Information processing by the nervous system depends on neurotransmitter release from synaptic vesicles (SVs) at the presynaptic active zone. Molecular components of the cytomatrix at the active zone (CAZ) regulate the final stages of the SV cycle preceding exocytosis and thereby shape the efficacy and plasticity of synaptic transmission. Part of this regulation is reflected by a physical association of SVs with filamentous CAZ structures via largely unknown protein interactions. The very C-terminal region of Bruchpilot (Brp), a key component of the Drosophila melanogaster CAZ, participates in SV tethering. Here, we identify the conserved SNARE regulator Complexin (Cpx) in an in vivo screen for molecules that link the Brp C terminus to SVs. Brp and Cpx interact genetically and functionally. Both proteins promote SV recruitment to the Drosophila CAZ and counteract short-term synaptic depression. Analyzing SV tethering to active zone ribbons of cpx3 knockout mice supports an evolutionarily conserved role of Cpx upstream of SNARE complex assembly.

Invertebrates such as Drosophila melanogaster have proven to be a valuable model organism for studies of the nervous system. In order to control neuronal activity, optogenetics has evolved as a powerful technique enabling non-invasive stimulation using light. This requires light sources that can deliver patterns of light with high temporal and spatial precision. Currently employed light sources for stimulation of small invertebrates, however, are either limited in spatial resolution or require sophisticated and bulky equipment. In this work, we used smartphone displays for optogenetic control of Drosophila melanogaster. We developed an open-source smartphone app that allows time-dependent display of light patterns and used this to activate and inhibit different neuronal populations in both larvae and adult flies. Characteristic behavioural responses were observed depending on the displayed colour and brightness and in agreement with the activation spectra and light sensitivity of the used channelrhodopsins. By displaying patterns of light, we constrained larval movement and were able to guide larvae on the display. Our method serves as a low-cost high-resolution testbench for optogenetic experiments using small invertebrate species and is particularly appealing to application in neuroscience teaching labs.

Background Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that transduces extracellular signals in virtually all eukaryotic cells. The soluble Beggiatoa photoactivatable adenylyl cyclase (bPAC) rapidly raises cAMP in blue light and has been used to study cAMP signaling pathways cell-autonomously. But low activity in the dark might raise resting cAMP in cells expressing bPAC, and most eukaryotic cyclases are membrane-targeted rather than soluble. Our aim was to engineer a plasma membrane-anchored PAC with no dark activity (i.e., no cAMP accumulation in the dark) that rapidly increases cAMP when illuminated. Results Using a streamlined method based on expression in Xenopus oocytes, we compared natural PACs and confirmed bPAC as the best starting point for protein engineering efforts. We identified several modifications that reduce bPAC dark activity. Mutating a phenylalanine to tyrosine at residue 198 substantially decreased dark cyclase activity, which increased 7000-fold when illuminated. Whereas Drosophila larvae expressing bPAC in mechanosensory neurons show nocifensive-like behavior even in the dark, larvae expressing improved soluble (e.g., bPAC(R278A)) and membrane-anchored PACs exhibited nocifensive responses only when illuminated. The plasma membrane-anchored PAC (PACmn) had an undetectable dark activity which increased >4000-fold in the light. PACmn does not raise resting cAMP nor, when expressed in hippocampal neurons, affect cAMP-dependent kinase (PKA) activity in the dark, but rapidly and reversibly increases cAMP and PKA activity in the soma and dendrites upon illumination. The peak responses to brief (2 s) light flashes exceed the responses to forskolin-induced activation of endogenous cyclases and return to baseline within seconds (cAMP) or ~10 min (PKA). Conclusions PACmn is a valuable optogenetic tool for precise cell-autonomous and transient stimulation of cAMP signaling pathways in diverse cell types.

Adhesion-type GPCRs (aGPCRs) participate in a vast range of physiological processes. Their frequent association with mechanosensitive functions suggests that processing of mechanical stimuli may be a common feature of this receptor family. Previously, we reported that the Drosophila aGPCR CIRL sensitizes sensory responses to gentle touch and sound by amplifying signal transduction in low-threshold mechanoreceptors (Scholz et al., 2017). Here, we show that Cirl is also expressed in high-threshold mechanical nociceptors where it adjusts nocifensive behaviour under physiological and pathological conditions. Optogenetic in vivo experiments indicate that CIRL lowers cAMP levels in both mechanosensory submodalities. However, contrasting its role in touch-sensitive neurons, CIRL dampens the response of nociceptors to mechanical stimulation. Consistent with this finding, rat nociceptors display decreased Cirl1 expression during allodynia. Thus, cAMP-downregulation by CIRL exerts opposing effects on low-threshold mechanosensors and high-threshold nociceptors. This intriguing bipolar action facilitates the separation of mechanosensory signals carrying different physiological information.

Orexin-A and orexin-B (Ox-A, Ox-B) are neuropeptides produced by a small number of neurons that originate in the hypothalamus and project widely in the brain. Only discovered in 1998, the orexins are already known to regulate several behaviours. Most prominently, they help to stabilise the waking state, a role with demonstrated significance in the clinical management of narcolepsy and insomnia. Orexins bind to G-protein-coupled receptors (predominantly postsynaptic) of two subtypes, OXR and OXR. The primary effect of Ox-OXR binding is a direct depolarising influence mediated by cell membrane cation channels, but a wide variety of secondary effects, both pre- and postsynaptic, are also emerging. Given that inhibitory GABAergic neurons also influence orexin-regulated behaviours, crosstalk between the two systems is expected, but at the cellular level, little is known and possible mechanisms remain unidentified. Here, we have used an expression system approach to examine the feasibility, and nature, of possible postsynaptic crosstalk between Ox-A and the GABA receptor (GABAR), the brain's main inhibitory neuroreceptor. When HEK293 cells transfected with OXR and the α, β, and γ subunits of GABAR were exposed to Ox-A, GABA-induced currents were inhibited, in a calcium-dependent manner. This inhibition was associated with increased phosphorylation of the β subunit of GABAR, and the inhibition could itself be attenuated by (1) kinase inhibitors (of protein kinase C and CaM kinase II) and (2) the mutation, to alanine, of serine 409 of the β subunit, a site previously identified in phosphorylation-dependent regulation in other pathways. These results are the first to directly support the feasibility of postsynaptic crosstalk between Ox-A and GABAR, indicating a process in which Ox-A could promote phosphorylation of the β subunit, reducing the GABA-induced, hyperpolarising current. In this model, Ox-A/GABAR crosstalk would cause the depolarising influence of Ox-A to be boosted, a type of positive feedback that could, for example, facilitate the ability to abruptly awake.

The γ-aminobutyric acid type A receptor (GABAAR) is negatively modulated by two structurally similar neurosteroids, pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS). This study attempted to ascertain the molecular mechanisms of inhibition of the GABA-ergic current by neurosteroids. We demonstrated that the presence of the γ subunit in GABAAR enhances the efficacy of DHEAS without altering its binding affinity. A saturating concentration of DHEAS blocked approximately 75 % of currents mediated by GABAAR, which is composed of human α1, β1, and γ2S subunits, whereas the inhibition was only 35 % in GABAAR containing only α1 and β1 subunits. The IC50 values of DHEAS with and without the γ subunit were almost identical. In contrast to DHEAS, neither the affinity nor the efficacy of PS was altered by the γ subunit. When Val256 of α1 subunit was mutated to Ser, the mutant channel became resistant to inhibition by both DHEAS and PS. PS exerted its inhibitory effect by enhancing the desensitization kinetics of GABAAR possibly through promoting the interaction between the M2-M3 linker and extracellular loop 7/loop 2. Mutant α1, containing double Cys in loop 2/loop 7 and the M2-M3 linker, formed disulfide bonds three times as much fast, when treated with saturating GABA+PS, compared with GABA alone or with GABA+DHEAS. We demonstrated that PS, but not DHEAS, mediates GABA-ergic inhibition by promoting collisions between the structural elements involved in receptor desensitization, i.e., loop 2, loop 7, and the M2-M3 linker, thus following different inhibitory mechanisms.

1 Adhesion-type G protein-coupled receptors (aGPCRs) participate in a vast range of 2 physiological processes. Correspondingly, these receptors are associated with diverse human 3 diseases, such as developmental disorders, defects of the nervous system, allergies and 4 cancer. Several aGPCRs have recently been linked to mechanosensitive functions suggesting 5 that processing of mechanical stimuli may be a common feature of this receptor family. CIRL 6 (ADGRL/Latrophilin, LPHN), one of the oldest members of the aGPCR family, sensitizes 7 sensory responses of larval Drosophila to gentle touch and sound by amplifying 8 mechanosensory signal transduction in low-threshold mechanoreceptors (Scholz et al., 2015; 9 2017). In the present study, we show that Cirl is also expressed in high-threshold mechanical 10 nociceptors where it adjusts nocifensive behaviour under physiological and pathophysiological 11 conditions. Optogenetic in vivo experiments indicate that CIRL quenches cAMP levels in both 12 mechanosensory submodalities. However, contrasting its effect in touch sensitive neurons, 13 CIRL dampens the response of nociceptors to mechanical stimulation. Consistent with this 14 finding, rat nociceptors display a drop in Cirl1 expression during allodynia. Taken together, 15 these results demonstrate that CIRL exerts opposing modulatory functions in low-threshold 16 mechanosensors and high-threshold nociceptors. This intriguing bipolar action likely facilitates 17 the separation of mechanosensory signals carrying different physiological information. 18 19 20 2 Mechanosensation encompasses the distinct submodalities of touch, proprioception, and 3 mechanical nociception. Touch plays an important discriminative role and contributes to social 4 interactions (Abraira and Ginty, 2013; McGlone et al., 2014). Nociception reports incipient or 5 potential tissue damage. It triggers protective behaviours and can give rise to pain sensations 6 (Basbaum et al., 2009). Thus, physically similar signals can carry fundamentally different 7 physiological information, depending on stimulus intensity. Whereas innocuous touch 8 sensations rely on low-threshold mechanosensory neurons, noxious mechanical stimuli 9 activate high-threshold mechanosensory neurons, i.e. nociceptors. While mechanisms to 10 differentiate these mechanosensory submodalities are essential for survival, little is known how 11 this is achieved at cellular and molecular levels.12 13 The activity of nociceptors can be increased through sensitization, e.g. upon inflammation, and 14 decreased through antinociceptive processes, leading to pain relief. In both cases, G protein-15 coupled receptors (GPCRs) play an important modulatory role. Receptors that couple to 16 heterotrimeric Gq/11 or Gs proteins, like the prostaglandin EP2 receptor, increase the excitability 17 of nociceptors by activating phospholipase C and adenylate cyclase pathways, respectively. 18 In contrast, Gi/o-coupled receptors, which are gated by soluble ligands like morphine and 19 endogenous opioid neu...

No abstract