John C. Chang

Modulation of neural network activity by patterning

Using neuronal cultures on microelectrode arrays, researchers have shown that recordable electrical activity can be influenced by chemicals in the culture environment, thus demonstrating potential applicability to biosensors or drug screening. Since practical success requires the design of robust networks with repeatable, reliable responses understanding the sources of variation is important. In this report, we used lithographic technologies to confine neurons to highly defined patterns (40 microm wide stripes); in turn these patterns gave us a measure of control over the local density of neurons (100-500 cells/mm(2)). We found that the apparent electrical activity of the network, as measured by the fraction of electrodes from which signals were recordable, increases 8-10-fold with greater local density. Also, average-firing rates of the active neurons increased 3-5-fold. We conclude that patterned networks offer one means of controlling and enhancing the responsiveness of cultured neural networks.

A modified microstamping technique enhances polylysine transfer and neuronal cell patterning

Macromolecular microstamping with polydimethylsiloxane (PDMS) stamps has been demonstrated to transfer proteins onto glassy substrates for antigen or antibody detection and for cell patterning. For many applications, including neuronal cell patterning, it is important to assure reliable transfer of sufficient quantity of protein. Research has shown that protein transfer is enhanced with the selection of the proper protein-stamp-substrate combination. In addition, detergent studies have shown that detergent-protein complexes detach from surfaces to a greater extent than proteins alone. Therefore, we hypothesized that stamp surface modification (termed here a release layer) can enhance polylysine transfer and benefit cell growth on microstamped substrates. We found unmodified stamps to transfer insufficient polylysine to support good cell survival of hippocampal neurons in a widely used serum-free, reduced-glia cell culture system. However, with modified stamps neuronal growth was reliably good. This enhanced cell growth can be attributed to the increased polylysine transfer due to the release layer rather than increased loading onto the stamp. This enhancement was found to be even greater for two-month old stamps that were stored in water. Furthermore, the physicochemical properties of the release layer can modulate the loading process. Thus, our data supports the conclusions that the release layer: (1) modulates polylysine loading, (2) enhances polylysine transfer, (3) enhances cellular growth on microstamped substrates, and (4) extends the durability (defined as the number of times a stamp can be reused) of PDMS microstamps.

Gold-coated microelectrode array with thiol linked self-assembled monolayers for engineering neuronal cultures

We report the use of a gold coating on microelectrode arrays (MEAs) to enable the use of the relatively reliable surface modification chemistry afforded by alkanethiol self-assembled monolayers (SAMs). The concept is simple and begins with planar MEAs, which are commercially available for neuronal cell culture and for brain slice studies. A gold film, with an intermediate adhesive layer of titanium, is deposited over the insulation of an existing MEA in a manner so as to be thin enough for transmission light microscopy as well as to avoid electrical contact to the electrodes. The alkanethiol-based linking chemistry is then applied for the desired experimental purpose. Here we show that polylysine linked to alkanethiol SAM can control the geometry of an in vitro hippocampal neuronal network grown on the MEA. Furthermore, recordings of neuronal action potentials from random and patterned networks suggest that the gold coating does not significantly alter the electrode properties. This design scheme may be useful for increasing the number of neurons located in close proximity to the electrodes. Realization of in vitro neuronal circuits on MEAs may significantly benefit basic neuroscience studies, as well as provide the insight relevant to applications such as neural prostheses or cell-based biosensors. The gold coating technique makes it possible to use the rich set of thiol-based surface modification techniques in combination with MEA recording.

Microelectrode Array Recordings of Patterned Hippocampal Neurons for Four Weeks

Recent advances in cell biology and surface patterning make possible the construction of in vitro neural networks for long-term, multichannel recording studies. Towards this goal, we have demonstrated the recording of spontaneous electrical activity from rat embryonic hippocampal neurons confined to parallel lines which overlay the microelectrode array. The neurons adhered to adsorbed poly-D-lysine patterns and remained alive on the pattern for up to one month. Recordable, extracellular electrical activity began as early as 6 days in vitro and continued for the duration of the culture. Average amplitude of detected action potentials ranged between 70 μ V to 150 μ V measured from baseline to peak, consistent with results from unpatterned culture technologies.

Neuronal network structuring induces greater neuronal activity through enhanced astroglial development

The confluence of micropatterning, microfabricated multielectrode arrays, and low-density neuronal culture techniques make possible the growth of patterned neuronal circuits overlying multielectrode arrays. Previous studies have shown synaptic interaction within patterned cultures which was more active on average than random cultures. In our present study, we found patterned cultures to have up to five times more astrocytes and three times more neurons than random cultures. In addition, faster development of synapses is also seen in patterned cultures. Together, this yielded greater overall neuronal activity as evaluated by the number of active electrodes. Our finding of astrocytic proliferation within serum-free culture is also novel.

Pattern Technologies for Structuring Neuronal Networks on MEAs

Much progress has been made over the past several centuries regarding the understanding of the human central nervous system. This progress has taken us from knowing the brain as a functionally homogeneous organ to one with interconnected networks of specialized neurons. The initiating study was conducted by Broca who examined a patient with a left-sided posterior frontal lobe lesion which resulted in dysphasia. Broca concluded that the ability to generate coherent speech resided in the dominant posterior frontal lobe and theorized that distinct regions of the brain functioned uniquely. This theory was further developed by Wernicke who speculated that distinct regions of the brain intercommunicated to achieve the observed complex human behavior. Findings from both electrical stimulation and seizure studies support this theory as motor and sensory functions can be localized to specific regions of the brain (Kandel et al., 2000). These experiments provided the foundation for the interconnected network theory, the details of which are being expounded daily by the numerous functional imaging studies reported in the literature (Kandel et al., 2000). Although the progress in understanding the regional interaction between brain regions has been exceptional, the detailed understanding of the neuronal firing patterns, either in aggregate as in electroencephalography (EEG) or in simultaneous multi-site monitoring as in cortical probes, is desirable. The advantage of understanding the patterns lies in the ability to diagnose diseased states or in cognitive control of exogenous actuators such as robotic arms or input devices to computers (Kandel et al., 2000; Nicolelis, 2001). Diagnosis of seizures is usually confirmed when third-person observation provides suitable symptoms such as absence, tonic or clonic muscular activities, and loss of consciousness, but EEGs can be diagnostic when proper neuronal activities are detected (Kandel et al., 2000). On the other hand, neuronal firing patterns can be harvested to control electronic gadgets as experiments with trained monkeys have shown (Carmena et al., 2003). Thus, knowledge of the patterns of neuronal firing can allow one to decode and predict the state of an organism’s neurological function.

Solitary Intercostal Arterial Trunk: A Previously Unreported Anatomical Variant

During embryonic life, events may alter the normal growth and fusion of the endocardial tube, the paired dorsal aortae, and the vitelline plexus, leading to anomalies and variants of the mature axial arterial system.1 Many of these are well known, especially those associated with the aorta, heart, and umbilical artery.1,2 Vitelline variants have also been found that are either associated with Meckel diverticulum or caudal regression syndrome.3,4 We present a case of anomalous intercostal arterial supply, which, to our knowledge, has not been reported previously. The aberrant intercostal trunk is associated with aberrant descending thoracic aorta position, a replaced common hepatic artery from the superior mesenteric artery, and an isolated splenic artery origin from the aorta. A 27-year-old man with a medical history of irritable bowel syndrome and stage I hypertension presented for renal vasculature evaluation. A renal arterial magnetic resonance …

Recording of patterned neural networks

The authors have accomplished long-term recording from patterned neural networks (parallel lines of varying width and length) in serum-free media from cultures as old as one month. This is an important milestone toward the goal of creating and studying sparse, precisely defined neural networks.

An enhanced microstamping technique for controlled deposition of proteins

It has been demonstrated that protein adsorbed onto poly-dimethyl-siloxane (PDMS) stamps is transferred to substrates in the process of microstamping, and it has been assumed that this amount is sufficient for supporting cellular growth on activated glass substrates. Currently, there exists no characterization of the microstamping process and no method to control the amount of protein deposited onto substrate surfaces. In this report, we demonstrate a new technique to vary the protein loaded onto the stamp and hence the amount transferred onto substrates. Using fluorescence, we characterized this new microstamping process with regard to the amount of time needed for complete transfer of protein and the variation of protein transfer with pressure and stamp age. Furthermore, we compared growth of cells on protein film (poly-D-lysine, 70 to 150 kDa) transferred with this new process and that of the traditional process and concluded: 1) a minimum of 7 /spl mu/g of PDL/cm/sup 2/ (20 minute loading time) is necessary for bare minimum growth of neurons and 2) good cellular growth can be obtained with 10-12 /spl mu/g of PDL/cm/sup 2/ (60 minute loading time).