276 W
accumulation especially in the CA2 CA3 region and the DMSO, therefore a final concentration of 1 DMSO was
stratum lacunosum moleculare of the hippocampus. In present in all experiments. In an earlier study it was found
contrast, IBMX, a non-selective PDE inhibitor, increased that 1 DMSO had no effect on cGMP levels in hip-
cGMP levels in varicose fibers and astrocytes throughout pocampal slices unpublished results.
the hippocampus [11]. Thus, the choice of the PDE inhibitor is a very important factor when cyclic nucleotide
2.2.1. Radioimmunoassay levels are studied in complex tissues. In this respect it is
cGMP and cAMP levels were determined in individual striking that in the canine proximal colon only a combina-
hippocampal slices, incubated as described above, using a tion of zaprinast and IBMX was effective in inhibiting
radioimmunoassay as published previously [9]. Briefly, the NO-mediated cGMP in smooth muscle cells [40].
incubations were terminated by placing the slices into a In order to study the possibilities of locally increasing
solution of 5 trichloroacetic acid. Subsequently, the cyclic nucleotide levels using selective inhibitors of PDE
samples were sonicated and centrifuged. The supernatant activity [11], we measured the effects on cGMP and cAMP
was used for the determination of the cGMP and cAMP accumulation in hippocampal slices, after the incubation
content, measured by a radioimmunoassay according to with a number of PDE inhibitors with different selectivity
Steiner and coworkers [44]. The pellet was used for the profiles. In addition, we visualized the effect of these
analysis of the protein content according to Lowry and inhibitors on the accumulation of NO-mediated cGMP
coworkers [30]. synthesis using cGMP immunocytochemistry. A compari-
Each condition was measured in two different slices per son was made between the determination of cGMP levels
animal and for each PDE inhibitor three different rats were by the use of a radioimmunoassay and by the evaluation of
tested. In each slice, both cGMP and cAMP levels were cGMP immunofluorescence intensity using an image anal-
measured in triplicate and the median was taken for each ysis system.
sample. In the radioimmunoassay, a level of 0.6 fmol cGMP and 5 fmol cAMP could be detected. cGMP and
cAMP levels were corrected for the protein content of each
2. Materials and methods
slice. 2.1. Animals
2.3. Immunocytochemistry Experiments were performed on hippocampal slices
obtained from adult male Lewis rats 200–240 g. The After the incubation, slices were fixed with ice-cold
effect of zaprinast on two different rat strains was studied fixative solution of 4 freshly prepared depolymerised
in female Lewis and Wistar rats obtained from Charles paraformaldehyde in 0.1 M phosphate buffer pH 7.4 for
River. The animals were housed under standard conditions 30 min at 48C. The slices were then fixed for another 90
at the local animal facility. All experiments were approved min with 4 paraformaldehyde containing 10 sucrose.
by the committee on animal welfare according to Dutch After washing overnight at 48C in 0.1 M phosphate buffer
governmental rules. pH 7.4 containing 10 sucrose, the slices were frozen in
CO . Cryostat sections 10 mm were cut, thawed onto
2
2.2. Tissue preparation chrome–alumn gelatin coated slides and processed for
immunocytochemistry. Animals were decapitated and their brains were immedi-
Frozen sections were dried for 20 min at room tempera- ately removed. Brains were transferred to ice-cold Krebs–
ture, followed by three 5 min washes with Tris-buffered Ringer bicarbonate buffer Krebs incubation buffer of the
saline TBS. Sections were incubated overnight at 48C following composition: 121.1 mM NaCl, 1.87 mM KCl,
with sheep anti-formaldehyde fixed cGMP, diluted 1:4000 1.17 mM KH PO , 1.15 mM MgSO .7H O, 24.9 mM
in TBS containing 0.3 Triton X-100 TBS-T. The
2 4
4 2
NaHCO , 2.0 mM CaCl .2H O and 11.0 mM glucose. specificity of this antibody has been detailed elsewhere
3 2
2
Hippocampal slices 400 mm were prepared as de- [10]. Washing consisted of 10 min of TBS, followed by
scribed previously [10]. Slices were incubated in Krebs TBS-T and another 10 min of washing with TBS at room
incubation buffer at 368C, under an atmosphere of 95 O temperature. The primary antibody was visualized by the
2
and 5 CO at pH 7.4. The incubations lasted 40 min; incubation of sections for 1 h at room temperature with
2
when appropriate, the last 10 min in the presence of the Fluorescein FITC–conjugated rabbit anti-sheep immuno-
NO donor sodium nitroprusside SNP in a concentration globulins Jackson, diluted 1:30 in TBS-T. After having
of 0.1 mM. The PDE inhibitors were present from the start been washed, sections were mounted and studied with an
of the incubation and were added in different concen- Olympus AX-70 microscope, equipped with a narrow band
trations to the slices. Vinpocetine, calmidazolium, EHNA, MNIBA-type FITC filter, or a MNG filter for Cy3 fluores-
SKF 96231, SKF 95654, rolipram, dipyridamole and cence Chroma Technology Corporation. These filters
zaprinast had to be dissolved in dimethylsulfoxide made it possible to photograph FITC or Cy3 fluorescence
W .C.G. van Staveren et al. Brain Research 888 2001 275 –286
277
without any bleeding of the other fluorescent marker in the presence of 0.1 mM SNP mean6S.E.M.; sig-
through the filter [10]. nificantly different from control 0 M without SNP,
Glial fibrillary acidic protein GFAP was stained with Student t-test, P,0.01. As shown in Fig. 1, in the absence
mouse anti-GFAP serum Innogenetics, diluted 1:10 in of SNP the cGMP content in hippocampal slices was
TBS-T and visualised with donkey anti-mouse Cy3 Jack- increased
by IBMX
1 mM,
EHNA 100
mM, son, diluted 1:800 in TBS-T.
dipyridamole 100 mM and vinpocetine 1 mM and higher compared to its controls. No changes in cGMP
2.4. Image analysis levels were found when slices were incubated with roli-
pram or zaprinast. For the semi-quantitative measurement of cGMP, sec-
Incubation of slices with 0.1 mM SNP in the presence of tions were stained for cGMP as described above and the
different concentrations of IBMX, EHNA or dipyridamole, primary antibody was visualised by the incubation of
resulted in a concentration dependent increase of cGMP sections for 1 h at room temperature with the Alexa 488
levels. Rolipram increased cGMP levels significantly at 1 donkey anti-sheep IgG H1L conjugate Molecular
mM and 100 mM and the cGMP content was only raised in Probes, diluted 1:100 in TBS-T. Each condition was tested
the presence of the highest dose of zaprinast 100 mM. On in hippocampal slices obtained from three different ani-
a molar basis EHNA and dipyridamole appeared to be the mals and from each slice, three different sections of the
most potent inhibitors. In the presence of SNP we did not hippocampal area were studied per animal. All sections
find a significant increase in cGMP accumulation by were stained and analysed at the same time under standard
vinpocetine Fig. 1 or calmidazolium not shown. conditions. Pictures of the stratum lacunosum moleculare
and the CA1 area were made at a magnification of 203 3.2. cAMP levels in hippocampal slices after incubation
using a Sony Power HAD 3CCD Color Video Camera. All with different PDE inhibitors
pictures were analysed with the computer program analy- SIS Vers. 3.0. For each image, a color separation of the
As shown in Fig. 2, in the absence of PDE inhibitors the green image was done and the mean grayvalue of each
basal cAMP
content of
hippocampal slices
was area was estimated as a measure for the cGMP content of
37.9464.94 pmol mg protein determined in six animals. the hippocampal area. All measurements were corrected
This is in the range of reported hippocampal levels [17]. for control sections which were incubated without the
Rolipram strongly increased cAMP levels while no effect primary antibody.
on the cAMP content was found in the presence of zaprinast, dipyridamole or IBMX. There was no effect of
2.5. Statistical analysis SNP on cAMP levels regardless of the PDE inhibitors
being present. Incubation of hippocampal slices with 10 To determine whether different concentrations of the
mM forskolin an activator of adenylyl cyclase or 10 mM PDE inhibitor tested, differed from its control, a Student
noradrenalin, both in the presence of IBMX, resulted in Newman–Keuls test and a Student t-test were used.
large increases of cAMP levels. 2.6. Chemicals
3.3. cGMP immunostaining after incubation with different PDE inhibitors
IBMX was
from Janssen
Chimica; zaprinast,
dipyridamole and EHNA from Sigma; rolipram and cal- When hippocampal slices were incubated in vitro with-
midazolium from RBI; vinpocetine from Tocris; SKF out PDE inhibitors, cGMP immunostaining was nearly
96231 and SKF 95654 were kindly donated by absent Fig. 3A. No effect on cGMP immunocyto-
SmithKline Beecham.
L
-NAME and SNP were obtained chemistry was observed when slices were treated with
from Fluka. vinpocetine, calmidazolium, SKF 95654, SKF 96231
or zaprinast data not shown. In the presence of 1 mM IBMX isolated fibers were observed distributed at random
3. Results throughout the hippocampal slice, with a cluster of thin,
