Paraformaldehyde (PFA) is the most commonly used fixative for immunostaining of cells, but has been associated with various problems, ranging from loss of antigenicity to changes in morphology during fixation. We show here that the small dialdehyde glyoxal can successfully replace PFA. Despite being less toxic than PFA, and, as most aldehydes, likely usable as a fixative, glyoxal has not yet been systematically tried in modern fluorescence microscopy. Here, we tested and optimized glyoxal fixation and surprisingly found it to be more efficient than PFA‐based protocols. Glyoxal acted faster than PFA, cross‐linked proteins more effectively, and improved the preservation of cellular morphology. We validated glyoxal fixation in multiple laboratories against different PFA‐based protocols and confirmed that it enabled better immunostainings for a majority of the targets. Our data therefore support that glyoxal can be a valuable alternative to PFA for immunostaining.

Interleukin-6 (IL-6) is a key mediator of inflammation. Inhibitors of IL-6 or of its signal transducing receptor gp130 constitute a novel class of anti-inflammatory drugs, which raise great hopes for improved treatments of painful inflammatory diseases such as rheumatoid arthritis. IL-6 and gp130 may enhance pain not only indirectly through their proinflammatory actions but also through a direct action on nociceptors (i.e., on neurons activated by painful stimuli). We found indeed that the IL-6/gp130 ligand-receptor complex induced heat hypersensitivity bothin vitroandin vivo. This process was mediated by activation of PKC-δ via Gab1/2/PI₃K and subsequent regulation of TRPV1, a member of the transient receptor potential (TRP) family of ion channels. To assess the relevance of this direct pain promoting effect of IL-6, we generated conditional knock-out mice, which lackgp130specifically in nociceptors, and tested them in models of inflammatory and tumor-induced pain. These mice showed significantly reduced levels of inflammatory and tumor-induced pain but no changes in immune reactions or tumor growth. Our results uncover the significance of gp130 expressed in peripheral pain sensing neurons in the pathophysiology of major clinical pain disorders and suggest their use as novel pain relieving agents in inflammatory and tumor pain.

Significance: Hearing relies on temporally precise and indefatigable transmission of sensory information mediated by inner hair cell (IHC) ribbon synapses. Synaptic ribbons are electron-dense structures that tether numerous vesicles at the active zone (AZ) and drive neural encoding of acoustical signals by neurotransmitter release. The mechanisms of ribbon formation, AZ targeting and subsequent functional maturation in murine IHCs remain elusive. By combining various high-resolution imaging techniques, we analyzed multiple developmental stages and provide a new hypothesis for the assembly of auditory ribbon synapses from the apical cochlear turn. Our findings suggest that fusion of presynaptic ribbons contributes to the morphological AZ development that is essential for the functional maturation of afferent synaptic transmission within the cochlea.

Levetiracetam (LEV) is a prominent antiepileptic drug that binds to neuronal synaptic vesicle glycoprotein 2A protein and has reported effects on ion channels, but with a poorly defined mechanism of action. We investigated inhibition of voltage-dependent Ca²⁺ (Ca_V) channels as a potential mechanism through which LEV exerts effects on neuronal activity. We used electrophysiological methods to investigate the effects of LEV on cholinergic synaptic transmission and Ca_V channel activity in superior cervical ganglion neurons (SCGNs). In parallel, we investigated the effects of the inactive LEV R-enantiomer, (R)-α-ethyl-2-oxo-1-pyrrolidine acetamide (UCB L060). LEV but not UCB L060 (each at 100 μM) inhibited synaptic transmission between SCGNs in long-term culture in a time-dependent manner, significantly reducing excitatory postsynaptic potentials after a ≥30-min application. In isolated SCGNs, LEV pretreatment (≥1 h) but not short-term application (5 min) significantly inhibited whole-cell Ba²⁺ current (I_Ba) amplitude. In current-clamp recordings, LEV reduced the amplitude of the afterhyperpolarizing potential in a Ca²⁺-dependent manner but also increased the action potential latency in a Ca²⁺-independent manner, which suggests additional mechanisms associated with reduced excitability. Intracellular LEV application (4–5 min) caused rapid inhibition of I_Ba amplitude, to an extent comparable to that seen with extracellular LEV pretreatment (≥1 h). Neither pretreatment nor intracellular application of UCB L060 produced any inhibitory effects on I_Ba amplitude. These results identify a stereospecific intracellular pathway through which LEV inhibits presynaptic Ca_V channels; resultant reductions in neuronal excitability are proposed to contribute to the anticonvulsant effects of LEV.

Virus-mediated expression of channelrhodopsin enables optogenetic stimulation of auditory pathways and restoration of auditory-driven behavior in deaf adult gerbils.

Optogenetic hearing restoration in rodents by LED-based multichannel optical cochlear implants provides improved spectral selectivity.