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Eukaryotic cells maintain proteostasis through mechanisms that require cytoplasmic and mitochondrial translation. Genetic defects affecting cytoplasmic translation perturb synapse development, neurotransmission, and are causative of neurodevelopmental disorders, such as Fragile X syndrome. In contrast, there is little indication that mitochondrial proteostasis, either in the form of mitochondrial protein translation and/or degradation, is required for synapse development and function. Here we focus on two genes deleted in a recurrent copy number variation causing neurodevelopmental disorders, the 22q11.2 microdeletion syndrome. We demonstrate that SLC25A1 and MRPL40, two genes present in the microdeleted segment and whose products localize to mitochondria, interact and are necessary for mitochondrial ribosomal integrity and proteostasis. Our Drosophila studies show that mitochondrial ribosome function is necessary for synapse neurodevelopment, function, and behavior. We propose that mitochondrial proteostasis perturbations, either by genetic or environmental factors, are a pathogenic mechanism for neurodevelopmental disorders. SIGNIFICANCE STATEMENT The balance between cytoplasmic protein synthesis and degradation, or cytoplasmic proteostasis, is required for normal synapse function and neurodevelopment. Cytoplasmic and mitochondrial ribosomes are necessary for two compartmentalized, yet interdependent, forms of proteostasis. Proteostasis dependent on cytoplasmic ribosomes is a well-established target of genetic defects that cause neurodevelopmental disorders, such as autism. Here we show that the mitochondrial ribosome is a neurodevelopmentally regulated organelle whose function is required for synapse development and function. We propose that defective mitochondrial proteostasis is a mechanism with the potential to contribute to neurodevelopmental disease.

A common electrophysiology technique used in neuroscience is patch clamp: a method in which a glass pipette electrode facilitates single cell electrical recordings from neurons. Typically, patch clamp is done manually in which an electrophysiologist views a brain slice under a microscope, visually selects a neuron to patch, and moves the pipette into close proximity to the cell to break through and seal its membrane. While recent advances in the field of patch clamping have enabled partial automation, the task of detecting a healthy neuronal soma in acute brain tissue slices is still a critical step that is commonly done manually, often presenting challenges for novices in electrophysiology. To overcome this obstacle and progress towards full automation of patch clamp, we combined the differential interference microscopy optical technique with an object detection-based convolutional neural network (CNN) to detect healthy neurons in acute slice. Utilizing the YOLOv3 convolutional neural network architecture, we achieved a 98% reduction in training times to 18 min, compared to previously published attempts. We also compared networks trained on unaltered and enhanced images, achieving up to 77% and 72% mean average precision, respectively. This novel, deep learning-based method accomplishes automated neuronal detection in brain slice at 18 frames per second with a small data set of 1138 annotated neurons, rapid training time, and high precision. Lastly, we verified the health of the identified neurons with a patch clamp experiment where the average access resistance was 29.25 M$$\Omega$$ Ω (n = 9). The addition of this technology during live-cell imaging for patch clamp experiments can not only improve manual patch clamping by reducing the neuroscience expertise required to select healthy cells, but also help achieve full automation of patch clamping by nominating cells without human assistance.

In Alzheimer's disease (AD), a multitude of genetic risk factors and early biomarkers are known. Nevertheless, the causal factors responsible for initiating cognitive decline in AD remain controversial. Toxic plaques and tangles correlate with progressive neuropathology, yet disruptions in circuit activity emerge before their deposition in AD models and patients. Parvalbumin (PV) interneurons are potential candidates for dysregulating cortical excitability as they display altered action potential (AP) firing before neighboring excitatory neurons in prodromal AD. Here, we report a novel mechanism responsible for PV hypoexcitability in young adult familial AD mice. We found that biophysical modulation of K

Motor learning involves neural circuit modifications in the cerebellar cortex, likely through re-weighting of parallel fiber inputs onto Purkinje cells (PCs). Climbing fibers instruct these synaptic modifications when they excite PCs in conjunction with parallel fiber activity, a pairing that enhances climbing fiber-evoked Ca2+ signaling in PC dendrites. In vivo, climbing fibers spike continuously, including during movements when parallel fibers are simultaneously conveying sensorimotor information to PCs. Whether parallel fiber activity enhances climbing fiber Ca2+ signaling during motor behaviors is unknown. In mice, we found that inhibitory molecular layer interneurons (MLIs), activated by parallel fibers during practiced movements, suppressed parallel fiber enhancement of climbing fiber Ca2+ signaling in PCs. Similar results were obtained in acute slices for brief parallel fiber stimuli. Interestingly, more prolonged parallel fiber excitation revealed latent supralinear Ca2+ signaling. Therefore, the balance of parallel fiber and MLI input onto PCs regulates concomitant climbing fiber Ca2+ signaling.

In many neurons, subthreshold depolarization in the soma can transiently increase action potential (AP)-evoked neurotransmission via analog-to-digital facilitation. The mechanisms underlying this form of short-term synaptic plasticity are unclear, in part, due to the relative inaccessibility of the axon to direct physiological interrogation. Using voltage imaging and patch-clamp recording from presynaptic boutons of cerebellar stellate interneurons, we observed that depolarizing somatic potentials readily spread into the axon, resulting in AP broadening, increased spike-evoked Ca2+ entry, and enhanced neurotransmission strength. Kv3 channels, which drive AP repolarization, rapidly inactivated upon incorporation of Kv3.4 subunits. This leads to fast susceptibility to depolarization-induced spike broadening and analog facilitation independent of Ca2+-dependent protein kinase C signaling. The spread of depolarization into the axon was attenuated by hyperpolarization-activated currents (Ih) in the maturing cerebellum, precluding analog facilitation. These results suggest that analog-to-digital facilitation is tempered by development or experience in stellate cells.

Excitatory amino acid transporters (EAATs) are responsible for extracellular glutamate uptake within the retina, and are expressed by retinal neurons and Muller cells. Their role within glutamatergic synapses is not completely understood. In the salamander retina, five distinct EAAT-encoding genes have been cloned, making the amphibian retina an excellent system to study EAAT function. This study focused on sEAAT2, which is expressed in photoreceptor terminals and Off-bipolar cells in two isoforms, sEAAT2A and sEAAT2B. Using whole-cell patch-clamp recording, florescence imaging and antibody labelling methods, we systematically studied the functions of these two isoforms at the synapse between photoreceptors and bipolar cells, both in dark and with photic stimulation. Both sEAAT2A and sEAAT2B were sensitive to dihydrokainic acid (DHKA), a known EAAT2-specific inhibitor. Each isoform of sEAAT2 was found to play a role in tonic glutamate uptake at the cone synapse in darkness. Furthermore, presynaptic sEAAT2A strongly suppressed the rapid, transient glutamate signal from cones following light-offset. This was achieved by quickly binding exocytosed glutamate, which subsequently limited glutamate spillover to adjacent receptors at postsynaptic sites. Since the intensity and duration of photic stimulation determine the magnitude of these cone transient signals, we postulate that presynaptic cone EAATs contribute to the encoding of contrast sensitivity in cone vision.

Purkinje cell dendrites convert excitatory climbing fiber input into signals that instruct plasticity and motor learning. Modulation of instructive signaling may increase the range in which learning is encoded, yet the mechanisms that allow for this are poorly understood. We found that optogenetic activation of molecular layer interneurons (MLIs) that inhibit Purkinje cells suppressed climbing-fiber-evoked dendritic Ca2+ spiking. Inhibitory suppression of Ca2+ spiking depended on the level of MLI activation and influenced the induction of associative synaptic plasticity, converting climbing-fiber-mediated potentiation of parallel fiber-evoked responses into depression. In awake mice, optogenetic activation of floccular climbing fibers in association with head rotation produced an adaptive increase in the vestibulo-ocular reflex (VOR). However, when climbing fibers were co-activated with MLIs, adaptation occurred in the opposite direction, decreasing the VOR. Thus, MLIs can direct a continuous spectrum of plasticity and learning through their influence on Purkinje cell dendritic Ca2+ signaling. MOE (Min. of Education, S’pore)

In axons, an action potential (AP) is thought to be broadcast as an unwavering binary pulse over its arbor driving neurotransmission uniformly at release sites. Yet, by recording from axons of cerebellar stellate cell (SC) interneurons, we show that AP width varies between presynaptic bouton sites, even within the same axon branch. The varicose geometry of SC boutons, alone, does not impose differences in spike duration. Rather, axonal patching revealed heterogeneous peak conductance densities of currents mediated mainly by fast-activating Kv3-type potassium channels, with clustered hot-spots at boutons and restricted expression at adjoining shafts. Blockade of Kv channels at individual boutons indicates that currents immediately local to a release site directs spike repolarization at that location. Thus, the clustered arrangement and variable expression density of Kv3 channels at boutons are key determinants underlying compartmentalized control of AP width in a near synapse-by-synapse manner, multiplying the signaling capacity of these structures.

The discrete arrangement of voltage-gated K+(Kv) channels in axons may impart functional advantages in action potential (AP) signaling yet, in compact cell types, the organization of Kvchannels is poorly understood. We find that in cerebellar stellate cell interneurons of mice, the composition and influence of Kvchannels populating the axon is diverse and depends on location allowing axonal compartments to differentially control APs in a local manner. Kv1 channels determine AP repolarization at the spike initiation site but not at more distal sites, limiting the expression of use-dependent spike broadening to the most proximal axon region, likely a key attribute informing spiking phenotype. Local control of AP repolarization at presynaptic boutons depends on Kv3 channels keeping APs brief, thus limiting Ca2+influx and synaptic strength. These observations suggest that AP repolarization is tuned by the local influence of distinct Kvchannel types, and this organization enhances the functional segregation of axonal compartments.

Taurine activates not only Cl(-)-permeable ionotropic receptors but also receptors that mediate metabotropic responses. The metabotropic property of taurine was revealed in electrophysiological recordings obtained after fully blocking Cl(-)-permeable receptors with an inhibitory "cocktail" consisting of picrotoxin, SR95531, and strychnine. We found that taurine's metabotropic effects regulate voltage-gated channels in retinal neurons. After applying the inhibitory cocktail, taurine enhanced delayed outward rectifier K(+) channels preferentially in Off-bipolar cells, and the effect was completely blocked by the specific PKC inhibitor, GF109203X. Additionally, taurine also acted through a metabotropic pathway to suppress both L- and N-type Ca(2+) channels in retinal neurons, which were insensitive to the potent GABA(B) receptor inhibitor, CGP55845. This study reinforces our previous finding that taurine in physiological concentrations produces a multiplicity of metabotropic effects that precisely govern the integration of signals being transmitted from the retina to the brain.