Synaptic potentiation facilitates memory-like attractor dynamics in cultured in vitro hippocampal networks

Collective rhythmic dynamics from neurons is vital for cognitive functions such as memory formation but how neurons self-organize to produce such activity is not well understood. Attractor-based models have been successfully implemented as a theoretical framework for memory storage in networks of neurons. Activity-dependent modification of synaptic transmission is thought to be the physiological basis of learning and memory. The goal of this study is to demonstrate that using a pharmacological perturbation on in vitro networks of hippocampal neurons that has been shown to increase synaptic strength follows the dynamical postulates theorized by attractor models. We use a grid of extracellular electrodes to study changes in network activity after this perturbation and show that there is a persistent increase in overall spiking and bursting activity after treatment. This increase in activity appears to recruit more "errant" spikes into bursts. Lastly, phase plots indicate a conserved activity pattern suggesting that the network is operating in a stable dynamical state

Messenger RNAs localized to distal projections of human stem cell derived neurons

The identification of mRNAs in distal projections of model organisms has led to the discovery of multiple proteins that are locally synthesized for functional roles such as axon guidance, injury signaling and regeneration. The extent to which local protein synthesis is conserved in human neurons is unknown. Here we used compartmentalized microfluidic chambers to characterize the transcriptome of distal projections of human embryonic stem cells differentiated using a protocol which enriched for glutamatergic neurons (hESC-neurons). Using gene expression analysis, we identified mRNAs proportionally enriched in these projections, representing a functionally unique local transcriptome as compared to the human neuronal transcriptome inclusive of somata. Further, we found that the most abundant mRNAs within these hESC-neuron projections were functionally similar to the axonal transcriptome of rat cortical neurons. We confirmed the presence of two well characterized axonal mRNAs in model organisms, β-actin and GAP43, within hESC-neuron projections using multiplexed single molecule RNA-FISH. Additionally, we report the novel finding that oxytocin mRNA localized to these human projections and confirmed its localization using RNA-FISH. This new evaluation of mRNA within human projections provides an important resource for studying local mRNA translation and has the potential to reveal both conserved and unique translation dependent mechanisms

The Role of Corticothalamic Projections (Prelimbic Cortex to Nucleus Reuniens) in Working Memory

Working memory (WM) is the ability to store information for short periods of time and is used to execute tasks WM has been understood to work via the medial prefrontal cortex (mPFC) and dorsal hippocampus (dHPC), but they do not directly project to each other The nucleus reuniens of the thalamus (Re) is a “middle man” between the mPFC and dHPC There are projections between the prelimbic cortex (PrL) and Re that may be used during WM To test the connection of the PrL to Re, a delayed nonmatch to position (DNMTP) task was performe

Transferable neuronal mini-cultures to accelerate screening in primary and induced pluripotent stem cell-derived neurons

The effort and cost of obtaining neurons for large-scale screens has limited drug discovery in neuroscience. To overcome these obstacles, we fabricated arrays of releasable polystyrene micro-rafts to generate thousands of uniform, mobile neuron mini-cultures. These mini-cultures sustain synaptically-active neurons which can be easily transferred, thus increasing screening throughput by >30-fold. Compared to conventional methods, micro-raft cultures exhibited significantly improved neuronal viability and sample-to-sample consistency. We validated the screening utility of these mini-cultures for both mouse neurons and human induced pluripotent stem cell-derived neurons by successfully detecting disease-related defects in synaptic transmission and identifying candidate small molecule therapeutics. This affordable high-throughput approach has the potential to transform drug discovery in neuroscience

Long-Term Dynamical Constraints on Pharmacologically Evoked Potentiation Imply Activity Conservation within In Vitro Hippocampal Networks.

This paper describes a long-term study of network dynamics from in vitro, cultured hippocampal neurons after a pharmacological induction of synaptic potentiation. We plate a suspension of hippocampal neurons on an array of extracellular electrodes and record electrical activity in the absence of the drugs several days after treatment. While previous studies have reported on potentiation lasting up to a few hours after treatment, to the best of our knowledge, this is the first report to characterize the network effects of a potentiating mechanism several days after treatment. Using this reduced, two-dimensional in vitro network of hippocampal neurons, we show that the effects of potentiation are persistent over time but are modulated under a conservation of spike principle. We suggest that this conservation principle might be mediated by the appearance of a resonant inter-spike interval that prevents the network from advancing towards a state of hyperexcitability

MMPs and soluble ICAM-5 increase neuronal excitability within in vitro networks of hippocampal neurons.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that are released from neurons in an activity dependent manner. Published studies suggest their activity is important to varied forms of learning and memory. At least one MMP can stimulate an increase in the size of dendritic spines, structures which represent the post synaptic component for a large number of glutamatergic synapses. This change may be associated with increased synaptic glutamate receptor incorporation, and an increased amplitude and/or frequency of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) mini excitatory post-synaptic currents (EPSCs). An associated increase in the probability of action potential occurrence would be expected. While the mechanism(s) by which MMPs may influence synaptic structure and function are not completely understood, MMP dependent shedding of specific cell adhesion molecules (CAMs) could play an important role. CAMs are ideally positioned to be cleaved by synaptically released MMPs, and shed N terminal domains could potentially interact with previously unengaged integrins to stimulate dendritic actin polymerization with spine expansion. In the present study, we have used multielectrode arrays (MEAs) to investigate MMP and soluble CAM dependent changes in neuronal activity recorded from hippocampal cultures. We have focused on intercellular adhesion molecule-5 (ICAM-5) in particular, as this CAM is expressed on glutamatergic dendrites and shed in an MMP dependent manner. We show that chemical long-term potentiation (cLTP) evoked changes in recorded activity, and the dynamics of action potential bursts in particular, are altered by MMP inhibition. A blocking antibody to β(1) integrins has a similar effect. We also show that the ectodomain of ICAM-5 can stimulate β(1) integrin dependent increases in spike counts and burst number. These results support a growing body of literature suggesting that MMPs have important effects on neuronal excitability. They also support the possibility that MMP dependent shedding of specific synaptic CAMs can contribute to these effects

Hippocampal neurons cultured on multi-electrode arrays.

<p>Left: A DIC image of cells plated on the MEA. Scale bar = 200 μm Right: Screen shot of raw, unfiltered data of spontaneous activity from the networks. Each box represents one second of activity.</p

Not all electrodes maintain an increase in spiking activity after synaptic potentiation.

<p>Number of spikes before and after treatment. Each symbol corresponds to an electrode from a different MEA. A) Synaptic potentiation experiments. Each symbol represents an electrode from each MEA. (N = 4) A uniform expansion is observed and takes the shape of an ellipsoid with principle axis of rotational symmetry about y = x. (one-way ANOVA, p <10^–5) B) Vehicle experiments. (N = 4) The change in the shape is negligible. (one-way ANOVA, p < 10^–6)</p

Log(ISI) histograms of activity within epochs of activity before and after synaptic potentiation.

<p>A-C: Activity from the pre-treatment MEAs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129324#pone.0129324.g002" target="_blank">Fig 2D</a> was equally divided and the ISI histograms are presented. There are no changes to the distribution as activity evolves over time. D-F: Activity from the first day after synaptic potentiation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129324#pone.0129324.g002" target="_blank">Fig 2E</a> was divided between epochs of high frequency activity and the epochs of lower activity. The numbers above <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129324#pone.0129324.g002" target="_blank">Fig 2E</a> define the epochs of activity presented here. A new peak in the distribution of the emerges around 100ms. This peak corresponds to firing rates in the range of rat hippocampal theta activity and is highly associated with hippocampal learning and memory (35). G-H: Activity from the fourth day after treatment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129324#pone.0129324.g002" target="_blank">Fig 2E</a> was divided between epochs of high frequency activity and the epochs of lower activity. This 100 ms peak in the distribution is still present. Note the y-axis scale change on day 5 (panels G and H).</p

There is a persistent increase in spiking activity after synaptic potentiation.

<p>A) Spike counts from all electrodes before and treatment. Most electrodes have an increase in activity with a large cluster displaying an increase of at least two orders of magnitude. (one-way ANOVA, p<10^–9) B) Spike counts from the DMSO-treated MEAs show no increase in activity. (one-way ANOVA, p<10^–7). Each symbol corresponds an electrode from a different MEA. Three MEAs were used for the vehicle and four MEAs were used for the synaptic potentiation studies. The diagonal line denotes the identity line, y = x. (Modified and reprinted with permission.)</p