The cellular basis of increased PET hypoxia tracer uptake in focal cerebral ischemia with comparison between [18F]FMISO and [64Cu]CuATSM.

PET hypoxia imaging can assess tissue viability in acute ischemic stroke (AIS). [18F]FMISO is an established tracer but requires substantial accumulation time, limiting its use in hyperacute AIS. [64Cu]CuATSM requires less accumulation time and has shown promise as a hypoxia tracer. We compared these tracers in a M2-occlusion model (M2CAO) with preserved collateral blood flow. Rats underwent M2CAO and [18F]FMISO (n = 12) or [64Cu]CuATSM (n = 6) examinations. [64Cu]CuATSM animals were also examined with MRI. Pimonidazole was used as a surrogate for [18F]FMISO in an immunofluorescence analysis employed to profile levels of hypoxia in neurons (NeuN) and astrocytes (GFAP). There was increased [18F]FMISO uptake in the M2CAO cortex. No increase in [64Cu]CuATSM activity was found. The pimonidazole intensity of neurons and astrocytes was increased in hypoxic regions. The pimonidazole intensity ratio was higher in neurons than in astrocytes. In the majority of animals, immunofluorescence revealed a loss of astrocytes within the core of regions with increased pimonidazole uptake. We conclude that [18F]FMISO is superior to [64Cu]CuATSM in detecting hypoxia in AIS, consistent with an earlier study. [18F]FMISO may provide efficient diagnostic imaging beyond the hyperacute phase. Results do not provide encouragement for the use of [64Cu]CuATSM in experimental AIS.

Selective Visualization of Administrated Arachidonic and Docosahexaenoic Acids in Brain Using Combination of Simple Stable Isotope-Labeling Technique and Imaging Mass Spectrometry.

We developed a new method for monitoring the distribution of administrated fatty acids in the body by combination of a stable isotope-labeling technique and imaging mass spectrometry (IMS). The developed stable isotope-labeling technique is very simple and able to adapt to all the fatty acid species. In this study, we synthesized stable isotope-labeled arachidonic acid (AA) and docosahexaenoic acid (DHA), and they were simultaneously administrated to mice to examine their migrations and distributions in the brain. The administrated AA and DHA have two more molecular weights compared to the originals and apparently were distinguished from the originally accumulated AA and DHA in the brain using IMS. As a result, we reveal that the administered AA and DHA first accumulated in the hippocampus and cerebellar cortex in the brain. This technique does not use radio isotopes and would appear to elucidate the role of all kinds of fatty acid species in the body.

Prolidase enzyme is required for extracellular matrix integrity and impacts on postnatal cerebellar cortex development.

The extracellular matrix is essential for brain development, lamination, and synaptogenesis. In particular, the basement membrane below the pial meninx (pBM) is required for correct cortical development. The last step in the catabolism of the most abundant protein in pBM, collagen Type IV, requires prolidase, an exopeptidase cleaving the imidodipeptides containing pro or hyp at the C-terminal end. Mutations impairing prolidase activity lead in humans to the rare disease prolidase deficiency characterized by severe skin ulcers and mental impairment. Thus, the dark-like (dal) mouse, in which the prolidase is knocked-out, was used to investigate whether the deficiency of prolidase affects the neuronal maturation during development of a brain cortex area. Focusing on the cerebellar cortex, thinner collagen fibers and disorganized pBM were found. Aberrant cortical granule cell proliferation and migration occurred, associated to defects in brain lamination, and in particular in maturation of Purkinje neurons and formation of synaptic contacts. This study deeply elucidates a link between prolidase activity and neuronal maturation shedding new light on the molecular basis of functional aspects in the prolidase deficiency.

Multizonal Cerebellar Influence Over Sensorimotor Areas of the Rat Cerebral Cortex.

The cerebral cortex requires cerebellar input for optimizing sensorimotor processing. However, how the sensorimotor cortex uses cerebellar information is far from understood. One critical and unanswered question is how cerebellar functional entities (zones or modules) are connected to distinct parts of the sensorimotor cortices. Here, we utilized retrograde transneuronal infection of rabies virus (RABV) to study the organization of connections from the cerebellar cortex to M1, M2, and S1 of the rat cerebral cortex. RABV was co-injected with cholera toxin ß-subunit (CTb) into each of these cortical regions and a survival time of 66-70 h allowed for third-order retrograde RABV infection of Purkinje cells. CTb served to identify the injection site. RABV+ Purkinje cells throughout cerebellar zones were identified by reference to the cerebellar zebrin pattern. All injections, including those into S1, resulted in multiple, zonally arranged, strips of RABV+ Purkinje cells. M1 injections were characterized by input from Purkinje cells in the vermal X-zone, medial paravermis (C1- and Cx-zones), and lateral hemisphere (D2-zone); M2 receives input from D2- and C3-zones; connections to S1 originate from X-, Cx-, C3-, and D2-zones. We hypothesize that individual domains of the sensorimotor cortex require information from a specific combination of cerebellar modules.

Neuroprotection by Heat Shock Factor-1 (HSF1) and Trimerization-Deficient Mutant Identifies Novel Alterations in Gene Expression.

Heat shock factor-1 (HSF1) protects neurons from death caused by the accumulation of misfolded proteins by stimulating the transcription of genes encoding heat shock proteins (HSPs). This stimulatory action depends on the association of trimeric HSF1 to sequences within HSP gene promoters. However, we recently described that HSF-AB, a mutant form of HSF1 that is incapable of either homo-trimerization, association with HSP gene promoters, or stimulation of HSP expression, protects neurons just as efficiently as wild-type HSF1 suggesting an alternative neuroprotective mechanism that is activated by HSF1. To gain insight into the mechanism by which HSF1 and HSF1-AB protect neurons, we used RNA-Seq technology to identify transcriptional alterations induced by these proteins in either healthy cerebellar granule neurons (CGNs) or neurons primed to die. When HSF1 was ectopically-expressed in healthy neurons, 1,211 differentially expressed genes (DEGs) were identified with 1,075 being upregulated. When HSF1 was expressed in neurons primed to die, 393 genes were upregulated and 32 genes were downregulated. In sharp contrast, HSF1-AB altered expression of 13 genes in healthy neurons and only 6 genes in neurons under apoptotic conditions, suggesting that the neuroprotective effect of HSF1-AB may be mediated by a non-transcriptional mechanism. We validated the altered expression of 15 genes by QPCR. Although other studies have conducted RNA-Seq analyses to identify HSF1 targets, our study performed using primary neurons has identified a number of novel targets that may play a special role in brain maintenance and function.

A simple and efficient toolset for analysing mitochondrial trafficking in neuronal cells.

Mitochondria are crucial for cells, supplying up to 90% of the energy requirements for neurons. Their correct localisation is crucial and ensured by a transport system. Mitochondrial trafficking in neurons is particularly critical, because mitochondria must leave the soma and travel along the axon and dendritic network to facilitate neuronal function. Abnormal mitochondrial trafficking has been reported in several neurological disorders, therefore the ability to quantify and analyse mitochondrial trafficking is vital to improving our understanding of their pathogenesis. Commercial software currently lacks an automated approach for performing such quantitation. Here we demonstrate the development of the Mitochondrial Trafficking and Distribution (MiTrakD) analysis toolset, which consists of simple and free-to-use instructions for mitochondrial trafficking analysis using time-lapse microscopy. MiTrakD utilises existing Fiji (ImageJ) tools for semi-automated, fast and efficient analysis of mitochondrial trafficking and distribution, including velocity, abundance, localisation and distance travelled in neurons. We document MiTrakD's efficiency and accuracy by analysing mitochondrial trafficking using two-dimensional fluorescence images of cortical neurons of wild type mice after 6 days (DIV6), 10 days (DIV10) and 14 days (DIV14) of in vitro incubation. Using MiTrakD we have demonstrated that neurons at all developmental stages exhibited the same percentage of mobile mitochondria, all of which travel in equidistance. Interestingly, the mitochondria in neurons at DIV10 were in greater abundance and were faster than those at DIV6 and DIV14. We can also conclude that MiTrakD is more efficient than manual analysis and is an accurate and reliable tool for performing mitochondrial trafficking analysis in neuronal cells.

The tree shrew cerebellum atlas: Systematic nomenclature, neurochemical characterization, and afferent projections.

The cerebellum is involved in the control of movement, emotional responses, and reward processing. The tree shrew is the closest living relative of primates. However, little is known not only about the systematic nomenclature for the tree shrew cerebellum but also about the detailed neurochemical characterization and afferent projections. In this study, Nissl staining and acetylcholinesterase histochemistry were used to reveal anatomical features of the cerebellum of tree shrews (Tupaia belangeri chinensis). The cerebellar cortex presented a laminar structure. The morphological characteristics of the cerebellum were comprehensively described in the coronal, sagittal, and horizontal sections. Moreover, distributive maps of calbindin-immunoreactive (-ir) cells in the Purkinje cell layer of the cerebellum of tree shrews were depicted using coronal, sagittal, and horizontal schematics. In addition, 5th cerebellar lobule (5Cb)-projecting neurons were present in the pontine nuclei, reticular nucleus, spinal vestibular nucleus, ventral spinocerebellar tract, and inferior olive of the tree shrew brain. The anterior part of the paramedian lobule of the cerebellum (PMa) received mainly strong innervation from the lateral reticular nucleus, inferior olive, pontine reticular nucleus, spinal trigeminal nucleus, pontine nuclei, and reticulotegmental nucleus of the pons. The present results provide the first systematic nomenclature, detailed atlas of the whole cerebellum, and whole-brain mapping of afferent projections to the 5Cb and PMa in tree shrews. Our findings provide morphological support for tree shrews as an alternative model for studies of human cerebellar pathologies.

Sensorimotor Integration and Amplification of Reflexive Whisking by Well-Timed Spiking in the Cerebellar Corticonuclear Circuit.

To test how cerebellar crus I/II Purkinje cells and their targets in the lateral cerebellar nuclei (CbN) integrate sensory and motor-related inputs and contribute to reflexive movements, we recorded extracellularly in awake, head-fixed mice during non-contact whisking. Ipsilateral or contralateral air puffs elicited changes in population Purkinje simple spike rates that matched whisking kinematics (∼1 Hz/1° protraction). Responses remained relatively unaffected when ipsilateral sensory feedback was removed by lidocaine but were reduced by optogenetically inhibiting the reticular nuclei. Optogenetically silencing cerebellar output suppressed movements. During puff-evoked whisks, both Purkinje and CbN cells generated well-timed spikes in sequential 2- to 4-ms windows at response onset, such that they alternately elevated their firing rates just before protraction. With spontaneous whisks, which were smaller than puff-evoked whisks, well-timed spikes were absent and CbN cells were inhibited. Thus, sensory input can facilitate millisecond-scale, well-timed spiking in Purkinje and CbN cells and amplify reflexive whisker movements.

Infrared laser ablation and capture of enzymes with conserved activity.

Infrared (IR) laser ablation at 3 µm wavelength was used to extract enzymes from tissue and quantitatively determine their activity. Experiments were conducted with trypsin, which was ablated, captured and then used to digest bovine serum albumin (BSA). BSA digests were evaluated using matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) and sequence coverage of 59% was achieved. Quantification was performed using trypsin and catalase standards and rat brain tissue by fluorescence spectroscopy. Both enzymes were reproducibly transferred with an efficiency of 75 ±â€¯8% at laser fluences between 10 and 30 kJ/m2. Trypsin retained 37 ±â€¯2% of its activity and catalase retained 50 ±â€¯7%. The activity of catalase from tissue was tested using three consecutive 50 µm thick rat brain sections. Two 4 mm2 regions were ablated and captured from the cortex and cerebellum regions. The absolute catalase concentration in the two regions was consistent with previously published data, demonstrating transfer of intact enzymes from tissue.

Altered expression and localization of synaptophysin in developing cerebellar cortex of neonatal rats due to maternal diabetes mellitus.

There is sufficient evidence that diabetes during pregnancy is associated with a higher risk of neurodevelopmental anomalies including learning deficits, behavioral problems and motor dysfunctions in the offspring. Synaptophysin (SYP) is an integral membrane protein of synaptic vesicles and is considered as a marker for synaptogenesis and synaptic density. This study aimed to examine the effects of maternal diabetes in pregnancy on the expression and localization of SYP in the developing rat cerebellum. Wistar female rats were maintained diabetic from a week before pregnancy through parturition and male offspring was euthanized at postnatal day (P) 0, 7, and 14. The results revealed a significant down-regulation in the mRNA expression of SYP in the offspring born to diabetic animals at both P7 and P14 (P < 0.05 each). One week after birth, there was a significant reduction in the localization of SYP expression in the external granular (EGL) and in the molecular (ML) layers of neonates born to diabetic animals (P < 0.05 each). We also found a marked decrease in the expression of SYP in all of the cerebellar cortical layers of STZ-D group pups at P14 (P < 0.05 each). Moreover, our results revealed no significant changes in either expression or localization of SYP in insulin-treated group pups when compared with the controls (P ≥ 0.05 each). The present study demonstrated that maternal diabetes has adverse effects on the synaptogenesis in the offspring's cerebellum. Furthermore, the rigid maternal blood glucose control in the most cases normalized these negative impacts.

Cerebral autoregulation in the microvasculature measured with near-infrared spectroscopy.

Cerebral autoregulation (CA) is the mechanism that allows the brain to maintain a stable blood flow despite changes in blood pressure. Dynamic CA can be quantified based on continuous measurements of systemic mean arterial pressure (MAP) and global cerebral blood flow. Here, we show that dynamic CA can be quantified also from local measurements that are sensitive to the microvasculature. We used near-infrared spectroscopy (NIRS) to measure temporal changes in oxy- and deoxy-hemoglobin concentrations in the prefrontal cortex of 11 human subjects. A novel hemodynamic model translates those changes into changes of cerebral blood volume and blood flow. The interplay between them is described by transfer function analysis, specifically by a high-pass filter whose cutoff frequency describes the autoregulation efficiency. We have used pneumatic thigh cuffs to induce MAP perturbation by a fast release during rest and during hyperventilation, which is known to enhance autoregulation. Based on our model, we found that the autoregulation cutoff frequency increased during hyperventilation in comparison to normal breathing in 10 out of 11 subjects, indicating a greater autoregulation efficiency. We have shown that autoregulation can reliably be measured noninvasively in the microvasculature, opening up the possibility of localized CA monitoring with NIRS.

Cerebral lipid accumulation in Chanarin-Dorfman Syndrome.

Chanarin-Dorfman Syndrome (CDS) is caused by a defect in the CGI-58/ABHD5 gene resulting in a deficiency of CGI-58 and in intracellular accumulation of triacylglycerol in skin and liver. Patients are mainly characterized by congenital ichthyosis, but the clinical phenotype is very heterogeneous. Distinct brain involvement has never been described. We present a clinical description of two patients with congenital ichthyosis. On suspicion of Sjögren-Larsson syndrome (SLS) single-voxel 1H-MR spectroscopy of the brain was performed and biochemical testing of fatty aldehyde dehydrogenase (FALDH) to establish this diagnosis gave normal results. Vacuolisation in a peripheral blood smear has led to the CDS suspicion. In both patients the diagnosis CDS was confirmed by ABHD5 mutation analysis. Interestingly, a clear lipid accumulation in the cerebral white matter, cortex and basal ganglia was demonstrated in both CDS-patients. These results demonstrate, for the first time, cerebral involvement in CDS and give new insights in the complex phenotype. Since the clinical implications of this abnormal cerebral lipid accumulation are still unknown, further studies are warranted.

Calbindin-D28K immunoreactivity in the human cerebellar cortex.

Calbindin-D28k (CB) is a calcium-binding protein largely distributed in the cerebellum of various species of vertebrates. As regards the human cerebellar cortex, precise data on the distribution of CB have not yet been reported. Aim of the present work was to analyze the distribution of CB in postmortem samples of human cerebellar cortex using light microscopy immunohistochemical techniques. Immunoreactivity to CB was detected within neuronal bodies and processes distributed in all cortex layers. In the molecular layer, the immunoreactivity was observed in subpopulations of stellate and basket neurons. In the Purkinje neuron layer, the immunoreactivity was observed in practically all the Purkinje neurons. In the granular layer, the immunoreactivity was observed in subpopulations of granules, of Golgi neurons, and also of other types of large neurons (candelabrum, Lugaro neurons, etc.). Immunoreactivity to CB was also observed in axon terminals distributed throughout the cortex according to layer-specific patterns of distribution. The qualitative and quantitative patterns of distribution of CB showed no difference among the different lobes of the cerebellar cortex. This study reports that CB is expressed by different neuron types, both inhibitory (GABAergic) and excitatory (glutamatergic), involved in both intrinsic and extrinsic circuits of the human cerebellar cortex. The study provides further insights on the functional role of CB and on the neuronal types of the cerebellar cortex in which it is expressed.

Elevated brain harmane (1-methyl-9H-pyrido[3,4-b]indole) in essential tremor cases vs. controls.

BACKGROUND: Harmane (1-methyl-9H-pyrido[3,4-ß]indole), a potent neurotoxin that has tremor-producing properties in animal models, is present in many foods; although we have demonstrated a difference in tissue harmane concentrations in ET cases vs. controls, all work to date has involved blood samples. OBJECTIVES: We quantified harmane concentrations in human cerebellum, a brain region of particular pathogenic interest in essential tremor (ET), comparing ET to control brains. METHODS: Cerebellar cortex was snap frozen and stored at -80°C in aliquots for biochemical analyses. Harmane concentration was assessed using high performance liquid chromatography. RESULTS: Geometric mean brain harmane concentrations (adjusted for postmortem interval [PMI] and freezer time) were higher in ET cases than controls: 1.0824 (95% confidence interval=0.9405-1.2457) vs. 0.8037 (0.6967-0.9272), p=0.004. Geometric mean of brain harmane concentrations (adjusting for PMI and freezer time) was highest in ET cases who reported other relatives with tremor (1.2005 [0.8712-1.6541]), intermediate in ET cases without family history (1.0312 ([0.8879-1.1976]), and both were significantly higher than controls (p=0.02). CONCLUSIONS: This study provides additional evidence of a possible etiological importance of this toxin in some cases of the human disease ET.

Heterogeneity of calretinin expression in the avian cerebellar cortex of pigeons and relationship with zebrin II.

The cerebellar cortex has a fundamental parasagittal organization that is reflected in the physiological responses of Purkinje cells, projections of Purkinje cells, afferent inputs from climbing and mossy fibres and the expression of several molecular markers. The most thoroughly studied of these molecular markers is zebrin II (ZII; a.k.a. aldolase C). ZII is differentially expressed in Purkinje cells, resulting in a pattern of sagittal stripes of high expression (ZII+ve) interdigitated with stripes of little or no expression (ZII-ve). The calcium binding protein calretinin (CR) is expressed heavily in mossy fibres terminating throughout the cerebellar cortex, but whether CR is heterogeneously expressed in parasagittal stripes, like ZII, is unknown. In this study, we examined CR expression in the cerebellum of pigeons and compared it to that of ZII. CR was expressed heavily in the granule layer in mossy fibres and their terminal rosettes. Moreover, CR is expressed heterogeneously in the granule layer such that there are sagittal stripes of heavy CR labelling (CR+ve) alternating with stripes of weaker labelling (CR-ve). The CR heterogeneity is most notable in folium IXcd and folia II-IV in the anterior lobe. In the anterior lobe, the central CR+ve stripe spanning the midline is aligned with the central ZII+ve stripe, whereas the other CR+ve stripes are aligned with the ZII-ve stripes. In IXcd, the CR+ve stripes are aligned with the ZII+ve stripes. This combination of aligned and unaligned CR+ve stripes, relative to ZII+ve stripes, differs from that of parvalbumin and other neurochemical markers, but the functional consequences of this is unclear. With respect to the posterior lobe, we suggest that the CR+ve mossy fibres to IXcd originate in two retinal recipient nuclei that are involved in the processing of optic flow.

Reevaluation of the beam and radial hypotheses of parallel fiber action in the cerebellar cortex.

The role of parallel fibers (PFs) in cerebellar physiology remains controversial. Early studies inspired the "beam" hypothesis whereby granule cell (GC) activation results in PF-driven, postsynaptic excitation of beams of Purkinje cells (PCs). However, the "radial" hypothesis postulates that the ascending limb of the GC axon provides the dominant input to PCs and generates patch-like responses. Using optical imaging and single-cell recordings in the mouse cerebellar cortex in vivo, this study reexamines the beam versus radial controversy. Electrical stimulation of mossy fibers (MFs) as well as microinjection of NMDA in the granular layer generates beam-like responses with a centrally located patch-like response. Remarkably, ipsilateral forepaw stimulation evokes a beam-like response in Crus I. Discrete molecular layer lesions demonstrate that PFs contribute to the peripherally generated responses in Crus I. In contrast, vibrissal stimulation induces patch-like activation of Crus II and GABAA antagonists fail to convert this patch-like activity into a beam-like response, implying that molecular layer inhibition does not prevent beam-like responses. However, blocking excitatory amino acid transporters (EAATs) generates beam-like responses in Crus II. These beam-like responses are suppressed by focal inhibition of MF-GC synaptic transmission. Using EAAT4 reporter transgenic mice, we show that peripherally evoked patch-like responses in Crus II are aligned between parasagittal bands of EAAT4. This is the first study to demonstrate beam-like responses in the cerebellar cortex to peripheral, MF, and GC stimulation in vivo. Furthermore, the spatial pattern of the responses depends on extracellular glutamate and its local regulation by EAATs.

Antigenic compartmentation of the cerebellar cortex in an Australian marsupial, the tammar wallaby Macropus eugenii.

The mammalian cerebellar cortex is apparently uniform in composition, but a complex heterogeneous pattern can be revealed by using biochemical markers such as zebrin II/aldolase C, which is expressed by a subset of Purkinje cells that form a highly reproducible array of transverse zones and parasagittal stripes. The architecture revealed by zebrin II expression is conserved among many taxa of birds and mammals. In this report zebrin II immunohistochemistry has been used in both section and whole-mount preparations to analyze the cerebellar architecture of the Australian tammar wallaby (Macropus eugenii). The gross appearance of the wallaby cerebellum is remarkable, with unusually elaborate cerebellar lobules with multiple sublobules and fissures. However, despite the morphological complexity, the underlying zone and stripe architecture is conserved and the typical mammalian organization is present.

Neurofilament protein levels: quantitative analysis in essential tremor cerebellar cortex.

Essential tremor (ET) is among the most prevalent neurological diseases. A substantial increase in the number of Purkinje cell axonal swellings (torpedoes) has been identified in ET brains. We recently demonstrated that torpedoes in ET contain an over-accumulation of disorganized neurofilament (NF) proteins. This now raises the question whether NF protein composition and/or phosphorylation state in cerebellar tissue might differ between ET cases and controls. We used a Western blot analysis to compare the levels and phosphorylation state of NF proteins and α-internexin in cerebellar tissue from 47 ET cases versus 26 controls (2:1 ratio). Cases and controls did not differ with respect to the cerebellar levels of NF-light (NF-L), NF-medium (NF-M), NF-heavy (NF-H), or α-internexin. However, SMI-31 levels (i.e., phosphorylated NF-H) and SMI-32 levels (i.e., non-phosphorylated NF-H) were significantly higher in ET cases than controls (1.28±0.47 vs. 1.06±0.32, p=0.02; and 1.38±0.75 vs. 1.00±0.42, p=0.006). Whether the abnormal phosphorylation state that we observed is a cause of defective axonal transport and/or function of NFs in ET is not known. NF abnormalities have been demonstrated in several neurodegenerative diseases. Regardless of whether these protein aggregates are the cause or consequence of these diseases, NF abnormalities have been shown to be an important factor in the cellular disruption observed in several neurodegenerative diseases. Therefore, further analyses of these NF abnormalities and their mechanisms are important to enhance our understanding of disease pathogenesis in ET.

Cerebellar amyloid-ß plaques: disturbed cortical circuitry in AßPP/PS1 transgenic mice as a model of familial Alzheimer's disease.

Cerebellar amyloid-ß (Aß) deposition in the form of neuritic plaques and Purkinje cell loss are common in certain pedigrees of familial Alzheimer's disease (FAD) mainly linked to PS1 mutations. AßPP/PS1 transgenic mice, here used as a model of FAD, show a few Aß plaques in the molecular layer of the cerebellum at 6 months, and which increase in number with age. Motor impairment is apparent in transgenic mice aged 12 months. Combined methods have shown degenerated parallel fibers as the main component of dystrophic neurites of Aß plaques, loss of synaptic contacts between parallel fibers and dendritic spines of Purkinje cells, and degeneration of granule cells starting at 12 months and increasing in mice 18/20 months old. In addition, abnormal mitochondria and focal loss of Purkinje and basket cells, together with occasional axonal torpedoes and increased collaterals of Purkinje cells in mice aged 18/20 months, is suggested to be a concomitant defect presumably related to soluble extracellular or intracellular Aß. These observations demonstrate serious deterioration of the neuronal circuitry in the cerebellum of AßPP/PS1 transgenic mice, and they provide support for the interpretation of similar alterations occurring in certain pedigrees with FAD.

Calcium influx measured at single presynaptic boutons of cerebellar granule cell ascending axons and parallel fibers.

Action potential-evoked calcium influx into presynaptic boutons is a key determinant of synaptic strength and function. Here, we have examined the calcium dynamics at individual presynaptic boutons of the cerebellar granule cells in the molecular layer of cerebellar slices and investigated whether different subpopulations of granule cell boutons exhibit different calcium dynamics. We found that a population of boutons with low basal calcium clearance rates may activate a second clearance mechanism and exhibit biphasic calcium decay on high calcium influx induced by bursts of action potentials. We also found that boutons on ascending axons and parallel fibers show similar calcium influx amplitudes and calcium clearance rates in response to action potentials. Lastly, we found that parallel fiber boutons located in the inner molecular layer have a higher calcium clearance rate than boutons located in the outer molecular layer. These results suggest that cerebellar granule cell boutons should not be regarded as a homogeneous population, but rather that different subpopulations of boutons may exhibit different properties. The heterogeneity of presynaptic boutons may allow different learned behaviors to be encoded in the same circuit without mutual interference and may be a general mechanism for increasing the computational capacity of the brain.