All experiments were conducted in compliance with the National Institutes of Health Guidelines for the Care and Use of Experimental Animals approved by the Institutional Animal Care and Use Committee at the University of California-Davis and Mount Sinai School of Medicine.
Six young adult (age range, 9–14 years old; mean, 10.8±0.8 years old; 4 females, 2 males) and 9 aged (age range, 22–35 years old; mean, 30.2±1.5 years old, 7 females, 2 males) Rhesus monkeys (Macaca mulatta) were used in the behavioral phase of this study and electron microscopic analyses, and a subset of nine (4 aged, 5 young) were used in the morphometric analyses of filled neurons (see below). Behavioral testing extended over approximately 2 years.
Delayed Response (DR) test
The DR testing was equivalent to the approach used in our previous studies (Hao et al., 2007
, Rapp, Morrison and Roberts, 2003
). Subjects watched from behind a transparent screen while one of the wells of the apparatus was baited with a food reward, and both wells were then covered. During initial training, the screen was raised immediately, allowing monkeys to displace the cover and retrieve the reward if the correct location was chosen. Testing continued until animals met a criterion of 90% correct or better across 9 consecutive blocks of 10 trials. For the next phase of testing, a 1 s retention interval was imposed between baiting the well and the opportunity to respond. Delays were implemented by lowering an opaque screen such that the monkey could no longer see the reward wells. Training with a 1 s delay continued until the monkey reached criterion (≥90% correct over 90 trials), and the demands on memory were then made progressively more difficult by imposing successively longer retention intervals of 5, 10, 15, 30, and 60 s. Each delay was tested for a total of 90 trials (30 trials/daily session; intertrial interval (ITI) = 20 s).
Delayed Non-Matching-To-Sample (DNMS) Test
DNMS testing was conducted as described in detail elsewhere (Rapp, Morrison and Roberts, 2003
, Rapp and Amaral, 1991
). Briefly, a sample object was placed over the baited central well of the test tray and after a response, the opaque barrier was lowered to impose a pre-designated retention interval. The same item was then presented simultaneously with a novel object that covered the food reward. Objects were drawn from a large enough pool such that the paired object was novel on every trial. During the acquisition phase, a 10 sec retention interval was used until the monkeys learned the non-matching rule to a criterion of 90% correct or better (across 100 trials, 20 trials/day, inter-trial interval = 30 sec throughout testing). Once the monkeys reached criterion, DNMS performance was measured by increasing the demand on recognition memory with successively longer delays of 15, 30, 60, and 120 sec (100 trials total at each delay, 20 trials/day) and 600 sec (50 trials total, 5 trials/day).
Perfusion and Tissue Preparation
Rhesus monkeys were perfused transcardially in groups of 2 or 3 as they completed their behavioral assessments. If all behavioral data had been acquired but perfusion was not able to be scheduled immediately, monkeys continued mock training up until the day prior to perfusion in order to avoid testing-induced variability. All animals were deeply anesthetized with ketamine hydrochloride (25 mg/kg) and pentobarbital (20–35 mg/kg, i.v.), intubated, and mechanically ventilated. The chest was opened to expose the heart and 1.5 ml of 0.5% sodium nitrate was injected into the left ventricle to facilitate vasodilation. The descending aorta was clamped immediately following intubation, and the monkeys were perfused transcardially with cold 1% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.2) for 2 min, followed by 4% paraformaldehyde in 0.1 M PB at 250 ml/min for 10 min, after which the flow rate was reduced to 100 ml/min for 50 min.
After perfusion, the brain was removed from the skull and dissected into standardized blocks, taking care to include the entire region surrounding the principal sulcus (Brodmann’s area 46) in the frontal block. The frontal block was post-fixed for 6 hours in 4% paraformaldehyde with 0.125% gluteraldehyde in PB and then cut serially on a Vibratome. Every 1.3 millimeter(mm) of tissue was divided into one 400 μm thick section, flanked by 200 μm sections on each side, with the remainder cut into 50 μm sections, with the same sequence repeated throughout the frontal block. Thereby, a series of 400 μm thick sections within area 46 were collected for intracellular injection of Lucifer Yellow (LY; Molecular Probes, Eugene, OR), while a systematic random set of 200 μm sections were embedded for electron microscopy (EM), and the 50 μm sections were stored at −20 degrees for light microscopic immunohistochemistry. Since the precise position of the first slab is random, this results in a systematic random series of at least 8 sections throughout area 46 available for cell loading. Multiple sections are used to obtain the total number of cells, such that the sampled neurons represent a random selection throughout area 46.
Quantitative analyses of spine density and spine morphology
Nine out of the 15 monkeys had undergone optimal perfusion and could be used for cell filling. Intracellular injection of layer III pyramidal cells with LY and quantitative analysis was performed according to methods previously described (Hao et al., 2007
). Printed datasets of dendritic tree maps were used to allow identification of dendritic segments chosen in a systematic-random fashion for spine analysis, as described in our previous studies (Hao et al., 2007
, Hao et al., 2006
). A clear acetate sheet, containing a series of scaled concentric rings 60 μms apart was placed over the working maps of neuronal reconstructions, with the cell soma at the center. Points of intersection between dendritic branches and the circles were used to identify those segments to be used for high magnification microscopic spine analysis. Consequently, although the somata of the sampled cells were always located in layer III, the dendritic segments chosen for analysis were located in layers I through III, with occasional basal dendrites extending into layer V. Confocal z-stacks of identified intersections were captured on a Zeiss LSM 410 (Oberkochen, Germany). Each designated segment (25–50 μms in length) was located in the microscope field, and confocal stacks of 100–300 digital images separated by a z-step of 0.1 um were captured using a Plan-Apochromat 100X/1.4 NA Zeiss oil-immersion objective (). LY was excited with an Ar/Kr laser at 488 nm (attenuation set at 10). All confocal stacks included at least 1 μm above and below the identified dendritic segment. Settings for pinhole size, aperture gain, and offset were optimized initially and then held constant throughout the study to ensure that all images were digitized under the same illumination conditions at a resolution of 256 X 256 pixels. An average of 12 high-magnification z-stacks were captured per neuron (total 39 neurons). The confocal stacks were deconvolved with AutoDeblur (Media Cybernetics, Silver Spring, MD) () and imported to NeuronStudio for 3-dimensional (3D) spine density, and spine size analysis (Rodriguez et al., 2008
) (). All spine measurements (total 21,000 spines) were performed in 3D from the z-stacks. The density was calculated by dividing the total number of spines present by the dendritic length of the segment. The automated measures of spine volume, spine head diameter and spine length were then imported into Matlab for classification of spines into thin and mushroom types based on categories previously established from serial section EM reconstruction of dendritic segments (Sorra and Harris, 2000
) (). A spine was labeled thin if its head was below 0.6 μm in diameter and had a max length that was at least twice as big as the head diameter. A spine was classified as mushroom if its head diameter exceeded 0.6 μm. The remainder of spines were classified as “other.” This group presumably contained primarily stubby spines. However, because stubby spines are classified based the absence of a neck (Sorra and Harris, 2000
) and because no data was available for this index, no further analysis was performed on this group of spines. All spines included in this study had a clear head, though in some instances the head swelling was more evident from an increase in fluorescence than an increase in diameter (see and inset). Thus it is unlikely that filopodia (long and thin protrusions without spine heads (Grutzendler, Kasthuri and Gan, 2002
)) contributed to the spine density and size measurements.
Figure 1 Dendritic segment analysis for spine quantification. A. Projection of a raw confocal z-stack of an imaged dendrite. B. Dendritic segment after deconvolution. The image is deblured by accounting for the point spread function in our system. Note that the (more ...)
Figure 6 Average spine head diameter and volume is significantly increased in aging, which is attributable to a selective loss of thin spines. All statistics were performed based on one aggregate (i.e. average) measure per animal. A. Mean spine head diameter is (more ...)
Figure 8 DNMS acquisition significantly correlates with synaptic indices, in particular thin spine head volume. A. The scatter plot of DNMS acquisition (trials required to reach 90% accuracy with a 10 second delay) versus EM synaptic density shows a moderate though (more ...)
Disector analysis of synapse density
100 μm thick sections chosen in a systematic random fashion and containing area 46 that had been postfixed with cold 4 % paraformaldehyde and 0.125% glutaraldehyde in PBS were embedded with Lowicryl Resin [Electron Microscopy Sciences(EMS), Hatfield, PA]. Consecutive ultrathin sections (90 nm thick) from layer III of area 46 were cut and mounted on formvar carbon-coated copper slot grids (EMS). Since the tissue sections were sampled in a systematic-random fashion, two consecutive ultrathin sections could be arbitrarily selected from the ultrathin sections and analyzed in an unbiased manner. The ultrathin sections were viewed on a JEOL 1200EX electron microscope (JEOL, Tokyo, Japan). To constitute physical disector pairs, the corresponding tissue areas from the two consecutive ultrathin sections were captured using the Advantage CCD camera (Advanced Microscopy Techniques Corporation, Danvers, MA).
The captured disector images were viewed using the IGL trace program (Harris Laboratory, Boston University, Boston). Axo-spinous and axo-dendritic synapses in area 46 were designated and counted with different markers by using the physical disector (), as previously described (Sterio, 1984
, de Groot and Bierman, 1986
, Tigges, Herndon and Rosene, 1996
, Adams et al., 2001
). In short, only those axo-spinous or axo-dendritic synapse profiles contained in the reference image but not in the corresponding look-up image were counted and the synapse profiles that appeared in both images were not counted. In order to increase sampling efficiency, the reference image and look-up image were reversed. The size of the disector area used was 80 μm2
and the average section thickness was 90 nm. Therefore, the height of the disector was 180 nm because the synapses were obtained by counting both ways. The volume of each disector pair was the area of the counting area multiplied by the height of the disector, which was 14.4 μm3
in the present study. On average, 20 disector pairs from each monkey were analyzed, generating a total volume of 288 μm3
. Approximately 100 synapses per monkey were counted. The criteria for inclusion as an axo-spinous synapse included the presence of synaptic vesicles in the presynaptic terminal and a distinct asymmetric postsynaptic density (PSD) separated by a clear synaptic cleft. The synapse density was calculated as the total number of counted synapses divided by the total volume of the disectors used.
Figure 2 Electron micrographs illustrating the disector method for synapse density. For synapse density measurements, two serial sections were used. One section was considered the reference (A) and the other the look-up (B). Only asymmetric axo-spinous (yellow (more ...)
Estimation of volume of area 46 with the Cavalieri method
A systematic random series of 50 μm sections was taken from the same prefrontal cortex used for electron microscopic analysis and provided for volume estimate. The interval between the sampled sections was 1.3 mm. The sampled sections were stained with the Nissl stain, and area 46 was outlined and measured using Stereoinvestigator software (MicroBrightField, Williston, VT) in each Nissl-stained PFC section. The volume of area 46 was estimated with the Cavalieri principle.
Differences in performance on DR and DNMS were assessed using a repeated measures ANOVA. Statistical analyses were performed using ANOVA to assess possible differences in the various morphometric parameters between groups. The values are shown as means ± SEM, calculated based on one aggregate (i.e. average) per animal. Observed power was calculated in all ANOVA to confirm that the sample size was sufficient to support the data. Pearson correlations were used to determine the relationships between morphological indices and acquisition and performance on DNMS and DR. The statistical significance level was set at p< 0.05.