to increased BFCN activity, thus giving rise to lower frequencies of resonance.
2. Materials and methods
2
.
1
. Subjects and surgical procedures Adult female Long – Evans rats n = 17, Etab-
lissements Janvier, Le Genest-St-Isle, France, weighing 215 – 295 g, were used in compliance
with Swiss guidelines for the care and use of laboratory animals and after receiving veterinary
governmental approval. The animals were subdi- vided into control n = 6, 192 IgG-saporin
treated n = 7 and NGF treated groups n = 4. Two animals received no lesions and contributed
to the control group. Nine animals received a unilateral ICV injection of either 5.4 mg of 192
IgG-saporin Chemicon International Inc., item no. MAB390 — 25 mg, 0.9 mgml dissolved in
sterile PBS containing 0.05 sodium azide n = 7 subjects in the SAP group, or either an equiva-
lent volume of vehicle n = 2 in the control group. The injections were performed via a 10-ml
Hamilton syringe in the left lateral ventricle, at about 1.2 mm lateral and 0.8 mm posterior from
bregma.
Six animals were implanted with an infusion cannula in the left lateral ventricle and then con-
nected to a miniosmotic pump Alzet, model 2002 either for NGF delivery n = 4 in the NGF
group following a procedure described elsewhere Williams et al., 1986; Fusco et al., 1989 or for
cytochrome c delivery, a protein that has similar physicochemical properties as NGF but lacks its
biological activity n = 2 in the control group. Recombinant human NGF 60 mg per pump
rhNGF, Genentech Inc. or cytochrome c, diluted with artificial cerebrospinal fluid, was infused at
the dose of about 4.3 mg per day for 2 weeks. Surgery for ICV injections or pump implantation
was
performed under
barbiturate anesthesia
Nembutal, 40 mgkg, IP. All operated rats were injected s.c. with Bactrim 1 mlkg the day of
surgery and then for the following 2 days.
2
.
2
. Postmortem procedures Electrolytical lesions for reconstruction of
recording sites were made at known depths by passing a current of about 8 mA for 10 s. Upon
completion of the recording session, animals were sacrified by cervical dislocation under deep anes-
thesia Nembutal, 120 mgkg body weight, IP. The brains were rapidly removed and a slice,
about 1.5 mm thick, was obtained by coronal sections of the forebrain between the level of the
rostral part of genu corpus callosum at about 10.5 mm from the interaural line and the optic
chiasm at about 9 mm from the interaural line. The medial septal area was dissected from this
slice and stored at − 80°C until biochemical as- says of ChAT were performed. In eight animals
we dissected also samples of the neocortex fron- talparietal anterior to the recording sites. The
activity of ChAT, which is a marker of BFCN, was measured Vantini et al., 1989 to validate the
effectiveness of the NGF treatment protocol used in this study of activating forebrain cholinergic
neurons. The part of the brain caudal to the optic chiasm
was postfixed
during 2
weeks 4
paraformaldehyde in phosphate buffer 0.1 M, pH 7.3 and used for histology. Reconstruction of the
electrode tracks was performed on standard cresyl violet-stained brain coronal sections, 50 mm thick.
2
.
3
. Electrophysiological procedures After 14 – 20 days of rhNGF infusion or after
14 – 20 days from the injection of the 192 IgG-sa- porin, the subjects were tested for an electrophys-
iological session in a sound-proof room. The anesthesia was induced by an IP injection 1.0
mlkg bw of a mixture of ketamine Ketalar; 30 mgkg and xylazine Rompun, 10 mgkg. Sup-
plementary doses 0.3 mlkg of this mixture were then administered every 90 min throughout the
recording session. The body temperature was con- tinuously monitored and regulated by a heating
pad maintained at 38°C. The rat was mounted in a stereotaxic apparatus without earbars. A hole
was drilled in the skull and the electrode penetra- tions were aimed to the temporal cortex areas
Te1, Te2, Te3.
LFPs were recorded from the temporal cortical regions by mean of four independently driven
glass-coated platinum-plated tungsten microelec- trodes impedance in the range 0.5 – 2 MV at 1
kHz which were advanced by 5 mm steps Villa et al., 1999. Two electrodes were inserted into the
cortex via one guide cannula, and were approxi- mately 200 mm apart from each other, and ap-
proximately
1000 mm
from the
two other
electrodes. High and low pass filter settings for LFPs were 7 – 100 Hz, respectively, with a 50 Hz
notch filter. Several sites were sampled across the entire cortical depth, but not the underlying white
matter.
2
.
4
. Analysis of local fields potentials Power spectra were computed by Fast Fourier
Transforms of 512 points. Data points were sam- pled at the rate of 512 sampless and we selected
epochs of LFPs lasting 1 s. Thus, the frequency resolution of our analysis was 1 Hz. In addition
to this analysis we used the third order cumulant analysis of the LFPs. This analysis was used to
measure the phase-coupled frequencies corre- sponding to non-linear coupling of spectral fre-
quency components, somewhat analogous to frequencies of resonance. In brief, this analysis
can be described as follows. Firstly, let us con- sider a case study where an analog signal from a
single channel, e.g. a local field potential xt, is recorded during N epochs of equal duration, such
that xt = S
N
a + b + ab where at = cos2p f
1
t + v
a
and bt = cos2pf
2
t + v
b
and f
1
, and f
2
represent two frequencies of periodic processes and v
a
and v
b
are phases randomly changed, i.e. uniformly distributed in [0, 2p], for each epoch.
Notice the non-linear interaction is represented by the term ab.
The spectral representation of this signal X f is obtained by the Fourier transform X f =
S
N
xt e
− it2pf
. The power spectrum is P
xx
f = X f
2
and its
shape will
show peaks
corresponding to frequencies f
l
, f
2
and f
3
= f
1
+ f
2
. This is not sufficient to determine if the peak at
frequency f
3
corresponds to a genuine non-linear interaction produced by the two oscillatory pro-
cesses that interact — and generate a third com- ponent — or if it corresponds to an independent
frequency. In order to resolve this ambiguity we compute the bispectrum. B
xxx
=
N
X f
1
X f
2
X f
1
+ f
2
, that will be near 0 in case of indepen- dence, and for the peaks in the bispectrum we
estimate the value of the interaction by the bico- herence
C
xxx
= B
xxx 2
P
xx
f
1
P
xx
f
2
P
xx
f
1
+ f
2
. In the case study xt an interaction exists
between f
1
, and f
2
-represented by the term ab-so that a significant value of the bicoherence is ob-
served for bifrequencies f
1
, f
2
. Let us now consider another signal yt,
recorded on a different channel, such that yt = S
N
c + d + cb where ct = cos2pf
1
t + v
c
and dt = cos2pf
2
t + v
d
and f
1
, and f
2
represent the same frequencies of signal xt and v
c
and v
d
are phases randomly changed in [0, 2p] for each
epoch. Notice the non-linear interaction repre- sented by the term cb The power spectra P
xx
f and P
yy
f are identical but in the case yt no interaction can be detected by the bispectrum B
yyy
because the term of interaction includes the com- ponent b with a phase, v
b
, that is absent from its linear components c and d.
The previous analysis can be extended to a bivariate case where xt and yt are recorded
simultaneously. In the cross-channel analysis the interactions are not symmetrical and one should
consider the influence of the generator processes of xt on yt and vice versa. Thus, for the
interaction x y we compute the cross-bispec- trum
B
xyx
= S
N
X f
1
Y f
2
X f
1
+ f
2
and the
cross-bicoherence C
xyx
= B
xyx 2
P
xx
f
1
P
yy
f
2
P
xx
f
1
+ f
2
. The non-linear interaction term of xt is ab
and it is independent from the linear terms of yt. Then, no significant peaks will be detected in
the crossbispectrum B
xyx
. The quantities B
yxy
and C
yxy
are derived in a similar way for the estima- tion of the interaction y x. In the case study the
interaction term cb contains the term b of xt and consequently the cross-bispectrum B
yxy
re- veals this non-linear interaction. In order to detect
the significant interactions at couples of frequen- cies f
1
and f
2
, we tested the hypothesis that the bispectrum was equal to zero Huber et al., 1971;
Brillinger and Irizarry, 1998 at the 99 confi- dence limit. Phase-coupled frequencies f
3
= f
1
+ f
2
were determined for corresponding significant bis- pectral analysis at couples of frequencies f
1
and f
2
.
3. Results
Kategori