110 A
mone primed female rats and rarely in cycling females. Sections were then rinsed with PBS, mounted onto gelatin-
The present study was conducted in cycling and ovariec- coated slides, dehydrated in graded alcohol and cover-
tomized rats, in order to identify the spinal distribution of slipped with Eukitt. Five sections of each spinal segment
neurons responding to VCS and to verify whether the were randomly selected and examined under a light
estrous cycle modulates c-fos expression in lumbosacral microscope. Fos-immunoreactive neurons characterized by
spinal segments that receive sensory afferents from the the presence of a brown reaction product in their nuclei
vaginocervical area. were counted. The mean number of Fos-positive neurons
was calculated for each labeled area in each studied spinal segment. Comparison between areas within a segment,
2. Materials between segments within a group and between groups was
performed with analysis of variance ANOVA and pair- Adult virgin female Sprague–Dawley rats Centre Jaber
wise multiple comparison was performed with the Stu- Ben El Hayan FSS-Marrakech weighing 190–220 g were
dent–Newman–Keuls methods. Differences were consid- used in this study. Rats were housed in animal facilities
ered significant for P,0.05. under a 12:12 h Light Dark cycle light on at 08:00 h
To determine the estrous stage of each female, vaginal with free access to food and water.
smears were colored with 10 Giemsa and analyzed under VCS was performed in an experimental group of
a light microscope. We have observed that the smears females, lightly anaesthetized with an intra-peritoneal
performed using the balloon or by a conventional tech- injection of 25 mg of ketamine chlorhydrate kg bw,
nique gentle swap gived similar results. In the ex- ˆ
´ IMALGENE 500, Rhone Merieux, Lyon, France. Anes-
perimental group, cycling females were assigned to one of thesia prevented animal motility and agitation during the
three groups: estrous, diestrous and proestrous. The num- stimulation which obliged the operator to apply unwanted
ber of rats at each stage was 5. Statistical analysis of Fos skin stimulation to maintain the animal. A latex balloon
data in cycling, non-stimulated control rats, revealed no was inserted through the vagina at the vaginocervical level
differences due to estrous cycle. Five rats were then and then inflated via a catheter with 0.5 ml of water 20
selected randomly to form the control group of cycling mmHg to distend the wall for 10 min. To record vag-
females. inocervical contractions, the catheter was connected to a
In order to verify the effect of ovariectomy, ten females pressure transducer Telco M52. Pressure signal was sent
were ovariectomized
bilaterally under
Ketamine to a paper chart recorder CS 600. At the end of the
chlorhydrate anesthesia 100 mg kg via lumbar incisions. stimulation, the balloon was deflated and used to perform a
After a recovery of 4 weeks, 5 females received vag- vaginal smear.
inocervical stimulation and the 5 others were used as Control rats were lightly anaesthetized and handled as
ovariectomized, non-stimulated controls. long as the experimental group. No VCS was applied. The
vaginal smear was performed just before perfusion. After a surviving period of 60 min after the end of the
3. Results
VCS, rats were killed by an overdose of anaesthetic Nembutal and perfused intracardially with 250 ml of
3.1. Vaginal and cervix activity during VCS phosphate buffered saline PBS, 0.1 M, pH 7.4, followed
by 500 ml of 4 paraformaldehyde in PBS. The spinal In anaesthetized female rats, vaginocervical contractions
cord was exposed and the L , L and S segments were were recorded during VCS. Fig. 1 represents a typical
5 6
1
removed and post-fixed overnight at 48C in the same recording of vaginocervical activity which showed periodic
fixative. Transverse sections 40 mm thick were cut on a contractions followed by a period of rest, corresponding to
vibratome, processed as free-floating sections and Fos the activity of smooth muscle of vaginocervical area. The
immunostained according to the avidin–biotin–peroxidase contractions
lasting 36.5067.26
s and
reaching method [23]. Sections were pre-incubated for 1 h in normal
12.4361.39 mmHg. The mean duration of the rest period goat serum 2 in PBS and 0.3 Triton X-100, followed
was 21.8067.82 s. by incubation for 48 h at 48C in the primary antibody,
raised against Fos protein, diluted at 1:1000 Oncogene 3.2. Fos-labeling in control and experimental cycling
Science Paris, France. Sections were then rinsed in PBS, females
incubated in biotinyled goat anti-rabbit antiserum Vector Laboratories, Burlingame, CA at 1:200 for 4 h, washed in
A preliminary study had shown that the most meaning- PBS and then incubated for 1 h 30 at room temperature in
ful c-fos expression in response to VCS was observed in avidin–biotin–peroxidase complex Vectastain Elite ABC
L –S segments in comparison with the other lumbar
5 1
Kit, Vector Laboratories, Burlingame, CA. The peroxidase segments. In the three spinal segments L , L and S , c-fos
5 6
1
activity was visualized with 0.05 of the chromogen expressing neurons in response to VCS were localized in:
diaminobenzidine DAB and 0.03 hydrogen peroxide. i the superficial layers and the medial part of the dorsal
A . Ghanima et al. Brain Research 880 2000 109 –117
111
Fig. 1. Typical recording of intravaginal pressure recorded during vaginocervical stimulation of ovariectomized female showing periodic contractions of nearly equal amplitude and duration.
horn DH, ii the intermediolateral cell column ILC, Fos-labeled neurons than diestrous and proestrous P,0.05
iii the dorsal gray commissure MGC and iv the for each.
ventral horn VH Fig. 2. The DH contained the greatest In L and L , estrous, diestrous and proestrous females
5 6
number of Fos-positive neurons 6067 of the total Fos- displayed significantly more Fos-positive neurons in re-
labeled nuclei, whereas the MGC, the VH and the ILC sponse to VCS, relative to cycling non stimulated controls
24
contained respectively 1864, 1764 and 562 Table respectively F3,16523.6, P,1.10
and F3,16521.2,
24
1. P,1.10
. In S , only estrous and diestrous females
1
One way ANOVA revealed significant differences in the significantly displayed a greater number of Fos-immuno-
total number of Fos-labeled neurons over the L –S spinal reactive neurons relative to control F3,16513.6, P,
5 1
24
portion between the four groups estrous, diestrous, 1.10
. Cycling females showed different quantities of proestrous and cycling non-stimulated controls F3,165
Fos-labeled neurons in response to VCS. Estrous females
24
31.7, P,1.10 . Student–Newman–Keuls test further
had a greater number of Fos-labeled neurons than proestr- revealed that stimulated rats displayed significantly more
ous in L 264.1620.0 and 131.0613.5, respectively,
5 23
Fos-labeled neurons than control rats Fig. 3 and among F2,12510.7, P52.2.10
, in L 238.2618.4 and
6 23
stimulated females, estrous rats had a greater number of 106.2613.4, respectively, F2,12511.9, P51.4.10
and in S 183.2628.7 and 90.7610.1, respectively,
1 22
F2,1255.7, P51.82.10 and than in diestrous in L
6
238.2618.4 and 172.4624.2 respectively, F2,12511.9,
23
P51.4.10 . The number of Fos-positive neurons in the
diestrous group was constantly greater than that of proestr- ous in L
202.5625.7 and 131.0613.5, respectively,
5 23
F2,12510.7, P52.2.10 , in L
172.4624.15 and
6 23
106.1613.7, respectively, F2,12511.9, P51.4.10 and in S
159.9617.2 and 90.7610.1, respectively,
1 22
F2,1255.7, P51.82 10 .
Data analysis of the different spinal segment areas revealed that the DH of estrous females contained sig-
nificantly more Fos-positive neurons than proestrous and diestrous in L , L and S P,0.05 for each. In diestrous
5 6
1
females, the DH displayed more Fos-labeled neurons than proestrous in L
and S P,0.05 for each. The ILC
5 1
presented significant differences only in the S segment
1
Fig. 2. Examples of transverse sections of the L , L and S spinal cord
5 6
1
between estrous and diestrous females vs proestrous P,
segments of an estrous female showing distribution of Fos-labeled
0.05 for each. In contrast, there was no difference in the
neurons in the dorsal horn DH, intermediolateral cell column ILC,
number of Fos-positive neurons in the MGC between the
dorsal grey commissure MGC and the ventral horn VH after vag-
different estrous cycle stages. The number of Fos-labeled
inocervical stimulation. CC: central canal. Each dot represents a Fos- positive cell 3 15.
neurons in the VH differed significantly in the L segment
6
112 A
Table 1 Mean number of Fos-positive neurons 6S.D. in the different spinal areas of the L -S spinal cord in the different groups
5 1
Control Cycling stimulated rats
OvX stimulated
c
Cycling OvX
Proestrous Estrous
Diestrous L
DH 35.1668.28
33.28610.71 75.12619.34
193.24619.43† 133.28619.42
127.32614.60
5
54 57
57 73
66 60
MGC 4.0863.62
2.2061.32 5.2061.84
10.0063.83 8.4868.26
8.8863.49 6
4 4
4 4
4 ILC
12.2064.34 11.4065.32
31.56613.47 33.7669.60
30.60619.43 33.7669.60
19 19
24 13
15 16
VH 13.2065.65
11.6863.52 19.1463.30
27.12619.46 30.12617.46
41.40622.24 21
20 15
10 15
20 L
DH 27.8467.17
27.0468.38 59.50614.72
152.52625.82† 86.68619.45
96.4868.93
6
51 48
56 64
50 56
MGC 1.2060.35
1.0860.72 5.5063.53
14.1262.50 12.92610.13
13.0864.95 2
2 5
6 7
8 ILC
11.7666.98 16.9665.81
21.95617.57 37.48615.88
34.20626.77 33.28617.09
21 30
21 16
20 19
VH 14.32610.42
11.2069.01 19.2764.07
34.1268.34 38.64610.76
29.40614.33 26
20 18
14 22
17 S
DH 22.7266.06
24.5265.83 52.68610.09
114.40631.95† 84.6465.28
79.40610.94
1
55 56
58 62
53 48
MGC 0.5260.66
1.0060.93 2.2461.65
14.2067.09 11.4865.12
9.7663.38 1
2 2
8 7
6 ILC
9.7264.68 10.3663.29
16.24611.81 29.08618.38
34.16616.40 44.12617.98
24 24
18 16
21 27
VH 8.0068.45
7.6066.39 19.5265.56
25.52615.04 29.64617.91
31.80612.53 20
18 22
14 19
19 Significantly higher than proestrous. P,0.05.
†
Significantly higher than diestrous. P,0.05.
c
OvX: ovariectomized.
between estrous and proestrous P,0.05 and between three spinal segments. The total number of Fos-positive
neurons over the L –S spinal segments observed in
diestrous and proestrous P,0.05 Fig. 4.
5 1
ovariectomized females, in response to VCS represents 80 of that of estrous females, 102 of diestrous females
3.3. Fos-labeling in ovariectomized females and 167 of proestrous females.
Data of different spinal segment areas indicate that In ovariectomized, female rats, the VCS induced a Fos-
differences between stimulated females of both ovariec- labeling distribution similar to that described in cycling
tomized and cycling groups, were restricted to differences females in the three spinal segments. The number of
in Fos-labeling in the DH. No difference occurred in the Fos-positive neurons was significantly higher in ovariec-
other spinal areas between groups. The number of Fos- tomized vaginocervical stimulated rats relative to ovariec-
labeled neurons in DH was greater in the estrous group and tomized non-stimulated controls in L
212.4615.2 and
5 24
smaller in the proestrous group, relative to the ovariectom- 58.664.7, respectively, F1,8592.3, P,1.10
, in L
6
ized vaginocervical-stimulated group in L F3,16534.8, 172.2612.5 and 56.366.4, respectively, F1,8567.9,
5 24
24 24
P,1.10 and in S 165.1619.2 and 43.164.3, respec-
P,1.10 ,
L F3,16522.8,
P,1.10 and
S
1 6
1 24
24
tively, F1,8537.9, P,1.10 .
F3,16510.1, P56.10 . Concerning other spinal areas,
There was no difference in the total number of Fos- the ILC of S in proestrous females contained a smaller
1
labeled neurons between control groups of both cycling number of Fos-positive neurons relative to ovariectomized
and ovariectomized rats at the three spinal segments. In vaginocervical-stimulated rats.
contrast, the number of Fos labeled neurons in responses to VCS was significantly higher in ovariectomized females
than that of proestrous but lower than that of estrous,
4. Discussion
