Prion diseases are characteristically accompanied by extensive synaptic pathology that can occur during the preclinical phase of the disease and, in animal models, correlates with the first decline of hippocampus-dependent cognitive functions. This pathology is defined by abnormally shaped synapses in which the postsynaptic membrane modifies its curvature and potentially engulfs the juxtaposed presynaptic membrane. Using the intrahippocampally injected ME7 prion model, we further detailed the structural alterations of the population of ostensibly intact synaptic compartments within the hippocampus during this period of extensive synaptic loss. A disease stage-dependent increase in the average PSD (postsynaptic density) area, the average length of the active zone and the average number of synaptic vesicles indicated that the synapses that were visualized as the animal progressed to end-stage disease were undergoing hypertrophy. Similar findings in samples from AD (Alzheimer's disease) patients, aged and senile individuals, and animal models of neurodegenerative diseases suggest synaptic swelling as synaptic loss is initiated and/or compensatory reaction to counteract the synaptic loss.

Introduction

Synaptic junctions play a pivotal role in communication and information processing, a fundamental prerequisite for neuronal circuit formation and maintenance. The vast majority of excitatory synapses within the CNS (central nervous system) are formed on dendritic spines [1], dynamic structures that maintain the potential for significant adaptive changes even in the fully differentiated adult CNS undergoing environmental, age- or disease-related responses [24]. Synaptic failure is an early correlate of the age-related cognitive decline [2] and a well-documented element in the initial phase of various neurodegenerative diseases such as AD (Alzheimer's disease) or prion diseases [57]. Enlargement of synaptic size as a compensatory response to synaptic loss in aging, dementia and AD is a well-documented phenomenon [24].

Prion diseases are a group of neurodegenerative disorders that are characterized by the presence of an aggregation-prone form {PrPSc [infectious form of PrPC (cellular prion protein)} of the normal PrPC, vacuolization in the grey matter and eventual neuronal death [8]. The timing and extent of synaptic loss has been described in the ME7 model [6,7], and some of the associated biochemical and morphological changes have been documented [7,9]. In particular, increased curvature of morphologically defined PSD (postsynaptic density) and the ensuing engulfment of the juxtaposed presynaptic specialization appear as a progressively increased feature of hippocampal synapses during prion disease. These profiles are identified in fields displaying apparently normal synaptic structures. Detailing membrane architecture across synaptic profiles should help refine understanding of the mechanisms that operate during synaptic loss.

We used morphometric analysis and demonstrated that there are structural changes in the apparently intact synapses found in diseased animals, indicating that they are significantly larger than their age-matched control. These observations are suggestive of structural changes that are a prelude to synapse loss or possibly a homoeostatic compensation.

Sample preparation

Normal C57 mouse brain (NBH) or ME7 prion agent-infected brain homogenate was injected slowly into 11–12-week-old C57BL/6J female mice and the mice were allowed to recover. All animal experimentation was conducted according to UK Home Office Licence, as described previously [7]. At 10, 12, 16 and 18 weeks p.i. (post-injection), mice were killed and prepared by perfusion fixation for TEM (transmission electron microscopy). Brains were then dissected and post-fixed in fresh fixative before cutting coronal sections and dissecting out the area of CA1 pyramidal layer and stratum radiatum. Tissue was mounted and trimmed to a trapezoid containing CA1 pyramidal cell bodies and dendritic arbour of stratum radiatum. Sections were stained, viewed by TEM and processed using Adobe Photoshop.

TEM of stratum radiatum synapses

Synapse identification

Synaptic profiles were analysed on the basis of ultrastructural parameters and classified as type I (asymmetric) or type II (symmetric) synapses [10]. Synapses with cross-, obliquely or tangentially sectioned prominent asymmetric PSD were considered as type I if the PSD and cleft material were present in the reference section and if synaptic vesicles were present in the reference or immediately adjacent sections. Intact presynaptic terminals (containing visible synaptic vesicles) with a postsynaptic terminal in typical membrane apposition were included in the study.

Measurements of PSD area and active zone length

Measurements were performed on asymmetric synapses at 10, 12, 16 and 18 weeks p.i. at ×20000 magnification. The area of a PSD was measured only when synaptic vesicles were present in the same section and typical membrane apposition between presynaptic and postsynaptic elements was present. At least 300 PSDs and active zones of 300 presynaptic terminals were measured for each time point from three different sections at least ten sections apart for both NBH and ME7 animals (minimum n=100 for each animal at each time point for each experimental group). The measurements were included only when synaptic terminal profiles were clearly visible. Measurements of PSD areas were performed using outline spline function (by encircling and determining the area of individual objects) within the Axiovision (Carl Zeiss) and its equivalent within ImageJ software (NIH; http://rsb.info.nih.gov/ij/download.html). The active zone length was measured using the freehand line function within the software as indicated above. The presynaptic membrane was deemed part of the active zone if there were vesicles in the proximity of 50 nm of the membrane or fusing with the membrane.

Vesicle number and integrity

The integrity of the vesicles in the presynaptic boutons of NBH and ME7 animals was evaluated in the following manner. Vesicles of normal size (~40 nm in diameter) and round, regular morphology were assigned a value of 3. Those synapses that consisted of a mixture of irregular shaped/pleiomorphic or swollen vesicles and normal-appearing vesicles were assigned a value of 2, and those synapses that were predominantly or completely made up of irregular vesicles both in size and shape were assigned a value of 1.

Demarcation of areas for quantification of synaptic vesicles in the presynaptic bouton was performed as described previously [11]. Briefly, zones 1 and 2 are each approximately the width of one vesicle of normal size (40 nm) and positioned such that zone 1 is immediately adjacent to the presynaptic terminal membrane, and zone 2 adjacent to zone 1. Zones 1 and 2 combined together contain rows of presynaptic vesicles that are physiologically termed the readily releasable pool. Zone 3, in the present study, contains the remaining vesicles further away from the presynaptic membrane (reserve pool) than zones 1 and 2. The total number of vesicles per synapse has been expressed as the sum of the vesicles in all three zones.

Statistical analysis

The identities of the coded blocks were revealed to the observer only after the data analysis was complete. Raw data from every block were collated using Microsoft Excel and two-tailed, unpaired Student's t tests (PSD area) or two-way ANOVA tests were performed to compare final data between NBH (n=3) and ME7 animals (n=3) at each time point using GraphPad Prism software.

Results and discussion

A morphometric analysis of NBH and ME7 animals revealed that during progressive, marked synaptic loss, where we have described accumulation of degenerating synaptic profiles [7], the population of the remaining, apparently normal synapses exhibits further structural changes in ME7 animals. At 10 weeks, before any measurable change in synaptic density, there were no detectable changes in the average area of PSDs. However, at 12 weeks p.i., the average PSD area was significantly smaller in ME7 animals relative to NBH animals. From 12 weeks onwards the remaining PSDs gradually increased in size and the mean area in ME7 animals was significantly bigger at 18 weeks compared with NBH animals [7] (Figure 1A). The area of the PSD is known to correlate with synaptic size and with the volume of the associated dendritic spine [12,13]; therefore we aimed to investigate whether other synaptic components were modified.

Quantification of the structural changes in type I synaptic compartments across different stages of disease progression

Figure 1
Quantification of the structural changes in type I synaptic compartments across different stages of disease progression

The areas of PSDs (A) and the active zone length (B) from either NBH animals (n=3) or ME7 animals (n=3) show differential changes at different time points p.i. The results of PSD area are means±S.E.M.; two-tailed, unpaired Student's t tests (*P<0.05, **P<0.01) were performed between individual p.i. time points of either NBH or ME7 homogenate; the final data are expressed from measurements of NBH animals set to 100. Note the appearance of groups of synapses with a longer active zone from 16 weeks onwards in ME7 animals (B) [two-way ANOVA with Bonferroni post-tests (**P<0.01)].

Figure 1
Quantification of the structural changes in type I synaptic compartments across different stages of disease progression

The areas of PSDs (A) and the active zone length (B) from either NBH animals (n=3) or ME7 animals (n=3) show differential changes at different time points p.i. The results of PSD area are means±S.E.M.; two-tailed, unpaired Student's t tests (*P<0.05, **P<0.01) were performed between individual p.i. time points of either NBH or ME7 homogenate; the final data are expressed from measurements of NBH animals set to 100. Note the appearance of groups of synapses with a longer active zone from 16 weeks onwards in ME7 animals (B) [two-way ANOVA with Bonferroni post-tests (**P<0.01)].

The active zone exhibited similar changes and in ME7 animals its average length was increased both 16 and 18 weeks p.i. (Figure 1B). Consistent with these findings the number of presynaptic vesicles present in the readily releasable pool (zones 1 and 2) and the reserve pool (zone 3) were also increased at 18 weeks. A trend (P=0.0614) detected in all the three zones corroborates the idea that enlarged presynaptic terminals in surviving synapses at the late disease stage contain on average more synaptic vesicles (Figures 2A–2D). Similar but converse changes were observed at 12 weeks p.i.; a significantly smaller PSD area [7] (Figure 1A) corresponded to a smaller number of synaptic vesicles (Figures 2A–2D).

Quantification of presynaptic vesicles within type I synapses across different stages of disease progression

Figure 2
Quantification of presynaptic vesicles within type I synapses across different stages of disease progression

Total pool of presynaptic vesicles (A) and vesicles in the demarcated zone 1 (B), zone 2 (C) and zone 3 (D) from either NBH animals (n=3) or ME7 animals (n=3) show differential changes at different time points p.i. The results are means±S.E.M.; two-way ANOVA with Bonferroni post-tests was performed to compare the final data. A trend of increase in the number of presynaptic vesicles was detected at late disease stage in ME7 animals.

Figure 2
Quantification of presynaptic vesicles within type I synapses across different stages of disease progression

Total pool of presynaptic vesicles (A) and vesicles in the demarcated zone 1 (B), zone 2 (C) and zone 3 (D) from either NBH animals (n=3) or ME7 animals (n=3) show differential changes at different time points p.i. The results are means±S.E.M.; two-way ANOVA with Bonferroni post-tests was performed to compare the final data. A trend of increase in the number of presynaptic vesicles was detected at late disease stage in ME7 animals.

In addition to the changes in synaptic size, a progressive decline in presynaptic vesicle integrity was also detected by us in ME7 animals. In NBH animals, the vast majority of the synaptic terminals in the stratum radiatum contained relatively small (<40 nm in diameter), regular, round vesicles [14]. As disease progressed we detected increasing numbers of synaptic terminals with seemingly intact presynaptic membrane and normal electron density, but they contained abnormal synaptic vesicles and intraterminal membrane structures. These changes included swollen or enlarged vesicles, and small synaptic-like vesicles of irregular shape were frequently observed (Figure 3A). These qualitative observations were quantified by classifying boutons as containing vesicles with normal morphology (3), predominantly abnormal morphology (1) or mixed morphology (2). At least 100 presynaptic boutons from each of NBH animals and ME7 animals were characterized. At 16 and 18 weeks of survival, the vesicle integrity of the remaining boutons was impaired relative to those seen in NBH animals (Figure 3B).

Quantification of presynaptic vesicle integrity across the different stages of disease progression

Figure 3
Quantification of presynaptic vesicle integrity across the different stages of disease progression

(A) Electron micrographs illustrating the morphology of presynaptic vesicles in NBH and ME7 animals at 18 weeks p.i. In ME7 animals (ME7 LS: late stage at 18 weeks p.i.), some presynaptic terminals are shrunken and filled with electron-dense cytoplasm, while others nearby appear normal; however, abnormally shaped and enlarged presynaptic vesicles are visible (arrows). Scale bars, 1 μm. (B) Quantification of structural changes of presynaptic vesicles across different stages of disease progression. The results are means±S.E.M.; two-way ANOVA with Bonferroni post-tests (**P<0.01, ***P<0.001) was used to compare the final data. Note the progressive decline in vesicle integrity in ME7 animals.

Figure 3
Quantification of presynaptic vesicle integrity across the different stages of disease progression

(A) Electron micrographs illustrating the morphology of presynaptic vesicles in NBH and ME7 animals at 18 weeks p.i. In ME7 animals (ME7 LS: late stage at 18 weeks p.i.), some presynaptic terminals are shrunken and filled with electron-dense cytoplasm, while others nearby appear normal; however, abnormally shaped and enlarged presynaptic vesicles are visible (arrows). Scale bars, 1 μm. (B) Quantification of structural changes of presynaptic vesicles across different stages of disease progression. The results are means±S.E.M.; two-way ANOVA with Bonferroni post-tests (**P<0.01, ***P<0.001) was used to compare the final data. Note the progressive decline in vesicle integrity in ME7 animals.

Our findings provide the first documented evidence for a reactive synaptic response during the prion disease progression, similar to the hypertrophy previously reported in aging or AD [24]. The detected increase in average synaptic parameters probably occurs due to a subpopulation of enlarged synaptic contacts representing either the junctions at the very start of their degeneration or a compensatory mechanism that has been previously suggested to counteract the reduction of synaptic number [4].

Synaptopathies: Dysfunction of Synaptic Function: A Biochemical Society Focused Meeting held at The Hotel Victoria, Newquay, U.K., 2–4 September 2009. Organized and Edited by Nils Brose (Max Planck Institute for Experimental Medicine, Göttingen, Germany), Vincent O'Connor (Southampton, U.K.) and Paul Skehel (Centre For Integrative Physiology, Edinburgh, U.K.)

Abbreviations

     
  • AD

    Alzheimer's disease

  •  
  • CNS

    central nervous system

  •  
  • p.i.

    post-injection

  •  
  • PrPC

    cellular prion protein

  •  
  • PSD

    postsynaptic density

  •  
  • TEM

    transmission electron microscopy

Funding

This work is funded by the Medical Research Council [grant number G0501636].

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