Glucocorticoid programming of dopamine neurons. Virdee et al.
Materials and Methods (full details)
All procedures were conducted under license in accordance with the United Kingdom
Animals (Scientific Procedures) Act of 1986.
Animals
Sprague-Dawley rats were purchased for timed matings (Harlan Olac, Blackthorn, Beicester,
Oxfordshire, UK) as described previously (McArthur et al., 2005; McArthur et al., 2006;
McArthur et al., 2007a). On arrival, males and females were caged separately and allowed to
acclimatize and recover from transport stress for 1 week before being housed together
overnight. The presence of vaginal plugs the following morning indicated mating (designated
gestational day 1, GD1). Pregnancy was confirmed approximately 6 days later by palpation.
Timed-pregnant rats were housed 5 per cage until GD 15, when they were individually
housed in solid-bottomed cages in preparation for the day of birth, designated postnatal day
one (P1). Offspring were weaned at P21, at which time males and females were housed
separately in standard cages and allowed to mature to young adulthood (3-4 months)
undisturbed, apart from standard husbandry, before commencement of the experimental
procedures. All animals were maintained under a 12/12 hour reversed light/dark cycle (lights
off at 7 am) and controlled temperature (21-230C) and humidity (63%) with standard rat
chow and drinking water available ad libitum.
Antenatal glucocorticoid treatment (AGT)
The synthetic GC, dexamethasone (dexamethasone sodium phosphate, Fauliding
Pharmaceuticals Plc., Royal Leamington Spa, UK), was added to the drinking water of
1
pregnant dams at a final concentration of 0.5 µg/ml on GD 16 - 19; control dams continued to
receive normal drinking water. This non-invasive, stress-free regimen delivers a dose of
dexamethasone to the mother (~ 0.075 mg/kg/day, based on an approximate water intake of
50 ml /day (McArthur et al., 2005)), which is approximately 3-fold lower than that used in
perinatal medicine (Ballard et al., 1995; Jobe and Soll, 2004), 4-fold less than that calculated
to mimic the physiological levels of GCs required to mature the fetal rat lung at GD 18-21
(Samtani et al., 2006a, b), insufficient to alter body weight at term (Kreider et al., 2006) and
has no significant effects on the outcome of the pregnancies or maternal behavior (McArthur
et al., 2006; McArthur et al., 2007a). Thus, offspring were reared by their biological mothers
in order to avoid potential effects of cross-fostering, which can alter both maternal behaviors
(Maccari et al., 1995) and DA transmission in adult offspring (Kane et al., 2004). Potential
effects of litter-of-origin were minimized by selecting no more than 2 offspring per litter to
contribute to the male and female AGT (dexamethasone exposure) and control (normal
drinking water) groups. Female subjects were not selected for specific stages of the estrous
cycle, which involves stressful processes of handling and probing to sample vaginal cytology;
daily handling of males would not mimic this invasive procedure and therefore has uncertain
equivalence as a control (Greenspan et al., 2007). Moreover, such stressors could potentially
interfere with the results of this study.
Immunohistochemistry
Immunohistochemical identification of tyrosine hydroxylase immunoreactivity (TH-IR) was
used to identify DA cell bodies in the VTA and SNc and their respective nerve terminals in
the ventral striatum (NAc, core and shell) and dorsal striatum (caudate-putamen, CPu). Adult
offspring were decapitated at 12 weeks of age between 09:00 and 10:00 (McArthur et al.,
2007b). Brains were rapidly removed and immersed in 4% formaldehyde in phosphate-
2
buffered saline (PBS), (0.1M NaH2PO4.2H2O, 0.1M Na2HPO4.12H2O, 0.15M NaCl (all
VWR International, UK), pH 7.4) for one week, cryoprotected by immersion in 20% sucrose
in PBS for 48h, then frozen and stored at -80°C. Serial coronal cryosections (20µm) were
colleceted and stored in an antifreeze solution (0.1M NaH2PO4.H2O, 0.05M Na2HPO4,
0.15mM NaCl, 50% v/v ethanediol (all VWR International), 1% w/v polyvinylpyrrolidone,
0.1% w/v NaN3 (both Sigma-Aldrich, UK)) at -20°C until required. Immunostaining was
carried out on free-floating sections (McArthur et al., 2007a). Briefly, sections were
permeabilized with 0.05% Triton X-100 in PBS; non-specific binding sites were saturated by
incubation for 1h with 10% normal goat serum (NGS) in PBS, and sections were incubated at
room temperature overnight under constant agitation with the rabbit polyclonal anti-tyrosine
hydroxylase (TH) (Millipore Ltd., UK) diluted 1:3000 in 1% NGS, as the primary antibody
for detection of the rate-limiting DA synthetic enzyme. Sections were washed in 1% NGS in
PBS and TH immunoreactivity TH-(IR) was visualized by incubation with AF594-conjugated
goat anti-rabbit IgG (Invitrogen Ltd., UK), diluted 1:500 in 1% NGS, for 1h at room
temperature. Sections were finally washed (3 x 10 min in PBS), mounted on glass microscope
slides and allowed to air-dry before being placed under cover-slips using an aqueous
mountant (Vectashield, Vector Laboratories, Peterborough, UK).
Estimates of DA neuron numbers were based on protocols for measuring the effects of sex
steroid hormones on cell numbers in specific brain nuclei (Balthazart et al., 2008; McArthur
et al., 2011; Yamamura et al., 2011). Images of the SNc and VTA were captured and
digitized using a CoolSNAP-Procf camera (Roper Scientific, Marlow, UK) attached to a
NikonEclipse E800 microscope (Media Cybernetics, UK), with an image analysis software
package (Image ProPlus 4.5, Media Cybernetics, Finchampstead, UK). At low magnification
(x35), which included the whole of the coronal plane of the SNc and VTA, regional
3
boundaries were outlined in digitized images, as defined by the presence of TH-IR neuronal
groups and neuroanatomical landmarks, which were the thalamus dorsally and the substantia
nigra pars reticulata ventrally for the SNc, and the parabrachial pigmented nucleus dorsally
and the interfascicular and interpeduncular nuclei medially for the VTA. The adjacent SNc
and VTA are also distinguished by the third cranial nerve tract running between the two
nuclei. As illustrated previously (McArthur et al., 2007a), this is most obvious at level B,
defined as -5.1 to -5.4 mm relative to bregma according to the parcellation scheme of Carman
et al (Carman et al., 1991)where the SNc/VTA are divided into four levels (A-D), each
spanning 300 µm from -4.8 mm to -6.0 mm with respect to bregma. Therefore, the present
study examined alternate sections within this region for estimation of TH-IR cell counts,
which we have shown consistently to be increased in AGT-exposed animals relative to
controls (McArthur et al., 2005; McArthur et al., 2007a). TH-IR cell counts and densities
were estimated by overlaying a counting grid (50 µm x 50 µm) on the regions of interest and
immunoreactive cells were maually tagged (x100) in systematically, randomly selected visual
fields within the grid; the number of TH-IR cells per field, grid-square area and number of
grid squares sampled were used to calculate cell densities. The level B volumes of the SNc
and VTA were calculated according to the method of Cavalieri, as detailed elsewhere
(Brodski et al., 2003; McArthur et al., 2007a). Briefly, the average cross sectional area for
both regions (defined by the presence of TH-IR neuronal groups, as above), measured in
every second section, was multiplied by the total number of sections and the section
thickness, with allowance for tissue shrinkage using an electronic microcator (McArthur et
al., 2007a). The total number of level B SNc and VTA TH-IR neurons was estimated by
relating their numerical cell density to the volume, and used to calculate the group average (n
= 6 rats per group). These methods provide cell estimates which are in good agreement with
4
others using sterological or non-sterological methods (Brodski et al., 2003; Dewing et al.,
2006; McArthur et al., 2007a; Balthazart et al., 2008; Yamamura et al., 2011).
For analysis of TH-IR fiber density in the CPu (innervated primarily by SNc DA neurons)
and NAc, (innervated primarily by VTA DA neurons) core and shell, images of a 1µm optical
section were captured using a Leica SP5 confocal microscope fitted with a 63x oil immersion
objective. Threshold masks were applied using ImageJ software, at a standardised intensity
for all samples. Percentage area cover was then measured by systematic random sampling of
matrix regions of the CPu and NAc, allowing for background correction, in at least 6
different sections per animal. The average fiber density for each region per animal was used
to calculate the group average (n = 6 rats per group).
Autoradiography
The DA transporter (DAT), D1-type (D1) and D2-type (D2) receptors were analyzed in the
CPu, NAc (core and shell) regions in order to assess the effects of AGT on pre- and postsynaptic markers of DA function. Male and female adult offspring of control and AGTexposed dams (n=6 per group) were decapitated at 12 weeks of age between 9-10am. Brains
were quickly removed (n=6 per group), frozen at -40 ºC, and stored at -80 ºC until required.
Serial coronal sections (20 µm) were cut using a cryostat (Bright Instruments Ltd) maintained
at -22°C. Sections were collected between +1.7 mm and +2.2 mm relative to bregma for the
NAc core and shell, whereas for the CPu serial sections were collected between -0.2 and
+1.6mm relative to bregma. Levels of DAT, D1 and D2 were quantified using binding of the
highly specific ligands [125I]-RTI 121, [3H]-SCH23390 and [3H]-raclopride, respectively,
following our protocol used previously for DAT autoradiography (McArthur et al., 2007b).
Briefly, after a pre-incubation period (30 min) adjacent sections were incubated for 1h in
5
0.1M PBS (pH 7.4) containing either radioligand alone (total binding) or radioligand plus
excess non-radioactive ligand (to assess non-specific binding). These were, respectively,
[125I]-RTI 121 (15pM; 2200Ci/mmol; PerkinElmer, UK) and GBR 12935 (10µM; Tocris,
UK) for DAT; [3H]-SCH23390 (200pM; 85Ci/mmol; PerkinElmer) and SCH23390 (20µM;
Tocris, UK) for D1-type receptors; [3H]-raclopride (3nM; 82.8Ci/mmol; PerkinElmer) and
sulpiride (300µM; Tocris, UK) for D2-type receptors. Binding buffer for D1-type receptor
assays also included 1µM CP809101 (Tocris, UK) to block potential 5-HT1C and 5-HT2C
receptor binding. Sections were then washed in 0.1M PBS at 4°C, allowed to air dry, and
exposed for 2 weeks ([125I]) or 6 weeks ([3H]) at -80°C to Kodak Biomax MS photographic
film (PerkinElmer, UK) in a lead-lined cassette, alongside microscale standards (ARC Inc., St
Louis, USA) impregnated with [125I] (0.18–93nCi/mg) or [3H] (0.14–489.1nCi/mg); signals
from [3H]-SCH23390 and [3H]-raclopride binding assays were amplified using phosphor
intensifying screens (PerkinElmer, UK). Films were developed and digitized using an MCID
Core system attached to a CoolSNAP Procf camera (Interfocus Imaging Ltd., Cambridge,
UK). Analysis within the defined brain regions (Paxinos and Watson, 1998) was performed
by systematic random sampling using NIH ImageJ, and densities were quantified as
femtomoles/mg equivalent. Average values from six different sections per region per animal
were used to calculate the group means (n=6 per group). Specific radioligand binding sites
were determined by subtraction of non-specific binding from total binding.
In-vivo microdialysis
Extracellular levels of DA and its metabolites were assessed by in vivo microdialysis coupled
with electrochemical detection using commercially-available microdialysis probes (MAB 6
15kDa cut-off PES, Microbiotech AB, Sweden). Probes were implanted stereotactically into
the NAc core through a burr hole in the skull (anterior-posterior: 1.2 mm forward of bregma;
6
lateral: ± 2 mm from the midline; ventral: -7.5 mm from the cortical surface) in rats
anaesthetized with urethane (1.2 g/kg i.p.). The probes were perfused continuously with
artificial CSF (in mM: 147 NaCl, 3 KCl, 1.3 CaCl2, 1 MgCl2, 0.2 NaH2PO4 and 1.3
Na2HPO4) for 3 h at a flow rate of 1 L/min prior to the collection of samples. After this
equilibration period, three 20 min baseline dialysate samples were collected (20 L) into
polypropylene vials. After three baseline collections, rats were injected with amphetamine
(0.8 mg/kg i.p) and sample collection (20 L/20 min) continued for an additional 2 h. All
samples were promptly frozen on dry ice before storage at -70oC pending analysis by HPLC
coupled with electrochemical detection. On completion of sample collection, rats were
sacrificed by cervical dislocation and their brains rapidly removed into formalin for
subsequent verification of probe placement. DA was determined in dialysate samples by
HPLC and electrochemical detection. Separation was obtained using a Hypersil analytical
column (100 x 4.6 mm inner diameter, 3 m; Phenomenex, UK) and a mobile phase
consisting of 8.82 g/L trisodium citrate, 2.03 gm/L NaH2PO4, 500 mg/L Na-1-octane sulfonic
acid, 22.5% methanol, 25 mg/L EDTA and 1 ml/L triethylamine, pH 2.7 adjusted using
orthophosphoric acid. DA was detected by oxidation using a Coulochem II detector (ESA
5014) equipped with a guard cell (+300 mV) and a dual electrode analytical cell (E1, -150
mV; E2 +150 mV). Chromatographic data were acquired and processed using Gyncosoft
V4.4 (Dionex, UK).
Behavioral analysis
Adult progeny of control and AGT dams were tested during the dark phase of the light-dark
cycle using the following range of established behavioral paradigms that have been shown to
depend on the integrity of midDA transmission.
7
Amphetamine-induced locomotor activity
Following habituation to the environment, each animal was subjected to a further five test
sessions of 120 min in the photocell testing chambers. On the day of testing, animals were
weighed and injected intraperitoneally with either vehicle (0.9% saline) or d-amphetamine at
varying doses (0.2, 0.4, 0.8 or 1.2 mg/kg free base weight in 0.9% saline), according to a
Latin square design. Immediately after injection, animals were placed in the centre of the
activity chambers and locomotor activity was assessed by recording the number of photocell
interruptions during the 2 h period.
Cocaine self-administration
Under isoflurane anaesthesia, chronic in-dwelling catheters were inserted into the right
jugular vein and mounted in the mid-scapular region, as detailed elsewhere (Caine et al.,
1992). Following surgery, subjects were housed individually with food and water made freely
available. Catheters were flushed daily with heparinized saline (0.15-0.2 ml) and after 7 days’
recovery, drug self-administration training commenced. Each operant chamber was equipped
with an active and inactive lever. Pressing the active lever activated an infusion pump to
deliver cocaine intravenously in a volume of 100 L 0.9% sterile saline over 3 seconds, and
simultaneously activated a cue light located above the active lever that remained illuminated
for 20 seconds during which time both levers were retracted (time out period). Responses on
the inactive lever were recorded but had no consequences. Training sessions were carried out
for 7 consecutive days at each dose on a fixed-ratio schedule of reinforcement with ascending
doses of cocaine (MacFarlan Smith, Edinburgh; 0.25, 0.5 and 1 mg/kg/100 L), and lever
presses were recorded. Sessions were terminated after a maximum of 50 cocaine infusions
had been self-administered or a period of 2h, whichever came first.
8
Spontaneous open field locomotor activity
Spontaneous locomotor activity in test-naïve animals was monitored in activity chambers
(259 mm x 476 mm x 209 mm; Allentown Inc) fitted with infrared photocell beams that were
enclosed in ventilated sound-proof boxes. Testing was conducted during the dark phase of the
light-dark cycle and beam breaks were recorded in bins of 5 min over a 90 min period using a
computer that was interfaced with the photocells and situated in an adjacent room.
Prepulse inhibition (PPI) of acoustic startle
The PPI test was conducted in adult rats (male and female control and AGT-exposed groups,
n=8) that had been drug- and test-free since birth in order to investigate the effects of AGT
and sex on baseline PPI values. Three Plexiglas startle chambers designed for rats (Kinder
Scientific, UK) were used and each chamber was housed in a ventilated sound-attenuating (35dB) cabinet. Acoustic stimuli were delivered through a speaker mounted on the ceiling of
the cabinet. A piezoelectric accelerometer mounted below the Plexiglas frame detected and
transduced motion from within the chamber. Acoustic stimulus intensities and response
sensitivities were calibrated to be near-identical in each startle chamber using the
manufacturer’s startle calibration system. For testing, rats were placed into the startle
chamber in a manner that prevented rearing but allowed free movement. Rats were subjected
to a 5 min habituation period of background broadband white noise of 65 dB before being
presented with a series of trials composed of mixture of three trial types (startle pulse-alone
trials, prepulse-pulse trials and no-stimulus trials). Each session started with six pulse-alone
trials, each involving exposure of the animal to a 40 ms burst of 120 dB noise. These pulsealone trials served to habituate and stabilize the startle response but were not used in the
subsequent analysis. These trials were followed by prepulse-pulse trials in which four levels
of prepulse (+3, +6, +12 and +16 dB above background for 20 ms) were presented 100 ms
9
(onset to onset) before the startle stimulus (120 dB, 40 ms). Each of the prepulse-pulse trials
were presented 5 times in a trial block that also included 10 ten presentations of the pulsealone trials, all delivered in a pseudorandom sequence. The average value of the startle
response obtained from the ten pulse-alone signals was used in the calculation of startle
reactivity and its inhibition by a prepulse. The discrete trial types were separated by a
variable inter-trial interval with a mean of 15 s (range 8-22 s). On no-stimulus trials, the
startle response was measured in the absence of an acoustic signal. No-stimulus trials were
intermixed between other trial types but were not used in the calculation of inter-trial
intervals. All PPI tests were performed in the dark phase of the light-dark cycle and prepulse
inhibition (PPI) was calculated as the % inhibition of the startle response obtained in the
pulse-alone trials at each of the four prepulse intensities, for each animal by the expression:
PPI = 100% x (1 – [mean reactivity in prepulse-pulse trials/mean reactivity in pulse-alone
trials]).
Pavlovian appetitive learning (autoshaping)
In the autoshaping experimental paradigm, learning results from the association of a
conditioned stimulus (CS+) that predicts food delivery and is measured relative to a second
identical stimulus (CS-) that is explicitly unpaired with food reward. With repeated exposures
animals acquire discriminated approach behavior to the CS+. Animals were trained using
automated chambers (Med Associates, USA; 29.5 x 32.5 x 23.5 cm) and the ‘lever
autoshaping’ software client (see http://whiskercontrol.com) as detailed elsewhere. Briefly,
rats that had been placed on a food-restricted regime sufficient to maintain body weight at no
less than 85% of free feeding weight were first habituated to the testing chambers for two
consecutive days for 20 min each day with sugar pellets (45 mg; Noyes dustless pellets;
Sandown Scientific, Hampton, UK) being placed in the central food magazine and the house
10
light illuminated. Autoshaping commenced on day three. In each chamber, one lever was
designated as the CS+ and the other as the CS-, with levers counterbalanced across animals
and across experimental groups. For each trial, the CS+ or CS- lever was inserted into the
chamber for a period of 15 sec and then retracted. A sugar pellet was delivered in the central
food magazine following the withdrawal of only the CS+ lever. The CS+ and CS- trials were
separated by an inter-trial interval that ranged between 45 and 75 s (average of 60 s) and the
order of presentation in each chamber was random, with no more than two trials of either
type occurring in sequence. Each autoshaping session consisted of 50 trials (25 CS+, 25 CS-)
and sessions were conducted for 7 consecutive days. For each trial only the first contact with
the CS, and not subsequent responses, was recorded as a lever press response, and contact
with either lever was interpreted as approach behaviour. Data were analyzed in blocks of 50
trials as mean CS+ and CS- approach scores. The mean difference between CS+ and CSscores was taken as a measure of discriminative learning.
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