With technologies rapidly evolving, many research institutions are now opting to invest in costly, high-quality, specialized microscopes which are shared by many researchers. As a consequence, the user may not have the ability to adapt a microscope to their specific needs and limitations in experimental design are introduced. A flexible work-horse microscopy system is a valuable tool in any laboratory to meet the diverse needs of a research team and promote innovation in experimental design. We have developed the Flexiscope; a multi-functional, adaptable, efficient and high-performance microscopy/electrophysiology system for everyday applications in a neurobiology laboratory. The core optical components are relatively constant in the three configurations described here: an upright configuration, an inverted configuration and an upright/electrophysiology configuration. We have provided a comprehensive description of the Flexiscope. We show that this method is capable of oblique infrared illumination imaging, multi-channel fluorescent imaging and automated three-dimensional scanning of larger specimens. Image quality is conserved across the three configurations of the microscope, and conversion between configurations is possible quickly and easily, while the motion control system can be repurposed to allow sub-micrometre computer-controlled micromanipulation. The Flexiscope provides similar performance and usability to commercially available systems. However, as it can be easily reconfigured for multiple roles, it can remove the need to purchase multiple microscopes, giving significant cost savings. The modular reconfigurable nature allows the user to customize the system to their specific needs and adapt/upgrade the system as challenges arise, without requiring specialized technical skills.

Over four decades ago, it was discovered that high-frequency stimulation of the dentate gyrus induces long-term potentiation (LTP) of synaptic transmission. LTP is believed to underlie how we process and code external stimuli before converting it to salient information that we store as 'memories'. It has been shown that rats performing spatial learning tasks display theta-frequency (3-12 Hz) hippocampal neural activity. Moreover, administering theta-burst stimulation (TBS) to hippocampal slices can induce LTP. TBS triggers a sustained rise in intracellular calcium [Ca2+]i in neurons leading to new protein synthesis important for LTP maintenance. In this study, we measured TBS-induced [Ca2+]i oscillations in thousands of cells at increasing distances from the source of stimulation. Following TBS, a calcium wave propagates radially with an average speed of 5.2 µm/s and triggers multiple and regular [Ca2+]i oscillations in the hippocampus. Interestingly, the number and frequency of [Ca2+]i fluctuations post-TBS increased with respect to distance from the electrode. During the post-tetanic phase, 18% of cells exhibited 3 peaks in [Ca2+]i with a frequency of 17 mHz, whereas 2.3% of cells distributed further from the electrode displayed 8 [Ca2+]i oscillations at 33 mHz. We suggest that these observed [Ca2+]i oscillations could lead to activation of transcription factors involved in synaptic plasticity. In particular, the transcription factor, NF-κB, has been implicated in memory formation and is up-regulated after LTP induction. We measured increased activation of NF-κB 30 min post-TBS in CA1 pyramidal cells and also observed similar temporal up-regulation of NF-κB levels in CA1 neurons following water maze training in rats. Therefore, TBS of hippocampal slice cultures in vitro can mimic the cell type-specific up-regulations in activated NF-κB following spatial learning in vivo. This indicates that TBS may induce similar transcriptional changes to spatial learning and that TBS-triggered [Ca2+]i oscillations could activate memory-associated gene expression.

Neuronal-glial interactions in the central nervous system are important for both normal function and response to pathological states. Differences in calcium processing between these cell types may be exploited to allow dynamic differentiation using calcium-imaging protocols without the need to fix and immunostain the study population. Mixed rat primary cortical cultures were grown on coverslips, incubated for 30 min in 2 microM fluo-3 AM and mounted in a devised, low volume imaging chamber. Calcium influx was measured over the duration of a 50s exposure to 30 microM glutamate in all cells. Cells were then fixed in situ, and immunostained for NeuN and GFAP. Direct comparison between live calcium dynamics and cell type markers were made. Over the duration of the glutamate exposure, those cells that subsequently stained for NeuN exhibited a sustained increase in intracellular calcium, whereas GFAP positive and non-staining cells exhibited a decline over the duration of the glutamate exposure. We found that examining the average calcium fluorescence over the last 10s of glutamate exposure allowed the identification of cells as neuronal if the average was >85% of the maximal calcium change, or non-neuronal if the average was <85% of the maximal calcium change. This technique compares very favourably to the established technique of immunocytochemical labeling for the identification of cell types; both techniques agreed in their classification of cells as neuronal or non-neuronal 96.83% of the time. However, this technique cannot reliably distinguish between non-neuronal cell types.

During cerebral ischemia, elevation of TNF-α and glutamate to pathophysiological levels in the hippocampus may induce dysregulation of normal synaptic processes, leading ultimately to cell death. Previous studies have shown that patients subjected to a mild transient ischemic attack within a critical time window prior to a more severe ischemic episode may show attenuation in the clinical severity of the stroke and result in a more positive functional outcome. In this study we have investigated the individual contribution of pre-exposure to TNF-α or glutamate in the development of 'ischemic tolerance' to a subsequent insult, using organotypic hippocampal cultures. At 6 days in vitro (DIV), cultures were exposed to an acute concentration of glutamate (30 μM) or TNF-α (5 ng/ml) for 30 min, followed by 24h recovery period. We then examined the effect of the pretreatments on calcium dynamics of the cells within the CA region. We found that pretreatment with TNF-α or glutamate caused in a significant reduction in subsequent glutamate-induced Ca(2+) influx 24h later (control: 100.0 ± 0.8%, n=7769 cells; TNF-α: 76.8 ± 1.0%, n=5543 cells; glutamate: 75.3 ± 1.4%, n=3859 cells; p<0.001). Antagonism of circulating TNF-α (using infliximab, 25 μg/ml), and inhibition of the p38 MAP kinase pathway (using SB 203580, 10 μM) completely reversed this effect. However glutamate preconditioning did not appear to be mediated by p38 MAP kinase signalling, or NMDAR activation as neither SB 203580 nor D-AP5 (100 μM) altered this effect. Glutamate and TNF-α preconditioning resulted in small yet significant alterations in resting Ca(2+) levels (control: 100.0 ± 0.9%, n=2994 cells; TNF-α: 109.7 ± 1.0%, n=2884 cells; glutamate; 93.3 ± 0.8%, n=2899 cells; p<0.001), TNF-α's effect reversed by infliximab and SB 203580. Both TNF-α and glutamate also resulted in the reduction of the proportion (P) of responsive cells within the CA region of the hippocampus (control; P=0.459, 0.451 ≤ x ≥ 0.467, n=14,968 cells, TNF-α; P=0.40, 0.392 ≤ x ≥ 0.407, n=15,218; glutamate; P=0.388, 0.303 ≤ x ≥ 0.396, n=13,919 cells), and in the depression of the frequency of spontaneous Ca(2+) events (vs. control: TNF-α: p>0.00001, D=0.0454; glutamate: p>0.0001, D=0.0534). Our results suggest that attenuation in resting Ca(2+) activity and Ca(2+) related responsiveness of cells within the CA region as a result of glutamate or TNF-α pre-exposure, may contribute to the development of ischemic tolerance.

Several cytokines and chemokines are now known to play normal physiological roles in the brain where they act as key regulators of communication between neurons, glia, and microglia. In particular, cytokines and chemokines can affect cardinal cellular and molecular processes of hippocampal-dependent long-term memory consolidation including synaptic plasticity, synaptic scaling and neurogenesis. The chemokine, CX3CL1 (fractalkine), has been shown to modulate synaptic transmission and long-term potentiation (LTP) in the CA1 pyramidal cell layer of the hippocampus. Here, we confirm widespread expression of CX3CL1 on mature neurons in the adult rat hippocampus. We report an up-regulation in CX3CL1 protein expression in the CA1, CA3 and dentate gyrus (DG) of the rat hippocampus 2 h after spatial learning in the water maze task. Moreover, the same temporal increase in CX3CL1 was evident following LTP-inducing theta-burst stimulation in the DG. At physiologically relevant concentrations, CX3CL1 inhibited LTP maintenance in the DG. This attenuation in dentate LTP was lost in the presence of GABAA receptor/chloride channel antagonism. CX3CL1 also had opposing actions on glutamate-mediated rise in intracellular calcium in hippocampal organotypic slice cultures in the presence and absence of GABAA receptor/chloride channel blockade. Using primary dissociated hippocampal cultures, we established that CX3CL1 reduces glutamate-mediated intracellular calcium rises in both neurons and glia in a dose dependent manner. In conclusion, CX3CL1 is up-regulated in the hippocampus during a brief temporal window following spatial learning the purpose of which may be to regulate glutamate-mediated neurotransmission tone. Our data supports a possible role for this chemokine in the protective plasticity process of synaptic scaling.

Activity of the transcription factor NF-kappaB is required for memory formation, but the identity and function of the genes it may regulate in this context remain obscure. Here, we comprehensively characterise NF-kappaB throughout the rat hippocampus following passive avoidance training and report significant subregion-specific increased activity across the dorsoventral axis 3h post-learning. Moreover, putative NF-kappaB binding motifs predominated in structural genes previously shown to regulate 3h following avoidance conditioning, the protein products of which may be involved in the subsequent synaptic remodelling required for consolidation. Finally, we assessed the influence of NF-kappaB-mediated transcription on neuritic structure and report that inhibition of NF-kappaB significantly decreases growth and branching of primary hippocampal neurons. These results suggest that NF-kappaB activity following hippocampal learning may contribute to consolidation-associated synaptic reorganisation.