPY results in transport of the neuropeptide
into both presumed glutamatergic (arrow heads in b and c) and GABAergic (arrows in e) nerve
terminals where it undergoes regulated release, but also into dendrites of transfected neurons (d2 in
d) where it may be constitutively secreted. Abbreviations: gr, granule cell layer; H, hilus; ml,
molecular layer. Scale bars: 20 mm (a); 0.1 µm (b); 0.2 µm (c-f).
Fig. S2 NPY release experiments and HPLC identification.
(a) NPY is released following KCl-induced depolarization to a much larger extent from
hippocampal slices of rAAV-NPY than rAAV-Empty injected rats (first panel in a). KCl-induced
NPY release in rAAV-NPY injected rats is Ca2+-dependent demonstrating its neuronal origin
(second panel in a). NPY content in hippocampal slices of rAAV-NPY rats was significantly higher
6
than in rAAV-Empty injected rats (rAAV-Empty, fmol/slice, 944.0 ± 125.0 (n=6 rats); rAAV-NPY,
10.230 ± 2.236 (n=6 rats), p<0.01 by Student’s t-test). (b) HPLC analysis of NPYimmunoreactivity in slices of rAAV-NPY or rAAV-Empty injected rats show that in both
experimental groups NPY-immunoreactivity co-migrates with synthetic rat NPY (indicated by
arrow).
Fig. S3 NPY-Y1 and -Y2 receptor binding.
(A) NPY-Y1 receptor binding in the hippocampus of rAAV-Empty and rAAV-NPY injected rats.
In rAAV-NPY injected rats, binding to NPY-Y1 receptors was significantly reduced in the
molecular layer of the dentate gyrus (DG) ipsilateral to the injection site (Ipsi). No changes in
binding were detected in brain areas outside the injected hippocampus, as exemplified in layers 1-2
of the frontoparietal cortex/somatosensory area (extFrSS). (B) NPY-Y2 receptor binding did not
differ in rAAV-NPY vs rAAV-Empty injected rats in hippocampal subfields, as exemplified in the
molecular layer of the dentate gyrus (DG) and in stratum oriens CA1 (CA1). **p<0.01 vs rAAVEmpty; °°p<0.05 vs contralateral non-injected side by Tukey’s test.
Fig. S4 Two-platform discrimination test.
We explored whether alterations in hippocampus-associated learning processes occurred in rAAVNPY treated normal rats using a spatial discrimination-learning test. Fig. S4 shows that spatial
discrimination learning is similar in rAAV-Empty and rAAV-NPY treated rats. Thus, panel A
shows that on the first day of acquisition the performance of both rAAV-Empty (n=7, blue triangle)
and rAAV-NPY (n=7, yellow square) injected rats was at chance (chance level = 50% correct
choices) while with training both groups improved their performance similarly, reaching about 80%
of corrected choices after the 4th day of training. Accordingly, the repeated measure ANOVA
indicates that rAAV-Empty and rAAV-NPY treated rats learned the task at similar rates (treatment
F(1,72) = 2.25, p = 0.16; days x treatment: F(6,72) = 0.39, p = 0.89). Panel B reports the average %
7
of correct choices over the 7 days of training. Panel C depicts that during all 7 days of training both
groups showed similar choice latency (mean ± SE over the 7 days of training: rAAV-Empty, 7.0 ±
0.4 sec; rAAV-NPY, 7.8 ± 1.0 sec). Errors of omissions were present only on the first day of
training and were equal in both groups (data not shown).
8
References
Carli M, Balducci C, Samanin R. Stimulation of 5-HT(1A) receptors in the dorsal raphe ameliorates
the impairment of spatial learning caused by intrahippocampal 7-chloro-kynurenic acid in
naive and pretrained rats. Psychopharmacology (Berl) 2001; 158: 39-47.
Morris RG, Hagan JJ, Rawlins JN. Allocentric spatial learning by hippocampectomised rats: a
further test of the "spatial mapping" and "working memory" theories of hippocampal
function. Q J Exp Psychol B 1986; 38: 365-395.
Paxinos G, Watson C. The rat brain in stereotaxic coordinates. Academic Press, New York, NY
1986.
Rizzi M, Monno A, Samanin R, Sperk G, Vezzani A. Electrical kindling of the hippocampus is
associated with functional activation of neuropeptide Y-containing neurons. Eur J Neurosci
1993; 5: 1534-1538.
Roder C, Schwarzer C, Vezzani A, Gobbi M, Mennini T, Sperk G. Autoradiographic analysis of
neuropeptide Y receptor binding sites in the rat hippocampus after kainic acid-induced
limbic seizures. Neuroscience 1996; 70: 47-55.
Schwarzer C, Sperk G, Samanin R, Rizzi M, Gariboldi M, Vezzani A. Neuropeptidesimmunoreactivity and their mRNA expression in kindling: functional implications for limbic
epileptogenesis. Brain Res Rev 1996; 22: 27-50.