Invertebrate Neurons Research Articles

KIF1A is the primary anterograde motor protein required for the axonal transport of dense-core vesicles in cultured hippocampal neurons

Dense-core vesicles (DCVs) are responsible for transporting, processing, and secreting neuropeptide cargos that mediate a wide range of biological processes, including neuronal development, survival, and learning and memory. DCVs are synthesized in the cell body and are transported by kinesin motor proteins along microtubules to pre- and postsynaptic release sites. Due to the dependence on kinesin-based transport, we sought to determine if the kinesin-3 family member, KIF1A, transports DCVs in primary cultured hippocampal neurons, as has been described for invertebrate neurons. Two-color, live-cell imaging showed that the DCV markers, chromogranin A-RFP and BDNF-RFP, move together with KIF1A-GFP in both the anterograde and retrograde directions. To demonstrate a functional role for KIF1A in DCV transport, motor protein expression in neurons was reduced using RNA interference (shRNA). Fluorescently tagged DCV markers showed a significant reduction in organelle flux in cells expressing shRNA against KIF1A. The transport of cargo driven by motors other than KIF1A, including mitochondria and the transferrin receptor, was unaffected in KIF1A shRNA expressing cells. Taken together, these data support a primary role for KIF1A in the anterograde transport of DCVs in mammalian neurons, and also provide evidence that KIF1A remains associated with DCVs during retrograde DCV transport.

Hypothermia translocates nitric oxide synthase from cytosol to membrane in snail neurons

Neuronal nitric oxide (NO) levels are modulated through the control of catalytic activity of NO synthase (NOS). Although signals limiting excess NO synthesis are being extensively studied in the vertebrate nervous system, our knowledge is rather limited on the control of NOS in neurons of invertebrates. We have previously reported a transient inactivation of NOS in hibernating snails. In the present study, we aimed to understand the mechanism leading to blocked NO production during hypothermic periods of Helix pomatia. We have found that hypothermic challenge translocated NOS from the cytosol to the perinuclear endoplasmic reticulum, and that this cytosol to membrane trafficking was essential for inhibition of NO synthesis. Cold stress also downregulated NOS mRNA levels in snail neurons, although the amount of NOS protein remained unaffected in response to hypothermia. Our studies with cultured neurons and glia cells revealed that glia-neuron signaling may inhibit membrane binding and inactivation of NOS. We provide evidence that hypothermia keeps NO synthesis "hibernated" through subcellular redistribution of NOS.

Mach–Zehnder interference microscopy optically records electrically stimulated cellular activity in unstained nerve cells

Dual-beam white light interference microscopy monitors changes in the optical density of the investigated object with high sensitivity. We report on the recording of dynamic changes in a neuron's optical density evoked by extracellular electrical stimulation. These recorded changes were analysed and unambiguously connected to the investigated object, an invertebrate neuron of the pond snail Lymnaea stagnalis. The results provide evidence for the method's applicability in visualizing cellular dynamics purely by evaluating changes in a cell's optical properties.

Action Potential Energy Efficiency Varies Among Neuron Types in Vertebrates and Invertebrates

The initiation and propagation of action potentials (APs) places high demands on the energetic resources of neural tissue. Each AP forces ATP-driven ion pumps to work harder to restore the ionic concentration gradients, thus consuming more energy. Here, we ask whether the ionic currents underlying the AP can be predicted theoretically from the principle of minimum energy consumption. A long-held supposition that APs are energetically wasteful, based on theoretical analysis of the squid giant axon AP, has recently been overturned by studies that measured the currents contributing to the AP in several mammalian neurons. In the single compartment models studied here, AP energy consumption varies greatly among vertebrate and invertebrate neurons, with several mammalian neuron models using close to the capacitive minimum of energy needed. Strikingly, energy consumption can increase by more than ten-fold simply by changing the overlap of the Na+ and K+ currents during the AP without changing the APs shape. As a consequence, the height and width of the AP are poor predictors of energy consumption. In the Hodgkin–Huxley model of the squid axon, optimizing the kinetics or number of Na+ and K+ channels can whittle down the number of ATP molecules needed for each AP by a factor of four. In contrast to the squid AP, the temporal profile of the currents underlying APs of some mammalian neurons are nearly perfectly matched to the optimized properties of ionic conductances so as to minimize the ATP cost.

Endoreplication: A Molecular Trick during Animal Neuron Evolution

The occurrence of endoreplication has been repeatedly reported in many organisms, including protists, plants, worms, arthropods, molluscs, fishes, and mammals. As a general rule, cells possessing endoreplicated genomes are large-sized and highly metabolically active. Endoreplication has not been frequently reported in neuronal cells that are typically considered to be fully differentiated and non-dividing, and which normally contain a diploid genome. Despite this general statement, various papers indicate that giant neurons in molluscs, as well as supramedullary and hypothalamic magnocellular neurons in fishes, contain DNA amounts larger than 2C. In order to study this issue in greater detail here, we review the available data about endoreplication in invertebrate and vertebrate neurons, and discuss its possible functional significance. As a whole, endoreplication seems to be a sort of molecular trick used by neurons in response to the high functional demands that they experience during evolution.

Adenosinergic modulation of neuronal activity in the pond snail Lymnaea stagnalis

Adenosine has been termed a retaliatory metabolite and its neuroprotective effects have been implicated in the hypoxia tolerance of several species; however, its role in the invertebrate CNS remains unclear. To determine if adenosine modulates neuronal activity in invertebrate neurons, we conducted whole-cell recordings from neurons in the central ring ganglia of the anoxia-tolerant pond snail Lymnaea stagnalis during exposure to adenosine and pharmacological compounds known to modulate the type I subclass of adenosine receptors (A(1)R). Action potential (AP) frequency and membrane potential (V(m)) were unchanged under control conditions, and addition of adenosine decreased AP frequency by 47% (from 1.08+/-0.22 to 0.57+/-0.14 Hz) and caused significant hyperpolarization of V(m). The A(1)R agonist cyclopentyladenosine (CPA) mimicked the results obtained with adenosine whereas antagonism of the A(1)R with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) had no effect on AP frequency or V(m) but prevented the adenosine and CPA-mediated decreases in neuronal activity. Furthermore, Ca(2+) measurements with fluo-4 revealed that A(1)R activation led to a 12% increase in intracellular Ca(2+) concentration and this elevation was also antagonized by DPCPX. Our results suggest that adenosine acting via the adenosine receptor (type I subclass) depresses neuronal activity in the adult L. stagnalis CNS and this depression is correlated with an increase in cytosolic Ca(2+) levels.

Antidepressant fluoxetine suppresses neuronal growth from both vertebrate and invertebrate neurons and perturbs synapse formation between Lymnaea neurons

Current treatment regimes for a variety of mental disorders involve various selective serotonin reuptake inhibitors such as Fluoxetine (Prozac). Although these drugs may 'manage' the patient better, there has not been a significant change in the treatment paradigm over the years and neither have the outcomes improved. There is also considerable debate as to the effectiveness of various selective serotonin reuptake inhibitors and their potential side-effects on neuronal architecture and function. In this study, using mammalian cortical neurons, a dorsal root ganglia cell line (F11 cells) and identified Lymnaea stagnalis neurons, we provide the first direct and unequivocal evidence that clinically relevant concentrations of Fluoxetine induce growth cone collapse and neurite retraction of both serotonergic and non-serotonergic neurons alike in a dose-dependent manner. Using intracellular recordings and calcium imaging techniques, we further demonstrate that the mechanism underlying Fluoxetine-induced effects on neurite retraction from Lymnaea neurons may involve lowering of intracellular calcium and a subsequent retardation of growth cone cytoskeleton. Using soma-soma synapses between identified presynaptic and postsynaptic Lymnaea neurons, we provide further direct evidence that clinically used concentrations of Fluoxetine also block synaptic transmission and synapse formation between cholinergic neurons. Our study raises alarms over potentially devastating side-effects of this antidepressant drug on neurite outgrowth and synapse formation in a developing/regenerating brain. Our data also demonstrate that drugs such as Fluoxetine may not just affect communication between serotonergic neurons but that the detrimental effects are widespread and involve neurons of various phenotypes from both vertebrate and invertebrate species.

NeuronBank: a tool for cataloging neuronal circuitry

The basic unit of any nervous system is the neuron. Therefore, understanding the operation of nervous systems ultimately requires an inventory of their constituent neurons and synaptic connectivity, which form neural circuits. The presence of uniquely identifiable neurons or classes of neurons in many invertebrates has facilitated the construction of cellular-level connectivity diagrams that can be generalized across individuals within a species. Homologous neurons can also be recognized across species. Here we describe NeuronBank.org, a web-based tool that we are developing for cataloging, searching, and analyzing neuronal circuitry within and across species. Information from a single species is represented in an individual branch of NeuronBank. Users can search within a branch or perform queries across branches to look for similarities in neuronal circuits across species. The branches allow for an extensible ontology so that additional characteristics can be added as knowledge grows. Each entry in NeuronBank generates a unique accession ID, allowing it to be easily cited. There is also an automatic link to a Wiki page allowing an encyclopedic explanation of the entry. All of the 44 previously published neurons plus one previously unpublished neuron from the mollusc, Tritonia diomedea, have been entered into a branch of NeuronBank as have 4 previously published neurons from the mollusc, Melibe leonina. The ability to organize information about neuronal circuits will make this information more accessible, ultimately aiding research on these important models.

The organisation of invertebrate brains: cells, synapses and circuits

AbstractMeinertzhagen, I.A. 2010. The organisation of invertebrate brains: cells, synapses and circuits. —Acta Zoologica (Stockholm) 91: 64–71Invertebrate brains are structurally diverse. Neuron numbers range from ∼102 to 108 in different groups, compared with larger numbers in vertebrate brains, ∼107 to 1014. The underpopulated brains of invertebrates are noted in their extreme cases for having few cells, and neurons that can be identified from animal to animal, many known in great detail. Although few in number, invertebrate neurons nevertheless comprise many classes. Correlated with the paucity of their number they are sparsely connected, many having ∼50 synapses or fewer. Synaptic densities, roughly 1 per μm3 of neuropile, differ little from those for much larger vertebrate neurons. Invertebrate neurons differ from their vertebrate counterparts in the position of their soma, generally in a cortex surrounding the neuropile that consequently occupies a relatively small volume. Their axons typically lack myelin and, supporting a range of conduction velocities, have diameters that differ over a wide range, from 103 to 10−1μm. Nerves with thousands of axons differ from neuropile fascicles, which typically have 20 or less. Unlike most vertebrate synapses, but like those of the vertebrate retina, synapses in many invertebrate groups – probably all ecdysozoans and possibly some lophotrochozoans – have synaptic contacts with multiple postsynaptic elements, dyads, triads and so on.

Circadian regulation of ion channels and their functions

Ion channels are the gatekeepers to neuronal excitability. Retinal neurons of vertebrates and invertebrates, neurons of the suprachiasmatic nucleus (SCN) of vertebrates, and pinealocytes of non-mammalian vertebrates display daily rhythms in their activities. The interlocking transcription-translation feedback loops with specific post-translational modulations within individual cells form the molecular clock, the basic mechanism that maintains the autonomic approximately 24-h rhythm. The molecular clock regulates downstream output signaling pathways that further modulate activities of various ion channels. Ultimately, it is the circadian regulation of ion channel properties that govern excitability and behavior output of these neurons. In this review, we focus on the recent development of research in circadian neurobiology mainly from 1980 forward. We will emphasize the circadian regulation of various ion channels, including cGMP-gated cation channels, various voltage-gated calcium and potassium channels, Na(+)/K(+)-ATPase, and a long-opening cation channel. The cellular mechanisms underlying the circadian regulation of these ion channels and their functions in various tissues and organisms will also be discussed. Despite the magnitude of chronobiological studies in recent years, the circadian regulation of ion channels still remains largely unexplored. Through more investigation and understanding of the circadian regulation of ion channels, the future development of therapeutic strategies for the treatment of sleep disorders, cardiovascular diseases, and other illnesses linked to circadian misalignment will benefit.

The Interscutularis Muscle Connectome

The complete connectional map (connectome) of a neural circuit is essential for understanding its structure and function. Such maps have only been obtained in Caenorhabditis elegans. As an attempt at solving mammalian circuits, we reconstructed the connectomes of six interscutularis muscles from adult transgenic mice expressing fluorescent proteins in all motor axons. The reconstruction revealed several organizational principles of the neuromuscular circuit. First, the connectomes demonstrate the anatomical basis of the graded tensions in the size principle. Second, they reveal a robust quantitative relationship between axonal caliber, length, and synapse number. Third, they permit a direct comparison of the same neuron on the left and right sides of the same vertebrate animal, and reveal significant structural variations among such neurons, which contrast with the stereotypy of identified neurons in invertebrates. Finally, the wiring length of axons is often longer than necessary, contrary to the widely held view that neural wiring length should be minimized. These results show that mammalian muscle function is implemented with a variety of wiring diagrams that share certain global features but differ substantially in anatomical form. This variability may arise from the dominant role of synaptic competition in establishing the final circuit.

The Regulative Role of Neurite Mechanical Tension in Network Development

A bewildering series of dynamical processes take part in the development of the nervous system. Neuron branching dynamics, the continuous formation and elimination of neural interconnections, are instrumental in constructing distinct neuronal networks, which are the functional building blocks of the nervous system. In this study, we investigate and validate the important regulative role of mechanical tension in determining the final morphology of neuronal networks. To single out the mechanical effect, we cultured relatively large invertebrate neurons on clean quartz surfaces. Applied to these surfaces were isolated anchoring sites consisting of carbon nanotube islands to which the cells and the neurites could mechanically attach. Inspection of branching dynamics and network wiring upon development revealed an innate selection mechanism in which one axon branch wins over another. The apparent mechanism entails the build-up of mechanical tension in developing axons. The tension is maintained by the attachment of the growth cone to the substrate or, alternatively, to the neurites of a target neuron. The induced tension promotes the stabilization of one set of axon branches while causing retraction or elimination of axon collaterals. We suggest that these findings represent a crucial, early step that precedes the formation of synapses and regulates neuronal interconnections. Mechanical tension serves as a signal for survival of the axonal branch and perhaps for the subsequent formation of synapses.

Axonal protein synthesis and the regulation of local mitochondrial function.

Axons and presynaptic nerve terminals of both invertebrate and mammalian SCG neurons contain a heterogeneous population of nuclear-encoded mitochondrial mRNAs and a local cytosolic protein synthetic system. Nearly one quarter of the total protein synthesized in these structural/functional domains of the neuron is destined for mitochondria. Acute inhibition of axonal protein synthesis markedly reduces the functional activity of mitochondria. The blockade of axonal protein into mitochondria had similar effects on the organelle's functional activity. In addition to mitochondrial mRNAs, SCG axons contain approximately 200 different microRNAs (miRs), short, noncoding RNA molecules involved in the posttranscriptional regulation of gene expression. One of these miRs (miR-338) targets cytochrome c oxidase IV (COXIV) mRNA. This nuclear-encoded mRNA codes for a protein that plays a key role in the assembly of the mitochondrial enzyme complex IV and oxidative phosphorylation. Over-expression of miR-338 in the axon markedly decreases COXIV expression, mitochondrial functional activity, and the uptake of neurotransmitter into the axon. Conversely, the inhibition of endogeneous miR-338 levels in the axon significantly increased mitochondrial activity and norepinephrine uptake into the axon. The silencing of COXIV expression in the axon using short, inhibitory RNAs (siRNAs) yielded similar results, a finding that indicated that the effects of miR-338 on mitochondrial activity and axon function were mediated, at least in part, through local COXIV mRNA translation. Taken together, recent findings establish that proteins requisite for mitochondrial activity are synthesized locally in the axon and nerve terminal, and call attention to the intimacy of the relationship that has evolved between the distant cellular domains of the neuron and its energy generating systems.

Neuronal Homeostasis: Does Form Follow Function or Vice Versa?

Nerve cells adjust their electrical excitability and the overall strength of synaptic connections to maintain their functional identity. A recent study suggests that they may also regulate dendritic branch patterns to compensate for the variability of synaptic contacts and help ensure appropriate connectivity in the brain.

Transient neurites of retinal horizontal cells exhibit columnar tiling via homotypic interactions

Sensory neurons with common function are often non-randomly arranged and form dendritic territories that exhibit little overlap or tiling. Repulsive homotypic interactions underlie such patterns in cell organization in invertebrate neurons. In mammalian retinal horizontal cells, however, it is unclear how dendro-dendritic repulsive interactions can produce a non-random distribution of cells and their spatial territories because mature horizontal cell dendrites overlap substantially. By imaging developing mouse horizontal cells, we found that upon reaching their final laminar positions, these cells transiently elaborate vertical neurites that form non-overlapping columnar territories. Targeted cell ablation revealed that the vertical neurites engage in homotypic interactions resulting in tiling of neighboring cells prior to establishment of their dendritic fields. This developmental tiling of transient neurites correlates with the emergence of a non-random distribution of the cells, and could represent a mechanism that organizes neighbor relationships and territories of neurons of the same type before circuit assembly.

Axonal degeneration and regeneration: a mechanistic tug‐of‐war

Axonal degeneration is a common hallmark of both nerve injury and many neurodegenerative conditions, including motor neuron disease, glaucoma, and Parkinson's, Alzheimer's, and Huntington's diseases. Degeneration of the axonal compartment is distinct from neuronal cell death, and often precedes or is associated with the appearance of the symptoms of the disease. A complementary process is the regeneration of the axon, which is commonly observed following nerve injury in many invertebrate neurons and in a number of vertebrate neurons of the PNS. Important discoveries, together with innovative imaging techniques, are now paving the way towards a better understanding of the dynamics and molecular mechanisms underlying these two processes. In this study, I will discuss these recent findings, focusing on the balance between axonal degeneration and regeneration.

Selective Spike Propagation in the Central Processes of an Invertebrate Neuron

Within a neuron, spike propagation can occur in a complex manner, with spikes propagating into some processes but not others. We study this phenomenon in an experimentally advantageous mechanoafferent in Aplysia, neuron B21. B21 has two processes within the CNS. One is ipsilateral to the soma and is referred to as the lateral process. The second travels into the contralateral hemiganglion and is referred to as the contralateral process. Previously we characterized spike propagation to the lateral process, which is an output region that contacts follower motor neurons. Spikes fail to actively propagate to the lateral process when B21 is peripherally activated at its resting potential. This propagation failure can be relieved if the medial regions of B21 are centrally depolarized during peripheral activation. This study examines spike propagation to the contralateral process. We show that, unlike the lateral process, active spike propagation in the contralateral process occurs when B21 is peripherally activated at its resting membrane potential. Thus spike propagation occurs selectively, favoring the contralateral process. Interestingly, the contralateral process of one B21 is immediately adjacent to the medial region of the bilaterally symmetrical cell. The B21 neurons are electrically coupled, suggesting that spikes propagating in the contralateral process of one cell could modify propagation in the sister neuron. Consistent with this idea, we show that lateral process propagation failures observed when a single B21 is peripherally activated can be relieved by central coactivation of the contralateral cell. These results imply that stimuli that coactivate the B21 neurons bilaterally are more apt to generate afferent activity that is transmitted to followers than stimuli that activate one cell.

Microtubules have opposite orientation in axons and dendrites of Drosophila neurons.

In vertebrate neurons, axons have a uniform arrangement of microtubules with plus ends distal to the cell body (plus-end-out), and dendrites have equal numbers of plus- and minus-end-out microtubules. To determine whether microtubule orientation is a conserved feature of axons and dendrites, we analyzed microtubule orientation in invertebrate neurons. Using microtubule plus end dynamics, we mapped microtubule orientation in Drosophila sensory neurons, interneurons, and motor neurons. As expected, all axonal microtubules have plus-end-out orientation. However, in proximal dendrites of all classes of neuron, approximately 90% of dendritic microtubules were oriented with minus ends distal to the cell body. This result suggests that minus-end-out, rather than mixed orientation, microtubules are the signature of the dendritic microtubule cytoskeleton. Surprisingly, our map of microtubule orientation predicts that there are no tracks for direct cargo transport between the cell body and dendrites in unipolar neurons. We confirm this prediction, and validate the completeness of our map, by imaging endosome movements in motor neurons. As predicted by our map, endosomes travel smoothly between the cell body and axon, but they cannot move directly between the cell body and dendrites.

Effects of Active Conductance Distribution over Dendrites on the Synaptic Integration in an Identified Nonspiking Interneuron

The synaptic integration in individual central neuron is critically affected by how active conductances are distributed over dendrites. It has been well known that the dendrites of central neurons are richly endowed with voltage- and ligand-regulated ion conductances. Nonspiking interneurons (NSIs), almost exclusively characteristic to arthropod central nervous systems, do not generate action potentials and hence lack voltage-regulated sodium channels, yet having a variety of voltage-regulated potassium conductances on their dendritic membrane including the one similar to the delayed-rectifier type potassium conductance. It remains unknown, however, how the active conductances are distributed over dendrites and how the synaptic integration is affected by those conductances in NSIs and other invertebrate neurons where the cell body is not included in the signal pathway from input synapses to output sites. In the present study, we quantitatively investigated the functional significance of active conductance distribution pattern in the spatio-temporal spread of synaptic potentials over dendrites of an identified NSI in the crayfish central nervous system by computer simulation. We systematically changed the distribution pattern of active conductances in the neuron's multicompartment model and examined how the synaptic potential waveform was affected by each distribution pattern. It was revealed that specific patterns of nonuniform distribution of potassium conductances were consistent, while other patterns were not, with the waveform of compound synaptic potentials recorded physiologically in the major input-output pathway of the cell, suggesting that the possibility of nonuniform distribution of potassium conductances over the dendrite cannot be excluded as well as the possibility of uniform distribution. Local synaptic circuits involving input and output synapses on the same branch or on the same side were found to be potentially affected under the condition of nonuniform distribution while operation of the major input-output pathway from the soma side to the one on the opposite side remained the same under both conditions of uniform and nonuniform distribution of potassium conductances over the NSI dendrite.

Physiology and Morphology of Shared and Specialized Spinal Interneurons for Locomotion and Scratching

Distinct types of rhythmic movements that use the same muscles are typically generated largely by shared multifunctional neurons in invertebrates, but less is known for vertebrates. Evidence suggests that locomotion and scratching are produced partly by shared spinal cord interneuronal circuity, although direct evidence with intracellular recording has been lacking. Here, spinal interneurons were recorded intracellularly during fictive swimming and fictive scratching in vivo and filled with Neurobiotin. Some interneurons that were rhythmically activated during both swimming and scratching had axon terminal arborizations in the ventral horn of the hindlimb enlargement, indicating their likely contribution to hindlimb motor outputs during both behaviors. We previously described a morphological group of spinal interneurons ("transverse interneurons" or T neurons) that were rhythmically activated during all forms of fictive scratching at higher peak firing rates and with larger membrane potential oscillations than scratch-activated spinal interneurons with different dendritic orientations. The current study demonstrates that T neurons are activated during both swimming and scratching and thus are components of the shared circuitry. Many spinal interneurons activated during fictive scratching are also activated during fictive swimming (scratch/swim neurons), but others are suppressed during swimming (scratch-specialized neurons). The current study demonstrates that some scratch-specialized neurons receive strong and long-lasting hyperpolarizing inhibition during fictive swimming and are also morphologically distinct from T neurons. Thus this study indicates that locomotion and scratching are produced by a combination of shared and dedicated interneurons whose physiological and morphological properties are beginning to be revealed.