A Perspective on Accelerated Aging Caused by the Genetic Deficiency of the Metabolic Protein, OPA1.

Autosomal Dominant Optic Atrophy (ADOA) is an ophthalmological condition associated primarily with mutations in the OPA1 gene. It has variable onset, sometimes juvenile, but in other patients, the disease does not manifest until adult middle age despite the presence of a pathological mutation. Thus, individuals carrying mutations are considered healthy before the onset of clinical symptoms. Our research, nonetheless, indicates that on the cellular level pathology is evident from birth and mutant cells are different from controls. We argue that the adaptation and early recruitment of cytoprotective responses allows normal development and functioning but leads to an exhaustion of cellular reserves, leading to premature cellular aging, especially in neurons and skeletal muscle cells. The appearance of clinical symptoms, thus, indicates the overwhelming of natural cellular defenses and break-down of native protective mechanisms.

OPA1 deficiency accelerates hippocampal synaptic remodelling and age-related deficits in learning and memory.

A healthy mitochondrial network is essential for the maintenance of neuronal synaptic integrity. Mitochondrial and metabolic dysfunction contributes to the pathogenesis of many neurodegenerative diseases including dementia. OPA1 is the master regulator of mitochondrial fusion and fission and is likely to play an important role during neurodegenerative events. To explore this, we quantified hippocampal dendritic and synaptic integrity and the learning and memory performance of aged Opa1 haploinsufficient mice carrying the Opa1Q285X mutation (B6; C3-Opa1Q285STOP ; Opa1+/- ). We demonstrate that heterozygous loss of Opa1 results in premature age-related loss of spines in hippocampal pyramidal CA1 neurons and a reduction in synaptic density in the hippocampus. This loss is associated with subtle memory deficits in both spatial novelty and object recognition. We hypothesize that metabolic failure to maintain normal neuronal activity at the level of a single spine leads to premature age-related memory deficits. These results highlight the importance of mitochondrial homeostasis for maintenance of neuronal function during ageing.

Opa1 Deficiency Leads to Diminished Mitochondrial Bioenergetics With Compensatory Increased Mitochondrial Motility.

Purpose: Retinal ganglion cells (RGCs) are susceptible to mitochondrial deficits and also the major cell type affected in patients with mutations in the OPA1 gene in autosomal dominant optic atrophy (ADOA). Here, we characterized mitochondria in RGCs in vitro from a heterozygous B6; C3-Opa1Q285STOP (Opa1+/-) mouse model to investigate mitochondrial changes underlying the pathology in ADOA. Methods: Mouse RGCs were purified from wild-type and Opa1+/- mouse retina by two-step immunopanning. The mitochondria in neurites of RGCs were labeled with MitoTracker Red for structure and motility measurement by time-lapse imaging. Mitochondrial bioenergetics were determined by the real-time measurement of oxygen consumption rate using a Seahorse XFe 96 Extracellular Flux Analyzer. Results: We observed a significant decrease in mitochondrial length in Opa1+/- RGCs with a remarkably higher proportion and density of motile mitochondria along the neurites. We also observed an increased transport velocity with a higher number of contacts between mitochondria in Opa1+/- RGC neurites. The oxygen consumption assays showed a severe impairment in basal respiration, Adenosine triphosphate-linked (ATP-linked) oxygen consumption, as well as reserve respiratory capacity, in RGCs from Opa1+/- mouse retina. Conclusions: Opa1 deficiency leads to significant fragmentation of mitochondrial morphology, activation of mitochondrial motility and impaired respiratory function in RGCs from the B6; C3-Opa1Q285STOP mouse model. This highlights the significant alterations in the intricate interplay between mitochondrial morphology, motility, and energy production in RGCs with Opa1 deficiency long before the onset of clinical symptoms of the pathology.

Optophysiological Characterisation of Inner Retina Responses with High-Resolution Optical Coherence Tomography.

Low coherence laser interferometry has revolutionised quantitative biomedical imaging of optically transparent structures at cellular resolutions. We report the first optical recording of neuronal excitation at cellular resolution in the inner retina by quantifying optically recorded stimulus-evoked responses from the retinal ganglion cell layer and comparing them with an electrophysiological standard. We imaged anaesthetised paralysed tree shrews, gated image acquisition, and used numerical filters to eliminate noise arising from retinal movements during respiratory and cardiac cycles. We observed increases in contrast variability in the retinal ganglion cell layer and nerve fibre layer with flash stimuli and gratings. Regions of interest were subdivided into three-dimensional patches (up to 5-15 µm in diameter) based on response similarity. We hypothesise that these patches correspond to individual cells, or segments of blood vessels within the inner retina. We observed a close correlation between the patch optical responses and mean electrical activity of the visual neurons in afferent pathway. While our data suggest that optical imaging of retinal activity is possible with high resolution OCT, the technical challenges are not trivial.

Enhancement of visual cortex plasticity by dark exposure.

Dark rearing is known to delay the time course of the critical period for ocular dominance plasticity in the visual cortex. Recent evidence suggests that a period of dark exposure (DE) may enhance or reinstate plasticity even after closure of the critical period, mediated through modification of the excitatory-inhibitory balance and/or removal of structural brakes on plasticity. Here, we investigated the effects of a week of DE on the recovery from a month of monocular deprivation (MD) in the primary visual cortex (V1) of juvenile mice. Optical imaging of intrinsic signals revealed that ocular dominance in V1 of mice that had received DE recovered slightly more quickly than of mice that had not, but the level of recovery after three weeks was similar in both groups. Two-photon calcium imaging showed no significant difference in the recovery of orientation selectivity of excitatory neurons between the two groups. Parvalbumin-positive (PV+) interneurons exhibited a smaller ocular dominance shift during MD but again no differences in subsequent recovery. The percentage of PV+ cells surrounded by perineuronal nets, a structural brake on plasticity, was lower in mice with than those without DE. Overall, DE causes a modest enhancement of mouse visual cortex plasticity.This article is part of the themed issue 'Integrating Hebbian and homeostatic plasticity'.

Adaptive-weighted bilateral filtering and other pre-processing techniques for optical coherence tomography.

This paper presents novel pre-processing image enhancement algorithms for retinal optical coherence tomography (OCT). These images contain a large amount of speckle causing them to be grainy and of very low contrast. To make these images valuable for clinical interpretation, we propose a novel method to remove speckle, while preserving useful information contained in each retinal layer. The process starts with multi-scale despeckling based on a dual-tree complex wavelet transform (DT-CWT). We further enhance the OCT image through a smoothing process that uses a novel adaptive-weighted bilateral filter (AWBF). This offers the desirable property of preserving texture within the OCT image layers. The enhanced OCT image is then segmented to extract inner retinal layers that contain useful information for eye research. Our layer segmentation technique is also performed in the DT-CWT domain. Finally we describe an OCT/fundus image registration algorithm which is helpful when two modalities are used together for diagnosis and for information fusion.

Non-invasive detection of early retinal neuronal degeneration by ultrahigh resolution optical coherence tomography.

Optical coherence tomography (OCT) has revolutionises the diagnosis of retinal disease based on the detection of microscopic rather than subcellular changes in retinal anatomy. However, currently the technique is limited to the detection of microscopic rather than subcellular changes in retinal anatomy. However, coherence based imaging is extremely sensitive to both changes in optical contrast and cellular events at the micrometer scale, and can generate subtle changes in the spectral content of the OCT image. Here we test the hypothesis that OCT image speckle (image texture) contains information regarding otherwise unresolvable features such as organelle changes arising in the early stages of neuronal degeneration. Using ultrahigh resolution (UHR) OCT imaging at 800 nm (spectral width 140 nm) we developed a robust method of OCT image analyses, based on spatial wavelet and texture-based parameterisation of the image speckle pattern. For the first time we show that this approach allows the non-invasive detection and quantification of early apoptotic changes in neurons within 30 min of neuronal trauma sufficient to result in apoptosis. We show a positive correlation between immunofluorescent labelling of mitochondria (a potential source of changes in cellular optical contrast) with changes in the texture of the OCT images of cultured neurons. Moreover, similar changes in optical contrast were also seen in the retinal ganglion cell- inner plexiform layer in retinal explants following optic nerve transection. The optical clarity of the explants was maintained throughout in the absence of histologically detectable change. Our data suggest that UHR OCT can be used for the non-invasive quantitative assessment of neuronal health, with a particular application to the assessment of early retinal disease.

Contribution of near-threshold currents to intrinsic oscillatory activity in rat medial entorhinal cortex layer II stellate cells.

The temporal lobe is well known for its oscillatory activity associated with exploration, navigation, and learning. Intrinsic membrane potential oscillations (MPOs) and resonance of stellate cells (SCs) in layer II of the entorhinal cortex are thought to contribute to network oscillations and thereby to the encoding of spatial information. Generation of both MPOs and resonance relies on the expression of specific voltage-dependent ion currents such as the hyperpolarization-activated cation current (I(H)), the persistent sodium current (I(NaP)), and the noninactivating muscarine-modulated potassium current (I(M)). However, the differential contributions of these currents remain a matter of debate. We therefore examined how they modify neuronal excitability near threshold and generation of near-threshold MPOs and resonance in vitro. We found that resonance mainly relied on I(H) and was reduced by I(H) blockers and modulated by cAMP and an I(M) enhancer but that neither of the currents exhibited full control over MPOs in these cells. As previously reported, I(H) controlled a theta-frequency component of MPOs such that blockade of I(H) resulted in fewer regular oscillations that retained low-frequency components and high peak amplitude. However, pharmacological inhibition and augmentation of I(M) also affected MPO frequencies and amplitudes. In contrast to other cell types, inhibition of I(NaP) did not result in suppression of MPOs but only in a moderation of their properties. We reproduced the experimentally observed effects in a single-compartment stochastic model of SCs, providing further insight into the interactions between different ionic conductances.

The range of intrinsic frequencies represented by medial entorhinal cortex stellate cells extends with age.

In both humans and rodents, the external environment is encoded in the form of cognitive maps. Neurons in the medial entorhinal cortex (mEC) represent spatial locations in a sequence of grid-like patterns scaled along the dorsal-ventral axis. The grid spacing correlates with the intrinsic resonance frequencies of stellate cells in layer II of mEC. We investigated the development of frequency preferences in these cells from weaning to adulthood using patch-clamp and sharp microelectrode recordings. We found that the dorsal-ventral gradient of stellate cell properties and frequency preferences exists before animals are able to actively explore their environment. In the transition to adulthood, cells respond faster and become less excitable, and the range of intrinsic resonance frequencies in the population expands in the dorsal direction. This is likely to reflect both the growth of the brain and the expansion of the internal representation caused by new exploratory experience.

Rhythms of the brain: an examination of mixed mode oscillation approaches to the analysis of neurophysiological data.

In the nervous system many behaviorally relevant dynamical processes are characterized by episodes of complex oscillatory states, whose periodicity may be expressed over multiple temporal and spatial scales. In at least some of these instances the variability in oscillatory amplitude and frequency can be explained in terms of deterministic dynamics, rather than being purely noise-driven. Recently interest has increased in studying the application of mixed-mode oscillations (MMOs) to neurophysiological data. MMOs are complex periodic waveforms where each period is comprised of several maxima and minima of different amplitudes. While MMOs might be expected to occur in brain kinetics, only a few examples have been identified thus far. In this article, we review recent theoretical and experimental findings on brain oscillatory rhythms in relation to MMOs, focusing on examples at the single neuron level but also briefly touching on possible instances of the phenomenon across local and global brain networks.

Spike timing-dependent synaptic depression in the in vivo barrel cortex of the rat.

Spike timing-dependent plasticity (STDP) is a computationally powerful form of plasticity in which synapses are strengthened or weakened according to the temporal order and precise millisecond-scale delay between presynaptic and postsynaptic spiking activity. STDP is readily observed in vitro, but evidence for STDP in vivo is scarce. Here, we studied spike timing-dependent synaptic depression in single putative pyramidal neurons of the rat primary somatosensory cortex (S1) in vivo, using two techniques. First, we recorded extracellularly from layer 2/3 (L2/3) and L5 neurons, and paired spontaneous action potentials (postsynaptic spikes) with subsequent subthreshold deflection of one whisker (to drive presynaptic afferents to the recorded neuron) to produce "post-leading-pre" spike pairings at known delays. Short delay pairings (<17 ms) resulted in a significant decrease of the extracellular spiking response specific to the paired whisker, consistent with spike timing-dependent synaptic depression. Second, in whole-cell recordings from neurons in L2/3, we paired postsynaptic spikes elicited by direct-current injection with subthreshold whisker deflection to drive presynaptic afferents to the recorded neuron at precise temporal delays. Post-leading-pre pairing (<33 ms delay) decreased the slope and amplitude of the PSP evoked by the paired whisker, whereas "pre-leading-post" delays failed to produce depression, and sometimes produced potentiation of whisker-evoked PSPs. These results demonstrate that spike timing-dependent synaptic depression occurs in S1 in vivo, and is therefore a plausible plasticity mechanism in the sensory cortex.

Rapid fluctuations in rat barrel cortex plasticity.

Neuronal populations in the sensory cortex exhibit fluctuations in excitability, and the present experiments tested the hypothesis that these variations coincide with peaks and troughs in cortical modifiability. The activity of multiunit neuronal clusters under light urethane anesthesia was recorded through 100-microelectrode arrays implanted in the infragranular layers of rat barrel cortex. Spontaneous activity was characterized by "bursts" of spikes, synchronized across the barrel cortex. This allowed activity at one selected electrode to be taken as a reliable monitor of widespread cortical bursts. We used spikes at the selected electrode to trigger stimulation of two pairs of whiskers during a 50 min conditioning procedure: (1) for the "burst-conditioned" whisker pair, each stimulus was delivered 1 msec after the triggering spike, activating cortex coincident with the burst; and (2) for the "interburst-conditioned" whisker pair, each stimulus was delivered 300 msec after the triggering spike, activating cortex during the trough between bursts. The cross-correlation between cortical neurons in the pairs of columns matching the stimulated whisker pairs was estimated after the termination of the conditioning procedure. Conditioning produced a twofold increase in the degree of co-firing between infragranular neurons in columns receiving burst-conditioned costimulation but no significant change in connectivity between infragranular neurons in columns receiving interburst-conditioned costimulation, although the two pairs of columns received an equal number of sensory inputs. These findings suggest that the strength of co-activity between columns in the barrel cortex can be modified by sensory input patterns during discrete, intermittent intervals time-locked to bursts.

Subthreshold resonance explains the frequency-dependent integration of periodic as well as random stimuli in the entorhinal cortex.

Neurons integrate subthreshold inputs in a frequency-dependent manner. For sinusoidal stimuli, response amplitudes thus vary with stimulus frequency. Neurons in entorhinal cortex show two types of such resonance behavior: stellate cells in layer II exhibit a prominent peak in the resonance profile at stimulus frequencies of 5-16 Hz. Pyramidal cells in layer III show only a small impedance peak at low frequencies (1-5 Hz) or a maximum at 0 Hz followed by a monotonic decrease of the impedance. Whether the specific frequency selectivity for periodic stimuli also governs the integration of non-periodic stimuli has been questioned recently. Using frozen-noise stimuli with different distributions of power over frequencies, we provide experimental evidence that the integration of non-periodic subthreshold stimuli is determined by the same subthreshold frequency selectivity as that of periodic stimuli. Differences between the integration of noise stimuli in stellate and pyramidal cells can be fully explained by the resonance properties of each cell type. Response power thus reflects stimulus power in a frequency-selective way. Theoretical predictions based on linear system's theory as well as on conductance-based model neurons support this finding. We also show that the frequency selectivity in the subthreshold range extends to suprathreshold responses in terms of firing rate. Cells in entorhinal cortex are representative examples of cells with resonant or low-pass filter impedance profiles. It is therefore likely that neurons with similar frequency selectivity will process input signals according to the same simple principles.

Effect of developmental sensory and motor deprivation on the functional organization of adult rat somatosensory cortex.

Most sensory systems are active, in the sense that the animal performs specific motor actions in order to collect information of interest-signals are not merely passively received. We, therefore, expect cortical development to depend not only correct sensory experience, but also on correct motor experience. In this study, we used the rat whisker system as a model to compare the importance of these factors. In one group of animals, we trimmed all whiskers starting from post-natal day 8 (P8). In a second group, we left the whiskers intact, but prevented "whisking" by sectioning the facial (VIIth cranial) nerve on P8. The first group had severely disrupted sensory experience but normal motor patterns ("whisker-cut" rats); the second group had normal sensory pathways within which temporal activity patterns were disrupted by motor impairment ("nerve-cut" rats). When they reached 3 months of age, we recorded multi-unit responses from the infragranular layers of primary somatosensory cortex in response to deflection of either single whiskers or pairs of whiskers in order to compare these two groups to a third group of rats that had normal sensory and motor experience. Cortical topographic organization was unaltered in whisker- and nerve-cut rats. Whisker-cut rats showed a smaller than normal difference between the response magnitudes for the principal and surrounding whiskers, as well as stronger than normal interactions between co-active whisker inputs. Responses in nerve-cut rats were nearly indistinguishable from those in normal animals. Thus, unexpectedly, neither pure sensory nor sensorimotor deprivation caused gross functional disruption of SI according to our measures. It appears that abnormal sensory experience leads to alterations in the fine-tuning of cortical properties, but cortex is unexpectedly resistant to the effects of abnormal sensory and sensorimotor experience.

Somatosensory cortical neuronal population activity across states of anaesthesia.

Experiments were carried out to learn about changes in sensory cortical processing associated with different levels of anaesthesia. Traditionally this question has been addressed by studying single neurons. Because state changes are likely to influence the relationships between neurons, the present experiments were undertaken to investigate the spatial and temporal firing patterns distributed across cortex. Using 5 x 5 or 10 x 10 microelectrode arrays, spontaneous and stimulus-evoked activity of multineuron clusters was recorded from rat somatosensory 'barrel' cortex (the whisker representation) during a light surgical stage of urethane anaesthesia, and after two supplemental doses of urethane which led to intermediate and deep levels of anaesthesia. At all depths of anaesthesia, spontaneously occurring action potentials at a single electrode tended to be clustered into 'bursts.' With increasing anaesthetic depth, bursts became more prominent and rhythmic, and increasingly synchronized between cortical barrel-columns. Burst frequency decreased and fewer spikes occurred outside bursts, leading to a decrease in the overall spontaneous firing rate. The cortical territory engaged by individual whiskers contracted with increasing depth of anaesthesia, leading to the spatial segregation of whisker representations. At all stages of anaesthesia, whisker stimulation produced the maximal cortical response when delivered close to burst onset. These observations show that ongoing spontaneous activity modulates sensory response properties and makes peripheral tactile information accessible to a cortical territory whose size is determined by the phase of burst cycle. The possible significance of the cyclic cortical responsiveness encountered during urethane anaesthesia to cortical processing in awake rats is considered.