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Deficits of neuronal density in CA1 and synaptic density in the dentate gyrus, CA3 and CA1, in a mouse model of Down syndrome
Brain Research 1022 (2004) 101 – 109 www.elsevier.com/locate/brainres
Research report
Deficits of neuronal density in CA1 and synaptic density in the dentate gyrus, CA3 and CA1, in a mouse model of Down syndrome M. Ayberk Kurta,*, M. Ilker Kafaa, Mara Dierssenb, D. Ceri Daviesc a Department of Anatomy, Uludag University Medical Faculty, Bursa, Turkey Program in Genes and Disease, Genomic Regulation Center, Barcelona 16059, Spain c Department of Anatomy and Developmental Biology, St George’s Hospital Medical School, London, UK b
Accepted 27 June 2004
Abstract Ts65Dn mice are partially trisomic for the distal region of MMU16, which is homologous with the obligate segment of HSA21 triplicated in Down syndrome (DS). Ts65Dn mice are impaired in learning tasks that require an intact hippocampus. In order to investigate the neural basis of these deficits in this mouse model of Down syndrome, quantitative light and electron microscopy were used to compare the volume densities of neurons and synapses in the hippocampus of adult Ts65Dn (n=4) and diploid mice (n=4). Neuron density was significantly lower in the CA1 of Ts65Dn compared to diploid mice ( pb0.01). Total synapse density was significantly lower in the dentate gyrus (DG; pb0.001), CA3 ( pb0.05) and CA1 ( pb0.001) of Ts65Dn compared to diploid mice. The synapse-to-neuron ratio was significantly lower in the DG ( pb0.001), CA3 ( pb0.01) and CA1 ( pb0.001) of Ts65Dn compared to diploid mice. When the data were broken down by synapse type, asymmetric synapse density was found to be significantly lower in the DG ( pb0.001), CA3 ( pb0.05) and CA1 ( pb0.001) of Ts65Dn compared to diploid mice, while such a difference in symmetric synapse density was only present in the DG ( pb0.01). The asymmetric synapse-to-neuron ratio was significantly lower in the DG ( pb0.001), CA3 ( pb0.01) and CA1 ( pb0.001) of Ts65Dn compared to diploid mice, but there were no such significant differences in symmetric synapse-to-neuron ratios. These results suggest that impaired synaptic connectivity in the hippocampus of Ts65Dn mice underlies, at least in part, their cognitive impairment. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Genetic models Keywords: Down syndrome; Ts65Dn mouse; Hippocampus; Neuron density; Synapse density; Stereology
1. Introduction Down syndrome (DS) is the most common known genetic cause of mental retardation in man and results from triplication of all or part of chromosome 21 [20]. A region of conserved synteny between human chromosome 21 and mouse chromosome 16 encompasses most of the DS critical region [28]. Therefore, mice with either a complete triplication of chromosome 16 (Ts16 [21]) or triplication of only a distal region of chromosome 16 * Corresponding author. Tel.: +90 224 442 88 08; fax: +90 224 441 75 35. E-mail address: [email protected] (M. Ayberk Kurt). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.06.075
(Ts65Dn [12], Ts1Cje [57], Ms1Ts65 [58]) have been developed as putative models of Down syndrome. Although complete murine Ts16 gives rise to some of the characteristics of DS [10], the value of this model is limited because mouse chromosome 16 contains many genes present on human chromosomes other than 21 and conversely, some genes on human chromosome 21 are located on murine chromosomes other than 16 [10,11]. More importantly, murine Ts16 is lethal in the perinatal period [10,11]. In order to overcome the viability problems associated with murine Ts16, three segmental Ts16 mouse lines (Ts65Dn, Ts1Cje and Ms1Ts65 mice) have been generated, with Ts65Dn mice showing most similarities to the DS phenotype [12,57,58].
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Ts65Dn mice exhibit behavioural abnormalities and hyperactivity during early development [31]. Adult Ts65Dn mice also display hyperactivity under certain experimental conditions, increased but ineffective exploration and impairment of learning and memory [16,17,22,23,26,31,32, 34,35,39,54]. Although the brains of Ts65Dn mice do not exhibit any overt neuropathology, morphological abnormalities have been reported in some brain regions of Ts65Dn mice, including reduced volume and neuronal density in the dentate gyrus [36], age-related degeneration of basal forebrain cholinergic neurons [25,26,31] and reduced cerebellar volume together with reduced thickness of the internal granule and molecular layers and reduced granule cell number [3], compared to diploid mice. However, despite the importance of both neuronal number and preserved synaptic integrity for learning and memory, little information is available about these subjects in Ts65Dn mice. Quantitative electron microscopy has revealed that the density of asymmetric (putative excitatory) synapses is lower in the temporal cortex of Ts65Dn mice than in diploid controls [41]. More recently, Dierssen et al. [18] found that cortical pyramidal neurons in Ts65Dn mice were considerably smaller and their dendritic arborisations less branched and less spinous than those of diploid mice. In order to investigate whether such impairments of neural connectivity could underlie the cognitive deficits present in Ts65Dn mice, a light and electron microscope study of synaptic connectivity in the hippocampus was performed, because of this brain structure’s involvement in learning and memory [48,47,49,50,56].
2. Materials and methods Ts65Dn mice were obtained by mating female carriers of the 1716 chromosome (B6C3H-Ts65Dn) with B6EiC3HF1 hybrid males, obtained by crossing C57BL/6JEi with C3H/ HeSnJ mice [12]. Four male Ts65Dn mice (three aged 16– 17 months and one aged 23 months) identified by karyotyping and four male diploid control mice (all aged 16–17 months) were given a lethal dose of anaesthetic and perfused transcardially with 4% paraformaldehyde, 0.1% glutaraldehyde and 2% picric acid in 0.1 M phosphate buffer at pH 7.3. Their brains were then removed and immersed in similar fixative. Each cerebrum was cut in the mid-coronal plane and 1-mm thick coronal slices were taken parallel to the cut surface of the posterior half of the left hemisphere throughout the entire hippocampus. Each slice was then trimmed to produce a rectangular block of tissue (approximately 1 mm wide) extending across the entire hippocampus and containing the DG, CA3 and CA1. Each tissue block was then post-fixed with 1% OsO4 in 0.1 M phosphate buffer for 1 h, dehydrated through a graded series of ethanols and embedded in Spurr’s resin (Agar Scientific, Stansted. UK). The unfolding systematic random sampling procedure [46] was then carried out on four randomly
selected tissue blocks from each animal. The blocks were coded so that subsequent analysis was performed blind. 2.1. Estimation of neuron volume density A semi-thin section (0.5 Am thick) was taken from each hippocampal block perpendicular to the long axis of the hippocampus and stained with a mixture of 1% toluidine blue and 1% borax in distilled water. The sections were examined at low magnification in a Zeiss Axioplan 2 light microscope to locate the apex of the stratum granulosum of the dentate gyrus and the central regions of the stratum pyramidale of CA3 and CA1. Five adjacent images were then captured along the neuronal layer of each of the three hippocampal fields using a Zeiss Axiocam 12106 pixel digital camera attached to the light microscope. A total of 20 images were captured for each hippocampal field per animal (five images per fieldfour tissue blocks). A counting frame of known area (7555 Am) was then created using ScionImage software (public domain) and superimposed on the digital image to be counted, after the appropriate calibrations. Neuronal nuclei were selected as the counting unit and distinguished from glial nuclei by their morphological features and staining patterns [42]. They were quantified according to the unbiased counting rule [44,63] and their perimeters were outlined using Scion Image’s tracing feature in order to obtain mean neuronal nucleus diameters as illustrated in Fig. 1. The volume densities of neurons (Nvn) were then estimated by applying the formula: ¯ n+t) where Nan is number of neurons per unit Nvn=Nan/(D ¯ area, D n is the mean nuclear diameter and t is the section thickness. The experimental codes were then broken and unpaired t-tests were used to compare the neuron volume density in the DG, CA3 and CA1 of Ts65Dn mice with those of diploid mice. 2.2. Estimation of synapse volume density An ultrathin section with a gold interference colour (approx. 80 nm thick) was cut from each hippocampal block, picked up on a copper grid and stained with 2% uranyl acetate and 0.2% lead citrate. The ultrathin sections were initially examined at 3000 in a Zeiss EM 900 electron microscope in order to locate the following areas for investigation: (1) the inner molecular layer of the dentate gyrus (~50–75 Am away from the stratum granulosum) that receives its principal input from the septum and diagonal band of Broca; (2) the stratum lucidum adjacent to the stratum pyramidale of CA3 that receives mossy fibre input and (3) the stratum radiatum and lacunosum of CA1 (~75– 100 Am away from the stratum pyramidale) that receives Schaffer collateral input. For each hippocampal region, images of neuropil were then captured from each corner of three adjacent grid squares parallel to the hippocampal stratum at a magnification of x12000, using a Sony CCD camera connected to an Apple Macintosh G3 computer.
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¯ s+t), where Nas is the stereological formula; Nvs=Nas/(H number of synapses per unit area, t is the section thickness and in the case of synapses, whose apposition zones are ¯ s=pD s/4, where D ¯s assumed to be flat circular discs [43], H ¯ s is the is the mean diameter of the synaptic contacts and H mean tangent diameter. The codes were then broken and the data assigned to either Ts65Dn or diploid groups. Unpaired t-tests were used to compare the volume density and mean synaptic apposition zone lengths of all synapses combined and asymmetric and symmetric synapses separately, from each hippocampal region of Ts65Dn and diploid control mice investigated. The synapse-to-neuron ratios in the DG, CA3 and CA1 were then calculated by dividing the volume density of synapses by that of neurons. The synapse-to-neuron rations of all synapses combined (asymmetric+symmetric) and
Fig. 1. Photomicrographs of examples of toulidine blue-stained semi-thin sections of the stratum pyramidale of CA1 from a Diploid (A) and a Ts65Dn (B) mouse used for determining neuron volume density. Neuronal nuclei (n) were used as the counting unit and could be distinguished from those of glia (g) by their morphological features. Neuronal nuclei (1) which crossed the upper and right margins of the counting frame (thin lines) of the counting frame were counted, whereas those which crossed the lower and left borders (E) of the counting frame (thick lines) were not counted. The diameters of neuronal nuclei were estimated from measurements of their circumference (dotted lines).
Thus, 48 images were captured for each hippocampal region per animal (12 per section from each of 4 blocks). The image analysis system was calibrated by capturing images of particles of known size. A counting frame of known area (3.52.5 Am) was then created using Scion-Image software and superimposed on the hippocampal image. The numbers of asymmetric and symmetric synapses present within each captured image and intersecting the right and superior edges of the frame were counted to obtain the area density. Only synapses with identifiable apposition zones and a presynaptic bouton containing synaptic vesicles were counted. Synapses with prominent post-synaptic densities and relatively wide synaptic clefts (approx. 20 nm) were counted as asymmetric synapses, while synapses with pre- and postsynaptic densities of equal thickness and narrower synaptic clefts (approx. 12 nm) were counted as symmetric synapses. Each synaptic apposition zone was then outlined using a mouse driven cursor and its length determined using ScionImage software. The volume density of synapses (Nvs; number of synapses/mm3) was estimated for each subject from the two-dimensional counts of synaptic profiles, using
Fig. 2. Transmission electron photomicrographs of examples of asymmetric (A) and symmetric (B) synapses in the stratum radiatum/lacunosum of CA1 from a Ts65Dn mouse. Arrows indicate the extent of the post-synaptic thickenings.
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Table 1 The mean neuronal densities, synaptic densities and synapse-to-neuron ratios in DG, CA3 and CA1 regions of individual diploid (D; n=4) and Ts65Dn (T; n=4) mice
D1 D2 D3 D4 T1 T2 T3 T4
Age (months)
Neuronal density N neu/mm3 DG
CA3
16–17 16–17 16–17 16–17 16–17 16–17 16–17 23
631,880 620,875 550,012 697,536 686,141 599,265 603,124 734,865
148,460 157,067 168,004 145,432 171,498 153,007 167,775 158,634
Synaptic density (109) N syn/mm3
Synapses per neuron
CA1
DG
CA3
CA1
DG
CA3
CA1
334,079 336,971 257,663 282,991 260,837 256,391 257,839 252,464
1.75 1.98 1.68 1.22 1.07 0.95 0.98 1.15
1.19 1.20 1.06 1.13 1.06 0.97 0.89 1.06
1.78 1.53 1.51 1.34 1.10 1.03 0.94 0.95
2800.52 3213.63 3064.61 1762.28 1575.50 1591.53 1708.04 1586.21
8242.33 7668.69 6705.60 7754.76 6093.83 6394.65 5441.90 6688.48
5398.40 4573.21 5931.19 4873.98 4227.09 4107.30 3701.46 3827.59
asymmetric and symmetric synapses separately in the three hippocampal fields of TS65Dn mice were then compared with those in diploid mice using unpaired t-tests.
3. Results The morphological preservation of the hippocampal tissue investigated was good and there was no apparent qualitative difference between Ts65Dn and diploid mice in either the light or electron microscopes (Figs. 1 and 2). There were no obvious signs of accelerated ageing or degeneration such as lipofuscin deposition, active microglia or astrocytosis in either Ts65Dn or diploid mice. Furthermore, there was no evidence of amyloid deposition or neurofibrillary degeneration in Ts65Dn mice. The volume densities of neurons and synapses (asymmetric and symmetric combined) as well as the synapse-toneuron ratios in the DG, CA3 and CA1 of individual Ts65Dn and diploid mice are given in Table 1. Pyramidal neuron volume density was significantly (t=3.64; pb0.01) lower in CA1 of Ts65Dn compared to diploid mice (Fig. 3). There was no significant difference between neuronal volume density in the DG or CA3 of Ts65Dn compared to diploid mice. The volume density of all synapses (asymmetric and symmetric combined) was significantly lower in the inner
molecular layer of the DG (t=6.36; pb0.001), the stratum lucidum of CA3 (t=2.48; pb0.05) and the stratum radiatum and lacunosum of CA1 (t=7.37; pb0.001), of Ts65Dn compared to diploid mice (Fig. 4). Breaking down the data by synapse type, asymmetric synapse volume density was significantly lower in the DG (t=4.53; pb0.001), CA3 (t=2.74; pb0.05) and CA1 (t=7.53; pb0.001) of Ts65Dn compared with diploid mice (Fig. 4). Although the volume density of symmetric synapses in the DG was significantly lower in Ts65Dn mice than in diploid mice (t=3.01; pb0.01), there were no significant differences between Ts65Dn and diploid mice in the volume densities of symmetric synapses in CA3 and CA1 (Fig. 4). The synapse-to-neuron ratio of all synapses (asymmetric and symmetric combined) was significantly lower in the DG (t=5.52; pb0.001), CA3 (t=3.08; pb0.01 and CA1 (t=3.88; pb0.001) of Ts65Dn compared to diploid mice (Fig. 5). Breaking down the data by synapse type, the ratio of asymmetric synapses-to-neurons was significantly lower in the DG (t=4.23; pb0.001), CA3 (t=3.11; pb0.01) and CA1 (t=4.02; pb0.001) of Ts65Dn compared with diploid mice (Fig. 5). However, no such significant differences were found between Ts65Dn and diploid mice in the ratios of symmetric synapses-to-neurons (Fig. 5). There were no significant differences between Ts65Dn and diploid mice in the mean synaptic apposition length of all synapses (asymmetric and symmetric combined) in the
Fig. 3. A bar diagram showing the mean volume density (FS.E.M.) of neurons in the stratum granulosum of the DG and the stratum pyramidale of CA3 and CA1 of diploid (n=4) and Ts65Dn (n=4) mice. **pb0.01.
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Fig. 4. A bar diagram showing the mean volume density (FS.E.M.) of asymmetric (Asym), symmetric (Sym) and all synapses combined (Asym+Sym) in the inner molecular layer of the dentate gyrus, the stratum lucidum of CA3 and the stratum radiatum/lacunosum of CA1 of diploid (n=4) and Ts65Dn (n=4) mice. *pb0.05, **pb0.01 and ***pb0.001.
DG, CA3 and CA1 (Fig. 6). When the data were broken down by synapse type, the mean apposition zone length of asymmetric synapses in CA1 of Ts65Dn mice was significantly larger (t= 2.07; pb0.046) than that of diploid mice. There were no significant differences between Ts65Dn and diploid mice in the mean lengths of asymmetric apposition zones in the DG or CA3. Neither were there any significant differences between Ts65Dn and diploid mice in the mean symmetric synapse apposition zone lengths in any of the three hippocampal regions investigated.
4. Discussion The results of the present study demonstrate a significantly lower neuronal density in CA1 and significantly lower synaptic densities and synapse-to-neuron ratios in the DG, CA3 and CA1 of Ts65Dn mouse hippocampus compared to those in diploid mice. The 37% lower synapse
density in the inner molecular layer of the DG cannot be attributed to a lower granule cell density, as this was unaffected in Ts65Dn mice. However, it may at least in part be attributable to a reduced input, because the inner molecular layer of the DG is principally innervated by cholinergic fibres from the septum and diagonal band of Broca [53] and a significant loss of cholinergic basal forebrain neurons and abnormal cholinergic function have been reported to occur in Ts65Dn mice from 6 months of age [25,26,31]. The results of the current study revealed a 14% lower synapse density in the stratum lucidum of CA3 in Ts65Dn compared to diploid mice. Since the terminals of mossy fibre axons of dentate granule cells terminate on the apical dendrites of pyramidal neurons in CA3 and neuronal density in the DG and CA3 is unaffected in Ts65Dn mice, the lower synapse density in Ts65Dn CA3 cannot be attributed to a reduction in projection and/or recipient neuron density. The Schaffer collaterals of CA3 pyramidal neurons synapse with the apical dendrites of CA1 pyramids
Fig. 5. A bar diagram showing synapse-to-neuron ratios for asymmetric (Asym), symmetric (Sym) and all synapses combined (Asym+Sym) in the inner molecular layer of the dentate gyrus, the stratum lucidum of CA3 and the stratum radiatum/lacunosum of CA1 of diploid (n=4) and Ts65Dn (n=4) mice. **pb0.01 and ***pb0.001.
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Fig. 6. A bar diagram showing the mean synaptic apposition zone length (FS.E.M.) of asymmetric (Asym), symmetric (Sym) and all synapses combined (Asym+Sym) in the inner molecular layer of the dentate gyrus, the stratum lucidum of CA3 and the stratum radiatum/lacunosum of CA1 of diploid (n=4) and Ts65Dn (n=4) mice. *pb0.05.
in the stratum radiatum and lacunosum. Approximately half of the 35% lower synapse density observed in this hippocampal region of Ts65Dn mice in the current study could be attributable to the 15% lower neuronal density in CA1 in these mice. However, the remainder of this synaptic deficit cannot be attributed to a lower efferent neuron density in CA3, which was unaffected in Ts65Dn mice. Thus, the results of the current and previous studies suggest that although lower densities of cholinergic neurons in the basal forebrain and pyramidal neurons in CA1 may contribute to some of the lower synapse densities in adult Ts65Dn mouse hippocampus, they cannot account for all of the synaptic deficits observed in the tri-synaptic circuit of the hippocampal formation of these mice. Therefore, the synaptic deficits in the hippocampus observed in the current study suggest that there is also some abnormality of axon terminal formation and/or dendritic arborisation in adult Ts65Dn mice during development, maturation or aging of hippocampal circuitry. It remains to be determined whether neurons in juvenile Ts65Dn mice fail to achieve the structural complexity of those in wild type mice, or whether they undergo greater age-related synaptic pruning. Our previous work [18] has revealed an impoverished pyramidal neuron phenotype in the M2 region of Ts65Dn mouse cerebral cortex. The dendrites of these neurons are smaller, less branched and less spinous compared to those of diploid mice. Furthermore, the dendrites of pyramidal neurons of Ts65Dn mice raised in an enriched environment were unable to respond to the enrichment by branching and becoming more spinous as did their diploid counterparts. These results support both a developmental retardation of neuronal morphology and a subsequent impairment of neuronal plasticity in Ts65Dn mice, which would both affect synaptic connectivity. The current findings of a significantly lower (15%) pyramidal neuronal density in CA1 of Ts65Dn compared to diploid mice, with no differences in granule cell density in
the DG or pyramidal neuron density in CA3, contrast with those of Insausti et al. [36] who reported significantly less granule cells in the DG and more pyramidal neurons in CA3 of Ts65Dn compared to diploid mice, but no differences in CA1 or CA2. The reasons for these apparent discrepancies are not clear, but may be due to a variety of factors such as differences in the ages of the animals investigated, tissue preparation techniques and methods of quantification. Insausti et al. [36] investigated 20–28-week-old mice, whereas in the current study, all mice were at least 16 months old. Individual neuronal populations in Ts65Dn mice may be differentially affected by age. This view is supported by the fact that atrophy and progressive loss of basal forebrain cholinergic neurons is not detected in Ts65Dn mice until after 6 months of age [31]. Similarly, DS individuals are born with a normal septo-hippocampal cholinergic system [37], which degenerates with aging [7,72]. Therefore, the neuronal deficit observed in CA1 of Ts65Dn mice in the current study may be age-dependant. However, the density of CA1 pyramidal neurons in the 23month-old Ts65Dn mouse (T4) was similar to that in the 16– 17-month-old Ts65Dn mice (T1–3; Table 1). In addition, although the proliferation and migration of CA3 and CA1 pyramidal neurons end before birth [1], the neurogenesis of dentate granule cells continues during adult life [5,51]. Therefore, the reduced number of granule cells in the 20– 28-week old Ts65Dn DG reported by Insausti et al. [36] may be ameliorated by continuing granule cell proliferation in later life. The nature of the quantification may also have had a bearing on the results of the two studies. Insausti et al. [36] used the doptical dissectorT [69] to estimate the total number of neurons in fields of the Ts65Dn hippocampal formation, whereas in the current study, the unfolding method [46] was used to determine neuronal density in order to estimate synapse-to-neuron ratios as indices of neuronal connectivity in the Ts65Dn hippocampus. However, Insausti et al. [36] did not find any difference between
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Ts65Dn and diploid mice in the volumes of the DG, CA3 or CA1 and the ddissectorT and dunfoldingT method have been shown to produce similar results when applied to determine the numerical density of neurons and synapses in the cerebral cortex [6,45,46,67]. The results of the current study demonstrated a significant deficit of asymmetric synapses in the DG (32%), CA3 (15%) and CA1 (35%) of Ts65Dn mouse hippocampal formation, while a significant deficit (45%) of symmetric synapses was restricted to the Ts65Dn dentate gyrus. Previous investigations have revealed significantly fewer (30%) asymmetric synapses in the temporal cortex of Ts65Dn compared to diploid mice, whereas the density of symmetric synapses appeared to be unaffected [41]. Since asymmetric and symmetric synapses are considered to mediate excitatory and inhibitory transmission respectively [2,8,19], excitatory synaptic transmission appears to be more affected than inhibitory transmission in the central nervous system of Ts65Dn mice. This view is supported by the fact that excitatory amino acids levels are significantly lower (approx. 50%) in the parahippocampal gyrus of DS adults compared to normal subjects [55]. Since a reciprocal relationship between synapse number and post-synaptic density size has been previously reported for both rodent [29] and human brain [15,59], in the current study, the mean apposition zone lengths of asymmetric and symmetric synapses in Ts65Dn mice were compared with those in diploid mice. Despite the marked deficits observed in the densities of asymmetric synapses in the DG, CA3 and CA1 of Ts65Dn mice, a significant difference was only found in the synaptic apposition lengths of asymmetric synapses in CA1. Although the reason for the 5% greater asymmetric synaptic apposition zone lengths in the Ts65Dn compared to diploid CA1 is not clear, it might reflect an attempt to compensate for the neuronal deficit that was present in this region of Ts65Dn mouse hippocampus. The results of a number of studies suggest abnormalities in the mechanisms that govern development and maturation of neuronal circuitry in DS. Abnormalities of synaptic development are present in foetuses [52] and children [71,70] with DS and dendritic atrophy has been reported to occur in adults with the condition [64]. Decreased levels of protein markers of dendritic spines such as synaptosomal associated protein 25 [27] and drebrin [60,68] also suggest impaired synaptogenesis or neuronal loss in DS. Abnormal dendritic spine morphology has also been reported to occur in both the DG and CA1 of the Ts65Dn hippocampus [4]. More recently, the basal dendrites of layer III pyramidal cells in the Ts65Dn mouse frontal cortex of have been shown to be smaller, less branched and less spinous than those of diploid controls [18]. The current finding of a widespread synaptic deficit in the hippocampal formation of Ts65Dn mice, without a similar deficit in neuronal density, adds weight to the suggestion that Ts65Dn mice model the dendritic and synaptic abnormalities characteristic of DS.
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There is a strong relationship between synapse number and cerebral function. Rats reared in complex housing conditions show an increase in cortical synapse-to-neuron ratio [66] and motor learning leads to an increase in synapse number in the rat motor cortex [38]. In contrast, a number of studies have associated age-related memory impairment with synapse loss, particularly from the hippocampus [13,24]. Furthermore, the cognitive decline in Alzheimer’s disease correlates more closely with synapse loss than with any other neuropathological feature of the disease [14,65]. Ts65Dn mice demonstrate impaired spatial learning and memory [16,17,22,23,26,31,32,34,35,39,54] and it is possible that the synaptic deficits in the DG, CA3 and CA1 of these mice underlie at least some of this impairment, because the hippocampal formation plays an important role in spatial learning [48,47,49,50,56]. Furthermore, long-term potentiation has been shown to be significantly reduced in the hippocampus of Ts65Dn mice, whereas long-term depression is increased [40,61,62]. Since there is evidence to suggest that long-term potentiation and long-term depression are implicated in learning and memory, their abnormality in Ts65Dn mice may reflect a reduced efficacy of neuronal networks underlying these processes that contributes to the learning impairments observed in Ts65Dn mice. The results of the present study may have important implications for alleviation of the behavioural and learning deficits in Ts65Dn mice and possibly the mental retardation in DS. Any therapeutic strategy that enhances synaptogenesis or inhibits synapse loss may reasonably be expected to improve their cognitive capability. Nerve growth factor may be one possible candidate, because it has been shown to correct developmental impairments and age-related neuronal atrophy of trisomy 16 mouse basal cholinergic neurons [9,30] and NGF levels decline significantly with age in the basal forebrain and hippocampus of Ts65Dn mice [33]. However, the efficacy of this and other prospective strategies remains to be tested.
Acknowledgments The work in Uludag University was supported by ¨ BI˙TAK (The Scientific and Technical Research Council TU of Turkey, Grant no: SBAG: 2422) and Uludag University (Grant no: 2001/9). We thank to Mr. R.F. Moss for his invaluable technical assistance. Mara Dierssen was supported by the Jeroˆme Lejeune Foundation.
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Research report
Deficits of neuronal density in CA1 and synaptic density in the dentate gyrus, CA3 and CA1, in a mouse model of Down syndrome M. Ayberk Kurta,*, M. Ilker Kafaa, Mara Dierssenb, D. Ceri Daviesc a Department of Anatomy, Uludag University Medical Faculty, Bursa, Turkey Program in Genes and Disease, Genomic Regulation Center, Barcelona 16059, Spain c Department of Anatomy and Developmental Biology, St George’s Hospital Medical School, London, UK b
Accepted 27 June 2004
Abstract Ts65Dn mice are partially trisomic for the distal region of MMU16, which is homologous with the obligate segment of HSA21 triplicated in Down syndrome (DS). Ts65Dn mice are impaired in learning tasks that require an intact hippocampus. In order to investigate the neural basis of these deficits in this mouse model of Down syndrome, quantitative light and electron microscopy were used to compare the volume densities of neurons and synapses in the hippocampus of adult Ts65Dn (n=4) and diploid mice (n=4). Neuron density was significantly lower in the CA1 of Ts65Dn compared to diploid mice ( pb0.01). Total synapse density was significantly lower in the dentate gyrus (DG; pb0.001), CA3 ( pb0.05) and CA1 ( pb0.001) of Ts65Dn compared to diploid mice. The synapse-to-neuron ratio was significantly lower in the DG ( pb0.001), CA3 ( pb0.01) and CA1 ( pb0.001) of Ts65Dn compared to diploid mice. When the data were broken down by synapse type, asymmetric synapse density was found to be significantly lower in the DG ( pb0.001), CA3 ( pb0.05) and CA1 ( pb0.001) of Ts65Dn compared to diploid mice, while such a difference in symmetric synapse density was only present in the DG ( pb0.01). The asymmetric synapse-to-neuron ratio was significantly lower in the DG ( pb0.001), CA3 ( pb0.01) and CA1 ( pb0.001) of Ts65Dn compared to diploid mice, but there were no such significant differences in symmetric synapse-to-neuron ratios. These results suggest that impaired synaptic connectivity in the hippocampus of Ts65Dn mice underlies, at least in part, their cognitive impairment. D 2004 Elsevier B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Genetic models Keywords: Down syndrome; Ts65Dn mouse; Hippocampus; Neuron density; Synapse density; Stereology
1. Introduction Down syndrome (DS) is the most common known genetic cause of mental retardation in man and results from triplication of all or part of chromosome 21 [20]. A region of conserved synteny between human chromosome 21 and mouse chromosome 16 encompasses most of the DS critical region [28]. Therefore, mice with either a complete triplication of chromosome 16 (Ts16 [21]) or triplication of only a distal region of chromosome 16 * Corresponding author. Tel.: +90 224 442 88 08; fax: +90 224 441 75 35. E-mail address: [email protected] (M. Ayberk Kurt). 0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2004.06.075
(Ts65Dn [12], Ts1Cje [57], Ms1Ts65 [58]) have been developed as putative models of Down syndrome. Although complete murine Ts16 gives rise to some of the characteristics of DS [10], the value of this model is limited because mouse chromosome 16 contains many genes present on human chromosomes other than 21 and conversely, some genes on human chromosome 21 are located on murine chromosomes other than 16 [10,11]. More importantly, murine Ts16 is lethal in the perinatal period [10,11]. In order to overcome the viability problems associated with murine Ts16, three segmental Ts16 mouse lines (Ts65Dn, Ts1Cje and Ms1Ts65 mice) have been generated, with Ts65Dn mice showing most similarities to the DS phenotype [12,57,58].
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Ts65Dn mice exhibit behavioural abnormalities and hyperactivity during early development [31]. Adult Ts65Dn mice also display hyperactivity under certain experimental conditions, increased but ineffective exploration and impairment of learning and memory [16,17,22,23,26,31,32, 34,35,39,54]. Although the brains of Ts65Dn mice do not exhibit any overt neuropathology, morphological abnormalities have been reported in some brain regions of Ts65Dn mice, including reduced volume and neuronal density in the dentate gyrus [36], age-related degeneration of basal forebrain cholinergic neurons [25,26,31] and reduced cerebellar volume together with reduced thickness of the internal granule and molecular layers and reduced granule cell number [3], compared to diploid mice. However, despite the importance of both neuronal number and preserved synaptic integrity for learning and memory, little information is available about these subjects in Ts65Dn mice. Quantitative electron microscopy has revealed that the density of asymmetric (putative excitatory) synapses is lower in the temporal cortex of Ts65Dn mice than in diploid controls [41]. More recently, Dierssen et al. [18] found that cortical pyramidal neurons in Ts65Dn mice were considerably smaller and their dendritic arborisations less branched and less spinous than those of diploid mice. In order to investigate whether such impairments of neural connectivity could underlie the cognitive deficits present in Ts65Dn mice, a light and electron microscope study of synaptic connectivity in the hippocampus was performed, because of this brain structure’s involvement in learning and memory [48,47,49,50,56].
2. Materials and methods Ts65Dn mice were obtained by mating female carriers of the 1716 chromosome (B6C3H-Ts65Dn) with B6EiC3HF1 hybrid males, obtained by crossing C57BL/6JEi with C3H/ HeSnJ mice [12]. Four male Ts65Dn mice (three aged 16– 17 months and one aged 23 months) identified by karyotyping and four male diploid control mice (all aged 16–17 months) were given a lethal dose of anaesthetic and perfused transcardially with 4% paraformaldehyde, 0.1% glutaraldehyde and 2% picric acid in 0.1 M phosphate buffer at pH 7.3. Their brains were then removed and immersed in similar fixative. Each cerebrum was cut in the mid-coronal plane and 1-mm thick coronal slices were taken parallel to the cut surface of the posterior half of the left hemisphere throughout the entire hippocampus. Each slice was then trimmed to produce a rectangular block of tissue (approximately 1 mm wide) extending across the entire hippocampus and containing the DG, CA3 and CA1. Each tissue block was then post-fixed with 1% OsO4 in 0.1 M phosphate buffer for 1 h, dehydrated through a graded series of ethanols and embedded in Spurr’s resin (Agar Scientific, Stansted. UK). The unfolding systematic random sampling procedure [46] was then carried out on four randomly
selected tissue blocks from each animal. The blocks were coded so that subsequent analysis was performed blind. 2.1. Estimation of neuron volume density A semi-thin section (0.5 Am thick) was taken from each hippocampal block perpendicular to the long axis of the hippocampus and stained with a mixture of 1% toluidine blue and 1% borax in distilled water. The sections were examined at low magnification in a Zeiss Axioplan 2 light microscope to locate the apex of the stratum granulosum of the dentate gyrus and the central regions of the stratum pyramidale of CA3 and CA1. Five adjacent images were then captured along the neuronal layer of each of the three hippocampal fields using a Zeiss Axiocam 12106 pixel digital camera attached to the light microscope. A total of 20 images were captured for each hippocampal field per animal (five images per fieldfour tissue blocks). A counting frame of known area (7555 Am) was then created using ScionImage software (public domain) and superimposed on the digital image to be counted, after the appropriate calibrations. Neuronal nuclei were selected as the counting unit and distinguished from glial nuclei by their morphological features and staining patterns [42]. They were quantified according to the unbiased counting rule [44,63] and their perimeters were outlined using Scion Image’s tracing feature in order to obtain mean neuronal nucleus diameters as illustrated in Fig. 1. The volume densities of neurons (Nvn) were then estimated by applying the formula: ¯ n+t) where Nan is number of neurons per unit Nvn=Nan/(D ¯ area, D n is the mean nuclear diameter and t is the section thickness. The experimental codes were then broken and unpaired t-tests were used to compare the neuron volume density in the DG, CA3 and CA1 of Ts65Dn mice with those of diploid mice. 2.2. Estimation of synapse volume density An ultrathin section with a gold interference colour (approx. 80 nm thick) was cut from each hippocampal block, picked up on a copper grid and stained with 2% uranyl acetate and 0.2% lead citrate. The ultrathin sections were initially examined at 3000 in a Zeiss EM 900 electron microscope in order to locate the following areas for investigation: (1) the inner molecular layer of the dentate gyrus (~50–75 Am away from the stratum granulosum) that receives its principal input from the septum and diagonal band of Broca; (2) the stratum lucidum adjacent to the stratum pyramidale of CA3 that receives mossy fibre input and (3) the stratum radiatum and lacunosum of CA1 (~75– 100 Am away from the stratum pyramidale) that receives Schaffer collateral input. For each hippocampal region, images of neuropil were then captured from each corner of three adjacent grid squares parallel to the hippocampal stratum at a magnification of x12000, using a Sony CCD camera connected to an Apple Macintosh G3 computer.
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¯ s+t), where Nas is the stereological formula; Nvs=Nas/(H number of synapses per unit area, t is the section thickness and in the case of synapses, whose apposition zones are ¯ s=pD s/4, where D ¯s assumed to be flat circular discs [43], H ¯ s is the is the mean diameter of the synaptic contacts and H mean tangent diameter. The codes were then broken and the data assigned to either Ts65Dn or diploid groups. Unpaired t-tests were used to compare the volume density and mean synaptic apposition zone lengths of all synapses combined and asymmetric and symmetric synapses separately, from each hippocampal region of Ts65Dn and diploid control mice investigated. The synapse-to-neuron ratios in the DG, CA3 and CA1 were then calculated by dividing the volume density of synapses by that of neurons. The synapse-to-neuron rations of all synapses combined (asymmetric+symmetric) and
Fig. 1. Photomicrographs of examples of toulidine blue-stained semi-thin sections of the stratum pyramidale of CA1 from a Diploid (A) and a Ts65Dn (B) mouse used for determining neuron volume density. Neuronal nuclei (n) were used as the counting unit and could be distinguished from those of glia (g) by their morphological features. Neuronal nuclei (1) which crossed the upper and right margins of the counting frame (thin lines) of the counting frame were counted, whereas those which crossed the lower and left borders (E) of the counting frame (thick lines) were not counted. The diameters of neuronal nuclei were estimated from measurements of their circumference (dotted lines).
Thus, 48 images were captured for each hippocampal region per animal (12 per section from each of 4 blocks). The image analysis system was calibrated by capturing images of particles of known size. A counting frame of known area (3.52.5 Am) was then created using Scion-Image software and superimposed on the hippocampal image. The numbers of asymmetric and symmetric synapses present within each captured image and intersecting the right and superior edges of the frame were counted to obtain the area density. Only synapses with identifiable apposition zones and a presynaptic bouton containing synaptic vesicles were counted. Synapses with prominent post-synaptic densities and relatively wide synaptic clefts (approx. 20 nm) were counted as asymmetric synapses, while synapses with pre- and postsynaptic densities of equal thickness and narrower synaptic clefts (approx. 12 nm) were counted as symmetric synapses. Each synaptic apposition zone was then outlined using a mouse driven cursor and its length determined using ScionImage software. The volume density of synapses (Nvs; number of synapses/mm3) was estimated for each subject from the two-dimensional counts of synaptic profiles, using
Fig. 2. Transmission electron photomicrographs of examples of asymmetric (A) and symmetric (B) synapses in the stratum radiatum/lacunosum of CA1 from a Ts65Dn mouse. Arrows indicate the extent of the post-synaptic thickenings.
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Table 1 The mean neuronal densities, synaptic densities and synapse-to-neuron ratios in DG, CA3 and CA1 regions of individual diploid (D; n=4) and Ts65Dn (T; n=4) mice
D1 D2 D3 D4 T1 T2 T3 T4
Age (months)
Neuronal density N neu/mm3 DG
CA3
16–17 16–17 16–17 16–17 16–17 16–17 16–17 23
631,880 620,875 550,012 697,536 686,141 599,265 603,124 734,865
148,460 157,067 168,004 145,432 171,498 153,007 167,775 158,634
Synaptic density (109) N syn/mm3
Synapses per neuron
CA1
DG
CA3
CA1
DG
CA3
CA1
334,079 336,971 257,663 282,991 260,837 256,391 257,839 252,464
1.75 1.98 1.68 1.22 1.07 0.95 0.98 1.15
1.19 1.20 1.06 1.13 1.06 0.97 0.89 1.06
1.78 1.53 1.51 1.34 1.10 1.03 0.94 0.95
2800.52 3213.63 3064.61 1762.28 1575.50 1591.53 1708.04 1586.21
8242.33 7668.69 6705.60 7754.76 6093.83 6394.65 5441.90 6688.48
5398.40 4573.21 5931.19 4873.98 4227.09 4107.30 3701.46 3827.59
asymmetric and symmetric synapses separately in the three hippocampal fields of TS65Dn mice were then compared with those in diploid mice using unpaired t-tests.
3. Results The morphological preservation of the hippocampal tissue investigated was good and there was no apparent qualitative difference between Ts65Dn and diploid mice in either the light or electron microscopes (Figs. 1 and 2). There were no obvious signs of accelerated ageing or degeneration such as lipofuscin deposition, active microglia or astrocytosis in either Ts65Dn or diploid mice. Furthermore, there was no evidence of amyloid deposition or neurofibrillary degeneration in Ts65Dn mice. The volume densities of neurons and synapses (asymmetric and symmetric combined) as well as the synapse-toneuron ratios in the DG, CA3 and CA1 of individual Ts65Dn and diploid mice are given in Table 1. Pyramidal neuron volume density was significantly (t=3.64; pb0.01) lower in CA1 of Ts65Dn compared to diploid mice (Fig. 3). There was no significant difference between neuronal volume density in the DG or CA3 of Ts65Dn compared to diploid mice. The volume density of all synapses (asymmetric and symmetric combined) was significantly lower in the inner
molecular layer of the DG (t=6.36; pb0.001), the stratum lucidum of CA3 (t=2.48; pb0.05) and the stratum radiatum and lacunosum of CA1 (t=7.37; pb0.001), of Ts65Dn compared to diploid mice (Fig. 4). Breaking down the data by synapse type, asymmetric synapse volume density was significantly lower in the DG (t=4.53; pb0.001), CA3 (t=2.74; pb0.05) and CA1 (t=7.53; pb0.001) of Ts65Dn compared with diploid mice (Fig. 4). Although the volume density of symmetric synapses in the DG was significantly lower in Ts65Dn mice than in diploid mice (t=3.01; pb0.01), there were no significant differences between Ts65Dn and diploid mice in the volume densities of symmetric synapses in CA3 and CA1 (Fig. 4). The synapse-to-neuron ratio of all synapses (asymmetric and symmetric combined) was significantly lower in the DG (t=5.52; pb0.001), CA3 (t=3.08; pb0.01 and CA1 (t=3.88; pb0.001) of Ts65Dn compared to diploid mice (Fig. 5). Breaking down the data by synapse type, the ratio of asymmetric synapses-to-neurons was significantly lower in the DG (t=4.23; pb0.001), CA3 (t=3.11; pb0.01) and CA1 (t=4.02; pb0.001) of Ts65Dn compared with diploid mice (Fig. 5). However, no such significant differences were found between Ts65Dn and diploid mice in the ratios of symmetric synapses-to-neurons (Fig. 5). There were no significant differences between Ts65Dn and diploid mice in the mean synaptic apposition length of all synapses (asymmetric and symmetric combined) in the
Fig. 3. A bar diagram showing the mean volume density (FS.E.M.) of neurons in the stratum granulosum of the DG and the stratum pyramidale of CA3 and CA1 of diploid (n=4) and Ts65Dn (n=4) mice. **pb0.01.
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Fig. 4. A bar diagram showing the mean volume density (FS.E.M.) of asymmetric (Asym), symmetric (Sym) and all synapses combined (Asym+Sym) in the inner molecular layer of the dentate gyrus, the stratum lucidum of CA3 and the stratum radiatum/lacunosum of CA1 of diploid (n=4) and Ts65Dn (n=4) mice. *pb0.05, **pb0.01 and ***pb0.001.
DG, CA3 and CA1 (Fig. 6). When the data were broken down by synapse type, the mean apposition zone length of asymmetric synapses in CA1 of Ts65Dn mice was significantly larger (t= 2.07; pb0.046) than that of diploid mice. There were no significant differences between Ts65Dn and diploid mice in the mean lengths of asymmetric apposition zones in the DG or CA3. Neither were there any significant differences between Ts65Dn and diploid mice in the mean symmetric synapse apposition zone lengths in any of the three hippocampal regions investigated.
4. Discussion The results of the present study demonstrate a significantly lower neuronal density in CA1 and significantly lower synaptic densities and synapse-to-neuron ratios in the DG, CA3 and CA1 of Ts65Dn mouse hippocampus compared to those in diploid mice. The 37% lower synapse
density in the inner molecular layer of the DG cannot be attributed to a lower granule cell density, as this was unaffected in Ts65Dn mice. However, it may at least in part be attributable to a reduced input, because the inner molecular layer of the DG is principally innervated by cholinergic fibres from the septum and diagonal band of Broca [53] and a significant loss of cholinergic basal forebrain neurons and abnormal cholinergic function have been reported to occur in Ts65Dn mice from 6 months of age [25,26,31]. The results of the current study revealed a 14% lower synapse density in the stratum lucidum of CA3 in Ts65Dn compared to diploid mice. Since the terminals of mossy fibre axons of dentate granule cells terminate on the apical dendrites of pyramidal neurons in CA3 and neuronal density in the DG and CA3 is unaffected in Ts65Dn mice, the lower synapse density in Ts65Dn CA3 cannot be attributed to a reduction in projection and/or recipient neuron density. The Schaffer collaterals of CA3 pyramidal neurons synapse with the apical dendrites of CA1 pyramids
Fig. 5. A bar diagram showing synapse-to-neuron ratios for asymmetric (Asym), symmetric (Sym) and all synapses combined (Asym+Sym) in the inner molecular layer of the dentate gyrus, the stratum lucidum of CA3 and the stratum radiatum/lacunosum of CA1 of diploid (n=4) and Ts65Dn (n=4) mice. **pb0.01 and ***pb0.001.
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M. Ayberk Kurt et al. / Brain Research 1022 (2004) 101–109
Fig. 6. A bar diagram showing the mean synaptic apposition zone length (FS.E.M.) of asymmetric (Asym), symmetric (Sym) and all synapses combined (Asym+Sym) in the inner molecular layer of the dentate gyrus, the stratum lucidum of CA3 and the stratum radiatum/lacunosum of CA1 of diploid (n=4) and Ts65Dn (n=4) mice. *pb0.05.
in the stratum radiatum and lacunosum. Approximately half of the 35% lower synapse density observed in this hippocampal region of Ts65Dn mice in the current study could be attributable to the 15% lower neuronal density in CA1 in these mice. However, the remainder of this synaptic deficit cannot be attributed to a lower efferent neuron density in CA3, which was unaffected in Ts65Dn mice. Thus, the results of the current and previous studies suggest that although lower densities of cholinergic neurons in the basal forebrain and pyramidal neurons in CA1 may contribute to some of the lower synapse densities in adult Ts65Dn mouse hippocampus, they cannot account for all of the synaptic deficits observed in the tri-synaptic circuit of the hippocampal formation of these mice. Therefore, the synaptic deficits in the hippocampus observed in the current study suggest that there is also some abnormality of axon terminal formation and/or dendritic arborisation in adult Ts65Dn mice during development, maturation or aging of hippocampal circuitry. It remains to be determined whether neurons in juvenile Ts65Dn mice fail to achieve the structural complexity of those in wild type mice, or whether they undergo greater age-related synaptic pruning. Our previous work [18] has revealed an impoverished pyramidal neuron phenotype in the M2 region of Ts65Dn mouse cerebral cortex. The dendrites of these neurons are smaller, less branched and less spinous compared to those of diploid mice. Furthermore, the dendrites of pyramidal neurons of Ts65Dn mice raised in an enriched environment were unable to respond to the enrichment by branching and becoming more spinous as did their diploid counterparts. These results support both a developmental retardation of neuronal morphology and a subsequent impairment of neuronal plasticity in Ts65Dn mice, which would both affect synaptic connectivity. The current findings of a significantly lower (15%) pyramidal neuronal density in CA1 of Ts65Dn compared to diploid mice, with no differences in granule cell density in
the DG or pyramidal neuron density in CA3, contrast with those of Insausti et al. [36] who reported significantly less granule cells in the DG and more pyramidal neurons in CA3 of Ts65Dn compared to diploid mice, but no differences in CA1 or CA2. The reasons for these apparent discrepancies are not clear, but may be due to a variety of factors such as differences in the ages of the animals investigated, tissue preparation techniques and methods of quantification. Insausti et al. [36] investigated 20–28-week-old mice, whereas in the current study, all mice were at least 16 months old. Individual neuronal populations in Ts65Dn mice may be differentially affected by age. This view is supported by the fact that atrophy and progressive loss of basal forebrain cholinergic neurons is not detected in Ts65Dn mice until after 6 months of age [31]. Similarly, DS individuals are born with a normal septo-hippocampal cholinergic system [37], which degenerates with aging [7,72]. Therefore, the neuronal deficit observed in CA1 of Ts65Dn mice in the current study may be age-dependant. However, the density of CA1 pyramidal neurons in the 23month-old Ts65Dn mouse (T4) was similar to that in the 16– 17-month-old Ts65Dn mice (T1–3; Table 1). In addition, although the proliferation and migration of CA3 and CA1 pyramidal neurons end before birth [1], the neurogenesis of dentate granule cells continues during adult life [5,51]. Therefore, the reduced number of granule cells in the 20– 28-week old Ts65Dn DG reported by Insausti et al. [36] may be ameliorated by continuing granule cell proliferation in later life. The nature of the quantification may also have had a bearing on the results of the two studies. Insausti et al. [36] used the doptical dissectorT [69] to estimate the total number of neurons in fields of the Ts65Dn hippocampal formation, whereas in the current study, the unfolding method [46] was used to determine neuronal density in order to estimate synapse-to-neuron ratios as indices of neuronal connectivity in the Ts65Dn hippocampus. However, Insausti et al. [36] did not find any difference between
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Ts65Dn and diploid mice in the volumes of the DG, CA3 or CA1 and the ddissectorT and dunfoldingT method have been shown to produce similar results when applied to determine the numerical density of neurons and synapses in the cerebral cortex [6,45,46,67]. The results of the current study demonstrated a significant deficit of asymmetric synapses in the DG (32%), CA3 (15%) and CA1 (35%) of Ts65Dn mouse hippocampal formation, while a significant deficit (45%) of symmetric synapses was restricted to the Ts65Dn dentate gyrus. Previous investigations have revealed significantly fewer (30%) asymmetric synapses in the temporal cortex of Ts65Dn compared to diploid mice, whereas the density of symmetric synapses appeared to be unaffected [41]. Since asymmetric and symmetric synapses are considered to mediate excitatory and inhibitory transmission respectively [2,8,19], excitatory synaptic transmission appears to be more affected than inhibitory transmission in the central nervous system of Ts65Dn mice. This view is supported by the fact that excitatory amino acids levels are significantly lower (approx. 50%) in the parahippocampal gyrus of DS adults compared to normal subjects [55]. Since a reciprocal relationship between synapse number and post-synaptic density size has been previously reported for both rodent [29] and human brain [15,59], in the current study, the mean apposition zone lengths of asymmetric and symmetric synapses in Ts65Dn mice were compared with those in diploid mice. Despite the marked deficits observed in the densities of asymmetric synapses in the DG, CA3 and CA1 of Ts65Dn mice, a significant difference was only found in the synaptic apposition lengths of asymmetric synapses in CA1. Although the reason for the 5% greater asymmetric synaptic apposition zone lengths in the Ts65Dn compared to diploid CA1 is not clear, it might reflect an attempt to compensate for the neuronal deficit that was present in this region of Ts65Dn mouse hippocampus. The results of a number of studies suggest abnormalities in the mechanisms that govern development and maturation of neuronal circuitry in DS. Abnormalities of synaptic development are present in foetuses [52] and children [71,70] with DS and dendritic atrophy has been reported to occur in adults with the condition [64]. Decreased levels of protein markers of dendritic spines such as synaptosomal associated protein 25 [27] and drebrin [60,68] also suggest impaired synaptogenesis or neuronal loss in DS. Abnormal dendritic spine morphology has also been reported to occur in both the DG and CA1 of the Ts65Dn hippocampus [4]. More recently, the basal dendrites of layer III pyramidal cells in the Ts65Dn mouse frontal cortex of have been shown to be smaller, less branched and less spinous than those of diploid controls [18]. The current finding of a widespread synaptic deficit in the hippocampal formation of Ts65Dn mice, without a similar deficit in neuronal density, adds weight to the suggestion that Ts65Dn mice model the dendritic and synaptic abnormalities characteristic of DS.
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There is a strong relationship between synapse number and cerebral function. Rats reared in complex housing conditions show an increase in cortical synapse-to-neuron ratio [66] and motor learning leads to an increase in synapse number in the rat motor cortex [38]. In contrast, a number of studies have associated age-related memory impairment with synapse loss, particularly from the hippocampus [13,24]. Furthermore, the cognitive decline in Alzheimer’s disease correlates more closely with synapse loss than with any other neuropathological feature of the disease [14,65]. Ts65Dn mice demonstrate impaired spatial learning and memory [16,17,22,23,26,31,32,34,35,39,54] and it is possible that the synaptic deficits in the DG, CA3 and CA1 of these mice underlie at least some of this impairment, because the hippocampal formation plays an important role in spatial learning [48,47,49,50,56]. Furthermore, long-term potentiation has been shown to be significantly reduced in the hippocampus of Ts65Dn mice, whereas long-term depression is increased [40,61,62]. Since there is evidence to suggest that long-term potentiation and long-term depression are implicated in learning and memory, their abnormality in Ts65Dn mice may reflect a reduced efficacy of neuronal networks underlying these processes that contributes to the learning impairments observed in Ts65Dn mice. The results of the present study may have important implications for alleviation of the behavioural and learning deficits in Ts65Dn mice and possibly the mental retardation in DS. Any therapeutic strategy that enhances synaptogenesis or inhibits synapse loss may reasonably be expected to improve their cognitive capability. Nerve growth factor may be one possible candidate, because it has been shown to correct developmental impairments and age-related neuronal atrophy of trisomy 16 mouse basal cholinergic neurons [9,30] and NGF levels decline significantly with age in the basal forebrain and hippocampus of Ts65Dn mice [33]. However, the efficacy of this and other prospective strategies remains to be tested.
Acknowledgments The work in Uludag University was supported by ¨ BI˙TAK (The Scientific and Technical Research Council TU of Turkey, Grant no: SBAG: 2422) and Uludag University (Grant no: 2001/9). We thank to Mr. R.F. Moss for his invaluable technical assistance. Mara Dierssen was supported by the Jeroˆme Lejeune Foundation.
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