Abnormal larval neuromuscular junction morphology and physiology in Drosophila Prickle isoform mutants with defective axonal transport and adult seizure behavior

Previous studies have demonstrated that mutations of the Drosophila planar cell polarity gene prickle (pk) result in altered microtubule-mediated vesicular transport in larval motor axons, as well as adult neuronal circuit hyperexcitability and epileptic behavior. It is also known that mutant alleles of the prickle-prickle (pkpk) and prickle-spiny-legs (pksple) isoforms differ in phenotype but display isoform counterbalancing effects in heteroallelic pkpk/pksple flies to ameliorate adult motor circuit and behavioral hyperexcitability. We have further investigated the larval neuromuscular junction (NMJ) and uncovered robust phenotypes in both pkpk and pksple alleles (heretofore referred to as pk and sple alleles, respectively), including synaptic terminal overgrowth, as well as irregular motor axon terminal excitability, poor vesicle release synchronicity, and altered efficacy of synaptic transmission. We observed significant increase in whole-cell excitatory junctional potential (EJP) in pk homozygotes, which was restored to near WT level in pk/sple heterozygotes. We further examined motor terminal excitability sustained by presynaptic Ca2+ channels, under the condition of pharmacological blockade of Na+ and K+ channel function. Such manipulation revealed extreme Ca2+ channel-dependent nerve terminal excitability in both pk and sple mutants. However, when combined in pk/sple heterozygotes, such terminal hyper-excitability was restored to nearly normal. Focal recording from individual synaptic boutons revealed asynchronous vesicle release in both pk and sple homozygotes, which nevertheless persisted in pk/sple heterozygotes without indications of isoform counter-balancing effects. Similarly, the overgrowth at NMJs was not compensated in pk/sple heterozygotes, exhibiting an extremity comparable to that in pk and sple homozygotes. Our observations uncovered differential roles of the pk and sple isoforms and their distinct interactions in the various structural and functional aspects of the larval NMJ and adult neural circuits.

Investigations into modifications of neuromuscular physiology by axonal transport disruptions in Drosophila SOD1 mutants

Cu/Zn superoxide dismutase (SOD1) is a cytoplasmic antioxidant enzyme, which, when mutant in humans, is linked to familial cases of the motor neurodegenerative disease amyotrophic lateral sclerosis (ALS). The Drosophila SOD1 gene (Sod) shares a highly conserved sequence with the human homolog, and this study includes examinations of the established hypomorphic n108 allele (Sodn108), alongside a knock-in construct of the G85R allele found in human ALS patients (SodG85R). In addition to previously documented decreased adult lifespan and attenuated motor function, we show that Sod mutant Drosophila can display significant mortality during larval and pupal development. Immunostaining of neuronal membrane at neuromuscular synapses in Sod mutant larvae revealed presynaptic terminals of abnormal morphology, with incompletely segregated and enlarged synaptic boutons along the motor terminal branches, in which vital staining indicated mitochondrial aggregation. We demonstrate strong genetic interactions between SodG85R and the axon transport-linked Pk mutants PkPk and PkSple in larval NMJ morphology and neuromuscular transmission. Intracellular recordings of larval excitatory junction potentials (EJPs) demonstrate enhanced EJP size in the double-mutant of PkPk and SodG85R. To examine synaptic terminal excitability, maintained by Ca2+ channel action and independent of Na+ channel function, we used the NaV blocker TTX, along with the KV1 blocker 4-aminopyridine (4-AP) and the commonly used broad-spectrum K+ channel blocker tetraethylammonium (TEA). We were able to induce prolonged “plateau-like” EJPs, which were further extended in Pk mutants and Pk;Sod double-mutants. These observations were corroborated with focal EJP recording from individual boutons. Altogether, this study highlights alterations in synaptic morphology and function at a developmental stage prior to neurodegeneration and death of Sod mutant organisms, along with a potential role of axonal transport in the maintenance of neuronal health.

Ultrastructural view of astrocyte-astrocyte and astrocyte-synapse contacts within the hippocampus

SummaryAstrocytes branch out and make contact at their interfaces. However, the ultrastructural interactions of astrocytes and astrocytes with their surroundings, including the spatial-location selectivity of astrocyte-synapse contacts, remain unknown. Here, the branching architecture of three neighboring astrocytes, their contact interfaces, and their surrounding neurites and synapses have been traced and 3D reconstructed using serial block-face scanning electron microscopy (SBF-SEM). Our reconstructions reveal extensive reflexive, loop-like processes that serve as scaffolds to neurites and give rise to spongiform astrocytic morphology. At the astrocyte-astrocyte interface, a cluster of process-process contacts were identified, which biophysically explains the existence of low inter-astrocytic electrical resistance. Additionally, we found that synapses uniformly made contact with the entire astrocyte, from soma to terminal processes, and can be ensheathed by two neighboring astrocytes. Lastly, in contrast to densely packed vesicles at the synaptic boutons, vesicle-like structures were scant within astrocytes. Together, these ultrastructural details should expand our understanding of functional astrocyte-astrocyte and astrocyte-neuron interactions.

Synaptic Organization of the Human Temporal Lobe Neocortex as Revealed by High-Resolution Transmission, Focused Ion Beam Scanning, and Electron Microscopic Tomography

Modern electron microscopy (EM) such as fine-scale transmission EM, focused ion beam scanning EM, and EM tomography have enormously improved our knowledge about the synaptic organization of the normal, developmental, and pathologically altered brain. In contrast to various animal species, comparably little is known about these structures in the human brain. Non-epileptic neocortical access tissue from epilepsy surgery was used to generate quantitative 3D models of synapses. Beside the overall geometry, the number, size, and shape of active zones and of the three functionally defined pools of synaptic vesicles representing morphological correlates for synaptic transmission and plasticity were quantified. EM tomography further allowed new insights in the morphological organization and size of the functionally defined readily releasable pool. Beside similarities, human synaptic boutons, although comparably small (approximately 5 µm), differed substantially in several structural parameters, such as the shape and size of active zones, which were on average 2 to 3-fold larger than in experimental animals. The total pool of synaptic vesicles exceeded that in experimental animals by approximately 2 to 3-fold, in particular the readily releasable and recycling pool by approximately 2 to 5-fold, although these pools seemed to be layer-specifically organized. Taken together, synaptic boutons in the human temporal lobe neocortex represent unique entities perfectly adapted to the “job” they have to fulfill in the circuitry in which they are embedded. Furthermore, the quantitative 3D models of synaptic boutons are useful to explain and even predict the functional properties of synaptic connections in the human neocortex.

Synaptic plasticity induced by differential manipulation of tonic and phasic motoneurons in Drosophila

Structural and functional plasticity induced by neuronal competition is a common feature of developing nervous systems. However, the rules governing how postsynaptic cells differentiate between presynaptic inputs are unclear. In this study we characterized synaptic interactions following manipulations of Ib tonic or Is phasic glutamatergic motoneurons that co-innervate postsynaptic muscles at Drosophila neuromuscular junctions (NMJs). After identifying drivers for each neuronal subtype, we performed ablation or genetic manipulations to alter neuronal activity and examined the effects on synaptic innervation and function. Ablation of either Ib or Is resulted in decreased muscle response, with some functional compensation occurring in the tonic Ib input when Is was missing. In contrast, the phasic Is terminal failed to show functional or structural changes following loss of the co-innervating Ib input. Decreasing the activity of the Ib or Is neuron with tetanus toxin light chain resulted in structural changes in muscle innervation. Decreased Ib activity resulted in reduced active zone (AZ) number and decreased postsynaptic subsynaptic reticulum (SSR) volume, with the emergence of filopodial-like protrusions from synaptic boutons of the Ib input. Decreased Is activity did not induce structural changes at its own synapses, but the co-innervating Ib motoneuron increased the number of synaptic boutons and AZs it formed. These findings indicate tonic and phasic neurons respond independently to changes in activity, with either functional or structural alterations in the tonic motoneuron occurring following ablation or reduced activity of the co-innervating phasic input, respectively.Significance StatementBoth invertebrate and vertebrate nervous systems display synaptic plasticity in response to behavioral experiences, indicating underlying mechanisms emerged early in evolution. How specific neuronal classes innervating the same postsynaptic target display distinct types of plasticity is unclear. Here, we examined if Drosophila tonic Ib and phasic Is motoneurons display competitive or cooperative interactions during innervation of the same muscle, or compensatory changes when the output of one motoneuron is altered. We established a system to differentially manipulate the motoneurons and examined the effects of cell-type specific changes to one of the inputs. Our findings indicate Ib and Is motoneurons respond differently to activity mismatch or loss of the co-innervating input, with the tonic subclass responding robustly compared to phasic motoneurons.

Synapses: Multitasking Global Players in the Brain

Synapses are key elements in the communication between neurons in any given network of the normal adult, developmental and pathologically altered brain. Synapses are composed of nearly the same structural subelements: a presynaptic terminal containing mitochondria with an ultrastructurally visible density at the pre- and postsynaptic apposition zone. The presynaptic density is composed of a cocktail of various synaptic proteins involved in the binding, priming and docking of synaptic vesicles inducing synaptic transmission. Individual presynaptic terminals (synaptic boutons) contain a couple of hundred up to thousands of synaptic vesicles. The pre- and postsynaptic densities are separated by a synaptic cleft. The postsynaptic density, also containing various synaptic proteins and more importantly various neurotransmitter receptors and their subunits specifically composed and arranged at individual synaptic complexes, reside at the target structures of the presynaptic boutons that could be somata, dendrites, spines or initial segments of axons.Beside the importance of the network in which synapses are integrated, their individual structural composition critically determines the dynamic properties within a given connection or the computations of the entire network, in particular, the number, size and shape of the active zone, the structural equivalent to a functional neurotransmitter release site, together with the size and organization of the three functionally defined pools of synaptic vesicles, namely the readily releasable, the recycling and the resting pool, are important structural subelements governing the ‘behavior’ of synaptic complexes within a given network such as the cortical column.In the late last century, neuroscientists started to generate quantitative 3D-models of synaptic boutons and their target structures that is one possible way to correlate structure with function, thus allowing reliable predictions about their function. The re-introduction of electron microscopy (EM) as an important tool achieved by modern high-end, high-resolution transmission-EM, focused ion beam scanning-EM, CRYO-EM and EM-tomography have enormously improved our knowledge about the synaptic organization of the brain not only in various animal species, but also allowed new insights in the ‘microcosms’ of the human brain in health and disease.

O-GlcNAcase contributes to cognitive function in Drosophila

O-GlcNAcylation is an abundant post-translational modification in neurons. In mice, an increase in O-GlcNAcylation leads to defects in hippocampal synaptic plasticity and learning. O-GlcNAcylation is established by two opposing enzymes: O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). To investigate the role of OGA in elementary learning, we generated catalytically inactive and precise knockout Oga alleles (OgaD133N and OgaKO, respectively) in Drosophila melanogaster. Adult OgaD133N and OgaKO flies lacking O-GlcNAcase activity showed locomotor phenotypes. Importantly, both Oga lines exhibited deficits in habituation, an evolutionarily conserved form of learning, highlighting that the requirement for O-GlcNAcase activity for cognitive function is preserved across species. Loss of O-GlcNAcase affected a number of synaptic boutons at the axon terminals of larval neuromuscular junction. Taken together, we report behavioral and neurodevelopmental phenotypes associated with Oga alleles and show that Oga contributes to cognition and synaptic morphology in Drosophila.