Uncovering the Cellular and Molecular Mechanisms of Synapse Formation and Functional Specificity Using Central Neurons of Lymnaea stagnalis.

All functions of the nervous system are contingent upon the precise organization of neuronal connections that are initially patterned during development, and then continually modified throughout life. Determining the mechanisms that specify the formation and functional modulation of synaptic circuitry are critical to advancing both our fundamental understanding of the nervous system as well as the various neurodevelopmental, neurological, neuropsychiatric, and neurodegenerative disorders that are met in clinical practice when these processes go awry. Defining the cellular and molecular mechanisms underlying nervous system development, function, and pathology has proven challenging, due mainly to the complexity of the vertebrate brain. Simple model system approaches with invertebrate preparations, on the other hand, have played pivotal roles in elucidating the fundamental mechanisms underlying the formation and plasticity of individual synapses, and the contributions of individual neurons and their synaptic connections that underlie a variety of behaviors, and learning and memory. In this Review, we discuss the experimental utility of the invertebrate mollusc Lymnaea stagnalis, with a particular emphasis on in vitro cell culture, semi-intact and in vivo preparations, which enable molecular and electrophysiological identification of the cellular and molecular mechanisms governing the formation, plasticity, and specificity of individual synapses at a single-neuron or single-synapse resolution.

A novel bio-mimicking, planar nano-edge microelectrode enables enhanced long-term neural recording.

Our inability to accurately monitor individual neurons and their synaptic activity precludes fundamental understanding of brain function under normal and various pathological conditions. However, recent breakthroughs in micro- and nano-scale fabrication processes have advanced the development of neuro-electronic hybrid technology. Among such devices are three-dimensional and planar electrodes, offering the advantages of either high fidelity or longer-term recordings respectively. Here, we present the next generation of planar microelectrode arrays with "nano-edges" that enable long-term (≥1 month) and high fidelity recordings at a resolution 15 times higher than traditional planar electrodes. This novel technology enables better understanding of brain function and offers a tremendous opportunity towards the development of future bionic hybrids and drug discovery devices.

Effect of planar microelectrode geometry on neuron stimulation: finite element modeling and experimental validation of the efficient electrode shape.

BACKGROUND: Microelectrode arrays have been used successfully for neuronal stimulation both in vivo and in vitro. However, in most instances currents required to activate the neurons have been in un-physiological ranges resulting in neuronal damage and cell death. There is a need to develop electrodes which require less stimulation current for neuronal activation with physiologically relevant efficacy and frequencies. NEW METHOD: The objective of the present study was to examine and compare the stimulation efficiency of different electrode geometries at the resolution of a single neuron. We hypothesized that increasing the electrode perimeter will increase the maximum current density at the edges and enhance stimulation efficiency. To test this postulate, the neuronal stimulation efficacy of common circular electrodes (smallest perimeter) was compared with star (medium perimeter), and spiral (largest perimeter with internal boundaries) electrodes. We explored and compared using both a finite element model and in vitro stimulation of neurons isolated from Lymnaea central ganglia. RESULTS: Interestingly, both the computational model and the live neuronal stimulation experiments demonstrated that the common circular microelectrode requires less stimulus to activate a cell compared to the other two electrode shapes with the same surface area. Our data further revealed that circular electrodes exhibit the largest sealing resistance, stimulus transfer, and average current density among the three types of electrodes tested. COMPARISON WITH EXISTING METHODS: Average current density and not the maximum current density at the edges plays an important role in determining the electrode stimulation efficiency. CONCLUSION: Circular shaped electrodes are more efficient in inducing a change in neuronal membrane potential.

Trophic factor-induced activity 'signature' regulates the functional expression of postsynaptic excitatory acetylcholine receptors required for synaptogenesis.

Highly coordinated and coincidental patterns of activity-dependent mechanisms ("fire together wire together") are thought to serve as inductive signals during synaptogenesis, enabling neuronal pairing between specific sub-sets of excitatory partners. However, neither the nature of activity triggers, nor the "activity signature" of long-term neuronal firing in developing/regenerating neurons have yet been fully defined. Using a highly tractable model system comprising of identified cholinergic neurons from Lymnaea, we have discovered that intrinsic trophic factors present in the Lymnaea brain-conditioned medium (CM) act as a natural trigger for activity patterns in post- but not the presynaptic neuron. Using microelectrode array recordings, we demonstrate that trophic factors trigger stereotypical activity patterns that include changes in frequency, activity and variance. These parameters were reliable indicators of whether a neuron expressed functional excitatory or inhibitory nAChRs and synapse formation. Surprisingly, we found that the post- but not the presynaptic cell exhibits these changes in activity patterns, and that the functional expression of excitatory nAChRs required neuronal somata, de novo protein synthesis and voltage gated calcium channels. In summary, our data provides novel insights into trophic factor mediated actions on neuronal activity and its specific regulation of nAChR expression.