Natural light is regarded as a key regulator of biological systems and typically serves as a Zeitgeber for biological rhythms. As a natural abiotic factor, it is recognized to regulate multiple behavioral and physiological processes in animals. Disruption of the natural light regime due to light pollution may result in significant effects on animal learning and memory development. Here, we investigated whether sensitivity to various photoperiods or light intensities had an impact on intermediate-term memory (ITM) and long-term memory (LTM) formation in the pond snail Lymnaea stagnalis. We also investigated the change in the gene expression level of molluscan insulin-related peptide II (MIP II) is response to the given light treatments. The results show that the best light condition for proper LTM formation is exposure to a short day (8 h light) and low light intensity (1 and 10 lx). Moreover, the more extreme light conditions (16 h and 24 h light) prevent the formation of both ITM and LTM. We found no change in MIP II expression in any of the light treatments, which may indicate that MIP II is not directly involved in the operant conditioning used here, even though it is known to be involved in learning. The finding that snails did not learn in complete darkness indicates that light is a necessary factor for proper learning and memory formation. Furthermore, dim light enhances both ITM and LTM formation, which suggests that there is an optimum since both no light and too bright light prevented learning and memory. Our findings suggest that the upsurge of artificial day length and/or night light intensity may also negatively impact memory consolidation in the wild.

In previous works, we identified a RNA-binding protein in presynaptic terminal of squid neurons, which is likely involved in local mRNA processing. Evidences indicate this strongly basic protein, called p65, is an SDS-stable dimer protein composed of ~ 37kDa hnRNPA/B-like subunits. The function of p65 in presynaptic regions is not well understood. In this work, we showed p65 and its subunit p37 are concentrated in RNA-enriched regions in synaptosomes. We performed in vitro binding studies with a recombinant protein and showed its propensity to selectively bind actin mRNA at the squid presynaptic terminal. Biochemical analysis using lysed synaptosomes suggested RNA integrity may affect p65 and p37 functions. Mass spectrometry analysis of oligo(dT) pull down indicated squid hnRNPA1, hnRNPA1-like 2, hnRNPA3 and ELAV-like proteins as candidates to interact with p65 and p37 forming a ribonucleoprotein complex, suggesting a role of squid hnRNPA/B-like proteins in site-specific RNA processing.

Conditioned taste aversion (CTA) in the freshwater pulmonate Lymnaea stagnalis can be formed by presenting ten pairings of sucrose as the conditioned stimulus (CS) and KCl as the unconditioned stimulus (US). The CTA is consolidated to long-term memory (LTM) lasting longer than a month. In the present study, we examined the time course of protein synthesis-dependent period during the consolidation of Lymnaea CTA to LTM by pharmacological inhibition of transcription or translation. The robustness for CTA-LTM was then examined by extinction trials, i.e., repeated presentations of the CS alone. Furthermore, we evaluated the effects of the interstimulus interval (ISI) between the presentation of the CS and US. Our findings indicated that the protein synthesis-dependent period coincides with the CTA training. Repeated presentations of the CS alone after establishment of CTA did not extinguish the CTA, demonstrating the robustness of the CTA-LTM. The ISI ranged from 10s to a few minutes, and there was no inverted U-shaped function between the ISI and the conditioned response (i.e., suppression of feeding). Thus, CTA still formed even when the presentation of the US was delayed. These features of Lymnaea CTA complement the knowledge for mammalian CTA.

Scorpion pectines detect chemical and physical stimuli via thousands of peg sensilla on ground-facing teeth. Each sensillum has multiple neurons that detect stimuli and transmit neural impulses to the subesophageal ganglion (SEG) in the central nervous system. Anatomically, the organization of the pectinal neuropil in the SEG reflects the arrangement of pectinal teeth, suggesting conservation of information about stimulus location in the SEG. In this study, neural impulses from the pectinal nerve of the striped bark scorpion (Centruroides vittatus) were recorded in the pecten, abdominal cavity, and SEG using electrophysiology. Recordings from the right pectinal nerve in the pecten showed that right tooth stimulations elicit sensory activity milliseconds before motor feedback, while left tooth stimulations only evoked motor spikes, suggesting no contralateral afferent communication between pectines. In the abdominal cavity recordings, two distinct waveforms (PN1 and PN2) were detected in baseline activity; PN1 seemed to enhance the likelihood that PN2 would fire, suggesting a possible excitatory interaction. Recordings from the SEG showed that mechanically stimulating different pectinal teeth evoked different compound neural activity patterns in the same SEG location. Stimulating the same tooth in succession showed mechanosensory adaptation. These recordings show promise for continued electrophysiological investigations of the scorpion SEG, and, in particular, how information from the pectines is processed and used for orientation.

The metabotropic glutamate receptors (mGluRs) are a class of G-protein-coupled receptor that undergo extensive interactions with scaffolding proteins, and this is intrinsic to their function as an important group of neuromodulators at glutamatergic synapses. The Caenorhabditis elegans nervous system expresses three metabotropic glutamate receptors, MGL-1, MGL-2 and MGL-3. Relatively little is known about how the function and signalling of these receptors is organised in C. elegans. To identify proteins that scaffold the MGL-1 receptor, we have conducted a yeast two-hybrid screen. Three of the interacting proteins, MPZ-1, NRFL-1 and PTP-1, displayed motifs characteristic of mammalian mGluR scaffolding proteins. Using cellular co-expression criterion, we show mpz-1 and ptp-1 exhibited overlapping expression patterns with subsets of mgl-1 neurons. This included neurones in the pharyngeal nervous system that control the feeding organ of the worm. The mGluR agonist L-CCG-I inhibits the activity of this network in wild-type worms, in an MGL-1 and dose-dependent manner. We utilised L-CCG-I to identify if MGL-1 function was disrupted in mutants with deletions in the mpz-1 gene. The mpz-1 mutants displayed a largely wild-type response to L-CCG-I, suggesting MGL-1 signalling is not overtly disrupted consistent with a non-obligatory modulatory function in receptor scaffolding. The selectivity of the protein interactions and overlapping expression identified here warrant further investigation of the functional significance of scaffolding of metabotropic glutamate receptor function.

Pulmonate gastropods provide unique opportunities to examine physiological and biochemical adaptation strategies when cellular metabolic activity is reduced. In this study, cytochemical changes in metacerebral neurons of the cerebral ganglia were investigated in the garden snail Cornu aspersum during the hibernation phase. The immunocytochemical expression of three cytoskeletal markers: microtubule-associate protein 2-like (MAP-2-li), phosphorylated form of tau-like (P-Tau-li) and heavy subunit of neurofilaments-like (NF-H-li), and of two calcium-binding proteins: calmodulin-like (CaM-li) and parvalbumin-like (PV-li) was compared in active and hibernated snails. The immunopositivity for all the markers increased during hibernation versus activity in metacerebral neurons, with the notable exception of PV-li, which remained highly expressed during the whole annual cycle. Strongly positive aggregates of MAP-2-li and P-Tau-li were detected in the somata of hibernated snail neurons. P-Tau-li aggregates co-localized with CaM-li-labelled masses during hibernation. In addition, increased labelling of NF-H-li epitopes was associated with enhancement of CaM immunopositivity. These changes may reflect neural plasticity mechanisms mainly mediated by microtubule-associated proteins and CaM. Moreover, neuroprotective strategies may allow neurons to endure the prolonged hypometabolic conditions, taking into account that many of the functions controlled by the metacerebrum, such as feeding and movement, are suspended during hibernation. In this context, the molluscan ganglia model offers an easy opportunity to understand the molecular mechanisms behind these life cycle changes in cell physiology and to investigate possible cytological similarities among distantly related animals that adapt to the same environmental challenges through hibernation.

The giant freshwater prawn, Macrobrachium rosenbergii, is an economically important crustacean species which has also been extensively used as a model in neuroscience research. The crustacean central nervous system is a highly complex structure, especially the brain. However, little information is available on the brain structure, especially the three-dimensional organization. In this study, we demonstrated the three-dimensional structure and histology of the brain of M. rosenbergii together with the distribution of serotonin (5-HT) in the brain and ovary as well as its effects on ovarian steroidogenesis. The brain of M. rosenbergii consists of three parts: protocerebrum, deutocerebrum and tritocerebrum. Histologically, protocerebrum comprises of neuronal clusters 6-8 and prominent anterior and posterior medial protocerebral neuropils (AMPN/PMPN). The protocerebrum is connected posteriorly to the deutocerebrum which consists of neuronal clusters 9-13, medial antenna I neuropil, a paired lateral antenna I neuropils and olfactory neuropils (ON). Tritocerebrum comprises of neuronal clusters 14-17 with prominent pairs of antenna II (AnN), tegumentary and columnar neuropils (CN). All neuronal clusters are paired structures except numbers 7, 13 and 17 which are single clusters located at the median zone. These neuronal clusters and neuropils are clearly shown in three-dimensional structure of the brain. 5-HT immunoreactivity (-ir) was mostly detected in the medium-sized neurons and neuronal fibers of clusters 6/7, 8, 9, 10 and 14/15 and in many neuropils of the brain including anterior/posterior medial protocerebral neuropils (AMPN/PMPN), protocerebral tract, protocerebral bridge, central body, olfactory neuropil (ON), antennal II neuropil (Ann) and columnar neuropil (CN). In the ovary, the 5-HT-ir was light in the oocyte step 1(Oc1) and very intense in Oc2-Oc4. Using an in vitro assay of an explant of mature ovary, it was shown that 5-HT was able to enhance ovarian estradiol-17β (E2) and progesterone (P4) secretions. We suggest that 5-HT is specifically localized in specific brain areas and ovary of this prawn and it plays a pivotal role in ovarian maturation via the induction of female sex steroid secretions, in turn these steroids may enhance vitellogenesis resulting in oocyte growth and maturation.

The crustacean stomatogastric nervous system (STNS) is a well-known model for investigating neuropeptidergic control of rhythmic behavior. Among the peptides known to modulate the STNS are the C-type allatostatins (AST-Cs). In the lobster, Homarus americanus, three AST-Cs are known. Two of these, pQIRYHQCYFNPISCF (AST-C I) and GNGDGRLYWRCYFNAVSCF (AST-C III), have non-amidated C-termini, while the third, SYWKQCAFNAVSCFamide (AST-C II), is C-terminally amidated. Here, antibodies were generated against one of the non-amidated peptides (AST-C I) and against the amidated isoform (AST-C II). Specificity tests show that the AST-C I antibody cross-reacts with both AST-C I and AST-C III, but not AST-C II; the AST-C II antibody does not cross-react with either non-amidated peptide. Wholemount immunohistochemistry shows that both subclasses (non-amidated and amidated) of AST-C are distributed throughout the lobster STNS. Specifically, the antibody that cross-reacts with the two non-amidated peptides labels neuropil in the CoGs and the stomatogastric ganglion (STG), axons in the superior esophageal (son) and stomatogastric (stn) nerves, and ~14 somata in each commissural ganglion (CoG). The AST-C II-specific antibody labels neuropil in the CoGs, STG and at the junction of the sons and stn, axons in the sons and stn, ~42 somata in each CoG, and two somata in the STG. Double immunolabeling shows that, except for one soma in each CoG, the non-amidated and amidated peptides are present in distinct sets of neuronal profiles. The differential distributions of the two AST-C subclasses suggest that the two peptide groups are likely to serve different modulatory roles in the lobster STNS.

Cephalopods are the sole invertebrates included in the list of regulated species following the Directive 2010/63/EU. According to the Directive, achieving competence through adequate training is a requisite for people having a role in the different functions (article 23) as such carrying out procedures on animals, designing procedures and projects, taking care of animals, killing animals. Cephalopod Biology and Care Training Program is specifically designed to comply with the requirements of the "working document on the development of a common education and training framework to fulfil the requirements under the Directive 2010/63/EU". The training event occurred at the ICM-CSIC in Barcelona (Spain) where people coming from Europe, America and Asia were instructed on how to cope with regulations for the use of cephalopod molluscs for scientific purposes. The training encompasses discussion on the guidelines for the use and care of animals and their welfare with particular reference to procedures that may be of interest for neuroscience. Intensive discussion has been carried out during the training sessions with focus on behavioural studies and paradigms, welfare assessment, levels of severity of scientific procedures, animal care, handling, transport, individual identification and marking, substance administration, anaesthesia, analgesia and humane killing.

Earthworms are segmented invertebrates that belong to the phylum Annelida. The segments can be divided into the anterior, clitellar and posterior parts. If the anterior part of the earthworm, which includes the brain, is amputated, the worm would essentially survive even in the absence of the brain. In these brain amputee-derived worms, the nerve cord serves as the primary control center for neurological function. In this current work, we studied changes in the expression levels of anti-acetylated tubulin and serotonin as the indicators of neuro-regenerative processes. The data reveal that the blastemal tissues express the acetylated tubulin and serotonin from day four and that the worm amputated at the 7th segment takes 30days to complete the regeneration of brain. The ability of self-assemblage is one of the specific functions of the earthworm's brain. The brain amputee restored the ability of self-assemblage on the eighth day.