1 Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT 06520, USA, 2 Department of Neurobiology, Yale University School of Medicine, New Haven, CT 06520, USA, 3 Department of Psychology, Yale University, New Haven, CT 06520, USA, 4 Institute of Anatomy, University of Hamburg, D-20246 Hamburg, Germany
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Abstract |
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Key Words: electron microscopic unbiased stereological calculation, glial fibrillary acidic protein, orchidectomy, pyramidal cell dendrite, sexual differences, vervet monkey
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Introduction |
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Materials and Methods |
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Thereafter, the chest was opened, the descending aorta clamped, the right atrium opened and after rinsing the vascular system via transcardial perfusion using a motorized peristaltic pump (Masterflex L/S; Cole Palmer, San Francisco, CA) with 1000 ml 0.9% heparinized (1 unit/ml) saline, animals were further perfused for 10 min with 1500 ml of fixative containing 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.35; PB). The temperature of both solutions was 20°C. Brains were postfixed at 4 0C for 2 h in the same fixative and stored in PB at 4 °C. The hypothalami and the nigro-striatal systems of these animals were used in other studies. The hippocampi were dissected out and divided into three equal pieces along its longitudinal axis. Thereafter, small 23 mm long blocks were cut (perpendicular to the longitudinal axis) out from the middle portion of these tissue samples and 50 and 200 µm thick sections were cut perpendicular to the longitudinal axis of the hippocampal formation on the vibratome for light microscopic immunostaining and electron microscopic statistical analyses, respectively.
Immunostaining for Glial Fibrillary Acidic Protein (GFAP)
In order to eliminate unbound aldehydes, the 50 µm vibratome sections were pretreated with 1% sodium borohydride in PBS for 30 min (Sigma, St Louis, MO) and washed 3 x 10 min in PB. Thereafter, sections were incubated in the primary antibody rabbit anti-GFAP (DAKO, Carpinteria, CA), 1:1000 in PB containing 2% normal goat serum (ICN Biomedicals, Aurora, OH) and 0.5% Triton X-100 (Acros Organics, Fairlawn, NJ) overnight at room temperature. Sections were washed 3 x 10 min in PB and further incubated in the secondary antibody biotinylated goat anti-rabbit IgG (Vector Laboratories Burlingame, CA), 1:500 in PB for 2 h at room temperature followed by another rinse in PB (3 x 10 min) and an incubation in the ABC Elite reagents (1:500; Vector Laboratories Burlingame, CA). After washing in PB (3 x 10min), the tissue bound peroxidase was visualized using nickeldiaminobenzidine (NiDAB: 15 mg DAB, 12 mg ammonium chloride, 0.12 mg glucose oxidase, 600 µl 0.05 M nickel ammonium sulphate and 600 µl 10% ß-D-glucose in 40 ml PB) for 8 min. Sections were mounted on gelatin-coated slides, dehydrated and mounted in Permount. To ensure that sections taken from the gonadectomized and control animals were treated under identical conditions, they were incubated together throughout the procedures.
Light Microscopic Analysis
The stratum radiatum of the hippocampal CA1 subfield was examined. Data analysis was performed as described recently (Lam and Leranth, 2003). Briefly, from each GFAP-immunostained section (25/animal), non-overlapping photographs (five per section) of the region were taken using 100x oil immersion and 20x objective lenses for process and cell counting, respectively, under an Olympus BX60 microscope equipped with a Zeiss AxioCam digital camera. The location of the first photograph was randomly selected, then the other four were taken from areas located on the left- or the right-hand side of the first photographed field, depending on the location of the first photograph. For analysis, images were further processed using Adobe Photoshop 6.0 and printed out at a final magnification of 160x (process counting) or 32x (cell counting) on a Brother HL-1450 laser printer. For process counting, a 100 x 80 µm large grid, composed of square grid lines 10 µm apart, was laid on the print. Immunoreactive processes in focus crossing the grid lines were counted to determine the relative density of processes as described previously (Horvath et al., 1999
; Lam and Leranth, 2003
). Twenty pictures from each animal were analyzed and the mean for each animal was calculated from these data. For cell counting, only GFAP positive cells in focus with clearly distinguishable somata were counted in a total area of 150 x 500 µm on each print; in this case 10 pictures were analyzed and used to determine the mean per animal. In order to avoid a biased analysis, sections were encoded prior to the analysis.
Electron Microscopic Analysis
For electron microscopic examination, the 200 µm vibratome sections (taken for the aforementioned three major areas along the longitudinal axis of the hippocampus) were post-osmicated (1% OsO4 in PB, for 30 min), dehydrated in increasing concentrations of ethanol (the 70% bath contained 1% uranyl acetate, for 30 min) and embedded in Araldite. Consecutive serial sections prepared from the middle third of the CA1 stratum radiatum (see Fig. 1 in Leranth et al., 2002) of each vibratome section were collected on Formvar-coated single slot grids. Sections were analyzed and photographed in a Tecnai 12 electron microscope equipped with an AMT Advantage 4.00 HR/HR-B CCD camera system (Advanced Microscopy Techniques, Danvers, MA).
Because synaptic density could be affected by changes in the volume of the tissue, a correction factor was first calculated by following the steps of a widely accepted protocol (Rusakov et al., 1997). This method is based on the assumption that the hormonal manipulations did not influence the total number of CA1 pyramidal cells. As performed previously (Leranth et al., 2000
, 2002), six or seven disector pairs (pairs of adjacent 2 µm semithin sections; reference and look-up sections) which contained the CA1 pyramidal cell layer were cut on the ultratome from Araldite blocks (three blocks, taken from three different areas along the longitudinal axis of the hippocampal formation, see above), of all hippocampi and placed on slides. Sections were stained with toluidine blue. The cell layer was located in the field of view of the light microscope (Olympus BX60 microscope furnished with a Zeiss AxioCam digital camera connected to a computer) and observed also on the screen before being captured as a TIFF file. The slide was then moved to allow the adjacent area to be captured. This process was repeated for the entire CA1 pyramidal cell layer of both the reference and look-up sections. In each of the captured images, a pair of parallel lines (250 µm apart) perpendicular to the layer was placed to form a sampling window. The simultaneous observation of captured images on the screen allowed the alignment of sampling windows in the reference and look-up sections. Thereafter, the disector technique was applied to count the cell nuclei in both sections. Only those cell nuclei were counted that were present in one, but not on the other section and vice versa (Braendgaar and Gundersen, 1986
). The left hand window line served as exclusion line (see Figs 1A,B in Rusakov et al., 1997
). The cell density value (D) was calculated using the formula D = N/sT, where N is the mean disector score across all the sampling windows, T is the thickness of the sections (2 µm) and s stands for the length of the window. Based on these values, a dimensionless volume correction factor kv was introduced: kv = D/D1, where D1 is the mean density across the different animals.
Photographs (at 9900x magnification) were made from identical fields of pairs of consecutive serial ultrathin sections (reference and look-up sections) from areas located between the upper and middle third of the CA1 stratum radiatum (300500 µm from the pyramidal cell layer) with the observer blinded to the experimental treatment. As in the light microscopic analysis, the location of the first photograph was randomly selected. Thereafter, non-overlapping photographs were taken from the left- and right-hand sides of the first photographed area. Potentially interfering structures, such as blood vessels, large dendrites or glial cells, were intentionally avoided. In order to obtain a comparable measure of synaptic numbers, unbiased for possible changes in synaptic size, the disector technique (Braendgaar and Gundersen, 1986) was used. Thus, the density of spine synapses [which were identified by the lack of mitochondria, but the presence of spine apparatus and dark postsynaptic bar adjacent to the synaptic contact in the head of the spines (Gray type 2 synapses) and vesicle accumulation at the synaptic contact in the presynaptic bouton] of pyramidal cell apical dendrites was calculated with the help of a reference grid superimposed on the EM photographs. The disector volume (volume of reference) was the unit area of the reference grid multiplied by the distance between the upper faces of the reference and look-up sections. Section thickness (average 0.075 µm) was determined by using the electron scattering technique (Small, 1968
). Only those spine synapses were counted that were present on the reference section, but absent in the corresponding look-up section and vice versa (Fig. 1). The measured synaptic density values were divided by the volume correction factor kv. This correction provided a synaptic density estimate normalized with respect to the density of pyramidal cells and also accounted for possible changes in hippocampal volume.
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In addition to the spine synapse density calculation, a semiquantitative analysis was performed on the diameter of dendrites located in this area. On all prints used in the spine density calculation, the diameter of dendritic profiles (30 dendrites in randomly selected photos taken from each animal) were measured and compared.
Statistical Analyses
For all dependent variables (glia process density, dendritic diameter and spine synapse density), the means determined for each animal were used for the statistical analysis. The non-central F-statistic was used to test the difference in the ratios of means between groups and group means were compared using pairwise t-tests (two-tailed probabilities). A level of confidence of P < 0.05 in two-tailed tests was adopted. P- and t-values as well as the degrees of freedom (d.f.) are given in the figure legends. In the text, the mean ± SEM values are given.
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Results |
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There was a major difference between the levels of circulating T in the GDX and control monkeys. In GDX monkeys, the mean T level was <0.2 ng/ml (the minimum sensitivity of radioimmunoassay is <0.2 ng/ml). In control monkeys, the average serum T level was 1.134 ± 0.016 ng/ml (Table 1), which is the same as we measured previously in the serum of same-age St Kitts male monkeys and very similar to the lower end of T levels measured in human men.
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Astroglia processes could easily be detected due to the specific GFAP staining. Light microscopic observations already suggested a difference in the density of astroglial processes between the control and GDX monkeys. In the CA1 of GDX animals, the fiber density was increased compared to the control (Fig. 2). The semiquantitative analysis confirmed this optical impression (Fig. 3). In control animals, the mean density of glia processes was 108.8 ± 2.952/8000 µm2, whereas in the castrated animals it was 124.8 ± 3.926/8000 µm2 (Table 1). This relative increase (15%) in the density of glia processes was statistically significant (P < 0.031). In contrast, there was no significant difference in the density of GFAP-containing cell bodies in the CA1 regions of castrated and control animals (data not shown).
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No significant differences were observed in the density of pyramidal cell nuclei between the control (840 ± 73.901/mm2) and GDX animals (832 ± 64.663/mm2; Table 1). Furthermore, in the electron microscope, no obvious qualitative differences could be observed by comparing the ultrastructure of CA1 stratum radiatum of control and GDX monkeys. However, surveying all of the electron micrographs, it appeared that the density of small glia processes is higher in the material depicted from GDX monkeys than in the control animals. Because of technical difficulties and the extremely time-consuming nature of a meaningful electron microscopic statistical analysis on the density of these processes and having the data of the light microscopic surface density calculation of glia processes (see below), an unbiased electron microscopic stereological calculation was not performed.
The result of the unbiased electron microscopic stereological calculation on the spine synapse density demonstrated a major, significant (P < 0.001) difference in the density values of CA1 area pyramidal cell spine synapse density of the two groups. The total numbers of spine synapses/kv counted in a 475 µm3 volume of control monkeys were 259.825, 287.802, and 276.45 (0.343 ± 0.00674/µm3). Whereas, in GDX animals, these numbers were 176.675, 156.75 and 163.875 (0.578 ± 0.0171/µm3), in the same volume (Table 1 and Fig. 4), which represents a 40% decrease.
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The semiquantitative analysis on the diameter of dendritic shafts in the outer third of the stratum radiatum of the CA1 subfield indicated that the average diameter of these dendrites is significantly (P < 0.001) larger in the GDX animals (1.332 ± 0.03 µm) than those (1.025 ± 0.018 µm) of control monkeys (Table 1 and Fig. 5). Thus, castration resulted in a 30% increase of the apical dendritic shaft diameter.
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Discussion |
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In spite of the 40% loss of spine synapses in the CA1 area of GDX monkeys, there was no apparent difference in the density of pyramidal and glia cells. Thus, a change in the volume of the hippocampus could be excluded. Two compensatory factors may be responsible for this phenomenon: the significant sprouting of glia processes and the significant, 30% enlargement of the diameter of pyramidal cell apical dendritic shafts. Since the increase in glia process density (15%) was much less than the decrease in the spine synapse density (40%), the remaining vacant space may have been filled up by the increased mass of dendrites. Enlargement of dendritic processes has been observed in female monkeys following OVX and this has been assumed to result from incorporation of the cytoplasm of spines that are retracted into the dendrites (Leranth et al., 2002). The enlargement of dendritic processes seen here in male monkeys may be due to a similar spine retraction.
Functional Considerations
The major observation of this study is that, similar to gonadal hormone action on female rats (Gould et al., 1990; Woolley et al., 1990
, 1997; Woolley and McEwen, 1992
, 1993; Leranth and Shanabrough, 2001
; Leranth et al., 2000
), female monkeys (Leranth et al., 2002
) and male rats (Leranth et al., 2003
), experimental manipulation of gonadal hormone levels greatly influences the density of pyramidal cell spine synapses in the CA1 hippocampal subfield of male subhuman primates. Thus, it can be assumed that the male primate hippocampus, in addition to other gonadal hormone-targeted structures that have major roles in mnemonic processes, e.g. the mesencephalic dopamine system in conjunction with the prefrontal cortex (McCarthy et al., 1996
; Goldman-Rakic, 1998
), is a major participant in the system by which gonadal hormones influence mnemonic functions. However, the specific mnemonic consequences of the observed spine synapse density changes in male monkeys are unclear at the present time. In conjunction with the estrogenic facilitation of hippocampal LTP and neurogenesis seen in female rats (e.g. Wong and Moss, 1992
; Warren et al., 1995
; Tanapat et al., 1999
), it has been assumed that the increased spine density reported during proestrus in cycling rats or in ovariectomized rats after exogenous estrogen should lead to improvements in learning and memory. Indeed, increased spine density in the CA1 region of the hippocampus has been associated with spatial learning (Moser et al., 1994
, 1997). However, studies in intact cycling rats have failed to find fluctuations in spatial memory that mirror changes in spine density (Frye, 1995
; Berry et al., 1997
; Stackman et al., 1997
; Warren and Juraska, 1997
). Furthermore in male rats, exogenous testosterone has been shown to impair the acquisition of a spatial water maze task (Goudsmit et al., 1990
; Naghdi et al., 2001
). Nevertheless, several studies in humans have demonstrated a positive relationship between testosterone levels and several types of memory, particularly in older men (Gouchie and Kimura, 1991
; Janowsky et al., 1994
, 2001; Barrett-Connor et al., 1999
; Silverman et al., 1999
; Cherrier et al., 2001
; Moffat et al., 2002
). In several cases, men with lower T levels exhibited impaired memory relative to those with higher T (Barrett-Connor et al., 1999
; Moffat et al., 2002
). Because the cytoarchitecture is similar between humans and monkeys (e.g. Rosene and Van Hoesen, 1987
), it is possible that humans with low testosterone may have lower spine densities.
Sexual Differences
Data from previous experiments regarding the response of the pyramidal cell spine synapse density to the manipulation of gonadal hormone levels of male (Leranth et al., 2003) and female (Leranth et al., 2000
) rats, as well as female (Leranth et al., 2002
) and male monkeys can be directly compared. In rats, similar to monkeys, experimental manipulations of circulating gonadal hormone levels have no effect on pyramidal cell density in either female or male animals. In contrast, gonadectomy has a significant effect on the density of the dendritic spine synapses of pyramidal cells in both female and male rats, as well as female and male monkeys (Table 2).
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In light of these data, assumptions regarding spine density and memory may need to be reevaluated or reconsidered. It is generally accepted that hippocampal long-term potentiation (LTP) is associated with synaptic plasticity and memory (Cohen and Eichenbaum, 1991) and electrophysiological experiments have demonstrated that E-induced spine synapse density in OVX rats is associated with augmented CA1 long-term potentiation (LTP; Cordoba Montoya and Carrer, 1997
; Foy et al., 1984
; Good et al., 1999
; Ito et al., 1999
). In contrast, T administration to castrated male rats has been reported to have an opposite, negative effect on CA1 LTP (Harley et al., 2000
), in spite of the fact that T administration dramatically increases the spine synapse density in both male rats (50%; Leranth et al., 2003
) and monkeys (40%; recent study). However, sex differences favoring males have been observed in some hippocampal-dependent memory tasks (Williams et al., 1990
; Williams and Meck, 1991
; Roof and Havens, 1992
; Roof, 1993
; Markowska, 1999
). Exogenous T or E during early postnatal development can reverse this sex difference and promote the development of a male-like hippocampus in females (Williams et al., 1990
; Williams and Meck, 1991
; Roof and Havens, 1992
; Roof, 1993
). Because males excel at some hippocampal-dependent memory tasks, despite having fewer CA1 spine synapses than females, it is tempting to speculate that either spine density has little to do with hippocampal-dependent memory processing, or that both LTP and gonadal hormones induce synaptic sprouting without causal relationships. On the other hand, the organizational effects of steroid hormones on the brain may necessitate that females have a greater density of CA1 spine synapses to compensate for spine reductions or other alterations elsewhere in the brain. Alternatively, high numbers of spine synapses may not be beneficial to memory (e.g. by producing excess disruptive input to the dendrite) and the impairments shown by females in some spatial memory tasks may be the result of too many spine synapses. Correlations between spine synapse density and memory measurements performed in the same animals will be necessary to clearly elucidate this issue.
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Notes |
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Address correspondence to Csaba Leranth, Department of Obstetrics and Gynecology, Yale University, School of Medicine, 333 Cedar Street, FMB 312, New Haven, CT 06520-8063, USA. Email: csaba.leranth{at}yale.edu.
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