Changes in presynaptic calcium signalling accompany age-related deficits in hippocampal LTP and cognitive impairment.

The loss of cognitive function accompanying healthy aging is not associated with extensive or characteristic patterns of cell death, suggesting it is caused by more subtle changes in synaptic properties. In the hippocampal CA1 region, long-term potentiation requires stronger stimulation for induction in aged rats and mice and long-term depression becomes more prevalent. An age-dependent impairment of postsynaptic calcium homeostasis may underpin these effects. We have examined changes in presynaptic calcium signalling in aged mice using a transgenic mouse line (SyG37) that expresses a genetically encoded calcium sensor in presynaptic terminals. SyG37 mice showed an age-dependent decline in cognitive abilities in behavioural tasks that require hippocampal processing including the Barnes maze, T-maze and object location but not recognition tests. The incidence of LTP was significantly impaired in animals over 18 months of age. These effects of aging were accompanied by a persistent increase in resting presynaptic calcium, an increase in the presynaptic calcium signal following Schaffer collateral fibre stimulation, an increase in postsynaptic fEPSP slope and a reduction in paired-pulse facilitation. These effects were not caused by synapse proliferation and were of presynaptic origin since they were evident in single presynaptic boutons. Aged synapses behaved like younger ones when the extracellular calcium concentration was reduced. Raising extracellular calcium had little effect on aged synapses but altered the properties of young synapses into those of their aged counterparts. These effects can be readily explained by an age-dependent change in the properties or numbers of presynaptic calcium channels.

Corrigendum: Imaging Calcium in Hippocampal Presynaptic Terminals With a Ratiometric Calcium Sensor in a Novel Transgenic Mouse.

[This corrects the article DOI: 10.3389/fncel.2018.00209.].

Imaging Calcium in Hippocampal Presynaptic Terminals With a Ratiometric Calcium Sensor in a Novel Transgenic Mouse.

Genetically encoded calcium indicators (GECIs) have gained widespread use for measurement of neuronal activity but their low expression levels in transgenic mice tend to limit sensitivity. We have developed a transgenic mouse line (SyG37) that expresses a ratiometric calcium sensor, SyGCaMP2-mCherry, that is expressed throughout the brain but targeted to presynaptic terminals. Within the CA1 and CA3 regions of hippocampus of male and female mice, SyGaMP2 fluorescence responds linearly up to 10 electrical stimuli at frequencies up to 100 Hz and it can detect responses to a single stimulus. Responses in single boutons can be measured using multiphoton microscopy. The ensemble amplitude of SyGCaMP2 responses is a function of the number of stimuli applied and the number of contributing boutons. The peak responses and initial rates of calcium influx in single boutons in CA1 and CA3 were similar but the rate of calcium clearance from CA3 boutons after stimulation was significantly faster. In CA1, DNQX reduced SyGCaMP2 responses to Schaffer collateral stimulation to 86% of baseline indicating that 14% of the total response originated from presynaptic terminals of neurones synaptically driven via AMPA receptors. Theta burst stimulation induced long-term potentiation (LTP) of both SyGCaMP2 and fEPSP responses in both young and 18-month-old mice. The proportion of postsynaptically connected terminals increased significantly to 76% of the total after LTP induction. The SyG37 mouse allows stable optical detection of synaptic activation and connectivity at the single bouton level and can be used to characterize the contributions of presynaptic calcium to synaptic transmission and plasticity.

Programmable illumination and high-speed, multi-wavelength, confocal microscopy using a digital micromirror.

Confocal microscopy is routinely used for high-resolution fluorescence imaging of biological specimens. Most standard confocal systems scan a laser across a specimen and collect emitted light passing through a single pinhole to produce an optical section of the sample. Sequential scanning on a point-by-point basis limits the speed of image acquisition and even the fastest commercial instruments struggle to resolve the temporal dynamics of rapid cellular events such as calcium signals. Various approaches have been introduced that increase the speed of confocal imaging. Nipkov disk microscopes, for example, use arrays of pinholes or slits on a spinning disk to achieve parallel scanning which significantly increases the speed of acquisition. Here we report the development of a microscope module that utilises a digital micromirror device as a spatial light modulator to provide programmable confocal optical sectioning with a single camera, at high spatial and axial resolution at speeds limited by the frame rate of the camera. The digital micromirror acts as a solid state Nipkov disk but with the added ability to change the pinholes size and separation and to control the light intensity on a mirror-by-mirror basis. The use of an arrangement of concave and convex mirrors in the emission pathway instead of lenses overcomes the astigmatism inherent with DMD devices, increases light collection efficiency and ensures image collection is achromatic so that images are perfectly aligned at different wavelengths. Combined with non-laser light sources, this allows low cost, high-speed, multi-wavelength image acquisition without the need for complex wavelength-dependent image alignment. The micromirror can also be used for programmable illumination allowing spatially defined photoactivation of fluorescent proteins. We demonstrate the use of this system for high-speed calcium imaging using both a single wavelength calcium indicator and a genetically encoded, ratiometric, calcium sensor.

Dual involvement of G-substrate in motor learning revealed by gene deletion.

In this study, we generated mice lacking the gene for G-substrate, a specific substrate for cGMP-dependent protein kinase uniquely located in cerebellar Purkinje cells, and explored their specific functional deficits. G-substrate-deficient Purkinje cells in slices obtained at postnatal weeks (PWs) 10-15 maintained electrophysiological properties essentially similar to those from WT littermates. Conjunction of parallel fiber stimulation and depolarizing pulses induced long-term depression (LTD) normally. At younger ages, however, LTD attenuated temporarily at PW6 and recovered thereafter. In parallel with LTD, short-term (1 h) adaptation of optokinetic eye movement response (OKR) temporarily diminished at PW6. Young adult G-substrate knockout mice tested at PW12 exhibited no significant differences from their WT littermates in terms of brain structure, general behavior, locomotor behavior on a rotor rod or treadmill, eyeblink conditioning, dynamic characteristics of OKR, or short-term OKR adaptation. One unique change detected was a modest but significant attenuation in the long-term (5 days) adaptation of OKR. The present results support the concept that LTD is causal to short-term adaptation and reveal the dual functional involvement of G-substrate in neuronal mechanisms of the cerebellum for both short-term and long-term adaptation.

The effects of fear conditioning on cerebellar LTP and LTD.

Long-term potentiation (LTP) and depression (LTD) at parallel fibre-Purkinje cell synapses have been described in vitro in the cerebellar cortex, but the physiological roles of these two forms of plasticity have not been well defined. Here we show that, in cerebellar slices taken from rats that had undergone fear conditioning, there was a significant occlusion of electrically induced LTP at parallel fibre-Purkinje cell synapses. This effect was long-lasting and related to associative processes, as LTP was not occluded in unpaired animals. Notably, in conditioned animals the LTP-inducing protocol produced LTD in some cells instead of LTP. Conversely, synaptic depression induced by conjunctive stimulation of parallel fibers and climbing fibres was impaired in tissue taken immediately following aversive stimulation in both paired and unpaired subjects. This effect was not, however, long-lasting as the incidence and extent of LTD returned to normal levels 24 h after behavioural testing. These findings suggest that LTP takes part in the mechanisms underlying aversive associative memories in the cerebellum.

Simple Windows-based software for the control of laser scanning confocal microscopes.

Rapid advances in computer processing power and the appearance of low cost, high speed multifunction data acquisition hardware makes the control of confocal laser scanning microscopes (CLSMs) with standard laboratory hardware a potentially straightforward task. This paper describes software designed to control a Biorad MRC 600 scan head under Windows 2000 or XP. Using a single high speed, multifunction data acquisition board running under the Igor Pro software environment, waveforms required to drive the scan head galvanometers can be generated and up to two channels of images (768 x 512 pixels at 8 or 12 bit levels) captured live or at set intervals. Image averaging, zooming, panning and cropping are supported as is live region of interest measurements over time. The software can trigger or be triggered by external devices via TTL signals and, with the addition of a commercial focus controller, Z scans can also be made. Control of the original neutral density and emission filters of multiple laser-based systems is also supported via serial control. The software should be easily adaptable to control custom designed scanning systems or other older makes of CLSM and it can be integrated with additional acquisition boards for simultaneous electrophysiological recording.

Differential susceptibility to synaptic plasticity reveals a functional specialization of ascending axon and parallel fiber synapses to cerebellar Purkinje cells.

Granule cell axons, via their parallel fibers, form synapses with Purkinje cells across large areas of the cerebellar cortex. Evidence for uniform transmission along parallel fibers to Purkinje cells is controversial, however, leading to speculation that the ascending axonal segment plays a dominant role in cerebellar processing. We have compared the relative susceptibilities of ascending axon and parallel fiber synaptic inputs to several forms of synaptic plasticity. We demonstrate that ascending axon synapses have a limited capability to undergo forms of long-term depression and potentiation compared with parallel fiber synapses. These results demonstrate that these two segments of the same axon play fundamentally different roles in cerebellar signaling, and, as such, the synapses formed between granule cells and Purkinje cells should not be treated as a homogenous population.

Differences in transmission properties and susceptibility to long-term depression reveal functional specialization of ascending axon and parallel fiber synapses to Purkinje cells.

An understanding of the patterns of mossy fiber transmission to Purkinje cells, via granule cell axons, is fundamental to models of cerebellar cortical signaling and processing. Early theories assumed that mossy fiber input is widely disseminated across the cerebellar cortex along beams of parallel fibers, which spread for several millimeters across the cerebellar cortex. Direct evidence for this has, however, proved controversial, leading to the development of an alternative hypothesis that mossy fiber inputs to the cerebral cortex are in fact vertically organized such that the ascending segment of the granule axon carries a greater synaptic weight than the parallel fiber segment. Here, we report that ascending axon synapses are selectively resistant to cerebellar long-term depression and that they release transmitter with higher mean release probabilities and mean quantal amplitudes than parallel fiber synapses. This novel specialization of synapses formed by different segments of the same axon not only explains the reported patterns of granule cell--> Purkinje cell transmission across the cerebellar cortex but also reveals an additional level of functionality and complexity of cerebellar processing. Consequently, ascending axon synapses represent a new element of cortical signal processing that should be distinguished from parallel fiber synapses in future experimental and theoretical studies of cerebellar function.

Insulin-stimulated endothelial nitric oxide release is calcium independent and mediated via protein kinase B.

Insulin exerts a vasodilator effect by stimulating endothelial nitric oxide (NO) production. Studies in cultured cells suggest that insulin might activate endothelial nitric oxide synthase (eNOS) by an atypical, calcium-independent mechanism. This study investigates the mechanism of insulin-stimulated endothelial NO production in intact aortic wall. Real time fluorescence imaging with 4,5-diaminofluorescin diacetate (DAF-2 DA) or 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) and FURA 2-AM was used to simultaneously visualise NO and intracellular calcium concentrations at multiple locations in the endothelium and vascular smooth muscle of isolated rat and mouse aorta after exposure to insulin. Inhibitors of intracellular insulin signalling were used to determine the pathway for insulin-stimulated NO production. Unlike acetylcholine, which stimulated endothelial NO production with a typical increase in free intracellular calcium, insulin (10(-8) to 10(-6)M) stimulated endothelial NO production without elevating intracellular calcium levels. Insulin-stimulated NO production was concentration dependent and detected within 30s of application. Peak increases in NO occurred between 60 and 120 s and declined slowly thereafter. Separate measurements of NO production by fluorescence of 2,3-diaminonaphthalene (DAN) noted that selective inhibitors of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB) inhibited insulin-stimulated NO production, whereas these inhibitors alone did not alter NO production or acetylcholine-stimulated NO production. Insulin-stimulated NO production by endothelium is an acute calcium-independent effect mediated via the PI3K-PKB signalling pathway.

Parallel fiber plasticity.

Cerebellar long-term depression (LTD) is classically observed when climbing fibers, originating from the inferior olive, and parallel fibers, axons of granule cells, are activated repetitively and synchronously. On the basis that the climbing fiber signals errors in motor performance, LTD provides a mechanism of learning whereby inappropriate motor signals, relayed to the cerebellar cortex by parallel fibers, are selectively weakened through their repeated, close temporal association with climbing fiber activity. LTD therefore provides a cellular substrate for error-driven motor learning in the cerebellar cortex. In recent years, it has become apparent that depression at this synapse can also occur without the need for concurrent climbing fiber activation provided the parallel fibers are activated in such a way as to mobilize calcium within the Purkinje cell. A form of long-term potentiation (LTP) has also been uncovered at this synapse, which similarly relies only upon parallel fiber activation. In brain slice preparations and contrary to expectation, each of these forms of parallel fiber induced plasticity, as well as classical LTD, does not remain confined to activated parallel fibers as previously thought, but both depression and potentiation have the capacity to spread to neighboring parallel fiber synapses several tens of microns away from the activated fibers. Here, the cellular mechanisms responsible for the induction and heterosynaptic spread of parallel fiber LTP and LTD are compared to those involved in classical LTD and the physiological implications that the heterosynaptic spread of plasticity may have on cerebellar signal processing are discussed.