Worldwide, seagrasses face threats including climate change, disease and anthropogenic disturbance, with populations at the extremes of species’ distributions likely presaging future problems elsewhere in their geographical ranges. At the geographic limits of two marine macrophytes (Zostera marina and Posidonia oceanica) and under intense urbanization, seagrasses around Gibraltar are particularly vulnerable. However, the last published survey of Gibraltar seagrass meadows, in 1993, showed both species were abundant. We resurveyed this area and were unable to locate any seagrass in Gibraltar waters. Extensive coastal development and land reclamation make much former seagrass habitat in Gibraltar waters unsuitable, presenting substantial hurdles to any future restoration efforts.

To image synaptic activity within neural circuits, we tethered the genetically encoded calcium indicator (GECI) GCaMP2 to synaptic vesicles by fusion to synaptophysin. The resulting reporter, SyGCaMP2, detected the electrical activity of neurons with two advantages over existing cytoplasmic GECIs: it identified the locations of synapses and had a linear response over a wider range of spike frequencies. Simulations and experimental measurements indicated that linearity arises because SyGCaMP2 samples the brief calcium transient passing through the presynaptic compartment close to voltage-sensitive calcium channels rather than changes in bulk calcium concentration. In vivo imaging in zebrafish demonstrated that SyGCaMP2 can assess electrical activity in conventional synapses of spiking neurons in the optic tectum and graded voltage signals transmitted by ribbon synapses of retinal bipolar cells. Localizing a GECI to synaptic terminals provides a strategy for monitoring activity across large groups of neurons at the level of individual synapses.

The identification of mutations underlying monogenic, early-onset forms of deafness in humans has provided unprecedented insight into the molecular mechanisms of hearing in the peripheral auditory system. The molecules involved in the development and function of the cochlea eluded characterization until recently owing to the scarcity of the principal cell types present. The genetic approach has circumvented this problem and succeeded in identifying proteins and deciphering some of the molecular complexes that operate in these cells. In combination with mouse models, the genetic approach is now revealing some of the principles underlying the development and physiology of the cochlea. Focusing on the hair bundle, the mechanosensory device of the sensory hair cell, we highlight recent advances in understanding the way in which the hair bundle is formed, how it operates as a mechanotransducer and how it processes sound. In particular, we discuss how these findings confer a central role on the various hair-bundle links in these processes.

Information transfer at chemical synapses occurs when vesicles fuse with the plasma membrane and release neurotransmitter. This process is stochastic and its likelihood of occurrence is a crucial factor in the regulation of signal propagation in neuronal networks. The reliability of neurotransmitter release can be highly variable: experimental data from electrophysiological, molecular and imaging studies have demonstrated that synaptic terminals can individually set their neurotransmitter release probability dynamically through local feedback regulation. This local tuning of transmission has important implications for current models of single-neuron computation.

The ribbon synapses of auditory inner hair cells (IHCs) undergo morphological and electrophysiological transitions during cochlear development. Here we report that myosin VI (Myo6), an actin-based motor protein involved in genetic forms of deafness, is necessary for some of these changes to occur. By using post-embedding immunogold electron microscopy, we showed that Myo6 is present at the IHC synaptic active zone. In Snell's waltzer mutant mice, which lack Myo6, IHC ionic currents and ribbon synapse maturation proceeded normally until at least post-natal day 6. In adult mutant mice, however, the IHCs displayed immature potassium currents and still fired action potentials, as normally only observed in immature IHCs. In addition, the number of ribbons per IHC was reduced by 30%, and 30% of the remaining ribbons were morphologically immature. Ca2+-dependent exocytosis probed by capacitance measurement was markedly reduced despite normal Ca2+ currents and the large proportion of morphologically mature synapses, which suggests additional defects, such as loose Ca2+-exocytosis coupling or inefficient vesicular supply. Finally, we provide evidence that Myo6 and otoferlin, a putative Ca2+ sensor of synaptic exocytosis also involved in a genetic form of deafness, interact at the IHC ribbon synapse, and we suggest that this interaction is involved in the recycling of synaptic vesicles. Our findings thus uncover essential roles for Myo6 at the IHC ribbon synapse, in addition to that proposed in membrane turnover and anchoring at the apical surface of the hair cells.

Here we report the functional assessment of two novel deafness-associated gamma-actin mutants, K118N and E241K, in a spectrum of different situations with increasing biological complexity by combining biochemical and cell biological analysis in yeast and mammalian cells. Our in vivo experiments showed that while the K118N had a very mild effect on yeast behaviour, the phenotype caused by the E241K mutation was very severe and characterized by a highly compromised ability to grow on glycerol as a carbon source, an aberrant multi-vacuolar pattern and the deposition of thick F-actin bundles randomly in the cell. The latter feature is consistent with the highly unusual spontaneous tendency of the E241K mutant to form bundles in vitro, although this propensity to bundle was neutralized by tropomyosin and the E241K filament bundles were hypersensitive to severing in the presence of cofilin. In transiently transfected NIH3T3 cells both mutant actins were normally incorporated into cytoskeleton structures, although cytoplasmic aggregates were also observed indicating an element of abnormality caused by the mutations in vivo. Interestingly, gene-gun mediated expression of these mutants in cochlear hair cells results in no gross alteration in cytoskeletal structures or the morphology of stereocilia. Our results provide a more complete picture of the biological consequences of deafness-associated gamma-actin mutants and support the hypothesis that the post-lingual and progressive nature of the DFNA20/26 hearing loss is the result of a progressive deterioration of the hair cell cytoskeleton over time.

In this study we examined changes in a persistent sodium current (INaP) after behavioral aversive classical conditioning in the snail Helix pomatia. We trained animals by pairing food with a mild electric shock that triggered the whole-body withdrawal reflex. This aversive training resulted in transcription dependent long-term associative memory. Isolated central nervous system preparations were set up from trained, random control and naive animals and using two-electrode voltage clamp methods, INaP was activated and measured in identified body withdrawal interneurons RPa3 and LPa3. We show here that in preparations from conditioned animals INaP is increased, suggesting that modifications in intrinsic cellular properties contribute to the formation of the memory trace. Blocking RNA synthesis by systemic injection of actinomycin D (0.12 µM) suppressed both memory consolidation in intact animals and the learning-induced increase of INaP in withdrawal interneurons, suggesting that aversive classical conditioning affects sodium channel expression at the transcriptional level.

Hair cells in the inner ear provide the basis for the exquisite hearing capabilities of mammals. These cells transduce sound-induced displacements of their mechanosensitive hair bundle into electrical currents within a fraction of a millisecond and with nanometer fidelity. Excitatory displacements of the hair cell¿s bundle tense tip links that open transducer channels. These channels are located either at one or at both ends of the links, where the latter possibility was thought to compromise sensitivity via negative cooperativity, and discarded for quantitatively describing the transduction process. Here, we show instead that this series mode of activation accurately explains measured transduction in hair cells. It enhances both sensitivity and dynamic range of hair cell transduction, by one channel that is extremely sensitive at small displacements while the other responds best to larger stimuli. Our results provide a new framework for exploring the dynamics of hair cell activation.

RATIONALE: Atypical antipsychotic-induced weight gain is a significant impediment in the treatment of schizophrenia. OBJECTIVES: In a putative model of antipsychotic drug-induced weight gain, we investigated the effects of sub-chronic olanzapine on body weight, meal patterns, the expression of genes encoding for hypothalamic feeding-related neuropeptides and the contribution of hyperphagia to olanzapine-induced weight gain in rats. MATERIALS AND METHODS: In experiment 1, female rats received either olanzapine (1 mg/kg, p.o.) or vehicle, twice daily for 7 days, while meal patterns were recorded. At the end of the treatment regimen, we measured the levels of hypothalamic messenger RNAs (mRNAs) encoding neuropeptide-Y (NPY), hypocretin/orexin (HCRT), melanin concentrating hormone and pro-opiomelanocortin. NPY and HCRT mRNA levels were also assessed in a separate cohort of female rats treated acutely with olanzapine (1 mg/kg, p.o.). In experiment 2, we investigated the effect of a pair-feeding paradigm on sub-chronic (1 mg/kg, p.o.) olanzapine-induced weight gain. RESULTS: In experiment 1, sub-chronic olanzapine increased body weight, food intake and meal size. Hypothalamic neuropeptide mRNA levels were unchanged after both acute and sub-chronic olanzapine treatment. In experiment 2, the restriction of food intake to the level of vehicle-treated controls abolished the sub-chronic olanzapine-induced increase in body weight. CONCLUSIONS: Hyperphagia mediated by drug-induced impairments in satiety (as evidenced by increased meal size) is a key requirement for olanzapine-induced weight gain in this paradigm. However, olanzapine-induced hyperphagia and weight gain may not be mediated via alterations in the expression of the feeding-related hypothalamic neuropeptides examined in this study.

A recent study of how incompatible behaviors are suppressed during feeding in the medicinal leech Hirudo provides new insights into the mechanisms of decision-making in neuronal networks.

We previously developed a one-trial reward classical conditioning paradigm using amyl-acetate as the conditioned stimulus (CS) and sucrose as the unconditioned stimulus (US), to study the electrical and molecular changes underlying long-term memory formation in the feeding system of the pond snail Lymnaea. Here, we compare the features of one-trial reward conditioning with an alternative aversive conditioning paradigm where quinine was used as the US while the CS was the same (amyl acetate) as in reward conditioning. Here, a single pairing of CS and US led to aversive conditioning. An electrophysiological correlate of both types of conditioning was measured in semi-intact preparations. The same CS caused excitation or inhibition depending on the type of the training. The source of the inhibition in case of aversive conditioning appears to be in non-feeding ganglia in the rest of the brain. Our results indicate that the same CS can trigger alternative responses depending on the type of training (US). These two types of responses involve different pathways within the CNS. The sensitivity of the aversive and appetitive memory trace to NO, dopamine and octopamine blockers was also studied. We established that while NO and dopamine are important in the development of a long-term appetitive memory trace at an early stage after conditioning they do not play a role in aversive memory formation. Octopamine, on the other hand is not needed for appetitive memory formation, but seems to be the key transmitter needed shortly after aversive training.

The paired-domain transcription factor Pax2 is involved in many facets of inner ear development, hot relatively little is known about the expression or function of Pax2 in the mature ear. In this study, we have used immunohistochemical methods to characterize the expression patterns of Pax2 in the sensory organs of inner ears from posthatch chicks. Immunoreactivity for Pax2 was observed in the nuclei of most hair cells and supporting cells in the vestibular organs. In contrast, Pax2 expression in the chick cochlea was limited to hair cells located in the very distal (low frequency) region. We then used organotypic cultures of the chick utricle to examine changes in Pax2 expression in response to ototoxic in, jury and during hair cell regeneration. Treatment with streptomycin resulted in the loss or most Pax2 immunoreactivity from the lumenal (hair cell) stratum of the utricle. During the early phases of regeneration, moderate Pax2 expression was maintained in the nuclei of proliferating supporting cells. Expression of Pax2 in the hair cell stratum recovered in parallel with hair cell regeneration. The results indicate that Pax2 continues to he expressed in the mature avian ear, and that its expression pattern is correlated with a vestibular phenotype.

A population of glial cells in the central nervous system of the gastropod mollusk Lymnaea stagnalis produces a soluble protein that specifically binds acetylcholine. This protein is named the acetylcholine binding protein (AChBP). Experiments performed in vitro indicated that AChBP inactivates released acetylcholine at cholinergic synapses. On the basis of these observations, a similar in vivo role for AChBP was hypothesized. To fulfill this function, AChBP-expressing glia ought to be located in close proximity to cholinergic synapses in vivo. To examine this, we have analyzed the cellular and subcellular expression of AChBP in the intact CNS. Using a variety of molecular techniques, we demonstrate here that AChBP expression is confined to a subpopulation of glial cells located within the peripheral zone of each of the ganglia constituting the CNS. This zone contains the cell bodies of neurons, but few synapses. Conversely, glial cells that do not express the AChBP are predominantly located in the synapse-rich central neuropile zone but are rare in the cell body zone. Thus, our findings are not compatible with the previous conclusions drawn from in vitro studies and suggest that AChBP functions in vivo as a regulator of nonsynaptic cholinergic transmission.-Banks, G., Kemenes, I., Schofield, M., O'Shea, M., Korneev, S. A. Acetylcholine binding protein of mollusks is unlikely to act as a regulator of cholinergic neurotransmission at neurite-neurite synaptic sites in vivo.

We assessed the involvement of harmonin-b, a submembranous protein containing PDZ domains, in the mechanoelectrical transduction machinery of inner ear hair cells. Harmonin-b is located in the region of the upper insertion point of the tip link that joins adjacent stereocilia from different rows and that is believed to gate transducer channel(s) located in the region of the tip link's lower insertion point. In Ush1c (dfcr-2J/dfcr-2J) mutant mice defective for harmonin-b, step deflections of the hair bundle evoked transduction currents with altered speed and extent of adaptation. In utricular hair cells, hair bundle morphology and maximal transduction currents were similar to those observed in wild-type mice, but adaptation was faster and more complete. Cochlear outer hair cells displayed reduced maximal transduction currents, which may be the consequence of moderate structural anomalies of their hair bundles. Their adaptation was slower and displayed a variable extent. The latter was positively correlated with the magnitude of the maximal transduction current, but the cells that showed the largest currents could be either hyperadaptive or hypoadaptive. To interpret our observations, we used a theoretical description of mechanoelectrical transduction based on the gating spring theory and a motor model of adaptation. Simulations could account for the characteristics of transduction currents in wild-type and mutant hair cells, both vestibular and cochlear. They led us to conclude that harmonin-b operates as an intracellular link that limits adaptation and engages adaptation motors, a dual role consistent with the scaffolding property of the protein and its binding to both actin filaments and the tip link component cadherin-23.

A recent study of how food-seeking behavior in the sea slug Aplysia becomes compulsive provides new insights into the neural mechanisms of operant conditioning.