Animals, drugs, and behavior
The protocols were conducted in accordance with the National Institutes of Health, “Guidelines for the Humane Care and Use of Laboratory Animals” (NIH Publications No. 80-23) and were approved by in-house Institutional Animal Care and Use Committees at the Rosalind Franklin University of Medicine and Science. Eight male Sprague-Dawley rats (Harlan, Indianapolis, IN), initially weighing 125-139 g, were housed 4 to a cage, on a 12 h light-dark cycle (lights on at 0700 h), with food and water available ad libitum
. They were handled for 2 days prior to the start of experiments to minimize handling stress during treatment. illustrates the experimental design, which has been previously shown to produce robust locomotor sensitization (Morshedi and Meredith, 2007
; Robinson and Kolb, 1999
). One group of rats (n = 4) received repeated d
-AMPH sulfate (AMPH in 0.9% saline, i.p.) injections: 3 mg/kg for 5 days, except for the first and last day when they received 1.5 mg/kg, followed by 2 drug-free days and repeated for 3 weeks, and the second group of rats was administered repeated saline injections (vehicle in the same volume) following the same procedure as that in the first group. On the first and last injection days (day 1 and day 19), locomotor activity in the open field was measured immediately after the injection of either AMPH or saline. On all other injection days, animals were returned to their home cage immediately after the AMPH or saline injection. All injections were administered at the same time each day. Ten days following the injection period, all rats were anesthetized (see below) and given stereotaxically-placed injections of the retrograde tracer, cholera toxin, subunit B (CTB), into the LH and were then euthanized a further 10 days later ().
Diagram of the AMPH treatment protocol. Vehicle-treated animals were handled and treated in the same manner. Abbreviations: AMPH, amphetamine; CTB cholera toxin subunit B; PF, paraformaldehyde (n = 4 per group)
Surgical and perfusion procedures
Rats were anesthetized for surgery with equithesin (3 mL/kg, i.p.: 1% sodium pentobarbital, 4.25% chloral hydrate in 10% ethanol) and secured in a Stoelting stereotaxic apparatus. A Hamilton syringe (25G; Hamilton Company, Reno, NV) filled with 1% CTB (#104, List Biological Labs, Campbell, CA) in double distilled H2
O was positioned using stereotaxic coordinates taken from a rat brain atlas (Paxinos and Watson, 2005
) and aimed at the LH (relative to bregma: anterior-posterior [AP]: −2.9 mm, medial-lateral [ML]: −1.5 mm; relative to skull: dorsal-ventral [DV]: −8.1 mm). CTB extensively fills dendrites and spines (Meredith, 1995
) and, therefore, was selected as our retrograde tracer of choice for these ultrastructural studies. The tracer was pressure injected for 10 min at a rate of 10 nl/min (100 nl total volume) and the syringe left in place for 10 min to minimize leakage along the injection tract during removal.
At the conclusion of the experiment, rats were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and transcardially perfused with 0.1M phosphate buffered saline, followed by 50 ml of 3.75% acrolein in buffered 2% paraformaldehyde (PF), pH 7.4, and then by 200 ml of buffered 2% PF alone (Leranth and Pickel, 1989
). All solutions were made at the same time and the perfusions were of uniform duration. The brains were removed, post-fixed for 30 min in buffered 2% PF, and cut sagittally to separate the hemispheres. The hemisphere ipsilateral to the injection site was then cut through the mPFC and the CTB injection site in the LH on a Vibratome (The Vibratome Co., St. Louis, MO) into coronal sections (30 μm) that were collected serially into 24-well cell culture plates filled with 0.1M phosphate buffer (PB).
Immunohistochemistry and processing for the electron microscope (EM)
In a pilot study using sample tissue, the concentrations of antisera, normal sera, and Triton X-100 (Tx) were titrated for each of the immunohistochemical reactions in order to determine the optimal concentrations that would preserve the ultrastructure for the EM.
The sections from the experimental animals were processed on the same day and the solutions for each antibody reaction were prepared as a single batch. Sections were incubated in 1% sodium borohydride in 0.1M PB for 30 min then rinsed extensively before being blocked for 1 h in 3% normal serum and 0.05% Tx, and then incubated for 72 h at 4°C in goat anti-CTB (1:5000; #703, List Biological Labs) with 3% normal serum and 0.05% Tx. They were then incubated for 90 min in biotinylated horse anti-goat IgG (1:200; Vector Laboratories) with 3% normal serum and 0.05% Tx at room temperature (RT), followed by 1 h in avidin-biotin complex (ABC). The sections were then reacted with 0.05% nickel-enhanced 3,3′-diaminobenzidine with 0.01% H2O2 (DAB-Ni) and rinsed thoroughly before being processed for the EM.
Sections were fixed in 2% osmium tetroxide (diluted in 0.1M PB) for 30 min at room temperature in the dark, dehydrated, and flat embedded in resin (25:15:55 Embed 812:Araldite 502:dodecenyl succinic anhydride; Electron Microscopy Sciences, Hatfield, PA) between two sheets of aclar fluorohalocarbon film. The embedding resin was allowed to polymerize at 60°C for 72 hours. Blocks were cut with an ultramicrotome (EM UC6, Leica Microsystems Inc., Bannockburn, IL) into ultrathin sections (silver, ~60-70 nm), mounted on formvar-coated copper slot grids (Electron Microscopy Sciences), and stained with uranyl acetate and lead citrate prior to stereological analysis.
We counted synapses using the physical disector in a specific area of the mPFC that was defined by unambiguous anatomical boundaries (Gabbott et al., 2005
). We established the anterior boundary at +4.3 mm from bregma and the posterior boundary at +2.2 mm from bregma. Since the anterior-posterior extent of these mPFC regions measures 1500 μm, we collected 50 serial, thick sections (30 μm each) through each mPFC. A random number was generated for the first section of each mPFC, and then every 5th
section was collected, so that 10 equally spaced sections were selected for analysis (; Day et al., 2006
; Howard and Reed, 1998
; Ingham et al., 1998
; Ismail and Bedi, 2007
). We then rotated the 10 sections so that their medial walls were aligned along the dorsoventral axis. Each section was then outlined on paper and a small reference point, referred to as a tangential reference point (), was placed at the top left-hand tip of the section (see Gabbott et al., 2005
for details). The dorsal boundary of the prelimbic area was then established by measuring a set distance ventrally from the tangential reference point on each section; the ventral boundary was likewise established according to set distances from the reference point on each section (Gabbott et al., 2005
). Boundaries were also established for the adjacent series of 10 sections that were collected so that the volume of the mPFC could be estimated using the Cavalieri method. This set of sections was mounted onto poly-L-lysine slides from 0.05M Tris-HCl buffer, dried at 60°C overnight, stained with cresyl violet, dehydrated in an ascending series of alcohols, cleared in xylene, and coverslipped. The volume of the mPFC was estimated using a Nikon E400 microscope equipped with a motorized stage in 3 axes, a video camera, and StereoInvestigator software (MicroBrightField, Inc; Williston, VT). The mPFC (prelimbic and infralimbic regions) was outlined on each section and the reference volume was calculated as (Vref
where a is the mean area of this part of the mPFC, t is the thickness of the Vibratome sections (30μm) and s is the total number of sections through the defined region of the mPFC (50 sections).
Fig. 2 Diagram of coronal sections through the medial prefrontal cortex (mPFC) used for stereological analysis. Stereotaxic maps were reproduced, with permission from Elsevier Science, from the brain atlas of Paxinos and Watson (2005). The section farthest to (more ...)
The physical disector is a 3-dimensional stereological technique that allows the counting of objects (synapses in this case) from a pair of disector sections (ultramicrotome sections in this study), which can be viewed simultaneously (see Braengaard and Gundersen, 1986 and Mouton, 2002
for reviews). The sections are a known distance apart and the distance between sections is 30% of the average projected height of the objects (synapses) that are to be counted (Howard and Reed, 1998
; Ingham et al., 1998
). The sampling strategy for the physical disector is generally referred to as a systematic uniform random sampling procedure (Braengaard and Gundersen, 1986; Howard and Reed, 1998
). The analysis requires that reference and lookup sections are obtained from ultrathin sections cut from samples, taken randomly from the thick (30μm) sections for each animal (for details of this systematic random selection method, see Braendgaard and Gunderson, 1986
). We used a random number generator to generate x and y coordinates, and a rotational value, to create a scaled square (equivalent to 0.5 mm × 0.5 mm) on paper. That square was first dropped onto an atlas image () and then placed onto the corresponding area of the selected, embedded section. A piece of tissue, the size of the scaled square, was then cut out of the section, mounted on a block with resin, and resectioned with the ultramicrotome. As illustrates, each sample had an equal chance of being in the reference volume, as required for disector analysis (Coggeshall, 1992
). Ultrathin serial sections (~10 per grid) were examined with a JEOL 1230 electron microscope (JEOL, Peabody, MA). All EM images were captured with a Hamamatsu ORCA HR CCD camera (AMT XR-60 imaging system, Danvers, MA) at 25,000X magnification. Physical disectors were collected using a systematic random sampling method, whereby each pair of micrographs was spaced 2 widths of the EM screen from the previous pair (Day et al., 2006
; Hunter and Stewart, 1993
; Ingham et al., 1998
), and the analysis was performed on 15 pairs of electron micrographs per block.
We analyzed 90 disectors for each animal per treatment group (total of 360 disectors per group). The absolute number of synapses was estimated using a disector height of 60 nm (Day et al., 2006
; Ingham et al., 1998
) and a rectangular unbiased counting frame (27.33 μm2
), containing 2 inclusion (dashed) and 2 exclusion (solid, extending above and below the square to infinity) lines, which was placed over each electron micrograph in the pair (). Synapses that fell within the counting frame or on the inclusion, but not on the exclusion, lines were counted. We counted “tops” of synapses in each pair of ultrathin sections. The synapse “top” was counted in the reference section when it did not appear in the “look-up” section and vice versa
(). We counted from 218-247 synapse “tops” per animal. All structures, such as myelinated fibers, cell somata, and blood vessels were included in the analysis, but regions obscured by folds or contamination, were avoided. Slides, sections, and grids were always coded so that the investigator was blind to the treatment groups.
Figure 3A-B A pair of electron micrographs, taken from serial sections through the mPFC, in which synapses within the unbiased counting frame are labeled. Synaptic contacts are labeled according to their membrane specialization (as, asymmetric; ss, symmetric) and (more ...)
A synapse was identified by the accumulation of at least 3 presynaptic vesicles and by the presence of a widened synaptic cleft with parallel, thickened pre- and post- synaptic membranes. Asymmetric synapses had postsynaptic densities 2.5-3 times thicker than the presynaptic densities, while the densities of symmetric synapses were equal. The associated postsynaptic targets were identified as soma, dendrite, spine, or unknown using recognized criteria (Peters et al., 1991
). Somata were identified by the presence of a nucleus. Dendrites were identified by the presence of structures postsynaptic to the axon terminals, such as mitochondria, microtubules, and, with larger dendrites, rough endoplasmic reticulum. Dendritic spines often contained a spine apparatus and were usually smaller than dendrites, typically lacking mitochondria, microtubules, and rough endoplasmic reticulum. Boutons were classified as multisynaptic (MSB) if they contacted more than one completely separate, independent target, enclosed in its own plasma membrane.
Estimation of mean synaptic numerical density (Nv syn
): Synaptic number per μm3
was calculated using the following formula:
adapted from Sterio (1984)
and de Groot and Bierman (1986)
, where Q-syn
= synapses present in the reference section but not the lookup section (tops), h = height of the disector which is the distance (μm) between disector planes (section thickness), and A = sample area (μm2
Estimation of ultrathin section thickness (Small's minimal fold Method; Small, 1968)
: At least 3 folds from each of the sections used for analysis were photographed at 25,000X magnification. Section thickness (h) was estimated as half the mean width of the measured folds.
Estimation of absolute number of synapses (N):
The following formula was used:
where Nv syn
is the mean synaptic density and Vref
, the reference volume (see above for formulae for these).