Surface plasmon resonance imaging of excitable cells.

Surface plasmons (SPs) are surface charge density oscillations occuring at a metal/dieletric interface and are highly sensitive to refractive index variations adjacent to the surface. This sensitivity has been exploited successfully for chemical and biological assays. In these systems, a surface plasmon resonance (SPR)-based sensor detects temporal variations in the refractive index at a point. SPR has also been used in imaging systems where the spatial variations of refractive index in the sample provide the contrast mechanism. SPR imaging systems using high numerical aperture (NA) objective lenses have been designed to image adherent live cells with high magnification and near-diffraction limited spatial resolution. Addressing research questions in cell physiology and pharmacology often requires the development of a multimodal microscope where complementary information can be obtained. In this paper, we present the development of a multimodal microscope that combines SPR imaging with a number of additional imaging modalities including bright-field, epifluorescence, total internal reflection microscopy and SPR fluorescence microscopy. We used a high NA objective lens for SPR and TIR microscopy and the platform has been used to image live cell cultures demonstrating both fluorescent and label-free techniques. Both the SPR and TIR imaging systems feature a wide field of view (~300 µm) that allows measurements from multiple cells whilst maintaining a resolution sufficient to image fine cellular processes. The capability of the platform to perform label-free functional imaging of living cells was demonstrated by imaging the spatial variations in contractions from stem cell-derived cardiomyocytes. This technique shows promise for non-invasive imaging of cultured cells over very long periods of time during development.

Probabilistic encoding of stimulus strength in astrocyte global calcium signals.

Astrocyte calcium signals can range in size from subcellular microdomains to waves that spread through the whole cell (and into connected cells). The differential roles of such local or global calcium signaling are under intense investigation, but the mechanisms by which local signals evolve into global signals in astrocytes are not well understood, nor are the computational rules by which physiological stimuli are transduced into a global signal. To investigate these questions, we transiently applied receptor agonists linked to calcium signaling to primary cultures of cerebellar astrocytes. Astrocytes repetitively tested with the same stimulus responded with global signals intermittently, indicating that each stimulus had a defined probability for triggering a response. The response probability varied between agonists, increased with agonist concentration, and could be positively and negatively modulated by crosstalk with other signaling pathways. To better understand the processes determining the evolution of a global signal, we recorded subcellular calcium "puffs" throughout the whole cell during stimulation. The key requirement for puffs to trigger a global calcium wave following receptor activation appeared to be the synchronous release of calcium from three or more sites, rather than an increasing calcium load accumulating in the cytosol due to increased puff size, amplitude, or frequency. These results suggest that the concentration of transient stimuli will be encoded into a probability of generating a global calcium response, determined by the likelihood of synchronous release from multiple subcellular sites.

Responsive cell-material interfaces.

Major design aspects for novel biomaterials are driven by the desire to mimic more varied and complex properties of a natural cellular environment with man-made materials. The development of stimulus responsive materials makes considerable contributions to the effort to incorporate dynamic and reversible elements into a biomaterial. This is particularly challenging for cell-material interactions that occur at an interface (biointerfaces); however, the design of responsive biointerfaces also presents opportunities in a variety of applications in biomedical research and regenerative medicine. This review will identify the requirements imposed on a responsive biointerface and use recent examples to demonstrate how some of these requirements have been met. Finally, the next steps in the development of more complex biomaterial interfaces, including multiple stimuli-responsive surfaces, surfaces of 3D objects and interactive biointerfaces will be discussed.

Photochromic molecular implementations of universal computation.

Unconventional computing is an area of research in which novel materials and paradigms are utilised to implement computation. Previously we have demonstrated how registers, logic gates and logic circuits can be implemented, unconventionally, with a biocompatible molecular switch, NitroBIPS, embedded in a polymer matrix. NitroBIPS and related molecules have been shown elsewhere to be capable of modifying many biological processes in a manner that is dependent on its molecular form. Thus, one possible application of this type of unconventional computing is to embed computational processes into biological systems. Here we expand on our earlier proof-of-principle work and demonstrate that universal computation can be implemented using NitroBIPS. We have previously shown that spatially localised computational elements, including registers and logic gates, can be produced. We explain how parallel registers can be implemented, then demonstrate an application of parallel registers in the form of Turing machine tapes, and demonstrate both parallel registers and logic circuits in the form of elementary cellular automata. The Turing machines and elementary cellular automata utilise the same samples and same hardware to implement their registers, logic gates and logic circuits; and both represent examples of universal computing paradigms. This shows that homogenous photochromic computational devices can be dynamically repurposed without invasive reconfiguration. The result represents an important, necessary step towards demonstrating the general feasibility of interfacial computation embedded in biological systems or other unconventional materials and environments.

Stimulus discrimination in cerebellar Purkinje neurons.

Cerebellar Purkinje neurons fire spontaneously in the absence of synaptic input. Overlaid on this intrinsic activity, excitatory input from parallel fibres can add simple spikes to the output train, whereas inhibitory input from interneurons can introduce pauses. These and other influences lead to an irregular spike train output in Purkinje neurons in vitro and in vivo, supplying a variable inhibitory drive to deep cerebellar nuclear neurons. From a computational perspective, this variability raises some questions, as individual spikes induced by excitatory inputs are indistinguishable from intrinsic firing activity. Although bursts of high-frequency excitatory input could be discriminated unambiguously from background activity, granule neurons are known to fire in vivo over a wide range of frequencies. This would mean that much of the sensory information relayed through the cerebellar cortex would be lost within the random variation in background activity. We speculated that alternative mechanisms for signal discrimination may exist, and sought to identify characteristic motifs within the sequence of spikes that followed stimulation events. We found that under certain conditions, parallel fibre stimulation could reliably add a "couplet" of spikes with an unusually short interspike interval to the output train. Therefore, despite representing a small fraction of the total number of spikes, these signals can be reliably discriminated from background firing on a moment-to-moment basis, and could result in a differential downstream response.

High-throughput analysis of calcium signalling kinetics in astrocytes stimulated with different neurotransmitters.

Astrocytes express a wide range of receptors for neurotransmitters and hormones that are coupled to increases in intracellular Ca(2+) concentration, enabling them to detect activity in both neuronal and vascular networks. There is increasing evidence that astrocytes are able to discriminate between different Ca(2+)-linked stimuli, as the efficiency of some Ca(2+) dependent processes--notably release of gliotransmitters--depends on the stimulus that initiates the Ca(2+) signal. The spatiotemporal complexity of Ca(2+) signals is substantial, and we here tested the hypothesis that variation in the kinetics of Ca(2+) responses could offer a means of selectively engaging downstream targets, if agonists exhibited a "signature shape" in evoked Ca(2+) response. To test this, astrocytes were exposed to three different receptor agonists (ATP, glutamate and histamine) and the resultant Ca(2+) signals were analysed for systematic differences in kinetics that depended on the initiating stimulus. We found substantial heterogeneity between cells in the time course of Ca(2+) responses, but the variation did not correlate with the type or concentration of the stimulus. Using a simple metric to quantify the extent of difference between populations, it was found that the variation between agonists was insufficient to allow signal discrimination. We conclude that the time course of global intracellular Ca(2+) signals does not offer the cells a means for distinguishing between different neurotransmitters.

A reconfigurable real-time compressive-sampling camera for biological applications.

Many applications in biology, such as long-term functional imaging of neural and cardiac systems, require continuous high-speed imaging. This is typically not possible, however, using commercially available systems. The frame rate and the recording time of high-speed cameras are limited by the digitization rate and the capacity of on-camera memory. Further restrictions are often imposed by the limited bandwidth of the data link to the host computer. Even if the system bandwidth is not a limiting factor, continuous high-speed acquisition results in very large volumes of data that are difficult to handle, particularly when real-time analysis is required. In response to this issue many cameras allow a predetermined, rectangular region of interest (ROI) to be sampled, however this approach lacks flexibility and is blind to the image region outside of the ROI. We have addressed this problem by building a camera system using a randomly-addressable CMOS sensor. The camera has a low bandwidth, but is able to capture continuous high-speed images of an arbitrarily defined ROI, using most of the available bandwidth, while simultaneously acquiring low-speed, full frame images using the remaining bandwidth. In addition, the camera is able to use the full-frame information to recalculate the positions of targets and update the high-speed ROIs without interrupting acquisition. In this way the camera is capable of imaging moving targets at high-speed while simultaneously imaging the whole frame at a lower speed. We have used this camera system to monitor the heartbeat and blood cell flow of a water flea (Daphnia) at frame rates in excess of 1500 fps.

Hippocampal cell response to substrates with surface chemistry gradients.

Surface chemical gradients are valuable tools for the high-throughput screening of cell-surface interactions. However, it has yet to be shown if biological data obtained from gradient surfaces are transferable to substrates with uniform properties. To explore this question, the response of hippocampal neurons to three different sample formats was compared. We fabricated samples of uniform surface wettability and samples with a linear or radial gradient in surface wettability by depositing plasma-polymerized hexane (hydrophobic) on oxygen-etched glass (hydrophilic). Differences in cell density, growth and viability of the neural cultures are found between the uniform and the gradient samples. The nature of the gradient (linear or radial) has only a small effect on the cell density of adhered hippocampal neurons.

Lesions of the vestibular system disrupt hippocampal theta rhythm in the rat.

The hippocampus has a major role in memory for spatial location. Theta is a rhythmic hippocampal EEG oscillation that occurs at approximately 8 Hz during voluntary movement and that may have some role in encoding spatial information. We investigated whether, as part of this process, theta might be influenced by self-movement signals provided by the vestibular system. The effects of bilateral peripheral vestibular lesions, made > or = 60 days prior to recording, were assessed in freely moving rats. Power spectral analysis revealed that theta in the lesioned animals had a lower power and frequency compared with that recorded in the control animals. When the electroencephalography (EEG) was compared in epochs matched for speed of movement and acceleration, theta was less rhythmic in the lesioned group, indicating that the effect was not a result of between-group differences in this behavior. Blood measurements of corticosterone were also similar in the two groups indicating that the results could not be attributed to changes in stress levels. Despite the changes in theta EEG, individual neurons in the CA1 region of lesioned animals continued to fire with a periodicity of approximately 8 Hz. The positive correlation between cell firing rate and movement velocity that is observed in CA1 neurons of normal animals was also maintained in cells recorded from lesion group animals. These findings indicate that although vestibular signals may contribute to theta rhythm generation, velocity-related firing in hippocampal neurons is dependent on nonvestibular signals such as sensory flow, proprioception, or motor efference copy.

Vestibular influences on CA1 neurons in the rat hippocampus: an electrophysiological study in vivo.

Vestibular information is known to be important for accurate spatial orientation and navigation. Hippocampal place cells, which appear to encode an animal's location within the environment, are also thought to play an essential role in spatial orientation. Therefore, it can be hypothesized that vestibular information may influence cornu ammonis region 1 (CA1) hippocampal neuronal activity. To explore this possibility, the effects of electrical stimulation of the medial vestibular nucleus (MVN) on the firing rates of hippocampal CA1 neurons in the urethane-anesthetized rat were investigated using extracellular single unit recordings. The firing rates of CA1 complex spike cells (n=29), which most likely correspond to place cells, consistently increased during electrical stimulation of the MVN in a current intensity dependent manner. Stimulation applied adjacent to the MVN failed to elicit a response. Overall, the firing rates of non-complex spike cells (n=22) did not show a consistent response to vestibular stimulation, although in some cells clear responses to the stimulation were observed. These findings suggest that vestibular inputs may contribute to spatial information processing in the hippocampus.

Bilateral peripheral vestibular lesions produce long-term changes in spatial learning in the rat.

In order to investigate whether bilateral peripheral vestibular lesions cause long-term impairment of spatial learning, rats were tested in a reference memory radial arm maze learning task at least 5 weeks following a bilateral labyrinthectomy (BL) or sham control lesion. All control rats reached criterion (i.e., 1 error or less, averaged across 7 trials for 3 consecutive days of training) but only 4 of the 8 BL rats had reached criterion by day 21 of the training sessions. The control rats reached criterion more quickly than the lesioned rats (Control, 7.0 +/- 0.63 days, Lesioned, 15.8 +/- 1.4 days, t10= 5.84, p < 0.0001). This difference resulted from the greater number of errors made by the BL animals. However, the latency to respond was comparable as a result of the increased locomotor activity of the BL group (i.e., 'hyperkinesis), and the overall rate of acquisition of the task, as indicated by analysis of the exponential decrease in errors over the entire training period, was not significantly different between the 2 groups. The results of this study demonstrate that BL in rats produces long-term changes in performance in a spatial reference memory task, which are not simply due to the inability to move but may relate to the way that the brain uses vestibular information to create spatial representations and determines behavioural strategies on the basis of these representations.

Long-term effects of permanent vestibular lesions on hippocampal spatial firing.

The hippocampus is thought to be important for spatial representation processes that depend on the integration of both self-movement and allocentric cues. The vestibular system is a particularly important source of self-movement information that may contribute to this spatial representation. To test the hypothesis that the vestibular system provides self-movement information to the hippocampus, rats were given either a bilateral labyrinthectomy (n = 6) or a sham surgery (n = 6), and at least 60 d after surgery hippocampal CA1 neurons were recorded extracellularly while the animals foraged freely in an open arena. Recorded cells were classified as complex spiking (n = 80) or noncomplex spiking (n = 33) neurons, and their spatial firing fields (place fields) were examined. The most striking effect of the lesion was that it appeared to completely abolish location-related firing. The results of this and previous studies provide converging evidence demonstrating that vestibular information is processed by the hippocampus. The disruption of the vestibular input to the hippocampus may interfere with the reconciliation of internal self-movement signals with the changes to the external sensory inputs that occur as a result of that movement. This would disrupt the ability of the animal to integrate allocentric and egocentric information into a coherent representation of space.