94 E
seen in this akinetic disease, DA-containing cells of the River, Raleigh-Durham, NC housed in group cages of
nigrostriatal pathway are selectively destroyed by in- four rats each until the time of surgery when they were
tracerebral injection of the neurotoxin 6-OHDA. Typically, singly housed. Animals were maintained on a 12-h light
the lesion is unilateral, leaving a denervated and an intact dark cycle lights on at 07.00 h with food and water
side within the same animal. Subsequently, systemic continuously available.
challenge with DA agonists in this ‘hemi-parkinsonian’ rat produces a circling behavior that is specific to the type of
2.2. Startle apparatus DA agonist used. Indirect DA agonists such as amphet-
amine induce ipsilateral rotation [73] by releasing DA at Startle testing of animals was conducted using four
the level of the striatum from the intact side. In contrast, identical stabilimeter devices located in separate, custom-
direct DA agonists such as apomorphine [22] and SKF designed 90370370 cm sound-attenuating chambers. Ven-
38393 [48] induce contralateral rotation, presumably due tilation for each chamber was provided by the ventilation
to activation of supersensitized DA receptors in the unit from an Industrial Acoustics Minibooth Industrial
denervated striatum [72]. Interestingly, systemic adminis- Acoustic Company, Bronx, New York. Each stabilimeter
tration of the anti-Parkinson’s drug
L
-3,4-dihydroxyphenyl- consisted of a 9315315 cm Plexiglas and wire-mesh cage
alanine
L
-DOPA also induces contralateral rotation suspended between compression springs within a steel
[24,72], despite the fact that 6-OHDA lesions dramatically frame. The floor of each stabilimeter consisted of four 6.0
reduce the activity .80 of aromatic
L
-amino acid mm diameter stainless steel bars spaced 18 mm apart. Cage
decarboxylase AADC, i.e. the enzyme responsible for the movement resulted in displacement of an accelerometer
conversion of
L
-DOPA into DA [40]. Spared DA neurons PCB Piezotronics, Depew, NY where the resultant
[23], serotonergic neurons [3,25], or striatal interneurons voltage was proportional to the velocity of the cage
that contain AADC [40,51] may all be possible sites for displacement. The analog output of the accelerometer was
the conversion of
L
-DOPA to DA which can act at amplified PCB Piezotronics, Model 483B21 and digitized
sensitized striatonigral DA receptors to induce contralateral on a scale of 0–2500 units by an InstruNET device GW
rotation in 6-OHDA-lesioned animals. Instruments, Model 100B; Somerville, MA interfaced to a
In the present study, we have used the 6-OHDA lesion Macintosh G3 computer. Startle amplitude was defined as
preparation to examine the effects of the dopamine D the peak accelerometer voltage that occurred during the
1
receptor agonist SKF 82958 on startle in animals with first 200 ms after onset of the startle stimulus. A sur-
destruction of the DA-containing cells of the substantia veillance camera Burle, Model TC651B with a TC9907a
nigra pars compacta SNc. Because striatonigral neurons lens, Operational Security Systems, Atlanta, GA was
mediate the enhancement of startle by SKF 82958 [42], positioned behind each stabilimeter and connected to a TV
putative 6-OHDA-induced supersensitivity of D receptors monitor located outside the isolation chamber. A red light
1
located on these neurons should lead to a potentiated bulb 7.5 W was located on the ceiling of the startle box
startle response by SKF 82958. In addition, we tested the to provide the illumination for the cameras in the otherwise
effects of the indirect DA agonist
L
-DOPA on startle in completely dark box.
6-OHDA-lesioned animals. Previous studies have reported Constant wide band background noise 60 dB was
either no effect [19] or an enhanced startle response only produced by a General Radio noise generator Type 1390-
after high doses of
L
-DOPA [29] in normal animals. Like B and delivered through high frequency speakers Radio
SKF 82958, we predict that
L
-DOPA should potentiate the Shack Supertweeters, range 5–40 kHz located 5 cm from
startle response in 6-OHDA-lesioned animals via superse- the front of each cage. Startle responses were evoked by 50
nsitive striatonigral DA receptors. An additional goal of ms bursts of white-noise 5 ms rise-decay generated by
the present study was to measure any
L
-DOPA-induced the Macintosh G3 computer 0–22 kHz, amplified by a
changes in the activity of caudate–putamen CPu cells Radio Shack amplifier 100 W; Model MPA-200, and
using c-Fos immunohistochemistry, a well-established delivered through the same speakers. Sound level measure-
¨ marker for neuronal activation [64]. Several studies have
ments SPL were made with a Bruel and Kjaer model reported a dramatic increase in c-Fos expression in the
2235 sound-level meter A scale; random input with the CPu of unilateral 6-OHDA-lesioned animals after
L
-DOPA microphone Type 4176 located 10 cm from the center of
treatment [11,61] that may reflect a supersensitive response the speaker, which approximates the distance of the rat’s
by striatal receptors to DA converted from exogenous ear from the speaker during testing. The presentation and
L
-DOPA. sequencing of all stimuli were under the control of the
Macintosh G3 computer using specially designed software The Experimenter, Glassbeads Inc., Newton, CT.
2. Materials and methods
2.3. Bilateral 6-OHDA lesions 2.1. Animals
Rats were anesthetized with Nembutal 50 mg kg, The animals were male Sprague–Dawley rats Charles
intraperitoneally, i.p. and placed in a Kopf stereotaxic
E .G. Meloni, M. Davis Brain Research 879 2000 93 –104
95
instrument Model 900 with blunt ear bars. The skin was measurements were made by sampling cage movement
retracted and bilateral holes were drilled in the skull above 200 ms duration 15 s after the onset of each startle
the SNc. A 28 g stainless steel needle attached to a stimulus.
Hamilton microsyringe 10 ml by polyethylene tubing was lowered into the brain using the following coordinates:
2.4.3. SKF 82958 and startle 25.3 and 26.5 mm posterior to bregma two injections
One week after surgery, half of the animals in the side, 62.1 mm lateral to the midline, 27.3 mm ventral to
6-OHDA and SHAM groups n54 group received either dura. A Harvard Apparatus Model 22 infusion pump was
SKF 82958 [6]-chloro-APB HBr, 0.05 mg kg or saline used to deliver 0.5 ml injection of 6-OHDA 2.5 mg in
0.9 and were immediately placed in the startle cages 0.9 saline containing 0.1 ascorbic acid; Sigma, St
and received a test session as described above. The dose of Louis, MO directly into the SNc at a rate of 0.25 ml min
SKF 82958 was chosen based on its weak startle enhanc- for 2 min followed by a 1 min diffusion wait. SHAM
ing effects determined in previous studies [43]. All in- animals received injections of vehicle into the SNc using
jections were subcutaneous s.c. and SKF 82958 Re- the procedure described above. After the injection, the
search Biochemical International; Natick, MA was dis- skull holes were packed with sterile Gelfoam and the
solved in 0.9 saline. Three days later, the drug order was wound was closed with surgical clips.
reversed so that systemic treatment with SKF 82958 was a Because 6-OHDA-lesioned animals showed consummat-
within-subjects measure. ory deficits after recovery from surgery, in addition to the
normal availability of food and water, these rats were 2.4.4.
L
-DOPA and startle given highly palatable foods such as cookies Chips Ahoy
One week after surgery, 6-OHDA animals received a and ground rat chow mixed with water and sucrose. A
matching test as described above and were divided into liquid supplement PediaLyte was also placed in a small
four groups n55 per group with equivalent levels of container on the floor of the cage. These methods for
startle. SHAM animals also received a matching test and maintaining the health of aphagic and adipsic 6-OHDA-
were divided into two groups n55 per group with lesioned animals, without the necessity for intragastric
equivalent levels of startle. Three days later, animals in feeding tubes, have been previously described [7,13,52]. In
each group were pretreated i.p. with the peripheral amino addition, because pilot studies have shown that 6-OHDA-
acid decarboxylase inhibitor benserazide 25 mg kg; lesioned rats tend to lose between 100 and 150 g in the few
Sigma followed after 20 min by injections i.p. of
L
- days after surgery, heavier rats 500–600 g were used as
DOPA Sigma: 6-OHDA animals 0, 1, 5 or 10 mg kg, these animals were more resilient to the weight-loss effects
SHAM animals 0 or 10 mg kg. Ten minutes later, all of bilateral 6-OHDA lesions. SHAM animals weighing
animals received a startle test as described above. Ben- between 400 and 450 g were used such that both groups of
serazide was dissolved in water and given in a volume of 1 rats had roughly equivalent weights at the time of testing.
ml kg and
L
-DOPA was dissolved in 1 N HCL, pH balanced to 7.0 and given in a volume of 2 ml kg.
2.4. Behavioral procedure 2.4.1. Matching
2.5. Statistical analysis Rats were placed in the startle cages and received a 5
min acclimation period followed by presentation of five Startle
amplitude data
were expressed
as the
habituating startle stimuli 95 dB, 30 s interstimulus mean6S.E.M. across startle stimuli at each of the five test
interval; ISI. Rats were then presented with 50 startle intensities or as data points collapsed across intensity for
stimuli, 10 at each of five different intensities 80, 85, 90, an analysis of drug effects across time. For SKF 82958
95 and 100 dB in a semirandom order with a 30 s ISI. effects on startle, significant differences were evaluated
Rats were assigned to the various treatment conditions using analysis of variance ANOVA followed by indi-
based on their startle responses such that the mean startle vidual comparisons using Student’s t-tests. Lesion group
level for each group was comparable. In addition to startle 6-OHDA and SHAM was a between-groups factor and
measurements, baseline activity measurements were made drug treatment SKF 82958 and saline, intensity 80, 85,
by sampling cage movement 200 ms duration 15 s after 90, 95, 100 dB and time were within-groups factors.
the onset of each startle stimulus. Activity level differences were evaluated separately from
the startle amplitude data with an ANOVA. 2.4.2. Startle testing
For
L
-DOPA effects on startle and c-Fos cell counts, Rats were placed in the startle cages and received a 5
significant differences were evaluated using analysis of min acclimation period followed by presentation of five
variance ANOVA followed by individual comparisons habituating startle stimuli 95 dB, 30 s ISI. Rats were then
using Student’s t-tests. Lesion group 6-OHDA and presented with 100 startle stimuli, 20 at each of five
SHAM and
L
-DOPA dose 0, 1, 5, and 10 mg kg were different intensities 80, 85, 90, 95 and 100 dB in a
between-groups factors whereas intensity 80, 85, 90,95, semirandom order with a 30 s ISI. Baseline activity
100 dB and CPu subdivisions were within-groups factors.
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
