Axonal insulation is critical for efficient action potential propagation and normal functioning of the nervous system. In Drosophila, the underlying basis of nerve ensheathment is the axonal insulation by glial cells and the establishment of septate junctions (SJs) between glial cell membranes. However, the details of the cellular and molecular mechanisms underlying axonal insulation and SJ formation are still obscure. This study reports the characterization of axonal insulation in the Drosophila peripheral nervous system (PNS). Targeted expression of tau-green fluorescent protein in the glial cells and ultrastructural analysis of the peripheral nerves allow visualization the glial ensheathment of axons. Individual or a groups of axons are ensheathed by inner glial processes, which in turn are ensheathed by the outer perineurial glial cells. SJs are formed between the inner and outer glial membranes. Neurexin IV, Contactin, and Neuroglian are coexpressed in the peripheral glial membranes and these proteins exist as a complex in the Drosophila nervous system. Mutations in neurexin IV, contactin, and neuroglian result in the disruption of blood-nerve barrier function in the PNS, and ultrastructural analyses of the mutant embryonic peripheral nerves show loss of glial SJs. Interestingly, the murine homologs of Neurexin IV, Contactin, and Neuroglian are expressed at the paranodal SJs and play a key role in axon-glial interactions of myelinated axons. Together, these data suggest that the molecular machinery underlying axonal insulation and axon-glial interactions may be conserved across species (Banerjee, 2006a).

The localization of Nrx IV, Cont, and Nrg in the embryonic nervous system was studied. Nrx IV, Nrg, and Cont show colocalization at the nerve glial membranes. Nrx IV, Cont, and Nrg are interdependent for their epithelial SJ localization. Having established that Nrx IV, Cont, and Nrg colocalize in the peripheral nerves, whether these proteins are interdependent for their localization in the peripheral nerves was studied. The effect of the absence of each of these three proteins on the localization of the other two was studied in the embryonic peripheral nerves using nrx IV, cont, and nrg null mutants. nrx IV mutant embryos were stained with anti-Nrx IV, anti-Cont, and anti-Nrg. Cont and Nrg proteins showed a rather diffused localization when compared with their wild-type localization. In addition, Cont is present as puncta in the cytoplasm of the glial cells because of its failure to get targeted properly to the membrane in the absence of Nrx IV. Similarly, cont null mutant embryos also showed less defined distribution and reduction of Nrx IV and Nrg in the glial membrane. nrg null mutant embryos display significant reduction in Nrx IV and Cont localization in the glial membranes. These results demonstrate that Nrx IV, Cont, and Nrg are interdependent for their proper localization in the embryonic peripheral nerves (Banerjee, 2006a).

Ultrastructural analyses of the embryonic epithelia in nrx IV, cont, and nrg mutants showed that these genes are required for the formation and/or organization of epithelial SJs. Nrx IV, Cont, and Nrg are interdependent for their proper localization both in the epithelia and in the peripheral nerves. These phenotypic similarities raised an interesting possibility that these proteins are part of a macromolecular protein complex that exists in the nervous system. To determine whether these three proteins form a biochemical complex in the nervous system, Drosophila heads, which are a rich source of both neurons and glial cells, were used. Coimmunoprecipitation experiments using Nrx IV, Cont, and Nrg antibodies efficiently coprecipitated Nrx IV, Cont, and Nrg. Interestingly, both isoforms of Nrg (180 kDa neuronal and 167 kDa epithelial) were immunoprecipitated by Nrx IV and Cont antibodies, suggesting that both isoforms are part of a protein complex that includes Nrx IV and Cont. However, at this point, it is not possible to differentiate whether isoform-specific complexes are formed or whether both isoforms are in the same complex. In addition, sucrose density gradient analysis of the fly head lysates was performed to determine how Nrx IV, Cont, and Nrg distribute in buoyant density gradients. Nrx IV and Cont cosediment in overlapping fractions. Nrg shows distribution in the lighter sucrose density fractions that do not overlap with Nrx IV and Cont but partially overlaps with that of Nrx IV and Cont, indicating that these proteins are associated with subcellular structures of the same density and may associate into a biochemical complex that is partially maintained during subcellular fractionation (Banerjee, 2006a).

The immunofluorescence analysis of nrx IV, cont, and nrg null mutants showed that in each of these mutants, the other two proteins show qualitatively reduced fluorescence intensities under identical confocal settings. The possible explanation for reduced fluorescence intensity could be that loss of any of these proteins affects the localization or stability of the other proteins. Whether the levels of the other two proteins had changed in nrx IV, cont, and nrg null mutants was examined using immunoblot analysis. Nrx IV protein levels did not seem to be affected in cont and nrg mutants when compared with wild type. Cont protein levels were severely affected in nrx IV mutants compared with wild-type and nrg mutant embryos. Nrg protein levels were also affected in nrx IV mutants but showed no change in cont mutants. At this stage, it cannot be rule out whether the change in Cont and Nrg protein levels in nrx IV mutants are attributable to reduced stability or less synthesis of these proteins. Together, the biochemical data indicate that Nrx IV, Cont, and Nrg form a protein complex in the nervous system and that, in the absence of Nrx IV, the stability of Cont and Nrg is severely affected (Banerjee, 2006a).

Inner glial membrane processes are involved in the ensheathment of either an individual axon or a group of axons. This glial ensheathment not only provides insulation of axons but also generates unique junctions between either glial membranes or between axons and glial membranes. Nrx IV, Cont, and Nrg are involved in the establishment of glial–glial SJs. Although additional components involved in these interactions need to be identified, these findings provide a basis for additional analysis of neuronal SJs, which would be relevant to the understanding of their vertebrate counterparts: the paranodal axo-glial SJs (Bhat, 2003; Hortsch, 2003; Salzer, 2003; Banerjee, 2006a and references therein).

The fundamental basis of axonal ensheathment in any species is to faithfully transmit neuronal signals along the nerve fibers and optimize desired cellular responses. To maximize the speed of conduction and/or to minimize the loss of nerve signals, many species evolved mechanisms in which axonal lengths remained short (as seen in insects) by increasing the diameter of the axons or by clustering voltage-gated Na⁺ channels to discrete unmyelinated regions of the axon, the node of Ranvier, as seen in myelinated nerve fibers of vertebrates. Most invertebrate species use some type of glial cells to ensheath their axons without generating a myelin sheath. The insulation is contiguous without any breaks, suggesting that primitive nodal structures or clustering of voltage-gated Na⁺ channels may not exist in invertebrates. However, recent reports have challenged some of these notions. In copepod crustaceans, ultrastructural analysis of the first antenna and the CNS has revealed extensive myelination of sensory and motor axons. These studies have raised some fundamental questions about axonal insulation and origins of myelination (Banerjee, 2006a and references therein).

In invertebrates like Drosophila, two types of glial cells are involved in insulation. The inner peripheral glial cells are involved in axonal ensheathment, and the outer (perineurial) glial cells wrap around the inner glial cells to provide another level of ensheathment. This two-cell ensheathment in Drosophila peripheral nerves may be advantageous to ensure that high K⁺ containing hemolymph does not interfere with action potential propagation. Although the cellular aspects of axonal insulation are being unraveled, the molecular mechanisms underlying the axonal ensheathment remain to be investigated. Most importantly, what are the protein constituents of the insulating membranes, and whether some of the vertebrate myelin proteins have their homologs in invertebrates? A detailed molecular analysis of the nature of the glial cells that ensheath axons as in Drosophila or produce myelin-like structures and the type of myelin in copepods may provide insights into whether myelinating glial cells arose from a common ancestor. However, a genetic dissection of the axonal ensheathment in Drosophila will uncover some of the basic aspects of the neuron-glial interactions that lead to ensheathment of nerve fibers across species (Banerjee, 2006a).

Cell adhesion molecules play a pivotal role in establishing intercellular junctions [e.g., cadherins and associated catenins form a protein scaffold that establish adhesion contacts at the adherens junctions and link them to the actin cytoskeleton. Similarly, claudins and associated cellular scaffolding proteins are required for establishing TJs. In both of these examples, transmembrane proteins bring two opposing membranes together to establish junctions through homophilic and/or heterophilic interactions (Banerjee, 2006a and references therein).

The finding that nrg null mutant nerves display increased spacing between the outer and inner glial membranes suggest that Nrg may be involved in cell–cell interactions and cell–cell adhesion between glial membranes. In contrast, the observation that loss of nrx IV and cont does not affect the membrane spacing between the inner and outer glial membranes suggests that Nrx IV and Cont are not involved in membrane adhesion or bringing the glial membranes in close apposition. Together, these data suggest that Nrg is critical for both the adhesion and SJ formation, whereas Nrx IV and Cont are critical for the formation of the septa at SJs. In addition, the missing axonal fascicles in nrg mutant nerves could result from axon fasciculation defects or axonal degeneration as a secondary consequence resulting from the loss of glial support. Axonal fasciculation defects have been observed in nrg mutants. Alternatively, axonal loss in nrg mutants might result from a disruption in axonal cytoskeleton, because Nrg possesses domains that could potentially interact with and stabilize the axonal cytoskeleton. Based on Nrg expression in S2 cells, Nrg is predicted to recruit membrane skeleton assembly within specialized domains of neurons in response to cell adhesion. Both Nrg protein forms Nrg¹⁶⁷ and Nrg¹⁸⁰ contain a short cytoplasmic domain as a binding site for ankyrin. Ankyrins are linker proteins that connect various membrane proteins with the actin-spectrin network in the cell. Loss of axons observed in nrg mutants is clearly suggestive of axon-glial interdependence that may alter axonal survival. In vertebrates, axolemmal-myelin interactions are critical for the formation of the paranodal axo-glial SJs (see Garcia-Fresco, 2006). This raises an interesting possibility that axon-glial interactions in Drosophila may use similar interactive mechanism for establishing axo-glial SJs. Thus, the current studies on Nrx IV, Cont, and Nrg suggest that these proteins are critical for the formation and/or organization of the SJs between either glial cells and possibly between axons and glial cells, which remains to be further investigated (Banerjee, 2006a).

Invertebrate axons are insulated from their salt-rich environment through a glial-dependent BBB, which plays a crucial role in electrical and chemical insulation. Ultrastructural studies have demonstrated SJs between perineurial glial cells and inner glial cell membrane to form the structural basis of BBB of some insects. Absence of SJs in nrx IV, cont, and nrg mutants, and a compromised BNB in the PNS as evidenced by dye exclusion analyses, provide additional confirmation in support of SJs as a prerequisite for blood-nerve barrier (BNB) formation. Not just in the PNS, recent reports on the BBB formation and function also in the CNS of Drosophila underscore the importance of SJs in proper sealing and insulation of the nerve cord (Schwabe, 2005), thereby supporting that insulation and establishment of functional SJs in both PNS and CNS go hand in hand. Although, G-protein-coupled receptor signaling pathway members have been identified to establish the BBB in Drosophila CNS, the signaling mechanisms that operate in the PNS still remain to be established (Banerjee, 2006a).

The axo-glial SJs in the myelinated axons share many anatomical features similar to those of invertebrate SJs, especially the electron-dense ladder-like transverse septa (Bhat, 2003; Banerjee, 2006a). Ensheathment of Drosophila axons by perineurial glial cells in the absence of myelin-producing glial cells would have predicted that the molecular components of the invertebrate SJs would be different from those of the vertebrate axo-glial SJs. Surprisingly, the fly SJ molecular components are present at the vertebrate axo-glial SJs and not at the TJs, which serve similar functions. Thus, the molecular similarities reflect an evolutionarily conserved function of creating an ionic barrier both at Drosophila SJs and at axo-glial SJs in the paranodal region. More importantly, Drosophila nerves contain a large number of axons, which are collectively held together by glial membranes not only to maintain nerve fasciculation but also to maintain the insulation of individual axons. It would be of significant interest to establish whether axo-glial SJs are present in Drosophila nerves and to identify downstream components of these junctions in Drosophila that link these junctions to the glial or axonal cytoskeleton. Identification of such molecules will provide insights into whether these junctions play a much broader role in axon-glial signal transduction. In summary, the molecular and functional similarities between the Drosophila SJs and vertebrate axo-glial SJs should allow a genetic and molecular dissection to be undertaken of the formation and function of these junctions in Drosophila (Banerjee, 2006a).

Neurexins have been proposed to function as major mediators of the coordinated pre- and postsynaptic apposition. However, key evidence for this role in vivo has been lacking, particularly due to gene redundancy. Null mutations have been obtained in the single Drosophila Neurexin gene. Nrx loss of function prevents the normal proliferation of synaptic boutons at glutamatergic neuromuscular junctions, while Nrx gain of function in neurons has the opposite effect. Nrx mostly localizes to the active zone of presynaptic terminals. Conspicuously, Nrx null mutants display striking defects in synaptic ultrastructure, with the presence of detachments between pre- and postsynaptic membranes, abnormally long active zones, and increased number of T bars. These abnormalities result in corresponding alterations in synaptic transmission with reduced quantal content. Together, these results provide compelling evidence for an in vivo role of neurexins in the modulation of synaptic architecture and adhesive interactions between pre- and postsynaptic compartments (Li, 2007).

Although cell adhesion molecules have long been postulated and in several cases have been shown to be major participants in synapse development and plasticity, the impact of their function and the molecular mechanisms that they activate remain a puzzle. Particularly intriguing is the function of neurexins, which may provide clues to our understanding of synapse organization. Null mutants were isolated in the single Drosophila dnrx gene. Nrx mutants were shown to have striking abnormalities in synapse development and function. A recent study reported that Drosophila neurexin is required for synapse formation in the adult CNS (Zeng, 2007). The current study demonstrates a primary role of Nrx in regulating the formation of synapses, and also reveal the crucial role of Nrx in the proper development of active zones and regulating synaptic function in an intact organism, thus providing insights into understanding the function of neurexins in vivo (Li, 2007).

These studies provide compelling evidence that Nrx plays a prime role during the expansion of the NMJ and, in particular, in defining the cytoarchitecture of the active zones within synaptic boutons. (1) In Nrx mutants, synaptic bouton proliferation is severely disrupted, and therefore NMJ expansion is significantly stunted. (2) Nrx gain of function promotes the formation of new boutons in a gene-dosage-dependent manner. (3) The ultrastructural analyses show that presynaptic densities (PRDs) are not apposed normally to PSDs displaying signs of abnormal adhesion to the postsynaptic density (PSD), although every PRD is exactly juxtaposed to the PSD. (4) In Nrx mutants, critical components of the presynaptic compartment, such as synaptic vesicle proteins and active zone components, are ectopically localized within axons. (5) The distribution of GluRs at the PSD is abnormally large, although this phenotype may arise as a consequence of the presynaptic defects observed in Nrx mutants (Li, 2007).

The great majority of abnormal phenotypes in Nrx mutants could be completely rescued by expressing a wild-type Nrx transgene in neurons; although in some instances the rescue was partial. However, even in the later case, expressing Nrx in both muscles and neurons did not further improve the residual abnormalities, suggesting that Nrx functions primarily if not exclusively in the presynaptic compartment (Li, 2007).

The partial rescue of some of the phenotypes, such as the defects in mEJPs and the morphology of active zones, might be due to the high sensitivity of these processes to the right levels and correct temporal expression of Nrx, which is not completely mimicked by the UAS/Gal4 system. This view is supported by the observation that overexpression of Nrx in a wild-type background also decreased quantal content, suggesting that increased Nrx dosage may have detrimental effects on synapse structure and/or function. However, the data strongly support that the abnormal phenotypes arise from the lack of Nrx: (1) all our experiments were carried out in mutants over a deficiency chromosome in an independent genetic background; (2) a precise excision of the P element did not show any of the mutant phenotypes. Together these data establish a specific role for Nrx in proper synaptic development (Li, 2007).

One of the important findings of this study is that Nrx mutants displayed defective active zones with larger PRD, and especially containing regions of detachment from the PSD. These detachment sites implicate Nrx as a mediator of cell adhesion between the pre- and the postsynaptic cell,. While a complete detachment of active zones is not observed, Nrx mutants have a significant decrease in the number of boutons. This raises the possibility that the phenotypes observed are from those boutons that are maintained and that a more drastic consequence is a failure to form synaptic boutons. Nevertheless, the lack of complete detachment of active zones in Nrx null mutants suggests that Nrx, although an important synapse-organization molecule, is not sufficient for trans-synaptic cell adhesion (Li, 2007).

Another notable phenotype in Nrx mutants was the presence of enlarged PRDs and increased number of T bars. A major feature of Drosophila larval NMJ is its ability to compensate for decreased postsynaptic responses by upregulating neurotransmitter release. For instance, a decrease in the number of postsynaptic GluRs results in an increase in neurotransmitter release, thus maintaining the amplitude of evoked responses. It is plausible that the enlarged PRDs and increase in number of T bars in Nrx mutants are a compensatory mechanism to adjust for defective presynaptic cell adhesion and/or reduced neurotransmitter release. In support of this notion, in Nrx mutants there was a 50% decrease in synaptic bouton number, but this was accompanied by a 2-fold increase in the number of T bars, such that the total number of T bars/NMJ remained constant, despite the changes in bouton number. Similarly, defective presynaptic cell adhesion and/or reduced neurotransmitter release could lead to an increase in GluR accumulation. In these studies, it was found that the length of the PSD was enlarged in Nrx mutants as well as the distribution of GluR clusters (Li, 2007).

The above structural abnormalities were accompanied by corresponding functional deficits. In Nrx mutants, the frequency of mEJPs was strikingly increased. Further, although the T bars were rescued by expression of a Nrx transgene, the length of the PRDs was not, and a similar lack of rescue was observed for mEJP frequency. Thus, there appears to be a notable correlation between the size of the PRD and spontaneous miniature excitatory potential (mEJP) frequency, perhaps due to increased probability of synaptic vesicle release with increased synapse size. In addition, a substantial increase was observed in mEJP amplitude. Two factors may contribute to this change; (1) the distribution of GluR clusters was enlarged, while the GluR intensity remained unchanged, suggesting that more GluRs were accumulated at mutant synapses; (2) an additional contributing factor is that mEJP frequency was increased, and instances of summation were observed (Li, 2007).

Overall, despite the increase in PRD size and the maintenance of overall T bar number, evoked events had a decrease in amplitude and quantal content. Recent studies have suggested that a major constituent of the T bars is Brunchpilot [BRP/CAST (Kittel, 2006; Wagh, 2006)]. In brp mutants, T bars fail to form, but PRDs appear unaltered. Further, although EJP amplitude is decreased, mEJP amplitude and frequency are normal. This has led to the model that T bars per se are not required for synaptic transmission but that they regulate the efficiency of transmission. In Nrx mutants, PRDs are disproportionately large, which could result in asynchronous release, leading to an EJP with decreased amplitude. It is also possible that the presynaptic membrane detachments observed in Nrx mutants could contribute to the functional impairment of neurotransmitter release (Li, 2007).

A recent study demonstrated that in Nrx mutant larvae associative learning is impaired in an olfactory choice paradigm (Zeng, 2007). However, in this study, larval locomotion was not assessed. The current study showing that locomotor behavior is impaired in Nrx mutants raises the possibility that the poor performance of mutant larvae in the conditioning assay might also result from the locomotor abnormalities. Zeng also reported that the number of T bars in the calyx of the mushroom bodies, the learning centers of the fly, was reduced in adult flies. In contrast, a significant increase was found in the number of T bar/bouton, and since Nrx mutants have fewer boutons, this translated in the maintenance of T bar number per NMJ. The differing results might be due to different mechanisms regulating T bar formation in the two tissues (Li, 2007).