Fluorescent fatty acid conjugates for live cell imaging of peroxisomes.

Peroxisomes are eukaryotic organelles that are essential for multiple metabolic pathways, including fatty acid oxidation, degradation of amino acids, and biosynthesis of ether lipids. Consequently, peroxisome dysfunction leads to pediatric-onset neurodegenerative conditions, including Peroxisome Biogenesis Disorders (PBD). Due to the dynamic, tissue-specific, and context-dependent nature of their biogenesis and function, live cell imaging of peroxisomes is essential for studying peroxisome regulation, as well as for the diagnosis of PBD-linked abnormalities. However, the peroxisomal imaging toolkit is lacking in many respects, with no reporters for substrate import, nor cell-permeable probes that could stain dysfunctional peroxisomes. Here we report that the BODIPY-C12 fluorescent fatty acid probe stains functional and dysfunctional peroxisomes in live mammalian cells. We then go on to improve BODIPY-C12, generating peroxisome-specific reagents, PeroxiSPY650 and PeroxiSPY555. These probes combine high peroxisome specificity, bright fluorescence in the red and far-red spectrum, and fast non-cytotoxic staining, making them ideal tools for live cell, whole organism, or tissue imaging of peroxisomes. Finally, we demonstrate that PeroxiSPY enables diagnosis of peroxisome abnormalities in the PBD CRISPR/Cas9 cell models and patient-derived cell lines.

Opposite, bidirectional shifts in excitation and inhibition in specific types of dorsal horn interneurons are associated with spasticity and pain post-SCI.

Spasticity, a common complication after spinal cord injury (SCI), is frequently accompanied by chronic pain. The physiological origin of this pain (critical to its treatment) remains unknown, although spastic motor dysfunction has been related to the hyperexcitability of motoneurons and to changes in spinal sensory processing. Here we show that the pain mechanism involves changes in sensory circuits of the dorsal horn (DH) where nociceptive inputs integrate for pain processing. Spasticity is associated with the DH hyperexcitability resulting from an increase in excitation and disinhibition occurring in two respective types of sensory interneurons. In the tonic-firing inhibitory lamina II interneurons, glutamatergic drive was reduced while glycinergic inhibition was potentiated. In contrast, excitatory drive was boosted to the adapting-firing excitatory lamina II interneurons while GABAergic and glycinergic inhibition were reduced. Thus, increased activity of excitatory DH interneurons coupled with the reduced excitability of inhibitory DH interneurons post-SCI could provide a neurophysiological mechanism of central sensitization and chronic pain associated with spasticity.

Maximum likelihood estimation of biophysical parameters of synaptic receptors from macroscopic currents.

Dendritic integration and neuronal firing patterns strongly depend on biophysical properties of synaptic ligand-gated channels. However, precise estimation of biophysical parameters of these channels in their intrinsic environment is complicated and still unresolved problem. Here we describe a novel method based on a maximum likelihood approach that allows to estimate not only the unitary current of synaptic receptor channels but also their multiple conductance levels, kinetic constants, the number of receptors bound with a neurotransmitter, and the peak open probability from experimentally feasible number of postsynaptic currents. The new method also improves the accuracy of evaluation of unitary current as compared to the peak-scaled non-stationary fluctuation analysis, leading to a possibility to precisely estimate this important parameter from a few postsynaptic currents recorded in steady-state conditions. Estimation of unitary current with this method is robust even if postsynaptic currents are generated by receptors having different kinetic parameters, the case when peak-scaled non-stationary fluctuation analysis is not applicable. Thus, with the new method, routinely recorded postsynaptic currents could be used to study the properties of synaptic receptors in their native biochemical environment.

Nociceptive neurons differentially express fast and slow T-type Ca²âº currents in different types of diabetic neuropathy.

T-type Ca²âº channels are known as important participants of nociception and their remodeling contributes to diabetes-induced alterations of pain sensation. In this work we have established that about 30% of rat nonpeptidergic thermal C-type nociceptive (NTCN) neurons of segments L4-L6 express a slow T-type Ca²âº current (T-current) while a fast T-current is expressed in the other 70% of these neurons. Streptozotocin-induced diabetes in young rats resulted in thermal hyperalgesia, hypoalgesia, or normalgesia 5-6 weeks after the induction. Our results show that NTCN neurons obtained from hyperalgesic animals do not express the slow T-current. Meanwhile, the fraction of neurons expressing the slow T-current did not significantly change in the hypo- and normalgesic diabetic groups. Moreover, the peak current density of fast T-current was significantly increased only in the neurons of hyperalgesic group. In contrast, the peak current density of slow T-current was significantly decreased in the hypo- and normalgesic groups. Experimental diabetes also resulted in a depolarizing shift of steady-state inactivation of fast T-current in the hyperalgesic group and slow T-current in the hypo- and normalgesic groups. We suggest that the observed changes may contribute to expression of different types of peripheral diabetic neuropathy occurring during the development of diabetes mellitus.

Different pools of postsynaptic GABAA receptors mediate inhibition evoked by low- and high-frequency presynaptic stimulation at hippocampal synapses.

Patterns of short-term synaptic plasticity could considerably differ between synapses of the same axon. This may lead to separation of synaptic receptors transmitting either low- or high-frequency signals and, therefore, may have functional consequences for the information transfer in the brain. Here, we estimated a degree of such separation at hippocampal GABAergic synapses using a use-dependent GABAA receptor antagonist, picrotoxin, to selectively suppress a pool of GABAA receptors monosynaptically activated during the low-frequency stimulation. The relative changes in postsynaptic responses evoked by the high-frequency stimulation before and after such block were used to estimate the contribution of this GABAA receptor pool to synaptic transmission at high frequencies. Using this approach, we have shown that IPSCs evoked by low-frequency (0.2 Hz) stimulation and asynchronous currents evoked by high-frequency (20-40 Hz) stimulation are mediated by different pools of postsynaptic GABAA receptors. Thus, our findings suggest that inhibition produced by a single hippocampal interneuron may be selectively routed to different postsynaptic targets depending on the presynaptic discharge frequency.

Specific functioning of Cav3.2 T-type calcium and TRPV1 channels under different types of STZ-diabetic neuropathy.

Streptozotocin (STZ)-induced type 1 diabetes in rats leads to the development of peripheral diabetic neuropathy (PDN) manifested as thermal hyperalgesia at early stages (4th week) followed by hypoalgesia after 8weeks of diabetes development. Here we found that 6-7 week STZ-diabetic rats developed either thermal hyper- (18%), hypo- (25%) or normalgesic (57%) types of PDN. These developmentally similar diabetic rats were studied in order to analyze mechanisms potentially underlying different thermal nociception. The proportion of IB4-positive capsaicin-sensitive small DRG neurons, strongly involved in thermal nociception, was not altered under different types of PDN implying differential changes at cellular and molecular level. We further focused on properties of T-type calcium and TRPV1 channels, which are known to be involved in Ca(2+) signaling and pathological nociception. Indeed, TRPV1-mediated signaling in these neurons was downregulated under hypo- and normalgesia and upregulated under hyperalgesia. A complex interplay between diabetes-induced changes in functional expression of Cav3.2 T-type calcium channels and depolarizing shift of their steady-state inactivation resulted in upregulation of these channels under hyper- and normalgesia and their downregulation under hypoalgesia. As a result, T-type window current was increased by several times under hyperalgesia partially underlying the increased resting [Ca(2+)]i observed in the hyperalgesic rats. At the same time Cav3.2-dependent Ca(2+) signaling was upregulated in all types of PDN. These findings indicate that alterations in functioning of Cav3.2 T-type and TRPV1 channels, specific for each type of PDN, may underlie the variety of pain syndromes induced by type 1 diabetes.

Efficient maximum likelihood estimation of kinetic rate constants from macroscopic currents.

A new method is described that accurately estimates kinetic constants, conductance and number of ion channels from macroscopic currents. The method uses both the time course and the strength of correlations between different time points of macroscopic currents and utilizes the property of semiseparability of covariance matrix for computationally efficient estimation of current likelihood and its gradient. The number of calculation steps scales linearly with the number of channel states as opposed to the cubic dependence in a previously described method. Together with the likelihood gradient evaluation, which is almost independent of the number of model parameters, the new approach allows evaluation of kinetic models with very complex topologies. We demonstrate applicability of the method to analysis of synaptic currents by estimating accurately rate constants of a 7-state model used to simulate GABAergic macroscopic currents.