Clinical and neuropathological diversity of tauopathy in MAPT duplication carriers.

Microduplications of the 17q21.31 chromosomal region encompassing the MAPT gene, which encodes the Tau protein, were identified in patients with a progressive disorder initially characterized by severe memory impairment with or without behavioral changes that can clinically mimic Alzheimer disease. The unique neuropathological report showed a primary tauopathy, which could not be unanimously classified in a given known subtype, showing both 4R- and 3R-tau inclusions, mainly within temporal cortical subregions and basal ganglia, without amyloid deposits. Recently, two subjects harboring the same duplication were reported with an atypical extrapyramidal syndrome and gait disorder. To decipher the phenotypic spectrum associated with MAPT duplications, we studied ten carriers from nine families, including two novel unrelated probands, gathering clinical (n = 10), cerebrospinal fluid (n = 6), MRI (n = 8), dopamine transporter scan (n = 4), functional (n = 5), amyloid (n = 3) and Tau-tracer (n = 2) PET imaging data as well as neuropathological examination (n = 4). Ages at onset ranged from 37 to 57 years, with prominent episodic memory impairment in 8/10 patients, associated with behavioral changes in four, while two patients showed atypical extrapyramidal syndrome with gait disorder at presentation, including one with associated cognitive deficits. Amyloid imaging was negative but Tau imaging showed significant deposits mainly in both mesiotemporal cortex. Dopaminergic denervation was found in 4/4 patients, including three without extrapyramidal symptoms. Neuropathological examination exclusively showed Tau-immunoreactive lesions. Distribution, aspect and 4R/3R tau aggregates composition suggested a spectrum from predominantly 3R, mainly cortical deposits well correlating with cognitive and behavioral changes, to predominantly 4R deposits, mainly in the basal ganglia and midbrain, in patients with prominent extrapyramidal syndrome. Finally, we performed in vitro seeding experiments in HEK-biosensor cells. Morphological features of aggregates induced by homogenates of three MAPT duplication carriers showed dense/granular ratios graduating between those induced by homogenates of a Pick disease and a progressive supranuclear palsy cases. These results suggest that MAPT duplication causes a primary tauopathy associated with diverse clinical and neuropathological features.

Human subiculo-fornico-mamillary system in Alzheimer's disease: Tau seeding by the pillar of the fornix.

In Alzheimer's disease (AD), Tau and Aß aggregates involve sequentially connected regions, sometimes distantly separated. These alterations were studied in the pillar of the fornix (PoF), an axonal tract, to analyse the role of axons in their propagation. The PoF axons mainly originate from the subicular neurons and project to the mamillary body. Forty-seven post-mortem cases at various Braak stages (Tau) and Thal phases (Aß) were analysed by immunohistochemistry. The distribution of the lesions showed that the subiculum was affected before the mamillary body, but neither Tau aggregation nor Aß deposition was consistently first. The subiculum and the mamillary body contained Gallyas positive neurofibrillary tangles, immunolabelled by AT8, TG3, PHF1, Alz50 and C3 Tau antibodies. In the PoF, only thin and fragmented threads were observed, exclusively in the cases with neurofibrillary tangles in the subiculum. The threads were made of Gallyas negative, AT8 and TG3 positive Tau. They were intra-axonal and devoid of paired helical filaments at electron microscopy. We tested PoF homogenates containing Tau AT8 positive axons in a Tau P301S biosensor HEK cell line and found a seeding activity. There was no Aß immunoreactivity detected in the PoF. We could follow microcryodissected AT8 positive axons entering the mamillary body; contacts between Tau positive endings and Aß positive diffuse or focal deposits were observed in CLARITY-cleared mamillary body. In conclusion, we show that non-fibrillary, hyperphosphorylated Tau is transported by the axons of the PoF from the subiculum to the mamillary body and has a seeding activity. Either Tau aggregation or Aß accumulation may occur first in this system: this inconstant order is incompatible with a cause-and-effects relationship. However, both pathologies were correlated and intimately associated, indicating an interaction of the two processes, once initiated.

Lipid markers and related transcripts during excitotoxic neurodegeneration in kainate-treated mice.

Lipid homeostasis is dysregulated in some neurodegenerative diseases and after brain injuries due to excess glutamate or lack of oxygen. However the kinetics and cell specificity of dysregulation in different groups of lipids during excitotoxic neuronal death are not clear. Here we examined the changes during excitotoxic neuronal death induced by injecting kainic acid (KA) into the CA1 region of mouse hippocampus. We compared neuronal loss and glial cell proliferation with changes in lipid-related transcripts and markers for different lipid groups, over 12 days after KA-treatment. As neurons showed initial signs of damage, transcripts and proteins linked to fatty acid oxidation were up-regulated. Cholesterol biosynthesis induced by transcripts controlled by the transcription factor Srebp2 seems to be responsible for a transient increase in neuronal free cholesterol at 1 to 2 days. In microglia, but not in neurons, Perilipin-2 associated lipid droplets were induced and properties of Nile red emissions suggest lipid contents change over time. After microglial expression of phagocytotic markers at 2 days, some neutral lipid deposits co-localized with lysosome markers of microglia and were detected within putative phagocytotic cups. These data delineate distinct lipid signals in neurons and glial cells during excitotoxic processes from initial neuronal damage to engagement of the lysosome-phagosome system.

Evidence for altered dendritic spine compartmentalization in Alzheimer's disease and functional effects in a mouse model.

Alzheimer's disease (AD) is associated with a progressive loss of synapses and neurons. Studies in animal models indicate that morphological alterations of dendritic spines precede synapse loss, increasing the proportion of large and short ("stubby") spines. Whether similar alterations occur in human patients, and what their functional consequences could be, is not known. We analyzed biopsies from AD patients and APP x presenilin 1 knock-in mice that were previously shown to present a loss of pyramidal neurons in the CA1 area of the hippocampus. We observed that the proportion of stubby spines and the width of spine necks are inversely correlated with synapse density in frontal cortical biopsies from non-AD and AD patients. In mice, the reduction in the density of synapses in the stratum radiatum was preceded by an alteration of spine morphology, with a reduction of their length and an enlargement of their neck. Serial sectioning examined with electron microscopy allowed us to precisely measure spine parameters. Mathematical modeling indicated that the shortening and widening of the necks should alter the electrical compartmentalization of the spines, leading to reduced postsynaptic potentials in spine heads, but not in soma. Accordingly, there was no alteration in basal synaptic transmission, but long-term potentiation and spatial memory were impaired. These results indicate that an alteration of spine morphology could be involved in the early cognitive deficits associated with AD.

Alzheimer's senile plaque as shown by microcryodissection, a new technique for dissociating tissue structures.

Extracellular accumulation of Aß peptides and intracellular aggregation of hyperphosphorylated tau proteins are the two hallmark lesions of Alzheimer disease (AD). The senile plaque is made of a core of extracellular Aß surrounded by phospho-tau positive neurites. It includes multiple components such as axons, synapses, glial fibers and microglia. To visualize the relationships of those elements, an original technique was developed, based on the dilation of interstitial water during freezing. Samples of neocortex, hippocampus and striatum were taken from formalin-fixed brains (one control case; three cases with severe Alzheimer disease). The samples were subjected to various numbers of freezing/thawing cycles (from 0 to 320) with an automated system we devised. The samples were embedded in paraffin, cut and stained with haematoxylin-eosin or immunostained against Aß, phospho-tau, and antigens enriched in axons, synapses, macrophages or astrocytes. Microcryodissection induced the dissociation of tissue components, especially in the grey matter where the neuropil formed an oriented "mesh". The size of the empty spaces separating the fiber bundles and cells increased with the number of cycles. The amyloid core of the senile plaque separated from its neuritic crown at around 300 freezing/thawing cycles. The dissected core remained associated with macrophages containing Aß in their cytoplasm. Phospho-tau positive axons were distinctly seen projecting from the neuritic crown to the isolated amyloid core, where they ended in large synapses. The microcryodissection showed astrocytic processes stuck directly to the core. The original method we developed-microcryodissection-helped understanding how histological components were assembled in the tissue.

Mature Purkinje cells require the retinoic acid-related orphan receptor-α (RORα) to maintain climbing fiber mono-innervation and other adult characteristics.

Neuronal maturation during development is a multistep process regulated by transcription factors. The transcription factor RORα (retinoic acid-related orphan receptor α) is necessary for early Purkinje cell (PC) maturation but is also expressed throughout adulthood. To identify the role of RORα in mature PCs, we used Cre-lox mouse genetic tools in vivo that delete it specifically from PCs between postnatal days 10-21. Up to 14 d of age, differences between mutant and control PCs were not detectable: both were mono-innervated by climbing fibers (CFs) extending along their well-developed dendrites with spiny branchlets. By week 4, mutant mice were ataxic, some PCs had died, and remaining PC soma and dendrites were atrophic, with almost complete disappearance of spiny branchlets. The innervation pattern of surviving RORα-deleted PCs was abnormal with several immature characteristics. Notably, multiple functional CF innervation was reestablished on these mature PCs, simultaneously with the relocation of CF contacts to the PC soma and their stem dendrite. This morphological modification of CF contacts could be induced even later, using lentivirus-mediated depletion of rora from adult PCs. These data show that the late postnatal expression of RORα cell-autonomously regulates the maintenance of PC dendritic complexity, and the CF innervation status of the PC (dendritic vs somatic contacts, and mono-innervation vs multi-innervation). Thus, the differentiation state of adult neurons is under the control of transcription factors; and in their absence, adult neurons lose their mature characteristics and acquire some characteristics of an earlier developmental stage.

Adaptive and non-adaptive changes in activity-deprived presynaptic terminals.

How the number of docked vesicles is regulated is still unclear. Following chronic activity blockade the number of docked vesicles increases, providing a model through which to address this issue. We tested the hypotheses that the number of docked vesicles is regulated with the size of the terminal, and by the level of Rab3-interacting molecule 1/2 (RIM1/2). We immobilized mouse hippocampal slice cultures by high-pressure freezing after 3 days of tetrodotoxin treatment and analysed them by electron microscopy. The number of docked vesicles, the size of the active zones and the amount of GluA2 were increased after activity blockade. However, there was no modification of either the total number of synaptic vesicles or the area of presynaptic profiles. Surprisingly, immunocytochemistry showed no change in the mean level of RIM1/2 per terminal but its distribution was modified. Additionally, there was no modification of the mean frequency or amplitude of miniature excitatory postsynaptic currents, but the distribution of amplitudes was modified. These results indicate a specific homeostatic regulation of the synaptic junction. The number of docked vesicles does not seem to be regulated by the amount of RIM1/2. The modification of the distribution, but not the amount, of RIM1/2 may explain the contradiction between the morphological and electrophysiological findings.

A role for vesicular glutamate transporter 1 in synaptic vesicle clustering and mobility.

Synaptic vesicles (SVs) from excitatory synapses carry vesicular glutamate transporters (VGLUTs) that fill the vesicles with neurotransmitter. Although the essential function of VGLUTs as glutamate transporters has been well established, the evidence for additional cell-biological functions is more controversial. Both VGLUT1 and VGLUT2 disruptions in mice result in a reduced number of SVs away from release sites, flattening of SVs, and the appearance of tubular structures. Therefore, we analysed the morphology, biochemical composition and trafficking of SVs at synapses of VGLUT1(-/-) mice in order to test for a function of VGLUTs in the formation or clustering of SVs. Analyses with high-pressure freezing immobilisation and electron tomography pointed to a role of VGLUT1 transport function in the tonicity of excitatory SVs, explaining the aldehyde-induced flattening of SVs observed in VGLUT1(-/-) synapses. We confirmed the steep reduction in the number of SVs previously observed in VGLUT1(-/-) presynaptic terminals, but did not observe accumulation of endocytotic intermediates. Furthermore, SV proteins of adult VGLUT1(-/-) mouse brain tissue were expressed at normal levels in all subcellular fractions, suggesting that they were not displaced to another organelle. We thus assessed the mobility of the recently documented superpool of SVs. Synaptobrevin2-enhanced green fluorescent protein time lapse experiments revealed an oversized superpool of SVs in VGLUT1(-/-) neurons. Our results support the idea that, beyond glutamate loading, VGLUT1 enhances the tonicity of excitatory SVs and stabilises SVs at presynaptic terminals.

Golgi localization of SARS-CoV-2 spike protein and interaction with furin in cerebral COVID-19 microangiopathy: a clue to the central nervous system involvement?

In a neuropathological series of 20 COVID-19 cases, we analyzed six cases (three biopsies and three autopsies) with multiple foci predominantly affecting the white matter as shown by MRI. The cases presented with microhemorrhages evocative of small artery diseases. This COVID-19 associated cerebral microangiopathy (CCM) was characterized by perivascular changes: arterioles were surrounded by vacuolized tissue, clustered macrophages, large axonal swellings and a crown arrangement of aquaporin-4 immunoreactivity. There was evidence of blood-brain-barrier leakage. Fibrinoid necrosis, vascular occlusion, perivascular cuffing and demyelination were absent. While no viral particle or viral RNA was found in the brain, the SARS-CoV-2 spike protein was detected in the Golgi apparatus of brain endothelial cells where it closely associated with furin, a host protease known to play a key role in virus replication. Endothelial cells in culture were not permissive to SARS-CoV-2 replication. The distribution of the spike protein in brain endothelial cells differed from that observed in pneumocytes. In the latter, the diffuse cytoplasmic labeling suggested a complete replication cycle with viral release, notably through the lysosomal pathway. In contrast, in cerebral endothelial cells the excretion cycle was blocked in the Golgi apparatus. Interruption of the excretion cycle could explain the difficulty of SARS-CoV-2 to infect endothelial cells in vitro and to produce viral RNA in the brain. Specific metabolism of the virus in brain endothelial cells could weaken the cell walls and eventually lead to the characteristic lesions of COVID-19 associated cerebral microangiopathy. Furin as a modulator of vascular permeability could provide some clues for the control of late effects of microangiopathy.

Alterations of Neuronal Lysosomes in Alzheimer's Disease and in APPxPS1-KI Mice.

BACKGROUND: The cellular and molecular alterations associated with synapse and neuron loss in Alzheimer's disease (AD) remain unclear. In transgenic mouse models that express mutations responsible for familial AD, neuronal and synaptic losses occur in populations that accumulate fibrillar amyloid-ß 42 (Aß42) intracellularly. OBJECTIVE: We aimed to study the subcellular localization of these fibrillar accumulations and whether such intraneuronal assemblies could be observed in the human pathology. METHODS: We used immunolabeling and various electron microscopy techniques on APP x presenilin1 - knock-in mice and on human cortical biopsies and postmortem samples. RESULTS: We found an accumulation of Aß fibrils in lipofuscin granule-like organelles in APP x presenilin1 - knock-in mice. Electron microscopy of human cortical biopsies also showed an accumulation of undigested material in enlarged lipofuscin granules in neurons from AD compared to age-matched non-AD patients. However, in those biopsies or in postmortem samples we could not detect intraneuronal accumulations of Aß fibrils, neither in the lipofuscin granules nor in other intraneuronal compartments. CONCLUSION: The intralysosomal accumulation of Aß fibrils in specific neuronal populations in APPxPS1-KI mice likely results from a high concentration of Aß42 in the endosome-lysosome system due to the high expression of the transgene in these neurons.

An emerging view of presynaptic structure from electron microscopic studies.

In response to calcium influx, some of the synaptic vesicles in presynaptic terminals fuse rapidly with the presynaptic membrane, allowing fast synaptic transmission. The regulated recycling of synaptic vesicles at the terminals is required for a sustained release of neurotransmitters. Localization of 'ready to be released' vesicles in close vicinities to voltage-gated calcium channels enables the rapid release of neurotransmitters. Thus, recycling vesicles must translocate from the sites of endocytosis to these release sites. However, the sub-cellular organization that supports this local vesicular traffic remains poorly understood. We will review the results of various electron microscopy studies, which have begun to unveil the structure of presynaptic terminals.

A common molecular basis for membrane docking and functional priming of synaptic vesicles.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes execute synaptic vesicle (SV) fusion. Vesicle fusion is preceded by an obligatory Munc13-dependent priming process that conveys fusion competence to SVs by facilitating SNARE complex assembly. Ultrastructural studies after chemical fixation indicated that vesicle docking to the plasma membrane is independent of Munc13s but these results may be misleading because aldehyde fixatives modify the localization of SVs with respect to the plasma membrane. To reinvestigate the role of Munc13s in vesicle docking, cultured hippocampal slices were immobilized using high-pressure freezing, which circumvents aldehyde artifacts. High-pressure freezing was combined with electron tomography to reach a resolution that allows the characterization of details of SV docking in a close-to-native state. In control slices, docked vesicles are not hemifused with the plasma membrane but linked to it and to dense material at the active zone by small strands. In slice cultures from Munc13-deficient mice, vesicles are not docked to the active zone plasma membrane. These results indicate that SV docking at the plasma membrane and functional priming are respective morphological and physiological manifestations of the same molecular process mediated by SNARE complexes and Munc13s.

Sparse force-bearing bridges between neighboring synaptic vesicles.

Most vesicles in the interior of synaptic terminals are clustered in clouds close to active zone regions of the plasma membrane where exocytosis occurs. Electron-dense structures, termed bridges, have been reported between a small minority of pairs of neighboring vesicles within the clouds. Synapsin proteins have been implicated previously, but the existence of the bridges as stable structures in vivo has been questioned. Here we use electron tomography to show that the bridges are present but less frequent in synapsin knockouts compared to wildtype. An analysis of distances between neighbors in wildtype tomograms indicated that the bridges are strong enough to resist centrifugal forces likely induced by fixation with aldehydes. The results confirm that the bridges are stable structures and that synapsin proteins are involved in formation or stabilization.

Three-dimensional architecture of presynaptic terminal cytomatrix.

Presynaptic terminals are specialized for mediating rapid fusion of synaptic vesicles (SVs) after calcium influx. The regulated trafficking of SVs likely results from a highly organized cytomatrix. How this cytomatrix links SVs, maintains them near the active zones (AZs) of release, and organizes docked SVs at the release sites is not fully understood. To analyze the three-dimensional (3D) architecture of the presynaptic cytomatrix, electron tomography of presynaptic terminals contacting spines was performed in the stratum radiatum of the rat hippocampal CA1 area. To preserve the cytomatrix, hippocampal slices were immobilized using high-pressure freezing, followed by cryosubstitution and embedding. SVs are surrounded by a dense network of filaments. A given vesicle is connected to approximately 1.5 neighboring ones. SVs at the periphery of this network are also linked to the plasma membrane, by longer filaments. More of these filaments are found at the AZ. At the AZ, docked SVs are grouped around presynaptic densities. Filaments with adjacent SVs emerge from these densities. Immunogold localizations revealed that synapsin is located in the presynaptic bouton, whereas Bassoon and CAST (ERC2) are at focal points next to the AZ. In synapsin triple knock-out mice, the number of SVs is reduced by 63%, but the size of the boutons is reduced by only 18%, and the mean distance of SVs to the AZ is unchanged. This 3D analysis reveals the morphological constraints exerted by the presynaptic molecular scaffold. SVs are tightly interconnected in the axonal bouton, and this network is preferentially connected to the AZ.

Magnetic resonance imaging and histological studies of corpus callosal and hippocampal abnormalities linked to doublecortin deficiency.

Mutated doublecortin (DCX) gives rise to severe abnormalities in human cortical development. Adult Dcx knockout mice show no major neocortical defects but do have a disorganized hippocampus. We report here the developmental basis of these hippocampal abnormalities. A heterotopic band of neurons was identified starting at E17.5 in the CA3 region and progressing throughout the CA1 region by E18.5. At neonatal stages, the CA1 heterotopic band was reduced, but the CA3 band remained unchanged, continuing into adulthood. Thus, in mouse, migration of CA3 neurons is arrested during development, whereas CA1 cell migration is retarded. On the Sv129Pas background, magnetic resonance imaging (MRI) also suggested abnormal dorsal hippocampal morphology, displaced laterally and sometimes rostrally and associated with medial brain structure abnormalities. MRI and cryosectioning showed agenesis of the corpus callosum in Dcx knockout mice on this background and an intermediate, partial agenesis in heterozygote mice. Wild-type littermates showed no callosal abnormalities. Hippocampal and corpus callosal abnormalities were also characterized in DCX-mutated human patients. Severe hippocampal hypoplasia was identified along with variable corpus callosal defects ranging from total agenesis to an abnormally thick or thin callosum. Our data in the mouse, identifying roles for Dcx in hippocampal and corpus callosal development, might suggest intrinsic roles for human DCX in the development of these structures.

Ultrastructural organization of presynaptic terminals.

In response to calcium influx, synaptic vesicles fuse very rapidly with the plasma membrane to release their neurotransmitter content. An important mechanism for sustained release includes the formation of new vesicles by local endocytosis. How synaptic vesicles are trafficked from the sites of endocytosis to the sites of release and how they are maintained at the release sites remain poorly understood. Recent studies using fast freezing immobilization and electron tomography have led to insights on the ultrastructural organization of presynaptic boutons and how these structural elements may maintain synaptic vesicles and organize their exocytosis at particular areas of the plasma membrane.

The development of hippocampal interneurons in rodents.

Interneurons are GABAergic neurons responsible for inhibitory activity in the adult hippocampus, thereby controlling the activity of principal excitatory cells through the activation of postsynaptic GABAA receptors. Subgroups of GABAergic neurons innervate specific parts of excitatory neurons. This specificity indicates that particular interneuron subgroups are able to recognize molecules segregated on the membrane of the pyramidal neuron. Once these specific connections are established, a quantitative regulation of their strength must be performed to achieve the proper balance of excitation and inhibition. We will review when and where interneurons are generated. We will then detail their migration toward and within the hippocampus, and the maturation of their morphological and neurochemical characteristics. We will finally review potential mechanisms underlying the development of GABAergic interneurons.

Analysis of synaptic ultrastructure without fixative using high-pressure freezing and tomography.

Electron microscopy allows the analysis of synaptic ultrastructure and its modifications during learning or in pathological conditions. However, conventional electron microscopy uses aldehyde fixatives that alter the morphology of the synapse by changing osmolarity and collapsing its molecular components. We have used high-pressure freezing (HPF) to capture within a few milliseconds structural features without aldehyde fixative, and thus to provide a snapshot of living synapses. CA1 hippocampal area slices from P21 rats were frozen at -173 degrees C under high pressure to reduce crystal formation, and synapses on dendritic spines were analysed after cryosubstitution and embedding. Synaptic terminals were larger than after aldehyde fixation, and synaptic vesicles in these terminals were less densely packed. Small filaments linked the vesicles in subgroups. The postsynaptic densities (PSDs) exhibited filamentous projections extending into the spine cytoplasm. Tomographic analysis showed that these projections were connected with the spine cytoskeletal meshwork. Using immunocytochemistry, we found as expected GluR1 at the synaptic cleft and CaMKII in the PSD. Actin immunoreactivity (IR) labelled the cytoskeletal meshwork beneath the filamentous projections, but was very scarce within the PSD itself. ProSAP2/Shank3, cortactin and Ena/VASP-IRs were concentrated on the cytoplasmic face of the PSD, at the level of the PSD projections. Synaptic ultrastructure after HPF was different from that observed after aldehyde fixative. The boutons were larger, and filamentous components were preserved. Particularly, filamentous projections were observed linking the PSD to the actin cytoskeleton. Thus, synaptic ultrastructure can be analysed under more realistic conditions following HPF.

Quantitative effects produced by modifications of neuronal activity on the size of GABAA receptor clusters in hippocampal slice cultures.

The number and strength of GABAergic synapses needs to be precisely adjusted for adequate control of excitatory activity. We investigated to what extent the size of GABA(A) receptor clusters at inhibitory synapses is under the regulation of neuronal activity. Slices from P7 rat hippocampus were cultured for 13 days in the presence of bicuculline or 4-aminopyridine (4-AP) to increase neuronal activity, or DNQX to decrease activity. The changes provoked by these treatments on clusters immunoreactive for the alpha1 and alpha2 subunits of the GABA(A) receptor or gephyrin were quantitatively evaluated. While an increase in activity augmented the density of these clusters, a decrease in activity provoked, in contrast, a decrease in their density. An inverse regulation was observed for the size of individual clusters. Bicuculline and 4-AP decreased whilst DNQX increased the mean size of the clusters. When the pharmacological treatments were applied for 2 days instead of 2 weeks, no effects on the size of the clusters were observed. The variations in the mean size of individual clusters were mainly due to changes in the number of small clusters. Finally, a regulation of the size of GABA(A) receptor clusters occurred during development in vivo, with a decrease of the mean size of the clusters between P7 and P21. This physiological change was also the result of an increase in the number of small clusters. These results indicate that neuronal activity regulates the mean size of GABA(A) receptor- and gephyrin-immunoreactive clusters by modifying specifically the number of synapses with small clusters of receptors.