On the mechanisms of cerebellar synaptic plasticity

Changes in the strength of signalling between neurones are thought to provide a cellular substrate for learning and memory. In the cerebellar cortex, raising the frequency and the strength of parallel fibre (PF) stimulation leads to a long-term depression (LTD) of the strength of signalling at the synapse between PFs and Purkinje cells (PCs), which spreads to distant synapses to the same cell via a nitric oxide (NO) dependent mechanism. At the same synapse, but under conditions of reduced post-synaptic calcium activity, raised frequency stimulation (RFS) of PFs triggers a long-term potentiation of synaptic transmission. The aims of the work described in this thesis were to investigate the conditions necessary for LTD and LTP at this synapse following RFS and to identify the origins and second messenger cascades involved in the induction and spread of LTP and LTD. In thin, parasagittal cerebellar slices whole cell patch clamp recordings were made from PCs and the effects of RFS of one of two, independent PF inputs to the same PC were examined under a range of experimental conditions. Under conditions designed to reduce post-synaptic calcium activity, RFS to a single PF input led to LTP and a decreases in paired pulse facilitation (PPF) in both pathways. This heterosynaptic potentiation was prevented by inhibition of protein kinase A (PKA) or by inhibition of NO synthase with either 7-nitroindazole (7-NI) or NG Nitro-L-argenine methyl ester. Inhibition of guanylate cyclase (GC) or protein kinase G (PKG) had no effect. A similar potentiation was observed upon application of the adenylyl cyclase (AC) activator forskolin or the NO donor spermine NONOate. Both of these treatments also resulted in an increase in the frequency of mEPSCs, which provides further evidence for a presynaptic origin of LTP. Forskolin induced potentiation and the increase in mEPSC frequency were blocked by 7-NI. The styryl dye FM1-43, a fluorescent reporter of endo- and exocytosis, was also used to further examine the possible pre-synaptic origins of LTP. RFS or forskolin application enhanced FM1-43 de-staining and NOS inhibitors blocked this effect. Application of NONOate also enhanced FM1-43 de-staining. When post-synaptic calcium activity was less strictly buffered, RFS to a single PF input led to a transient potentiation that was succeeded by LTD in both pathways. This LTD, which resembled previously described forms, was prevented by inhibition of the NO/cGMP/PKG cascade. Modification of the AC/cAMP/PKA cascade had no effect. In summary, the direction of synaptic plasticity at the PF-PC synapse in response to RFS depends largely on the level of post-synaptic calcium activity. LTP and LTD were non-input specific and both forms of plasticity were dependent on NOS activity. Induction of LTP was mediated by a presynaptic mechanism and depended on NO and cAMP production. LTD on the other hand was a post-synaptic process and required activity of the NO/cGMP/PKG signalling cascade.

The surface of Pseudomonas aeruginosa in cystic fibrosis lung infection

Chronic experimental lung infection in rats was induced by intratracheal inoculation of agar beads containing Pseudomonas aeruginosa. Bacteria were recovered directly without subculture from the lungs of rats at 14 days post-infection and the outer membrane (OM) antigens were studied. The results indicated that bacteria grew under iron-restricted conditions as revealed by the expression of several iron-regulated membrane proteins (IRMPs) which could also be observed when the isolate was grown under iron-depleted conditions in laboratory media. The antibody response to P. aeruginosa OM protein antigens was investigated by immunoblotting with serum and lung fluid from infected rats. These fluids contained antibodies to all the major OM proteins, including the IRMPs, and protein H1. Results obtained using immunoblotting and enzyme-linked immunosorbent assay indicated that lipopolysaccharide (LPS) was the major antigen recognised by antibodies in sera from infected rats. The animal model was used to follow the development of the immune response to P. aeruginosa protein and LPS antigens. Immunoblotting was used to investigate the antigens recognised by antibodies in sequential serum samples. An antibody response to the IRMPs and OM proteins D, E, G and H1 and alao to rough LPS was detected as early as 4 days post-infection. Results obtained using immunoblotting and crossed immunoelectrophoresis techniques indicated that there was a progressive increase in the number of P. aeruginosa antigens recognised by antibodies in these sera. Both iron and magnesium depletion influenced protein H1 production. Antibodies in sera from patients with infections due to P. aeruginosa reacted with this antigen. Results obtained using quantitative gas-liquid chromatographic analysis indicated that growth phase and magnesium and iron depletion also affected the amount of LPS fatty acids, produced by P. aeruginosa. The silver stained SDS-polyacrylamide gels of proteinase K digested whole cell lysates of P. aeruginosa indicated that the O-antigen and core LPS were both affected by growth phase and specific nutrient depletion.

Influence of iron on bacterial infections in leukaemia

The influence of iron metabolism, both on the invading bacterial pathogen and in the host is widespread and often appears to be crucial in determining the outcome of an infection. This study involved the investigation of leukaemia, a clinical disease where abnormal availability of iron may play a part in predisposing patients to bacterial infection. The iron status throughout a Gram-negative septicaemia and in 20 random, newly diagnosed leukaemic patients was assessed. The results revealed that the majority of the patients exhibited high serum iron levels and serum transferrin saturation often at 100%, with an inability to reduce the latter to within normal values during an infection episode. The antibody response to P.aeruginosa, E.coli and K.pneumoniae outer membrane protein (OMP) antigens were investigated by immunoblotting with sequential serum samples during infection in the leukaemic host. Antibodies to all the major OMPs, were observed, although recognition of iron-regulated membrane proteins (IRMPs) was in many cases weak. Results from the enzyme-linked immunosorbent assay indicated that in all patients antibody titre in response to infection was poor. Sub-MICs of mitomycin C significantly altered the surface characteristics of P.aeruginosa. The silver-stained SDS-PAGE gels of proteinase K digested whole cell lysates of strains PAO1, 6750, M7 and PAJ indicated that core LPS was affected in the presence of mitomycin C. In contrast, the rough strain AK1012 showed no observable differences. Results obtained using quantitative gas-liquid chromatographic analysis showed the amount of LPS fatty acids to be unaffected, however, the KDO and carbohydrate content in strains PAO1, 6750 and M7 under Fe+ and Fe- growth conditions were decreased by up to 4-fold in the presence of mitomycin C, indicating perturbed expression of LPS. The cell surface became significantly more hydrophobic in the P.aeruginosa strains, except AK1012 which was comparatively unaffected. The induction of protein G (OprG) in P.aeruginosa was found to be a sensitive indicator of media iron. The data indicated that expression of OprG can be modulated by growth rate/phase, availability of iron and by the presence of ciprofloxacin in the growth medium.

Ethosuximide modifies network excitability in the rat entorhinal cortex via an increase in GABA release

Ethosuximide is the drug of choice for treating generalized absence seizures, but its mechanism of action is still a matter of debate. It has long been thought to act by disrupting a thalamic focus via blockade of T-type channels and, thus, generation of spike-wave activity in thalamocortical pathways. However, there is now good evidence that generalized absence seizures may be initiated at a cortical focus and that ethosuximide may target this focus. In the present study we have looked at the effect ethosuximide on glutamate and GABA release at synapses in the rat entorhinal cortex in vitro, using two experimental approaches. Whole-cell patch-clamp studies revealed an increase in spontaneous GABA release by ethosuximide concurrent with no change in glutamate release. This was reflected in studies that estimated global background inhibition and excitation from intracellularly recorded membrane potential fluctuations, where there was a substantial rise in the ratio of network inhibition to excitation, and a concurrent decrease in excitability of neurones embedded in this network. These studies suggest that, in addition to well-characterised effects on ion channels, ethosuximide may directly elevate synaptic inhibition in the cortex and that this could contribute to its anti-absence effects. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.

Astroglial d-serine is the endogenous co-agonist at the presynaptic NMDA receptor in rat entorhinal cortex

Presynaptic NMDA receptors facilitate the release of glutamate at excitatory cortical synapses and are involved in regulation of synaptic dynamics and plasticity. At synapses in the entorhinal cortex these receptors are tonically activated and provide a positive feedback modulation of the level of background excitation. NMDA receptor activation requires obligatory occupation of a co-agonist binding site, and in the present investigation we have examined whether this site on the presynaptic receptor is activated by endogenous glycine or d-serine. We used whole-cell patch clamp recordings of spontaneous AMPA receptor-mediated synaptic currents from rat entorhinal cortex neurones in vitro as a monitor of presynaptic glutamate release. Addition of exogenous glycine or d-serine had minimal effects on spontaneous release, suggesting that the co-agonist site was endogenously activated and likely to be saturated in our slices. This was supported by the observation that a co-agonist site antagonist reduced the frequency of spontaneous currents. Depletion of endogenous glycine by enzymatic breakdown with a bacterial glycine oxidase had little effect on glutamate release, whereas d-serine depletion with a yeast d-amino acid oxidase significantly reduced glutamate release, suggesting that d-serine is the endogenous agonist. Finally, the effects of d-serine depletion were mimicked by compromising astroglial cell function, and this was rescued by exogenous d-serine, indicating that astroglial cells are the provider of the d-serine that tonically activates the presynaptic NMDA receptor. We discuss the significance of these observations for the aetiology of epilepsy and possible targeting of the presynaptic NMDA receptor in anticonvulsant therapy. © 2014 Elsevier Ltd. All rights reserved.

Depression of glutamate and GABA release by presynaptic GABAB receptors in the entorhinal cortex in normal and chronically epileptic rats

Presynaptic GABAB receptors (GABABR) control glutamate and GABA release at many synapses in the nervous system. In the present study we used whole-cell patch-clamp recordings of spontaneous excitatory and inhibitory synaptic currents in the presence of TTX to monitor glutamate and GABA release from synapses in layer II and V of the rat entorhinal cortex (EC)in vitro. In both layers the release of both transmitters was reduced by application of GABABR agonists. Quantitatively, the depression of GABA release in layer II and layer V, and of glutamate release in layer V was similar, but glutamate release in layer II was depressed to a greater extent. The data suggest that the same GABABR may be present on both GABA and glutamate terminals in the EC, but that the heteroreceptor may show a greater level of expression in layer II. Studies with GABABR antagonists suggested that neither the auto- nor the heteroreceptor was consistently tonically activated by ambient GABA in the presence of TTX. Studies in EC slices from rats made chronically epileptic using a pilocarpine model of temporal lobe epilepsy revealed a reduced effectiveness of both auto- and heteroreceptor function in both layers. This could suggest that enhanced glutamate and GABA release in the EC may be associated with the development of the epileptic condition. Copyright © 2006 S. Karger AG.

Diverse antiepileptic drugs increase the ratio of background synaptic inhibition to excitation and decrease neuronal excitability in neurones of the rat entorhinal cortex in vitro

Although most anti-epileptic drugs are considered to have a primary molecular target, it is clear that their actions are unlikely to be limited to effects on a single aspect of inhibitory synaptic transmission, excitatory transmission or voltage-gated ion channels. Systemically administered drugs can obviously simultaneously access all possible targets, so we have attempted to determine the overall effect of diverse agents on the balance between GABAergic inhibition, glutamatergic excitation and cellular excitability in neurones of the rat entorhinal cortex in vitro. We used an approach developed for estimating global background synaptic excitation and inhibition from fluctuations in membrane potential obtained by intracellular recordings. We have previously validated this approach in entorhinal cortical neurones [. Greenhill and Jones (2007a) Neuroscience 147:884-892]. Using this approach, we found that, despite their differing pharmacology, the drugs tested (phenytoin, lamotrigine, valproate, gabapentin, felbamate, tiagabine) were unified in their ability to increase the ratio of background GABAergic inhibition to glutamatergic excitation. This could occur as a result of decreased excitation concurrent with increased inhibition (phenytoin, lamotrigine, valproate), a decrease in excitation alone (gabapentin, felbamate), or even with a differential increase in both (tiagabine). Additionally, we found that the effects on global synaptic conductances agreed well with whole cell patch recordings of spontaneous glutamate and GABA release (our previous studies and further data presented here). The consistency with which the synaptic inhibition:excitation ratio was increased by the antiepileptic drugs tested was matched by an ability of all drugs to concurrently reduce intrinsic neuronal excitability. Thus, it seems possible that specific molecular targets among antiepileptic drugs are less important than the ability to increase the inhibition:excitation ratio and reduce overall neuronal and network excitability. © 2010 IBRO.

Syntheses of aza analogues of kainic acid

The kainate receptors are one of the three major groups of ionotropic glutamate receptors in the mammalian central nervous system. They are so named after their most potent agonist, kainic acid (KA), a natural product isolated from the seaweed Diginea simplex. This compound shows both neuroexcitatory and excitotoxic activities, and is an important pharmacological tool for neurophysiological studies. We predict that the more synthetically accessible aza analogues of kainic acid, could act as functional mimics of KA. These could be produced by the 1,3-dipolar cycloaddition of diazoalkanes with trans glutaconate esters. ^ 1,3-Dipolar cycloadditions have been shown to produce 1-pyrazolines that isomerize into 2-pyrazolines. The 1- and 2-pyrazolines can be precursors to aza analogs of kainoids. The regioselectivity, relative stereochemistry and isomerization of the 1-pyrazolines into 2-pyrazolines have been evaluated. Reductions of the 1- and 2-pyrazolines produced aza analogs of kainoids. TMS diazomethane was used as the dipole in 1,3-dipolar cycloaddition reactions leading to aza KA analogs via 2-pyrazolines. A systematic study of cycloaddition-isomerization processes involving TMS-diazomethane and various α, β-unsaturated dipolarophiles has been undertaken. 1H-NMR monitoring of the reaction mixture compositions during the cycloaddition reaction revealed evidence of retro-dipolar cycloaddition processes. Faster formation of 4,5- trans-1-pyrazoline at the beginning of the reaction and subsequent isomerization of this product into 4,5-cis-1-pyrazoline via a retro-dipolar cycloaddition has been observed. Increased reaction time and/or reaction temperature preferentially caused the irreversible isomerization of 4,5-cis-1-pyrazoline into 4,5-cis-2-pyrazoline, which led to high yields of 4,5-cis-2-pyrazolines in the overall process. ^ Two syntheses of the 5-unsubstituted aza-kainic acid have been performed; first, via the reduction of the TMS-eliminated 2-pyrazoline from TMS diazomethane; second by the direct reduction of 1-pyrazoline with Hg/Al-amalgam. 5-Phenyl aza-kainic acid has been produced by direct reduction of 1-pyrazoline, obtained in the reaction of phenyldiazomethane and dibenzyl glutaconate, with Hg/Al-amalgam. ^ Current responses to aza kainate analogs in Aplysia whole cell buccal ganglia indicate potent neuroexcitatory activity. The repetitive exposure of neuronal cells to the 5-unsubstituted aza-kainic acid led to non-desensitizing current responses, showing both binding affinity and neuronal ion-channel activation by the synthesized agonist compound. ^

Real-time biosensor for the assessment of nanotoxicity and cancer electrotherapy

Knowledge of cell electronics has led to their integration to medicine either by physically interfacing electronic devices with biological systems or by using electronics for both detection and characterization of biological materials. In this dissertation, an electrical impedance sensor (EIS) was used to measure the electrode surface impedance changes from cell samples of human and environmental toxicity of nanoscale materials in 2D and 3D cell culture models. The impedimetric response of human lung fibroblasts and rainbow trout gill epithelial cells when exposed to various nanomaterials was tested to determine their kinetic effects towards the cells and to demonstrate the biosensor's ability to monitor nanotoxicity in real-time. Further, the EIS allowed rapid, real-time and multi-sample analysis creating a versatile, noninvasive tool that is able to provide quantitative information with respect to alteration in cellular function. We then extended the application of the unique capabilities of the EIS to do real-time analysis of cancer cell response to externally applied alternating electric fields at different intermediate frequencies and low-intensity. Decreases in the growth profiles of the ovarian and breast cancer cells were observed with the application of 200 and 100 kHz, respectively, indicating specific inhibitory effects on dividing cells in culture in contrast to the non-cancerous HUVECs and mammary epithelial cells. We then sought to enhance the effects of the electric field by altering the cancer cell's electronegative membrane properties with HER2 antibody functionalized nanoparticles. An Annexin V/EthD-III assay and zeta potential were performed to determine the cell death mechanism indicating apoptosis and a decrease in zeta potential with the incorporation of the nanoparticles. With more negatively charged HER2-AuNPs attached to the cancer cell membrane, the decrease in membrane potential would thus leave the cells more vulnerable to the detrimental effects of the applied electric field due to the decrease in surface charge. Therefore, by altering the cell membrane potential, one could possibly control the fate of the cell. This whole cell-based biosensor will enhance our understanding of the responsiveness of cancer cells to electric field therapy and demonstrate potential therapeutic opportunities for electric field therapy in the treatment of cancer.

Development of a Lab-on-a-Chip Device for Rapid Nanotoxicity Assessment In Vitro

Increasing useof nanomaterials in consumer products and biomedical applications creates the possibilities of intentional/unintentional exposure to humans and the environment. Beyond the physiological limit, the nanomaterialexposure to humans can induce toxicity. It is difficult to define toxicity of nanoparticles on humans as it varies by nanomaterialcomposition, size, surface properties and the target organ/cell line. Traditional tests for nanomaterialtoxicity assessment are mostly based on bulk-colorimetric assays. In many studies, nanomaterials have found to interfere with assay-dye to produce false results and usually require several hours or days to collect results. Therefore, there is a clear need for alternative tools that can provide accurate, rapid, and sensitive measure of initial nanomaterialscreening. Recent advancement in single cell studies has suggested discovering cell properties not found earlier in traditional bulk assays. A complex phenomenon, like nanotoxicity, may become clearer when studied at the single cell level, including with small colonies of cells. Advances in lab-on-a-chip techniques have played a significant role in drug discoveries and biosensor applications, however, rarely explored for nanomaterialtoxicity assessment. We presented such cell-integrated chip-based approach that provided quantitative and rapid response of cellhealth, through electrochemical measurements. Moreover, the novel design of the device presented in this study was capable of capturing and analyzing the cells at a single cell and small cell-population level. We examined the change in exocytosis (i.e. neurotransmitterrelease) properties of a single PC12 cell, when exposed to CuOand TiO2 nanoparticles. We found both nanomaterials to interfere with the cell exocytosis function. We also studied the whole-cell response of a single-cell and a small cell-population simultaneously in real-time for the first time. The presented study can be a reference to the future research in the direction of nanotoxicity assessment to develop miniature, simple, and cost-effective tool for fast, quantitative measurements at high throughput level. The designed lab-on-a-chip device and measurement techniques utilized in the present work can be applied for the assessment of othernanoparticles' toxicity, as well.

Descending Control of Limb Movements in Drosophila melanogaster

Because the interactions between feedforward influences are inextricably linked during many motor outputs (including but not limited to walking), the contribution of descending inputs to the generation of movements is difficult to study. Here we take advantage of the relatively small number of descending neurons (DNs) in the Drosophila melanogaster model system. We first characterize the number and distribution of the DN populations, then present a novel load free preparation, which enables the study of descending control on limb movements in a context where sensory feedback can be is reduced while leaving the nervous system, musculature, and cuticle of the animal relatively intact. Lastly we use in-vivo whole cell patch clamp electrophysiology to characterize the role of individual DNs in response to specific sensory stimuli and in relationship to movement. We find that there are approximately 1100 DNs in Drosophila that are distributed across six clusters. Input from these DNs is not necessary for coordinated motor activity, which can be generated by the thoracic ganglion, but is necessary for the specific combinations of joint movements typically observed in walking. Lastly, we identify a particular cluster of DNs that are tuned to sensory stimuli and innervate the leg neuromeres. We propose that a multi-layered interaction between these DNs, other DNs, and motor circuits in the thoracic ganglia enable the diverse but well-coordinated range of motor outputs an animal might exhibit.

INVESTIGATING THE MECHANISM OF AUTOIMMUNE DISEASE ASSOCIATED-LQTS

The human ether-a-go-go-related gene (hERG) protein passes the rapidly activating delayed rectifier potassium channel (IKr), and malfunction of hERG protein/IKr is the primary cause of acquired long QT syndrome (LQTS). Autoimmune diseases are significantly correlated with prolonged QT intervals, for which autoantibodies have been implicated. The anti-Ro52 autoantibody is the most frequently evaluated, and importantly has been correlated with prolonged QT intervals. Pathological anti-Ro52-hERG interactions have been discussed as a mechanism for autoimmune disease-related LQTS. However, the mechanism is unclear, and it does not explain LQTS in autoimmune diseases which do not commonly express anti-Ro52. In this thesis, I investigated the effects of anti-Ro52 on hERG/IKr function. Through Western blot analysis, whole-cell patch-clamp, and immunofluorescence, I show that anti-Ro52 chronically (12 h) reduced hERG protein expression and hERG current by over 50%, but did not acutely block the channel. My work revealed a novel mechanism in which the Fc portion of anti-Ro52 interacts with the extracellular S5-pore linker of the channel to induce internalization through a tyrosine phosphorylation dependent pathway. This phenomenon extends beyond anti-Ro52 IgG, as other IgG, regardless of their antigen binding specificity, have the potential to reduce hERG expression/current. Rather, the ability of IgG to reduce hERG expression and current is dependent on the IgG subclass, as we show mouse IgG2A was the only mouse IgG subclass which reduced hERG expression. These results provide a novel explanation for autoimmune disease associated LQTS. It also has implications in the development of safe monoclonal antibody drugs.

Electrophysiological study of the effects of arginine vasopressin in the rat juxtacapsular nucleus of the bed nucleus of the stria terminalis

Arginine vasopressin (AVP), a nine amino acid neuropeptide (CYFQNCPRG- NH2) fulfills a dual function: (i) in the periphery, AVP acts as a peptide hormone and (ii) in the CNS, AVP is a neuromodulatory peptide. AVP produces its effects through 3 AVP receptors (AVPRs). AVPR1a and AVPR1b are expressed in the CNS and periphery, whilst AVPR2 is not found centrally but instead solely expressed in the kidneys. Recent evidence revealed a high density of AVP-binding sites in the juxtacapsular nucleus of the bed nucleus of the stria terminalis (jxBNST). While in other regions of the brain, AVP acts at AVPRs to regulate an array of biological processes, including male-typical social behaviours, social memory, stress adaptation, fear, anxiety, and fluid homeostasis, its role in the jxBNST remains elusive. Furthermore, the neurophysiological properties of AVP in the jxBNST are unknown so this study aimed to examine how AVP modulates synaptic transmission in the rat jxBNST. The BNST being one of the most notable sexually dimorphic brain regions and AVPR expression being influenced by gonadal steroids, we investigated the putative influence of sex on the modulatory effects of AVP in the jxBNST. Finally, due to AVP being released at a substantially higher concentration following periods of water deprivation, we examined changes in AVPs modulatory role following water deprivation. Male and female Long Evans rats were euthanized and brain slice whole-cell voltage-clamp electrophysiology was done in the jxBNST to measure the effects of AVP on synaptic transmission of GABA synapses. Exogenous application of AVP produced three responses; either postsynaptic long-term potentiation (LTP) of GABAA-inhibitory postsynaptic currents (IPSC), postsynaptic long-term depression (LTD) of GABAA-IPSC, or no change in GABAA-IPSC amplitudes. Interestingly, the proportion of neurons responding in each of these ways did not differ between sexes and within females was not estrous cycle-dependent. Finally, although not statistically significant, 24-hour water deprivation abolished GABAA-LTD, an effect that was not a consequence of social isolation. Taken together, our data show that AVP modulates GABAA synaptic transmission in the jxBNST in fluid homeostasis- but not sex-dependent manner.

Impaired GABA Plasticity at ovBNST Synapses Predicts Compulsive Drinking in Schedule-Induced Polydipsia

Schedule-Induced Polydipsia (SIP) is an animal model of adjunctive drinking induced when a hungry rat receives food on a fixed interval of time. This model has been implemented as a model of compulsive behaviour and may represent a powerful tool to understand the neural mechanisms of compulsion. The bed nucleus of the stria terminalis (BNST) is thought to translate challenges to energy homeostasis into consummatory behaviours, and is therefore likely to contribute to drinking behaviours displayed by food restricted rats in the SIP paradigm. Furthermore, the BNST seems implicated in various compulsive behaviors, including compulsive water drinking in rats. Therefore, the goal of this project was to determine whether compulsive drinking in the SIP paradigm was associated with alterations in transmission at oval BNST (ovBNST) synapses. Rats undergoing the SIP procedure had restricted food access (1-hours/day) for a total of 29 days. After 7 days of food restriction and for the next 21 consecutive days, the rats had daily 2-hour access to operant conditioning chambers where they were presented with a 45-mg food pellet every minute. Water consumed during these 2-hour sessions was measured and the rats that drank 15 ml or more water for a minimum of 3 consecutive days were considered High Drinkers (HD; n=17) or otherwise, Low Drinkers (LD; n=13). Brain slices whole-cell patch clamp recordings conducted 18-hours after the last SIP training showed that chronic food restriction changed low frequency stimulation (LFS) - induced long-term potentiation of ovBNST inhibitory synaptic transmission (iLTP) into LFS - induced long-term depression (iLTD) in a majority of neurons, regardless of drinking behaviours. However, ad libitum access to food between the last day of SIP training and brain slice recordings (18-hour refeed) rescued LFS-induced iLTP in LD but not in HD, suggesting that impaired bi-directional plasticity of ovBNST synapses may contribute to compulsive drinking in the SIP paradigm.

Calcium oscillations

Changes in cellular calcium concentration control a wide range of physiological processes, from the subsecond release of synaptic neurotransmitters, to the regulation of gene expression over months or years. Calcium can also trigger cell death through both apoptosis and necrosis, and so the regulation of cellular calcium concentration must be tightly controlled through the concerted action of pumps, channels and buffers that transport calcium into and out of the cell cytoplasm. A hallmark of cellular calcium signalling is its spatiotemporal complexity: stimulation of cells by a hormone or neurotransmitter leads to oscillations in cytoplasmic calcium concentration that can vary markedly in time course, amplitude, frequency, and spatial range. In this chapter we review some of the biological roles of calcium, the experimental characterisation of complex dynamic changes in calcium concentration, and attempts to explain this complexity using computational models. We consider the "toolkit" of cellular proteins which influence calcium concentration, describe mechanistic models of key elements of the toolkit, and fit these into the framework of whole cell models of calcium oscillations and waves. Finally, we will touch on recent efforts to use stochastic modelling to elucidate elementary calcium signal events, and how these may evolve into global signals.