The Use of Thioflavin T for the Estimation and Measurement of the Plasma Membrane Electric Potential Difference in Different Yeast Strains.

The use of the cationic, dye thioflavin T (ThT), to estimate the electric plasma membrane potential difference (PMP) via the fluorescence changes and to obtain its actual values from the accumulation of the dye, considering important correction factors by its binding to the internal components of the cell, was described previously for baker's yeast. However, it was considered important to explore whether the method developed could be applied to other yeast strains. Alternative ways to estimate the PMP by using flow cytometry and a multi-well plate reader are also presented here. The methods were tested with other strains of Saccharomyces cerevisiae (W303-1A and FY833), as well as with non-conventional yeasts: Debaryomyces hansenii, Candida albicans, Meyerozyma guilliermondii, and Rhodotorula mucilaginosa. Results of the estimation of the PMP via the fluorescence changes under different conditions were adequate with all strains. Consistent results were also obtained with several mutants of the main monovalent transporters, validating ThT as a monitor for PMP estimation.

Effect of the Ketone Body, D-ß-Hydroxybutyrate, on Sirtuin2-Mediated Regulation of Mitochondrial Quality Control and the Autophagy-Lysosomal Pathway.

Mitochondrial activity and quality control are essential for neuronal homeostasis as neurons rely on glucose oxidative metabolism. The ketone body, D-ß-hydroxybutyrate (D-BHB), is metabolized to acetyl-CoA in brain mitochondria and used as an energy fuel alternative to glucose. We have previously reported that D-BHB sustains ATP production and stimulates the autophagic flux under glucose deprivation in neurons; however, the effects of D-BHB on mitochondrial turnover under physiological conditions are still unknown. Sirtuins (SIRTs) are NAD+-activated protein deacetylases involved in the regulation of mitochondrial biogenesis and mitophagy through the activation of transcription factors FOXO1, FOXO3a, TFEB and PGC1α coactivator. Here, we aimed to investigate the effect of D-BHB on mitochondrial turnover in cultured neurons and the mechanisms involved. Results show that D-BHB increased mitochondrial membrane potential and regulated the NAD+/NADH ratio. D-BHB enhanced FOXO1, FOXO3a and PGC1α nuclear levels in an SIRT2-dependent manner and stimulated autophagy, mitophagy and mitochondrial biogenesis. These effects increased neuronal resistance to energy stress. D-BHB also stimulated the autophagic-lysosomal pathway through AMPK activation and TFEB-mediated lysosomal biogenesis. Upregulation of SIRT2, FOXOs, PGC1α and TFEB was confirmed in the brain of ketogenic diet (KD)-treated mice. Altogether, the results identify SIRT2, for the first time, as a target of D-BHB in neurons, which is involved in the regulation of autophagy/mitophagy and mitochondrial quality control.

Haploinsufficiency of Shank3 increases the orientation selectivity of V1 neurons.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder whose hallmarks are social deficits, language impairment, repetitive behaviors, and sensory alterations. It has been reported that patients with ASD show differential activity in cortical regions, for instance, increased neuronal activity in visual processing brain areas and atypical visual perception compared with healthy subjects. The causes of these alterations remain unclear, although many studies demonstrate that ASD has a strong genetic correlation. An example is Phelan-McDermid syndrome, caused by a deletion of the Shank3 gene in one allele of chromosome 22. However, the neuronal consequences relating to the haploinsufficiency of Shank3 in the brain remain unknown. Given that sensory abnormalities are often present along with the core symptoms of ASD, our goal was to study the tuning properties of the primary visual cortex to orientation and direction in awake, head-fixed Shank3+/- mice. We recorded neural activity in vivo in response to visual gratings in the primary visual cortex from a mouse model of ASD (Shank3+/- mice) using the genetically encoded calcium indicator GCaMP6f, imaged with a two-photon microscope through a cranial window. We found that Shank3+/- mice showed a higher proportion of neurons responsive to drifting gratings stimuli than wild-type mice. Shank3+/- mice also show increased responses to some specific stimuli. Furthermore, analyzing the distributions of neurons for the tuning width, we found that Shank3+/- mice have narrower tuning widths, which was corroborated by analyzing the orientation selectivity. Regarding this, Shank3+/- mice have a higher proportion of selective neurons, specifically neurons showing increased selectivity to orientation but not direction. Thus, the haploinsufficiency of Shank3 modified the neuronal response of the primary visual cortex.

An interactive time series image analysis software for dendritic spines.

Live fluorescence imaging has demonstrated the dynamic nature of dendritic spines, with changes in shape occurring both during development and in response to activity. The structure of a dendritic spine correlates with its functional efficacy. Learning and memory studies have shown that a great deal of the information stored by a neuron is contained in the synapses. High precision tracking of synaptic structures can give hints about the dynamic nature of memory and help us understand how memories evolve both in biological and artificial neural networks. Experiments that aim to investigate the dynamics behind the structural changes of dendritic spines require the collection and analysis of large time-series datasets. In this paper, we present an open-source software called SpineS for automatic longitudinal structural analysis of dendritic spines with additional features for manual intervention to ensure optimal analysis. We have tested the algorithm on in-vitro, in-vivo, and simulated datasets to demonstrate its performance in a wide range of possible experimental scenarios.

Altered Light Sensitivity of Circadian Clock in Shank3+/- Mouse.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairment in communication and social interaction, repetitive or stereotypical behaviors, altered sensory perception, and sleep disorders. In general, the causes of ASD remain unknown, but in Phelan-McDermid syndrome, it is known that the disorder is related to the haploinsufficiency of the Shank3 gene. We used an autism model with compromised glutamatergic signaling, the Shank3+/- mouse, to study the circadian rhythm architecture of locomotion behavior and its entrainment to light. We also analyzed the synapse between the retinohypothalamic tract (RHT) and the suprachiasmatic nucleus (SCN), employing tract tracing and immunohistochemical techniques. We found that Shank3+/- mice were not impaired in the SCN circadian clock, as indicated by a lack of differences between groups in the circadian architecture in entrained animals to either long or short photoperiods. Circadian rhythm periodicity (tau) was unaltered between genotypes in constant darkness (DD, dim red light). Similar results were obtained in the re-entrainment to shifts in the light-dark cycle and in the entrainment to a skeleton photoperiod from DD. However, Shank3+/- mice showed larger phase responses to light pulses, both delays and advances, and rhythm disorganization induced by constant bright light. Immunohistochemical analyses indicated no differences in the RHT projection to the SCN or the number of SCN neurons expressing the N-methyl-D-aspartate (NMDA) receptor subunit NR2A, whereas the Shank3+/- animals showed decreased c-Fos induction by brief light pulses at CT14, but increased number of vasoactive intestinal polypeptide (VIP)-positive neurons. These results indicate alterations in light sensitivity in Shank3+/- mice. Further studies are necessary to understand the mechanisms involved in such increased light sensitivity, probably involving VIP neurons.

Homeostatic Plasticity Scales Dendritic Spine Volumes and Changes the Threshold and Specificity of Hebbian Plasticity.

Information is encoded in neural networks through changes in synaptic weights. Synaptic learning rules involve a combination of rapid Hebbian plasticity and slower homeostatic synaptic plasticity that regulates neuronal activity through global synaptic scaling. Hebbian and homeostatic plasticity have been extensively investigated, whereas much less is known about their interaction. Here we investigated structural and functional consequences of homeostatic plasticity at dendritic spines of mouse hippocampal neurons. We found that prolonged activity blockade induced spine growth, paralleling synaptic strength increases. Following activity blockade, glutamate uncaging-mediated stimulation at single spines led to size-dependent structural potentiation: smaller spines underwent robust growth, whereas larger spines remained unchanged. Moreover, spines near the stimulated spine exhibited volume changes following homeostatic plasticity, indicating that there was a breakdown of input specificity following homeostatic plasticity. Overall, these findings demonstrate that Hebbian and homeostatic plasticity interact to shape neural connectivity through non-uniform structural plasticity at inputs.

Tracking-assisted Detection of Dendritic Spines in Time-Lapse Microscopic Images.

Detecting morphological changes of dendritic spines in time-lapse microscopy images and correlating them with functional properties such as memory and learning, are fundamental and challenging problems in neurobiology research. In this paper, we propose an algorithm for dendritic spine detection in time series. The proposed approach initially performs spine detection at each time point and improves the accuracy by exploiting the information obtained from tracking of individual spines over time. To detect dendritic spines in a time point image we employ an SVM classifier trained by pre-labeled SIFT feature descriptors in combination with a dot enhancement filter. Second, to track the growth or loss of spines, we apply a SIFT-based rigid registration method for the alignment of time-series images. This step takes into account both the structure and the movement of objects, combined with a robust dynamic scheme to link information about spines that disappear and reappear over time. Next, we improve spine detection by employing a probabilistic dynamic programming approach to search for an optimum solution to accurately detect missed spines. Finally, we determine the spine location more precisely by performing a watershed-geodesic active contour model. We quantitatively assess the performance of the proposed spine detection algorithm based on annotations performed by biologists and compare its performance with the results obtained by the noncommercial software NeuronIQ. Experiments show that our approach can accurately detect and quantify spines in 2-photon microscopy time-lapse data and is able to accurately identify spine elimination and formation.

Synaptic competition in structural plasticity and cognitive function.

Connections between neurons can undergo long-lasting changes in synaptic strength correlating with changes in structure. These events require the synthesis of new proteins, the availability of which can lead to cooperative and competitive interactions between synapses for the expression of plasticity. These processes can occur over limited spatial distances and temporal periods, defining dendritic regions over which activity may be integrated and could lead to the physical rewiring of synapses into functional groups. Such clustering of inputs may increase the computational power of neurons by allowing information to be combined in a greater than additive manner. The availability of new proteins may be a key modulatory step towards activity-dependent, long-term growth or elimination of spines necessary for remodelling of connections. Thus, the aberrant growth or shrinkage of dendritic spines could occur if protein levels are misregulated. Indeed, such perturbations can be seen in several mental retardation disorders, wherein either too much or too little protein translation exists, matching an observed increase or decrease in spine density, respectively. Cellular events which alter protein availability could relieve a constraint on synaptic competition and disturb synaptic clustering mechanisms. These changes may be detrimental to modifications in neural circuitry following activity.

Long lasting protein synthesis- and activity-dependent spine shrinkage and elimination after synaptic depression.

Neuronal circuits modify their response to synaptic inputs in an experience-dependent fashion. Increases in synaptic weights are accompanied by structural modifications, and activity dependent, long lasting growth of dendritic spines requires new protein synthesis. When multiple spines are potentiated within a dendritic domain, they show dynamic structural plasticity changes, indicating that spines can undergo bidirectional physical modifications. However, it is unclear whether protein synthesis dependent synaptic depression leads to long lasting structural changes. Here, we investigate the structural correlates of protein synthesis dependent long-term depression (LTD) mediated by metabotropic glutamate receptors (mGluRs) through two-photon imaging of dendritic spines on hippocampal pyramidal neurons. We find that induction of mGluR-LTD leads to robust and long lasting spine shrinkage and elimination that lasts for up to 24 hours. These effects depend on signaling through group I mGluRs, require protein synthesis, and activity. These data reveal a mechanism for long lasting remodeling of synaptic inputs, and offer potential insights into mental retardation.

Reactive oxygen species participate in the p38-mediated apoptosis induced by potassium deprivation and staurosporine in cerebellar granule neurons.

Apoptosis induced by low potassium (K5) or staurosporine in cerebellar granule neurons triggers an increase in reactive oxygen species (ROS) levels. ROS inhibition by antioxidants or inhibitors of the NADPH oxidase (NOX) activity reduces the apoptosis induced by both stimuli. It has been reported that JNK mediates the apoptosis induced by K5 but not by staurosporine. No information is available about the role of other signaling pathways such as p38 in staurosporine-induced apoptosis, and whether p38 activation could be related to ROS levels induced by both K5 and staurosporine. Here, we explored this possibility and found that K5 activates p38 and ATF2 and that the inhibition of p38 activity prevents the apoptosis induced by this treatment. We also found that p38 is downstream of ROS generation induced by K5. On the other hand, staurosporine promotes a sustained activation of p38. We found that p38 inhibition markedly decreases ROS generation, NOX activity and apoptosis induced by staurosporine. Furthermore, antioxidants inhibit p38 activation induced by staurosporine. These data indicate that apoptosis induced by both K5 and staurosporine is dependent on p38 activation, which is mediated by ROS. In addition, p38 activation by staurosporine induces a further production of ROS through NOX activation.

Role of oxidative stress and JNK pathway in apoptotic death induced by potassium deprivation and staurosporine in cerebellar granule neurons.

Several signaling pathways are differentially activated during apoptotic cell death. We have previously found that during apoptotic death of cerebellar granule neurons (CGN) induced by potassium deprivation (K5) and staurosporine there is an increase in the generation of reactive oxygen species (ROS). The inhibition of ROS generation reduces the extent of cell death. However, remain to be elucidated the mechanisms by which ROS participate in this apoptotic process. On the other hand, it is well known that c-Jun amino-terminal kinase (JNK) pathway plays a pivotal role in cell death of several cell types. In the present study we found that K5 activated the JNK pathway and that its inhibition with SP600125 markedly prevented caspase 3 activation, nuclear condensation and cell death induced by K5. In contrast, JNK pathway was not activated by staurosporine and the JNK inhibitor did not affect cell death induced by this stimulus. We also found that JNK inhibition did not affect ROS levels induced by K5 or staurosporine, suggesting that ROS are upstream of JNK pathway activation. Antioxidants increased ASK1 phosphorylation and decreased JNK1/2 and c-Jun phosphorylation induced by K5. According to these results, we suggest that apoptosis induced by K5 is JNK-dependent and mediated by ROS, but apoptosis induced by staurosporine is not dependent on JNK and that the observed ROS generation by staurosporine seems not to be involved in the activation of this signaling pathway.