31 research outputs found
Focusing light in biological tissue through a multimode optical fiber: refractive index matching
No full textControlling light propagation through a step-index multimode optical fiber (MMF) has several important applications, including biological imaging. However, little consideration has been given to the coupling of fiber and tissue optics. In this Letter, we characterized the effects of tissue-induced light distortions, in particular those arising from a mismatch in the refractive index of the pre-imaging calibration and biological media. By performing the calibration in a medium matching the refractive index of the brain, optimal focusing ability was achieved, as well as a gain in focus uniformity within the field-of-view. These changes in illumination resulted in a 30% improvement in spatial resolution and intensity in fluorescence images of beads and live brain tissue. Beyond refractive index matching, our results demonstrate that sample-induced aberrations can severely deteriorate images from MMF-based systems
An investigation of presynaptic plasticity mechanisms
No full textThe regulation of synaptic strength is thought to underlie the complex emergent dynamics of neural networks. The strength of a synapse is determined by its pre- and postsynaptic properties, both of which are under tight regulatory control orchestrated by ongoing neuronal activity. We now have a good understanding of the plasticity rules underlying the regulation of postsynaptic strength. The same set of rules has been imposed onto the regulation of presynaptic strength. However, the operation of pre- and postsynaptic terminals is fundamentally different, both on a mechanistic and functional level. I have therefore systematically investigated the mechanisms underlying presynaptic plasticity at the Schaffer collateral-CA1 synapse under varying conditions of synaptic activity. I have examined three general modes of regulation: (1) synaptic changes dependent on the concerted activity of pre- and postsynaptic neurone (homosynaptic plasticity); (2) changes dependent only on presynaptic activity; (3) changes dependent on postsynaptic activity alone (heterosynaptic plasticity). Using a combination of electrophysiological and optical techniques, I have monitored and manipulated the strengths of single synapses both pre- and postsynaptically, which allowed me to impose certain activity patterns and investigate resulting synaptic modifications. I found that heterosynaptic plasticity along local segments of dendrites is expressed at both synaptic loci and depends on the spatial arrangement of synapses. Pre- and postsynaptic strength changes were weakly correlated and pharmacologically dissociable. Next, I found that glutamate release suppresses both short- and long-term presynaptic function, which required the activation of presynaptic NMDA receptors. Lastly, I found that presynaptic long-term potentiation (LTP) is spike timing-dependent but does not rely on coincidence detection via postsynaptic NMDA receptors. My findings suggest that the presynaptic terminal is functionally distinct, which is reflected in parallel regulatory pathways. I suggest the synapse to be viewed as a two- compartment model, consisting of a presynaptic non-linear transformation followed by postsynaptic linear weighting
Imaging synaptic plasticity
No full textAbstract Over the past decade, the use and development of optical imaging techniques has advanced our understanding of synaptic plasticity by offering the spatial and temporal resolution necessary to examine long-term changes at individual synapses. Here, we review the use of these techniques in recent studies of synaptic plasticity and, in particular, long-term potentiation in the hippocampus.</p
Of sounds, photons and maps: in vivo optical characterisation of the auditory thalamocortical system
No full textAs information ascends up the sensory streams, the maps of receptor surface might be faithfully relayed from one brain structure to another, degraded or lost altogether. Such transformations can inform about the type of circuit computation carried out in each region. In vivo high-resolution imaging methods, like two-photon microscopy, are useful for characterising the functional architecture of neuronal circuits down to the micro-scale. To better understand the rules governing thalamocortical connectivity and the origin of cortical maps, I used in vivo two-photon calcium imaging to characterise the properties of thalamic axons innervating different layers and subfields of mouse auditory cortex. Although topographically organised at a global level, I found the frequency selectivity of individual thalamocortical axons to be surprisingly heterogeneous, even in the middle layers (L3b/4) of the primary cortical areas where the thalamic input is dominated by the lemniscal projection. Subsequently, I employed a dual-colour imaging approach to explore the spectral transformations taking place between the thalamocortical projection and granular and supragranular layers of auditory cortex. Some differences in local spectral properties between neurons in L4 and L2/3 were observed in awake, passively listening mice, confirming previous observations on the anaesthetised preparation. Finally, I present a demonstration of a novel fibre-based high-resolution fluorescence imaging method to optically investigate neuronal circuits in deep brain regions, with minimal invasiveness. In short, my work provides some new insights on the functional micro-organisation of the auditory thalamocortical system and constitutes a classical example of the power of optical methods for the study of neuronal circuits in vivo.</p
Extended range and aberration-free autofocusing via remote focusing and sequence-dependent learning
No full textRapid autofocusing over long distances is critical for tracking 3D topological variations and sample motion in real time. Taking advantage of a deformable mirror and Shack-Hartmann wavefront sensor, remote focusing can permit fast axial scanning with simultaneous correction of system-induced aberrations. Here, we report an autofocusing technique that combines remote focusing with sequence-dependent learning via a bidirectional long short term memory network. A 120 µm autofocusing range was achieved in a compact reflectance confocal microscope both in air and in refractive-index-mismatched media, with similar performance under arbitrary-thickness liquid layers up to 1 mm. The technique was validated on sample types not used for network training, as well as for tracking of continuous axial motion. These results demonstrate that the proposed technique is suitable for real-time aberration-free autofocusing over a large axial range, and provides unique advantages for biomedical, holographic and other related applications
Inhibition of lysosomal Ca<sup>2+</sup> signalling disrupts dendritic spine structure and impairs wound healing in neurons
No full textA growing body of evidence suggests that lysosomes, which have traditionally been regarded as degradative organelles, can function as Ca2+ stores, regulated by the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP). We previously demonstrated that in hippocampal pyramidal neurons, activity-dependent Ca2+ release from these stores triggers fusion of the lysosome with the plasma membrane. We found that the physiological role of this Ca2+-dependent fusion was to maintain the long-term structural enlargement of dendritic spines induced by synaptic activity. Here, we examined the pathophysiological consequences of lysosomal dysfunction in hippocampal pyramidal neurons by chronically inhibiting lysosomal Ca2+ signalling using the NAADP antagonist, NED-19. We found that within just 20 hours, inhibition of lysosomal function led to a profound intracellular accumulation of lysosomal membrane. This was accompanied by a significant change in dendritic spine structure, which included a lengthening of dendritic spines, an increase in the number of filipodia, and an overall decrease in spine number. Inhibition of lysosomal function also inhibited wound healing in neurons by preventing lysosomal fusion with the plasma membrane. Neurons were therefore more susceptible to injury. Our findings suggest that dysfunction in lysosomal Ca2+ signalling and lysosomal fusion with the plasma membrane may contribute to the loss of dendritic spines and neurons seen in neurological disorders, such as Niemann-Pick disease type C1, in which lysosomal function is impaired
Calcium stores in hippocampal synaptic boutons mediate short-term plasticity, store-operated Ca2+ entry, and spontaneous transmitter release.
No full textEvoked transmitter release depends upon calcium influx into synaptic boutons, but mechanisms regulating bouton calcium levels and spontaneous transmitter release are obscure. To understand these processes better, we monitored calcium transients in axons and presynaptic terminals of pyramidal neurons in hippocampal slice cultures. Action potentials reliably evoke calcium transients in axons and boutons. Calcium-induced calcium release (CICR) from internal stores contributes to the transients in boutons and to paired-pulse facilitation of EPSPs. Store depletion activates store-operated calcium channels, influencing the frequency of spontaneous transmitter release. Boutons display spontaneous Ca2+ transients; blocking CICR reduces the frequency of these transients and of spontaneous miniature synaptic events. Thus, spontaneous transmitter release is largely calcium mediated, driven by Ca2+ release from internal stores. Bouton store release is important for short-term synaptic plasticity and may also contribute to long-term plasticity
GluN2A- and GluN2B-containing pre-synaptic N -methyl- d -aspartate receptors differentially regulate action potential-evoked Ca 2+ influx via modulation of SK channels
No full textN-methyl-d-aspartate receptors (NMDARs) play a pivotal role in synaptic plasticity. While the functional role of post-synaptic NMDARs is well established, pre-synaptic NMDAR (pre-NMDAR) function is largely unexplored. Different pre-NMDAR subunit populations are documented at synapses, suggesting that subunit composition influences neuronal transmission. Here, we used electrophysiological recordings at Schaffer collateral-CA1 synapses partnered with Ca2+ imaging and glutamate uncaging at boutons of CA3 pyramidal neurones to reveal two populations of pre-NMDARs that contain either the GluN2A or GluN2B subunit. Activation of the GluN2B population decreases action potential-evoked Ca2+ influx via modulation of small-conductance Ca2+-activated K+ channels, while activation of the GluN2A population does the opposite. Critically, the level of functional expression of the subunits is subject to homeostatic regulation, bidirectionally affecting short-term facilitation, thus providing a capacity for a fine adjustment of information transfer. This article is part of a discussion meeting issue ‘Long-term potentiation: 50 years on’
Repeated imaging through a multimode optical fiber using adaptive optics
No full textMultimode optical fibers (MMF) have shown considerable potential for minimally invasive diffraction-limited fluorescence imaging of deep brain regions owing to their small size. They also look to be suitable for imaging across long time periods, with repeated measurements performed within the same brain region, which is useful to assess the role of synapses in normal brain function and neurological disease. However, the approach is not without challenge. Prior to imaging, light propagation through a MMF must be characterized in a calibration procedure. Manual repositioning, as required for repeated imaging, renders this calibration invalid. In this study, we provide a two-step solution to the problem consisting of (1) a custom headplate enabling precise reinsertion of the MMF implant achieving low-quality focusing and (2) sensorless adaptive optics to correct translational shifts in the MMF position enabling generation of high-quality imaging foci. We show that this approach achieves fluorescence imaging after repeated removal and reinsertion of a MMF
