Euan R. Brown

D-Aspartic Acid in the Nervous System of Aplysia limacina: Possible Role in Neurotransmission

In the marine mollusk Aplysia limacina, a substantial amount of endogenous D‐aspartic acid (D‐Asp) was found following its synthesis from L‐aspartate by an aspartate racemase. Concentrations of D‐Asp between 3.9 and 4.6 µmol/g tissue were found in the cerebral, abdominal, buccal, pleural, and pedal ganglia. In non nervous tissues, D‐Asp occurred at a very low concentration compared to the nervous system. Immunohistochemical studies conducted on cultured Aplysia neurons using an anti‐D‐aspartate antibody demonstrated that D‐Asp occurs in the soma, dendrites, and in synaptic varicosities. Synaptosomes and synaptic vesicles from cerebral ganglia were prepared and characterized by electron microscopy. HPLC analysis revealed high concentrations of D‐Asp together with L‐aspartate and L‐glutamate in isolated synaptosomes In addition, D‐Asp was released from synaptosomes by K+ depolarization or by ionomycin. D‐Asp was one of the principal amino acids present in synaptic vesicles representing about the 25% of total amino acids present in these cellular organelles. Injection of D‐Asp into live animals or addition to the incubation media of cultured neurons, caused an increase in cAMP content. Taken as a whole, these findings suggest a possible role of D‐Asp in neurotransmission in the nervous system of Aplysia limacina.

GABAergic synaptic transmission modulates swimming in the ascidian larva

To examine the role of the amino acid GABA in the locomotion of basal chordates, we investigated the pharmacology of swimming and the morphology of GABA‐immunopositive neurones in tadpole larvae of the ascidians Ciona intestinalis and Ciona savignyi. We verified that electrical recording from the tail reflects alternating muscle activity during swimming by correlating electrical signals with tail beats using high‐speed video recording. GABA reversibly reduced swimming periods to single tail twitches, while picrotoxin increased the frequency and duration of electrical activity associated with spontaneous swimming periods. Immunocytochemistry for GABA revealed extensive labelling throughout the larval central nervous system. Two strongly labelled regions on either side of the sensory vesicle were connected by an arc of labelled fibres, from which fibre tracts extended caudally into the visceral ganglion. Fibre tracts extended ventrally from a third, more medial region in the posterior sensory vesicle. Two rows of immunoreactive cell bodies in the visceral ganglion extended neurites into the nerve cord, where varicosities were seen. Thus, presumed GABAergic neurones form a network that could release GABA during swimming that is involved in modulating the time course and frequency of periods of spontaneous swimming. GABAergic and motor neurones in the visceral ganglion could interact at the level of their cell bodies and/or through the presumed GABAergic fibres that enter the nerve cord. The larval swimming network appears to possess some of the properties of spinal networks in vertebrates, while at the same time possibly showing a type of peripheral innervation resembling that in some protostomes.

AMPA/kainate and NMDA-like glutamate receptors at the chromatophore neuromuscular junction of the squid: role in synaptic transmission and skin patterning

Glutamate receptor types were examined at the chromatophore synapses of the squids Alloteuthis subulata and Loligo vulgaris, where nerve‐induced muscle contraction causes chromatophore expansion. Immunoblotting with antibody raised against a squid AMPA receptor (sGluR) demonstrated that AMPA/kainate receptors are present in squid skin. Application of l‐glutamate evoked chromatophore muscle contractions in both ventral and dorsal skins, while NMDA was only active on a subpopulation of dorsal chromatophores. In dorsal skin, neurotransmission was partly blocked by either AMPA/kainate receptor antagonists (CNQX and DNQX) or NMDA receptor antagonists (AP‐5 and MK‐801) or completely blocked by simultaneous application of both classes of antagonists. In isolated muscle fibres, ionophoretic application of l‐glutamate evoked fast inward CNQX‐ and DNQX‐sensitive currents with reversal potentials around +14 mV and a high conductance to Na+. In fibres from dorsal skin only, a slower outward glutamate‐sensitive current appeared at positive holding potentials. At negative potentials, currents were potentiated by glycine or by removing external Mg2+ and were blocked by AP‐5 and MK‐801. Glutamate caused a fast, followed by a slow, transient increase in cytoplasmic Ca2+. The slow component was increased in amplitude and duration by glycine or by lowering external Mg2+ and decreased by AP‐5 and MK‐801. In cells from ventral skin, no ‘NMDA‐like responses’ were detected. Thus, while AMPA/kainate receptors mediated fast excitatory synaptic transmission and rapid colour change over the whole skin, activation of both AMPA/kainate and NMDA‐like receptors in a subpopulation of dorsal chromatophores prolonged the postsynaptically evoked Ca2+ elevation causing temporally extended colour displays with behavioural significance.

Class I PI 3-kinases: Function and evolution

In many human cell types, the class I phosphoinositide 3-kinases play key roles in the control of diverse cellular processes including growth, proliferation, survival and polarity. This is achieved through their activation by many cell surface receptors, leading to the synthesis of the phosphoinositide lipid signal, PIP3, which in turn influences the function of numerous direct PIP3-binding proteins. Here we review PI3K pathway biology and analyse the evolutionary distribution of its components and their functions. The broad phylogenetic distribution of class I PI3Ks in metazoa, amoebozoa and choannoflagellates, implies that these enzymes evolved in single celled organisms and were later co-opted into metazoan intercellular communication. A similar distribution is evident for the AKT and Cytohesin groups of downstream PIP3-binding proteins, with other effectors and pathway components appearing to evolve later. The genomic and functional phylogeny of regulatory systems such as the PI3K pathway provides a framework to improve our understanding of the mechanisms by which key cellular processes are controlled in humans.

Highly conserved elements discovered in vertebrates are present in non-syntenic loci of tunicates, act as enhancers and can be transcribed during development

Co-option of cis-regulatory modules has been suggested as a mechanism for the evolution of expression sites during development. However, the extent and mechanisms involved in mobilization of cis-regulatory modules remains elusive. To trace the history of non-coding elements, which may represent candidate ancestral cis-regulatory modules affirmed during chordate evolution, we have searched for conserved elements in tunicate and vertebrate (Olfactores) genomes. We identified, for the first time, 183 non-coding sequences that are highly conserved between the two groups. Our results show that all but one element are conserved in non-syntenic regions between vertebrate and tunicate genomes, while being syntenic among vertebrates. Nevertheless, in all the groups, they are significantly associated with transcription factors showing specific functions fundamental to animal development, such as multicellular organism development and sequence-specific DNA binding. The majority of these regions map onto ultraconserved elements and we demonstrate that they can act as functional enhancers within the organism of origin, as well as in cross-transgenesis experiments, and that they are transcribed in extant species of Olfactores. We refer to the elements as ‘Olfactores conserved non-coding elements’.

Nitric Oxide Mediates the Glutamate-dependent Pathway for Neurotransmission in Sepia officinalis Chromatophore Organs

Chromatophore organs are complex and unique structures responsible for the variety of body coloration patterns used by cephalopods to communicate and camouflage. They are formed by a pigment-containing cytoelastic sacculus, surrounded by muscle fibers directly innervated from the brain. Muscle contraction and relaxation are responsible for expansion and retraction of the pigment-containing cell. Their functioning depends on glutamate and Phe-Met-Arg-Phe-NH2-related peptides, which induce fast and slow cell expansion, respectively, and 5-hydroxytryptamine, which induces retraction. Apart from these three substances and acetylcholine, which acts presynaptically, no other neuroactive compounds have so far been found to be involved in the neuroregulation of chromatophore physiology, and the detailed signaling mechanisms are still little understood. Herein, we disclose the role of nitric oxide (NO) as mediator in one of the signaling pathways by which glutamate activates body patterning. NO and nitric-oxide synthase have been detected in pigment and muscle fibers of embryo, juvenile, and adult chromatophore organs from Sepia officinalis. NO-mediated Sepia chromatophore expansion operates at slower rate than glutamate and involves cGMP, cyclic ADP-ribose, and ryanodine receptor activation. These results demonstrate for the first time that NO is an important messenger in the long term maintenance of the body coloration patterns in Sepia.

Natural variation of model mutant phenotypes in Ciona intestinalis.

Background The study of ascidians (Chordata, Tunicata) has made a considerable contribution to our understanding of the origin and evolution of basal chordates. To provide further information to support forward genetics in Ciona intestinalis, we used a combination of natural variation and neutral population genetics as an approach for the systematic identification of new mutations. In addition to the significance of developmental variation for phenotype-driven studies, this approach can encompass important implications in evolutionary and population biology. Methodology/Principal Findings Here, we report a preliminary survey for naturally occurring mutations in three geographically interconnected populations of C. intestinalis. The influence of historical, geographical and environmental factors on the distribution of abnormal phenotypes was assessed by means of 12 microsatellites. We identified 37 possible mutant loci with stereotyped defects in embryonic development that segregate in a way typical of recessive alleles. Local populations were found to differ in genetic organization and frequency distribution of phenotypic classes. Conclusions/Significance Natural genetic polymorphism of C. intestinalis constitutes a valuable source of phenotypes for studying embryonic development in ascidians. Correlating genetic structure and the occurrence of abnormal phenotypes is a crucial focus for understanding the selective forces that shape natural finite populations, and may provide insights of great importance into the evolutionary mechanisms that generate animal diversity.

Evolution of Skeletal Muscle Excitation-Contraction Coupling and the Appearance of Dihydropyridine-Sensitive Intramembrane Charge Movement

Excitation-contraction (E-C) coupling was studied in the striated muscle fibres of lower chordates and invertebrates. In the agnathan Lampetra, twitches in response to electrical stimulation were unchanged after Ca2+ influx was blocked by the addition of Co2+. In the invertebrates examined (Branchiostoma, Oikopleura, Doliolum, Diphyes, Patinopecten and Sagitta), Ca2+ influx was necessary to cause a twitch. Whole-cell voltage clamp on muscle fibres of Lampetra revealed that intramembrane charge movement was partly blocked by 5 μm nifedipine, a dihydropyridine (DHP) derivative. Charge movement in fibres from Branchiostoma and Patinopecten was insensitive to nifedipine, although the Ca2+ current was 75% blocked by 5 μm nifedipine. The results suggest that the vertebrate type of E-C coupling was acquired between cephalochordates and agnathans, and was accompanied by the appearance of the DHP-sensitive component of intramembrane charge movement.

Modulation of an AMPA-like glutamate receptor (SqGluR) gating by L- and D-aspartic acids

Summary.L- and D-aspartic acids (L-Asp and D-Asp) are present in the majority of nervous systems. In phylogeny, significant levels have been reported in mollusc brains, particularly cephalopods. To examine the role of L- and D-Asp on a cephalopod receptor, we studied ligand gating of a squid glutamate receptor (SqGluR) expressed in HEK 239 (human embryonic kidney) cells. Under voltage clamp, application of L-glutamate (L-Glu; 1–30 mM), but not D-glutamate (D-Glu), or L- or D-Asp, evoked an inward current of 0.1 nA. L- or D-Asp (200 µM) applied with 20 mM L-Glu, slowed the time course of activation and inactivation of the L-Glu gated current (time constant increased from 1 s (L-Glu alone) to 3 s (D-Asp and L-Glu) and to 19 s (L-Asp and L-Glu)). Our results suggest that in molluscan systems, aspartic acid could act as a neuromodulator during glutamatergic transmission and could significantly alter synaptic integration by slowing glutamate receptor gating.

Pre-and postsynaptic excitation and inhibition at octopus optic lobe photoreceptor terminals; implications for the function of the 'presynaptic bags'

Synaptic transmission was examined in the plexiform zone of Octopus vulgaris optic lobes using field‐potential recording from optic lobe slices. Stimulation of the optic nerve produced pre‐ and postsynaptic field potentials. Transmission was abolished in calcium‐free seawater, L‐ glutamate or the AMPA/Kainate receptor blocker CNQX (EC50, 40 µm), leaving an intact presynaptic field potential. ACh markedly reduced or blocked and d‐tubocurarine augmented both pre‐ and postsynaptic field potentials, while α‐bungarotoxin and atropine were without effect. Paired‐pulse stimulation showed short‐term depression of pre‐ and postsynaptic components with a half‐time of recovery of ∼ 500 ms. The depression was partially relieved in the presence of d‐tubocurarine (half‐time of recovery, 350 ms). No long‐term changes in synaptic strength were induced by repetitive stimulation. A polyclonal antibody raised against a squid glutamate receptor produced positive staining in the third radial layer of the plexiform zone. No positive staining was observed in the other layers. Taking into account previous morphological data and our results, we propose that the excitatory terminations of the photoreceptors are in the innermost layer of the plexiform zone where the transmitter is likely to be glutamate and postsynaptic receptors are AMPA/kainate‐like. Thus, the function of the terminal bags is to provide a location for a presynaptic cholinergic inhibitory shunt. The results imply that this arrangement provides a temporal filter for visual processing and enhances the perception of moving vs. stationary objects.