96 E
Activity level differences were evaluated separately from Cruz Biotechnology, Inc., Santa Cruz, CA diluted in
the startle amplitude data with an ANOVA. antibody medium 1:5000. The sections were washed with
PBS and incubated 1 h at room temperature with a 2.6. Tyrosine hydroxylase TH immunohistochemistry
biotinylated secondary antibody against rabbit immuno- globulin G Jackson Laboratories in the antibody medium
At the end of the experiment, the animals were over- 1:400. The sections were washed and incubated for 30
dosed with chloral hydrate and perfused intracardially with min with the avidin–biotin complex Vector Laboratories.
0.9 saline followed by 10 formalin. The brains were The sections were then incubated 10 min at room
removed from the skull and immersed in the fixative for 2 temperature in 3,39-diaminobenzidine H O Sigma as a
2 2
h at 48C. After fixation, the brains were stored for 4 days chromagen for visualization of c-Fos reaction product.
in a 30 sucrose 0.1 M PBS pH 7.4 solution at 48C and Sections were mounted on gelatin-coated slides and cover-
subsequently, 30 mm frozen coronal sections were cut slipped with Permount for immunohistological examina-
through the SNc and striatum. Every third section was tion.
collected in a glass vial for a total of 20 sections vial. The sections were immunostained using the avidin–biotin–
2.8. Microscopy and cell count peroxidase ABC method [26]. Briefly, the sections were
incubated in 0.6 hydrogen peroxide in 0.1 M PBS pH For each animal, a c-Fos immunostained section of the
7.4 for 30 min to eliminate endogenous peroxidases. To CPu corresponding to Plate 18 20.26 mm caudal to
reduce non-specific binding, the sections were preincu- bregma; Fig. 1 from the atlas of Paxinos and Watson [56]
bated in antibody medium 2 normal goat serum, 1 was visualized under bright-field microscopy with a Zeiss
bovine serum albumin, 0.3 Triton X-100 in 0.1 M PBS, Axioscope Oberkochen, Germany. Still frame, 310
pH 7.4 for 1 h followed by incubation 24 h at room images of the CPu were captured with a Sony DXC5000
temperature with a primary antibody; mouse monoclonal digital camera interfaced with a Macintosh G3 computer.
antibody against TH MAB 318, Chemicon, Temecula, To quantify the number of c-Fos-positive cells within the
2
CA diluted in antibody medium 1:1000. The sections CPu, 1 mm squares in the dorsolateral DL, dorsomedial
were washed with PBS and incubated 1 h at room DM, ventrolateral VL and ventromedial VM CPu see
temperature with a biotinylated secondary antibody Fig. 1 were affixed to each of the captured images using
against mouse immunoglobulin G Jackson Laboratories, Adobe Photoshop software Adobe System Incorporated,
West Grove, PA in the antibody medium 1:400. The Mountain View, CA. For each animal, neurons with
sections were washed and incubated for 30 min with the round oval nuclei clearly stained dark brown were counted
avidin–biotin complex Vector Laboratories, Burlingame, in each of the four CPu areas and averaged across each
CA. The sections were then incubated 10 min at room treatment group.
temperature in 3,39-diaminobenzidine H O Sigma Fast;
2 2
Sigma, St Louis, MO as a chromagen for visualization of TH reaction-product. Sections were mounted on gelatin-
3. Results
coated slides and coverslipped with Permount Fisher Scientific, Unionville, Ontario for immunohistological
3.1. 6-OHDA lesions examination of the extent of the 6-OHDA-induced lesion.
Fig. 2 shows the type of lesion produced after bilateral 2.7. c-Fos immunohistochemistry
6-OHDA injections into the SNc. By using two injections per side, severe loss of TH-containing cells and processes
Ninety minutes after
L
-DOPA injections, animals were TH-containing dendrites in the substantia nigra pars
sacrificed by an overdose of chloral hydrate and perfused reticulata; SNr was seen throughout the rostrocaudal
intracardially with 0.9 saline followed by 10 formalin extent of the SNc. In addition, most animals had nearly
for 20 min. The brains were removed from the skull and complete sparing of the ventral tegmental area VTA. Fig.
immersed in the fixative for 2 h at 48C. After fixation, the 1 shows intense TH-staining in the CPu in SHAM animals
brains were stored for 4 days in a 30 sucrose 0.1 M PBS Fig. 1A versus a dramatic loss of TH-staining in the CPu
pH 7.4 solution at 48C and subsequently, 30 mm frozen in animals that received bilateral 6-OHDA lesions of the
coronal sections were cut through the striatum and DR. SNc Fig. 1B. Taken together, these data suggest that the
Adjacent sections through each brain region were collected DA-containing cells of the SNc, and subsequently the DA
in a glass vials for a total of 20 sections vial. For c-Fos innervation of the CPu, were destroyed by 6-OHDA.
immunohistochemistry of the CPu, sections were immuno- Despite being adipsic and aphagic for the first couple
stained using the avidin–biotin–peroxidase ABC method days after 6-OHDA lesions, rats responded well to the
described above. Briefly, the sections were incubated 24 h highly palatable foods placed in their cages to stave weight
at room temperature with a rabbit polyclonal antibody loss and help restore their health. In general, 6-OHDA-
against a 62 kD nuclear phosphoprotein c-Fos4, Santa lesioned rats lost between 100 and 150 g of body weight in
E .G. Meloni, M. Davis Brain Research 879 2000 93 –104
97
recovery as these animals were more resilient to the few days of ‘food-deprivation’ as a result of the 6-OHDA
lesions.
3.2. Systemic SKF 82958 and startle Fig. 3 shows the effect of systemic administration of
SKF 82958 0.05 mg kg on startle in SHAM and 6- OHDA-lesioned rats. A two-way ANOVA of the effects of
SKF 82958 across intensity Fig. 3A revealed a significant main effect of group [F
59.5, P,0.005], intensity
3,12
[F 540.1, P,0.0001] and a group by intensity inter-
4,12
action [F 53.07, P,0.005]. Individual comparisons
12,48
revealed that this low dose of SKF 82958 produced a significant enhancement of startle at each intensity in
6-OHDA-lesioned animals. SHAM animals showed a trend for a higher startle response by SKF 82958, but this effect
was not significant at any intensity. An analysis of activity levels one-way ANOVA revealed a significant elevation
in activity by SKF 82958 in 6-OHDA-lesioned, but not SHAM, animals.
A two-way ANOVA of the effects of SKF 82958 on startle over time collapsed across intensity for the duration
of the test session; Fig. 3B, revealed a significant main effect of group [F
59.5, P,0.005] and time [F 5
3,12 19,12
2.0, P,0.01]. The group by time interaction was not significant. Linear contrast analyses showed that 6-OHDA-
lesioned animals that received SKF 82958 had a significant elevation in startle across time compared to those that
received vehicle [F
519.8, P,0.001]. SHAM animals
1,12
that received SKF 82958 were not significantly different from those that received vehicle.
3.3. Systemic
L
-DOPA and startle Fig. 4 shows that systemic administration of
L
-DOPA 0, 1, 5, and 10 mg kg produced a dose-dependent enhance-
ment of startle in 6-OHDA-lesioned rats. A two-way ANOVA revealed a significant main effect of dose [F
5
3,16
4.2, P,0.05], intensity [F 547.6, P,0.0001] and a
4,16
group by intensity interaction [F 52.7, P,0.005].
12,64
Fig. 1. Photomicrographs of coronal sections through the mid-striatum
Individual comparisons revealed that compared to vehicle-
stained for tyrosine hydroxylase black areas from representative SHAM
treated rats, the high dose of
L
-DOPA 10 mg kg pro-
A and 6-OHDA-lesioned B rats corresponding to Plate 18 20.26 mm
duced a significant enhancement of startle at each intensi-
posterior to bregma, C from the atlas of Paxinos and Watson [56]. The
ty. Because of large variances in the amplitude of their
caudate–putamen CPu was divided into four regions at this level:
startle responses, animals treated with 5 mg kg of
L
-DOPA
dorsolateral DL, dorsomedial DM, ventrolateral VL and ventrome-
2
dial VM and c-Fos positive cells were counted in 1 mm areas within
showed a significant elevation at the 80 dB intensity only.
each subdivision.
There was no significant effect on startle at any intensity in animals treated with the low dose of
L
-DOPA 1 mg kg. the first three to 4 days after surgery before reaching a
An analysis of activity levels one-way ANOVA revealed steady body weight. This observation is consistent with
a significant elevation in activity by
L
-DOPA 5 and 10 other reports that rats are able to recover from consummat-
mg kg doses in these 6-OHDA-lesioned rats. ory deficits caused by bilateral 6-OHDA lesions and
Fig. 5 shows the effect of the high dose of
L
-DOPA 10 increase their body weight in a regular fashion when cared
mg kg on startle in SHAM and 6-OHDA-lesioned ani- for in this manner [13]. In addition, the use of heavier rats
mals. A two-way ANOVA of the effects of
L
-DOPA across e.g. rats that have ample fat reserves helped with
intensity Fig. 5A revealed a significant main effect of
98 E
Fig. 2. Photomicrographs of coronal sections in millimeters posterior to bregma through the substantia nigra pars compacta SNc from representative SHAM A and 6-OHDA-lesioned B rats. Extensive loss of tyrosine hydroxylase TH-containing cells and processes black areas of the SNc, but not
the ventral tegmental area VTA, was seen after bilateral 6-OHDA-lesions of the SNc.
group [F 512.5, P,0.0005], intensity [F
5109.1, main effect of time, and the group by time interaction, was
3,16 4,16
P,0.0001] and a group by intensity interaction [F 5
not significant. Linear contrast analyses showed that
L
-
12,64
5.1, P,0.0001]. Individual comparisons revealed that this DOPA produced a significant elevation in startle across
dose of
L
-DOPA produced a significant enhancement of time in 6-OHDA-lesioned animals compared to 6-OHDA
startle at each intensity in 6-OHDA-lesioned animals. animals that received vehicle [F
517.4, P,0.001].
1,16
Startle was not significantly affected by
L
-DOPA in SHAM SHAM animals that received
L
-DOPA were not sig- animals at any intensity. An analysis of activity levels
nificantly different from those that received vehicle. one-way ANOVA revealed a significant elevation in
activity by
L
-DOPA in 6-OHDA-lesioned, but not SHAM, 3.4. c-Fos expression in the CPu
animals. A two-way ANOVA of the effects of
L
-DOPA 10 Fig. 6 shows representative digitized photomicrographs
mg kg on startle over time collapsed across intensity for of the CPu DL division from SHAM and 6-OHDA-
the duration of the test session; Fig. 5B, revealed a lesioned rats that received either vehicle or
L
-DOPA significant main effect of group [F
59.6, P,0.005]. The behavioral data presented in Fig. 5. There was heavy
3,16
E .G. Meloni, M. Davis Brain Research 879 2000 93 –104
99
Fig. 4. Dose-dependent enhancement of startle by
L
-DOPA in 6-OHDA- lesioned rats. Significant differences from vehicle response: P,0.05,
P,0.005.
a practical application to the study of Parkinson’s disease [72]. Animals with unilateral 6-OHDA lesions of the
nigrostriatal dopamine system show a gain of motor function expressed as a contralateral away from the
lesioned side turning behavior when challenged with direct DA agonists such as apomorphine mixed D D
1 2
agonist [22,79], SKF 38393 D agonist [49,63], or the
1
Fig. 3. Enhancement of startle by the dopamine D receptor agonist SKF
1
indirect DA agonist
L
-DOPA [72]. These effects are
82958 SKF; 0.05 mg kg in 6-OHDA-lesioned versus SHAM rats across
believed to represent an enhanced sensitivity supersen-
intensity A and time B. Significant differences from vehicle VEH
sitivity by DA receptors in the denervated striatum to DA
response: P,0.05, P,0.005.
agonists which subsequently leads to an enhanced be- havioral response [72].
expression of c-Fos throughout the CPu, in all divisions, in In the present study, we have found that the acoustic
6-OHDA-lesioned but not SHAM rats that received
L
- startle reflex is also sensitive to the effects of specific DA
DOPA 10 mg kg. Quantification of c-Fos-positive cells agonists in animals with 6-OHDA lesions of the nigros-
2
in each of the CPu subdivisions 1 mm grids for 6-
triatal pathway. Systemic administration of a threshold OHDA-lesioned and SHAM animals that received either
dose of the dopamine D agonist SKF 82958 0.05 mg kg
1
vehicle or
L
-DOPA is shown in Fig. 7. A two-way ANOVA produced a marked enhancement of startle in 6-OHDA-
revealed a significant main effect of group [F 562.6,
lesioned, but not SHAM animals. In fact, this enhancement
3,16
P,0.0001] and a group by intensity interaction [F 5
was roughly equal to that seen in normal animals using a
9,48
3.1, P,0.01]. The main effect of CPu subdivision was not 20-fold higher dose of SKF 82958 [43] and thus reflects a
significant. Individual comparisons showed that c-Fos shift-to-the-left in the dose–response curve. Because we
expression was significantly elevated by
L
-DOPA in each have shown that D receptors mediate SKF 82958-induced
1
CPu subdivision in 6-OHDA-lesioned animals. effects on startle [42], the enhanced behavioral response to
SKF 82958 in 6-OHDA-lesioned animals in the present study is consistent with the hypothesis that DA receptors,
4. Discussion specifically D receptors, are supersensitized as a result of
