L. B. Popova

Analysis of the Central Pattern Generator for Swimming in the Mollusk Clionea

Abstract: The pteropod mollusk Clione limacina swims by rhythmic movements of two wings. The central pattern generator (CPG) for swimming, located in the pedal ganglia, is formed by three groups of interneurons. The interneurons of the groups 7 and 8 are of crucial importance for rhythm generation. They are endogenous oscillators capable of generating rhythmic activity with a range of frequencies typical of swimming after extraction from the ganglia. This endogenous rhythmic activity is enhanced by serotonin. The interneurons 7 and 8 produce one prolonged action potential (about 100 ms in duration) per cycle. Prolonged action potentials contribute to determining the duration of the cycle phases. The interneurons of two groups inhibit one another determining their reciprocal activity. The putative transmitters of groups 7 and 8 interneurons are glutamate and acetylcholine, respectively. Transition from one phase to the other is facilitated by the plateau interneurons of group 12 that contribute to termination of one phase and to initiation of the next phase. Maintaining the rhythm generation and transition from one phase to the other is also promoted by postinhibitory rebound. The redundant organization of the swimming generator guarantees the high reliability of its operation. Generation of the swimming output persisted after the inhibitory input from interneurons 8 to 7 had been blocked by atropine. Activity of the swimming generator is controlled by a set of command neurons that activate, inhibit or modulate the operation of the swimming CPG in relation to a behaviorally relevant context.

Control of locomotion in marine mollusc Clione limacina. VI. Activity of isolated neurons of pedal ganglia.

SummaryIn the pteropodial mollusc Clione limacina, the rhythmic locomotor wing movements are controlled by the pedal ganglia. The locomotor rhythm is generated by two groups of interneurons (groups 7 and 8) which drive efferent neurons. In the present paper, the activity of isolated neurons, which were extracted from the pedal ganglia by means of an intracellular electrode, is described. The following results have been obtained: 1. Isolated type 7 and 8 interneurons preserved the capability for generation of prolonged (100–200 ms) action potentials. The frequency of these spontaneous discharges was usually within the limit of locomotor frequencies (0.5–5 Hz). By de- or hyperpolarizing a cell, one could usually cover the whole range of locomotor frequencies. This finding demonstrates that the locomotor rhythm is indeed determined by the endogenous rhythmic activity of type 7 and 8 interneurons. 2. Type 1 and 2 efferent neurons, before isolation, could generate single spikes as well as high-frequency bursts of spikes. These two modes of activity were also observed after isolating the cells. Thus, the bursting activity of type 1 and 2 neurons, demonstrated during locomotion, is determined by their own properties. Type 3 and 4 efferent neurons generated only repeated single spikes both before and after isolation. 3. The activity of the isolated axons of type 1 and 2 neurons did not differ meaningfully from the activity of the whole cells. Furthermore, in the isolated pedal commissure, we found units whose activity (rhythmically repeating prolonged action potentials) resembled the activity of type 7 and 8 interneurons. These units seemed to be the axons of type 7 and 8 interneurons. Thus, different parts of the cell membrane (soma and axons) have similar electric properties.

Molecular characterization of the first aromatic nutrient transporter from the sodium neurotransmitter symporter family

SUMMARY Nutrient amino acid transporters (NATs, subfamily of sodium neurotransmitter symporter family SNF, a.k.a. SLC6) represent a set of phylogenetically and functionally related transport proteins, which perform intracellular absorption of neutral, predominantly essential amino acids. Functions of NATs appear to be critical for the development and survival in organisms. However, mechanisms of specific and synergetic action of various NAT members in the amino acid transport network are virtually unexplored. A new transporter, agNAT8, was cloned from the malaria vector mosquito Anopheles gambiae (SS). Upon heterologous expression in Xenopus oocytes it performs high-capacity, sodium-coupled (2:1) uptake of nutrients with a strong preference for aromatic catechol-branched substrates, especially phenylalanine and its derivatives tyrosine and L-DOPA, but not catecholamines. It represents a previously unknown SNF phenotype, and also appears to be the first sodium-dependent B0 type transporter with a narrow selectivity for essential precursors of catecholamine synthesis pathways. It is strongly and specifically transcribed in absorptive and secretory parts of the larval alimentary canal and specific populations of central and peripheral neurons of visual-, chemo- and mechano-sensory afferents. We have identified a new SNF transporter with previously unknown phenotype and showed its important role in the accumulation and redistribution of aromatic substrates. Our results strongly suggest that agNAT8 is an important, if not the major, provider of an essential catechol group in the synthesis of catecholamines for neurochemical signaling as well as ecdysozoan melanization and sclerotization pathways, which may include cuticle hardening/coloring, wound curing, oogenesis, immune responses and melanization of pathogens.

Origin of signals conveyed by the ventral spino-cerebellar tract and spino-reticulo-cerebellar pathway

Summary(1)The “fictitious” scratch reflex was evoked in decerebrate curarized cats by pinna stimulation. Activity of neurons of the ventral spinocerebellar tract (VSCT) from the L4 and L5 segments of the spinal cord as well as of neurons of the spinoreticulo-cerebellar pathway (SRCP) from the lateral reticular nucleus of the medulla oblongata was recorded. Cooling and destruction of different parts of the lumbo-sacral enlargement of the spinal cord were performed.(2)Cooling of the L5 or L6 segment abolished the rhythmic activity in the greater part of the spinal hindlimb centre but did not affect the generation of rhythmic oscillations in the remaining (rostral) segments of the lumbo-sacral enlargement. Under these conditions, neither the rhythmic activity of VSCT neurons located rostral to the thermode nor that of SRCP neurons changed.(3)A normal rhythmic activity of SRCP neurons also persisted after destruction of grey matter in the L3 and L4 segments. It can be concluded that activity of these neurons is independent of whichever part of the enlargement generates rhythmic oscillations.(4)From these observations a hypothesis is advanced that the main content of signals conveyed by the VSCT and SRCP to the cerebellum is the information regarding activity of the generator of rhythmic oscillations that is located in the L3-L5 spinal segments.

The invertebrate B0 system transporter, D. melanogaster NAT1, has unique d-amino acid affinity and mediates gut and brain functions

The CG3252 gene product, DmNAT1, represents the first Nutrient Amino acid Transporter cloned from Drosophila. It absorbs a broader set of neutral amino acids versus earlier characterized insect NATs and mammalian NATs-B(0) system transporters from the Sodium Neurotransmitter symporter Family (SNF, a.k.a. solute carrier family 6, SLC6). In addition to B(0)-specific l-substrates, DmNAT1 equally or more effectively transports d-amino acids with sub-millimolar affinities and 1:1 sodium:amino acid transport stoichiometry. DmNAT1 is strongly transcribed in the absorptive and secretory regions of the larval alimentary canal and larval brain, revealing its roles in the primary absorption and redistribution of large neutral l-amino acids as well as corresponding d-isomers. The absorption of d-amino acids via DmNAT1 may benefit the acquisition of fermented and symbiotic products, and may support the unique capacity of fruit fly larvae to utilize a diet with substitution of essential amino acids by d-isomers. It also suggests a remarkable adaptive plasticity of NAT-SLC6 mechanisms via alterations of a few identifiable sites in the substrate-binding pocket. The strong transcription in the brain suggests roles for DmNAT1 in neuronal nutrition and clearance of l-neutral amino acids from the fly brain. In addition, neuronal DmNAT1 may absorb synaptic d-serine and modulate NMDA receptor-coupled signal transduction. The characterization of the first invertebrate B(0)-like transporter extends the biological roles of the SLC6 family, revealing adaptations for the absorption of d-isomers of the essential amino acids. These findings suggest that some members of the NAT-SLC6 subfamily are evolving specific properties which contribute to nutrient symbiotic relationships and neuronal functions.

Activity of C3-C4 propriospinal neurons during fictitious forelimb locomotion in the cat

The activity of C3-C4 propriospinal neurons was recorded during fictitious forelimb locomotion in immobilized decerebrated cats with the spinal cord transected at the lower thoracic level. The discharge frequency of most neurons was rhythmically modulated in relation to the cycle of fictitious stepping in spite of the absence of any rhythmic signals from the limb receptors. Thus, the intraspinal mechanisms present a powerful input to the C3-C4 propriospinal neurons.

Cloning and functional expression of the first eukaryotic Na+–tryptophan symporter, AgNAT6

SUMMARY The nutrient amino acid transporter (NAT) subfamily of the neurotransmitter sodium symporter family (NSS, also known as the solute carrier family 6, SLC6) represents transport mechanisms with putative synergistic roles in the absorption of essential and conditionally essential neutral amino acids. It includes a large paralogous expansion of insect-specific genes, with seven genes from the genome of the malaria mosquito, Anopheles gambiae. One of the An. gambiae NATs, AgNAT8, was cloned, functionally expressed and characterized in X. laevis oocytes as a cation-coupled symporter of aromatic amino acids, preferably l-phenylalanine, l-tyrosine and l-DOPA. To explore an evolutionary trend of NAT-SLC6 phenotypes, we have cloned and characterized AgNAT6, which represents a counterpart of AgNAT8 descending from a recent gene duplication (53.1% pairwise sequence identity). In contrast to AgNAT8, which preferably mediates the absorption of phenol-branched substrates, AgNAT6 mediates the absorption of indole-branched substrates with highest apparent affinity to tryptophan (K0.5Trp=1.3 μmol l–1 vs K0.5Phe=430 μmol l–1) and [2 or 1 Na+ or K+]:[aromatic substrate] stoichiometry. AgNAT6 is highly transcribed in absorptive and secretory regions of the alimentary canal and specific neuronal structures, including the neuropile of ventral ganglia and sensory afferents. The alignment of AgNATs and LeuTAa, a bacterial NAT with a resolved 3D structure, reveals three amino acid differences in the substrate-binding pocket that may be responsible for the indole- vs phenol-branch selectivity of AgNAT6 vs AgNAT8. The identification of transporters with a narrow selectivity for essential amino acids suggests that basal expansions in the SLC6 family involved duplication and retention of NATs, improving the absorption and distribution of under-represented essential amino acids and related metabolites. The identified physiological and expression profiles suggest unique roles of AgNAT6 in the active absorption of indole-branched substrates that are used in the synthesis of the neurotransmitter serotonin as well as the key circadian hormone and potent free-radical scavenger melatonin.

Synergy and specificity of two Na+-aromatic amino acid symporters in the model alimentary canal of mosquito larvae.

SUMMARY The nutrient amino acid transporter (NAT) subfamily is the largest subdivision of the sodium neurotransmitter symporter family (SNF; also known as SLC6; HUGO). There are seven members of the NAT population in the African malaria mosquito Anopheles gambiae, two of which, AgNAT6 and AgNAT8, preferably transport indole- and phenyl-branched substrates, respectively. The relative expression and distribution of these aromatic NATs were examined with transporter-specific antibodies in Xenopus oocytes and mosquito larval alimentary canal, representing heterologous and tissue expression systems, respectively. NAT-specific aromatic-substrate-induced currents strongly corresponded with specific accumulation of both transporters in the plasma membrane of oocytes. Immunolabeling revealed elevated expressions of both transporters in specific regions of the larval alimentary canal, including salivary glands, cardia, gastric caeca, posterior midgut and Malpighian tubules. Differences in relative expression densities and spatial distribution of the transporters were prominent in virtually all of these regions, suggesting unique profiles of the aromatic amino acid absorption. For the first time reversal of the location of a transporter between apical and basal membranes was identified in posterior and anterior epithelial domains corresponding with secretory and absorptive epithelial functions, respectively. Both aromatic NATs formed putative homodimers in the larval gut whereas functional monomers were over-expressed heterologously in Xenopus oocytes. The results unequivocally suggest functional synergy between substrate-specific AgNAT6 and AgNAT8 in intracellular absorption of aromatic amino acids. More broadly, they suggest that the specific selectivity, regional expression and polarized membrane docking of NATs represent key adaptive traits shaping functional patterns of essential amino acid absorption in the metazoan alimentary canal and other tissues.

Non-synaptic interaction between neurons in molluscs

Abstract 1. Isolated pedal ganglia of the pteropodial mollusc, Clione limacina , generate a locomotory rhythm. In 30% of the pedal ganglia preparations the locomotory rhythm was not regular, i.e. the locomotor generator worked in “bursts” alternating with periods of low activity. 2. The “locomotor bursts” were caused by spontaneous activation of command neurons located in the pedal ganglia. 3. A single neuron was extracted from burst-generating preparations by means of the intracellular microelectrode and then its soma was put back, into the initial place between the ganglion cells. Twenty-five percent of the isolated neurons renewed the bursts-related changes in their activity after the insertion into the ganglion. The neurons which were originally excited during the “locomotor bursts” continued to be excited after isolation, while those which were inhibited continued to be inhibited during the bursts. 4. It is suggested that the command neurons controlling the locomotor generator can exert action on the target cells in the absence of morphological synapses.

Vestibular compensation in lampreys: restoration of symmetry in reticulospinal commands

SUMMARY Removal of a vestibular organ (unilateral labyrinthectomy, UL) in the lamprey results in a loss of equilibrium, so that the animal rolls (rotates around its longitudinal axis) when swimming. Owing to vestibular compensation, UL animals gradually restore postural equilibrium and, in a few weeks, swim without rolling. Important elements of the postural network in the lamprey are the reticulospinal (RS) neurons, which are driven by vestibular input and transmit commands for postural corrections to the spinal cord. As shown previously, a loss of equilibrium after UL is associated with disappearance of vestibular responses in the contralateral group of RS neurons. Are these responses restored in animals after compensation? To answer this question, we recorded vestibular responses in RS neurons (elicited by rotation of the compensated animal in the roll plane) by means of chronically implanted electrodes. We found that the responses re-appeared in the compensated animals. This result supports the hypothesis that the loss of equilibrium after UL was caused by asymmetry in supraspinal motor commands, and the recovery of postural control in compensated animals was due to a restoration of symmetry.