The Wingless planar cell polarity pathway is essential for optimal activity-dependent synaptic plasticity.

From fly to man, the Wingless (Wg)/Wnt signaling molecule is essential for both the stability and plasticity of the nervous system. The Drosophila neuromuscular junction (NMJ) has proven to be a useful system for deciphering the role of Wg in directing activity-dependent synaptic plasticity (ADSP), which, in the motoneuron, has been shown to be dependent on both the canonical and the noncanonical calcium Wg pathways. Here we show that the noncanonical planar cell polarity (PCP) pathway is an essential component of the Wg signaling system controlling plasticity at the motoneuron synapse. We present evidence that disturbing the PCP pathway leads to a perturbation in ADSP. We first show that a PCP-specific allele of disheveled (dsh) affects the de novo synaptic structures produced during ADSP. We then show that the Rho GTPases downstream of Dsh in the PCP pathway are also involved in regulating the morphological changes that take place after repeated stimulation. Finally, we show that Jun kinase is essential for this phenomenon, whereas we found no indication of the involvement of the transcription factor complex AP1 (Jun/Fos). This work shows the involvement of the neuronal PCP signaling pathway in supporting ADSP. Because we find that AP1 mutants can perform ADSP adequately, we hypothesize that, upon Wg activation, the Rho GTPases and Jun kinase are involved locally at the synapse, in instructing cytoskeletal dynamics responsible for the appearance of the morphological changes occurring during ADSP.

A comparison of three different methods of eliciting rapid activity-dependent synaptic plasticity at the Drosophila NMJ.

The Drosophila NMJ is a system of choice for investigating the mechanisms underlying the structural and functional modifications evoked during activity-dependent synaptic plasticity. Because fly genetics allows considerable versatility, many strategies can be employed to elicit this activity. Here, we compare three different stimulation methods for eliciting activity-dependent changes in structure and function at the Drosophila NMJ. We find that the method using patterned stimulations driven by a K+-rich solution creates robust structural modifications but reduces muscle viability, as assessed by resting potential and membrane resistance. We argue that, using this method, electrophysiological studies that consider the frequency of events, rather than their amplitude, are the only reliable studies. We contrast these results with the expression of CsChrimson channels and red-light stimulation at the NMJ, as well as with the expression of TRPA channels and temperature stimulation. With both these methods we observed reliable modifications of synaptic structures and consistent changes in electrophysiological properties. Indeed, we observed a rapid appearance of immature boutons that lack postsynaptic differentiation, and a potentiation of spontaneous neurotransmission frequency. Surprisingly, a patterned application of temperature changes alone is sufficient to provoke both structural and functional plasticity. In this context, temperature-dependent TRPA channel activation induces additional structural plasticity but no further increase in the frequency of spontaneous neurotransmission, suggesting an uncoupling of these mechanisms.

Cnr2 Is Important for Ribbon Synapse Maturation and Function in Hair Cells and Photoreceptors.

The role of the cannabinoid receptor 2 (CNR2) is still poorly described in sensory epithelia. We found strong cnr2 expression in hair cells (HCs) of the inner ear and the lateral line (LL), a superficial sensory structure in fish. Next, we demonstrated that sensory synapses in HCs were severely perturbed in larvae lacking cnr2. Appearance and distribution of presynaptic ribbons and calcium channels (Cav1.3) were profoundly altered in mutant animals. Clustering of membrane-associated guanylate kinase (MAGUK) in post-synaptic densities (PSDs) was also heavily affected, suggesting a role for cnr2 for maintaining the sensory synapse. Furthermore, vesicular trafficking in HCs was strongly perturbed suggesting a retrograde action of the endocannabinoid system (ECs) via cnr2 that was modulating HC mechanotransduction. We found similar perturbations in retinal ribbon synapses. Finally, we showed that larval swimming behaviors after sound and light stimulations were significantly different in mutant animals. Thus, we propose that cnr2 is critical for the processing of sensory information in the developing larva.

Rearing methods and life cycle characteristics of Chironomus sp. Florida (Chironomidae: Diptera): A rapid-developing species for laboratory studies.

The species Chironomus sp. "Florida" has several qualities that make it a potential aquatic laboratory model to be used in Puerto Rico. Its use as such, however, requires a rearing protocol and life cycle description not previously reported. The present study addresses this lack of information by first describing a rearing method obtained through three years of observations. Next we describe and discuss the life cycle and the effects of temperature and feeding on development. The species has a short life cycle (typically 11 days) and larval stages easily identified using body measurements. Temperature affects the duration of the life cycle, with warm temperatures producing faster development than cold temperatures. The effects of different food concentrations vary: in large water volumes, concentrations of 2 mg/larva/day produce faster developmental times, but at low water volumes, small food concentrations of 0.5 mg/larva/day produce faster developmental times. The rearing protocol and life cycle parameters presented in this study are intended to promote the use of this species as a laboratory model. The fast development of Chironomus sp. "Florida" makes it ideal for toxicological studies.

Cortactin Is a Regulator of Activity-Dependent Synaptic Plasticity Controlled by Wingless.

Major signaling molecules initially characterized as key early developmental regulators are also essential for the plasticity of the nervous system. Previously, the Wingless (Wg)/Wnt pathway was shown to underlie the structural and electrophysiological changes during activity-dependent synaptic plasticity at the Drosophila neuromuscular junction. A challenge remains to understand how this signal mediates the cellular changes underlying this plasticity. Here, we focus on the actin regulator Cortactin, a major organizer of protrusion, membrane mobility, and invasiveness, and define its new role in synaptic plasticity. We show that Cortactin is present presynaptically and postsynaptically at the Drosophila NMJ and that it is a presynaptic regulator of rapid activity-dependent modifications in synaptic structure. Furthermore, animals lacking presynaptic Cortactin show a decrease in spontaneous release frequency, and presynaptic Cortactin is necessary for the rapid potentiation of spontaneous release frequency that takes place during activity-dependent plasticity. Most interestingly, Cortactin levels increase at stimulated synaptic terminals and this increase requires neuronal activity, de novo transcription and depends on Wg/Wnt expression. Because it is not simply the presence of Cortactin in the presynaptic terminal but its increase that is necessary for the full range of activity-dependent plasticity, we conclude that it probably plays a direct and important role in the regulation of this process.SIGNIFICANCE STATEMENT In the nervous system, changes in activity that lead to modifications in synaptic structure and function are referred to as synaptic plasticity and are thought to be the basis of learning and memory. The secreted Wingless/Wnt molecule is a potent regulator of synaptic plasticity in both vertebrates and invertebrates. Understanding the molecular mechanisms that underlie these plastic changes is a major gap in our knowledge. Here, we identify a presynaptic effector molecule of the Wingless/Wnt signal, Cortactin. We show that this molecule is a potent regulator of modifications in synaptic structure and is necessary for the electrophysiological changes taking place during synaptic plasticity.

Rab8, POSH, and TAK1 regulate synaptic growth in a Drosophila model of frontotemporal dementia.

Mutations in genes essential for protein homeostasis have been identified in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) patients. Why mature neurons should be particularly sensitive to such perturbations is unclear. We identified mutations in Rab8 in a genetic screen for enhancement of an FTD phenotype associated with ESCRT-III dysfunction. Examination of Rab8 mutants or motor neurons expressing a mutant ESCRT-III subunit, CHMP2B(Intron5), at the Drosophila melanogaster neuromuscular junction synapse revealed synaptic overgrowth and endosomal dysfunction. Expression of Rab8 rescued overgrowth phenotypes generated by CHMP2B(Intron5). In Rab8 mutant synapses, c-Jun N-terminal kinase (JNK)/activator protein-1 and TGF-ß signaling were overactivated and acted synergistically to potentiate synaptic growth. We identify novel roles for endosomal JNK-scaffold POSH (Plenty-of-SH3s) and a JNK kinase kinase, TAK1, in regulating growth activation in Rab8 mutants. Our data uncover Rab8, POSH, and TAK1 as regulators of synaptic growth responses and point to recycling endosome as a key compartment for synaptic growth regulation during neurodegenerative processes.

Engrailed alters the specificity of synaptic connections of Drosophila auditory neurons with the giant fiber.

We show that a subset of sound-detecting Johnston's Organ neurons (JONs) in Drosophila melanogaster, which express the transcription factors Engrailed (En) and Invected (Inv), form mixed electrical and chemical synaptic inputs onto the giant fiber (GF) dendrite. These synaptic connections are detected by trans-synaptic Neurobiotin (NB) transfer and by colocalization of Bruchpilot-short puncta. We then show that misexpressing En postmitotically in a second subset of sound-responsive JONs causes them to form ectopic electrical and chemical synapses with the GF, in turn causing that postsynaptic neuron to redistribute its dendritic branches into the vicinity of these afferents. We also introduce a simple electrophysiological recording paradigm for quantifying the presynaptic and postsynaptic electrical activity at this synapse, by measuring the extracellular sound-evoked potentials (SEPs) from the antennal nerve while monitoring the likelihood of the GF firing an action potential in response to simultaneous subthreshold sound and voltage stimuli. Ectopic presynaptic expression of En strengthens the synaptic connection, consistent with there being more synaptic contacts formed. Finally, RNAi-mediated knockdown of En and Inv in postmitotic neurons reduces SEP amplitude but also reduces synaptic strength at the JON-GF synapse. Overall, these results suggest that En and Inv in JONs regulate both neuronal excitability and synaptic connectivity.

Adar is essential for optimal presynaptic function.

RNA editing is a powerful way to recode genetic information. Because it potentially affects RNA targets that are predominantly present in neurons, it is widely hypothesized to affect neuronal structure and physiology. Across phyla, loss of the enzyme responsible for RNA editing, Adar, leads to behavioral changes, impaired locomotion, neurodegeneration and death. However, the consequences of a loss of Adar activity on neuronal structure and function have not been studied in detail. In particular, the role of RNA editing on synaptic development and physiology has not been investigated. Here we test the physiological and morphological consequences of the lack of Adar activity on the Drosophila neuromuscular junction (NMJ). Our detailed examination of synaptic transmission showed that loss of Adar increases quantal size, reduces the number of quanta of neurotransmitter released and perturbs the calcium dependence of synaptic release. In addition, we find that staining for several synaptic vesicle proteins is abnormally intense at Adar deficient synapses. Consistent with this finding, Adar mutants showed a major alteration in synaptic ultrastructure. Finally, we present evidence of compensatory changes in muscle membrane properties in response to the changes in presynaptic activity within the Adar mutant NMJs.

Synaptic homeostasis is consolidated by the cell fate gene gooseberry, a Drosophila pax3/7 homolog.

In a large-scale screening effort, we identified the gene gooseberry (gsb) as being necessary for synaptic homeostasis at the Drosophila neuromuscular junction. The gsb gene encodes a pair-rule transcription factor that participates in embryonic neuronal cell fate specification. Here, we define a new postembryonic role for gooseberry. We show that gsb becomes widely expressed in the postembryonic CNS, including within mature motoneurons. Loss of gsb does not alter neuromuscular growth, morphology, or the distribution of essential synaptic proteins. However, gsb function is required postembryonically for the sustained expression of synaptic homeostasis. In GluRIIA mutant animals, miniature EPSP (mEPSP) amplitudes are significantly decreased, and there is a compensatory homeostatic increase in presynaptic release that restores normal muscle excitation. Loss of gsb significantly impairs the homeostatic increase in presynaptic release in the GluRIIA mutant. Interestingly, gsb is not required for the rapid induction of synaptic homeostasis. Furthermore, gsb seems to be specifically involved in the mechanisms responsible for a homeostatic increase in presynaptic release, since it is not required for the homeostatic decrease in presynaptic release observed following an increase in mEPSP amplitude. Finally, Gsb has been shown to antagonize Wingless signaling during embryonic fate specification, and we present initial evidence that this activity is conserved during synaptic homeostasis. Thus, we have identified a gene (gsb) that distinguishes between rapid induction versus sustained expression of synaptic homeostasis and distinguishes between the mechanisms responsible for homeostatic increase versus decrease in synaptic vesicle release.

Transcriptional control of behavior: engrailed knock-out changes cockroach escape trajectories.

The cerci of the cockroach are covered with identified sensory hairs that detect air movements. The sensory neurons that innervate these hairs synapse with giant interneurons in the terminal ganglion that in turn synapse with interneurons and leg motor neurons in thoracic ganglia. This neural circuit mediates the animal's escape behavior. The transcription factor Engrailed (En) is expressed only in the medially born sensory neurons, which suggested that it could work as a positional determinant of sensory neuron identity. Previously, we used double-stranded RNA interference to abolish En expression and found that the axonal arborization and synaptic outputs of an identified En-positive sensory neuron changed so that it came to resemble a nearby En-negative cell, which was itself unaffected. We thus demonstrated directly that En controls synaptic choice, as well as axon projections. Is escape behavior affected as a result of this miswiring? We showed recently that adult cockroaches keep each escape unpredictable by running along one of a set of preferred escape trajectories (ETs) at fixed angles from the direction of the threatening stimulus. The probability of selecting a particular ET is influenced by wind direction. In this present study, we show that early instar juvenile cockroaches also use those same ETs. En knock-out significantly perturbs the animals' perception of posterior wind, altering the choice of ETs to one more appropriate for anterior wind. This is the first time that it has been shown that knock-out of a transcription factor controlling synaptic connectivity can alter the perception of a directional stimulus.

Dap160/intersectin scaffolds the periactive zone to achieve high-fidelity endocytosis and normal synaptic growth.

Dap160/Intersectin is a multidomain adaptor protein that colocalizes with endocytic machinery in the periactive zone at the Drosophila NMJ. We have generated severe loss-of-function mutations that eliminate Dap160 protein from the NMJ. dap160 mutant synapses have decreased levels of essential endocytic proteins, including dynamin, endophilin, synaptojanin, and AP180, while other markers of the active zone and periactive zone are generally unaltered. Functional analyses demonstrate that dap160 mutant synapses are unable to sustain high-frequency transmitter release, show impaired FM4-64 loading, and show a dramatic increase in presynaptic quantal size consistent with defects in synaptic vesicle recycling. The dap160 mutant synapse is grossly malformed with abundant, highly ramified, small synaptic boutons. We present a model in which Dap160 scaffolds both endocytic machinery and essential synaptic signaling systems to the periactive zone to coordinately control structural and functional synapse development.

FGF-2 modulates expression and distribution of GAP-43 in frog retinal ganglion cells after optic nerve injury.

Basic fibroblast growth factor (bFGF or FGF-2) has been implicated as a trophic factor that promotes survival and neurite outgrowth of neurons. We found previously that application of FGF-2 to the proximal stump of the injured axon increases retinal ganglion cell (RGC) survival. We determine here the effect of FGF-2 on expression of the axonal growth-associated phosphoprotein (GAP)-43 in retinal ganglion cells and tectum of Rana pipiens during regeneration of the optic nerve. In control retinas, GAP-43 protein was found in the optic fiber layer and in optic nerve; mRNA levels were low. After axotomy, mRNA levels increased sevenfold and GAP-43 protein was significantly increased. GAP-43 was localized in retinal axons and in a subset of RGC cell bodies and dendrites. This upregulation of GAP-43 was sustained through the period in which retinal axons reconnect with their target in the tectum. FGF-2 application to the injured nerve, but not to the eyeball, increased GAP-43 mRNA in the retina but decreased GAP-43 protein levels and decreased the number of immunopositive cell bodies. In the tectum, no treatment affected GAP-43 mRNA but FGF-2 application to the axotomized optic nerve increased GAP-43 protein in regenerating retinal projections. We conclude that FGF-2 upregulates the synthesis and alters the distribution of the axonal growth-promoting protein GAP-43, suggesting that it may enhance axonal regrowth.

Differential roles of engrailed paralogs in determining sensory axon guidance and synaptic target recognition.

The transcription factor Engrailed (En) controls axon pathfinding and synaptic target choice in an identified neuron (6m) of the cockroach cercal sensory system. Knock-out of En using double-stranded RNA interference (dsRNAi) transforms 6m so that it resembles a neighboring neuron that normally does not express the en gene, has a different arbor anatomy, and makes different connections. Like many animals, the cockroach has two En paralogs, Pa-En1 and Pa-En2. In this study we tested the hypothesis that the paralogs have different effects on axon guidance and synaptic target recognition, using RNAi to knock out each one individually. Using dye injections into 6m and intracellular recordings from target interneurons, we obtained evidence that both Pa-En1 and Pa-En2 determine the axonal arborization, but only Pa-En1 controls synaptic connections. However, because immunocytochemical quantification of En protein in 6m after RNAi showed that Pa-En1 represents 65% of the total En activity and Pa-En2 only 35%, our results could be caused by dosage effects. We measured the effects of diluting the mixture of both dsRNAs on the amounts of En protein. From this dose-response curve, we calculated the appropriate dilutions of the dsRNA mixture that would titrate total En protein to levels equivalent to knock-out of either paralog. RNAi using these dilutions showed that Pa-En1 and Pa-En2 both contribute toward the control of axonal guidance and confirmed that Pa-En1 has the paralog-specific function of controlling synaptic target recognition.

Persistent engrailed expression is required to determine sensory axon trajectory, branching, and target choice.

The transcription factor Engrailed (En) directs, in the cockroach cercal system, the shape of the axonal arborization and the choice of postsynaptic partners of an identified sensory neuron (6m). Knock-out of En using double-stranded RNA interference transforms 6m so that it resembles a neighboring neuron that normally does not express the en gene, has a different arbor anatomy, and makes different connections. We characterized the development of 6m and perturbed en expression at different stages. Our results show that En is not required before birth for 6m to become a neuron, but that it is required in the postmitotic neuron to control axonal arborization and synaptic specificity. Knock-out of En after 6m has entered the CNS does not change the axonal trajectory and has minor effects on axonal branches but causes the formation of synaptic connections typical of an En-negative cell. This suggests that En controls target recognition molecules independently from those guiding the axon. In contrast, double-stranded RNA injection 1 d later does not have any effects on the phenotype of 6m, suggesting that the period of synapse formation is over by the time En levels have fallen or, if synapse turnover occurs, that En is not required to maintain the specificity of synaptic connections. We conclude that persistent en expression is required to determine successive stages in the differentiation of the neuron, suggesting that it is not far upstream from those genes encoding axon guidance and synaptic recognition molecules.