A population of immature cerebellar parallel fibre synapses are insensitive to adenosine but are inhibited by hypoxia

The purine adenosine plays an important role in a number of physiological and pathological processes and is neuroprotective during hypoxia and ischemia. The major effect of adenosine is to suppress network activity via the activation of A1 receptors. Here we report that in immature cerebellar slices, the activation of A1 receptors has variable effects on parallel fibre synaptic transmission, ranging from zero depression to an almost complete abolition of transmission. Concentration–response curves suggest that the heterogeneity of inhibition stems from differences in A1 receptor properties which could include coupling to downstream effectors. There is less variation in the effects of adenosine at parallel fibre synapses in slices from older rats and thus adenosine signalling appears developmentally regulated. In the cerebellum, hypoxia increases the concentration of extracellular adenosine leading to the activation of A1 receptors (at adenosine-sensitive parallel fibre synapses) and the suppression of glutamate release. It would be predicted that the synapses that were insensitive to adenosine would be less depressed by hypoxia and thus maintain function during metabolic stress. However those synapses which were insensitive to adenosine were rapidly inhibited by hypoxia via a mechanism which was not reversed by blocking A1 receptors. Thus another mechanism must be responsible for the hypoxia-mediated depression at these synapses. These different mechanisms of depression may be important for cell survival and for maintenance of cerebellar function following oxygen starvation

Activity-dependent release of Adenosine: a critical re-evaluation of mechanism

Adenosine is perhaps the most important and universal modulator in the brain. The current consensus is that it is primarily produced in the extracellular space from the breakdown of previously released ATP. It is also accepted that it can be released directly, as adenosine, during pathological events primarily by equilibrative transport. Nevertheless, there is a growing realization that adenosine can be rapidly released from the nervous system in a manner that is dependent upon the activity of neurons. We consider three competing classes of mechanism that could explain neuronal activity dependent adenosine release (exocytosis of ATP followed by extracellular conversion to adenosine; exocytotic release of an unspecified transmitter followed by direct non-exocytotic adenosine release from an interposed cell; and direct exocytotic release of adenosine) and outline discriminatory experimental tests to decide between them. We review several examples of activity dependent adenosine release and explore their underlying mechanisms where these are known. We discuss the limits of current experimental techniques in definitively discriminating between the competing models of release, and identify key areas where technologies need to advance to enable definitive discriminatory tests. Nevertheless, within the current limits, we conclude that there is evidence for a mechanism that strongly resembles direct exocytosis of adenosine underlying at least some examples of neuronal activity dependent adenosine release

Biosensor measurement of purine release from cerebellar cultures and slices

We have previously described an action-potential and Ca2+-dependent form of adenosine release in the molecular layer of cerebellar slices. The most likely source of the adenosine is the parallel fibres, the axons of granule cells. Using microelectrode biosensors, we have therefore investigated whether cultured granule cells (from postnatal day 7–8 rats) can release adenosine. Although no purine release could be detected in response to focal electrical stimulation, purine (adenosine, inosine or hypoxanthine) release occurred in response to an increase in extracellular K+ concentration from 3 to 25 mM coupled with addition of 1 mM glutamate. The mechanism of purine release was transport from the cytoplasm via an ENT transporter. This process did not require action-potential firing but was Ca2+dependent. The major purine released was not adenosine, but was either inosine or hypoxanthine. In order for inosine/hypoxanthine release to occur, cultures had to contain both granule cells and glial cells; neither cellular component was sufficient alone. Using the same stimulus in cerebellar slices (postnatal day 7–25), it was possible to release purines. The release however was not blocked by ENT blockers and there was a shift in the Ca2+ dependence during development. This data from cultures and slices further illustrates the complexities of purine release, which is dependent on cellular composition and developmental stage

Adenosine A1 receptor activation mediates the developmental shift at layer 5 pyramidal cell synapses and is a determinant of mature synaptic strength

During the first postnatal month glutamatergic synapses between layer 5 pyramidal cells in the rodent neocortex switch from an immature state exhibiting high probability of neurotransmitter release, large unitary amplitude and synaptic depression to a mature state with decreased probability of release, smaller unitary amplitude and synaptic facilitation. Using paired recordings, we demonstrate that the developmental shift in release probability at synapses between rat somatosensory layer 5 thick-tufted pyramidal cells is due to a higher and more heterogeneous activation of presynaptic adenosine A1 receptors. Immature synapses under control conditions exhibited distributions of CV, failure rate and release probability that were almost coincident with the A1 receptor blocked condition; however, mature synapses under control conditions exhibited much broader distributions that spanned those of both the A1 receptor agonised and antagonised conditions. Immature and mature synapses expressed A1 receptors with no observable difference in functional efficacy and therefore the heterogeneous A1 receptor activation seen in the mature neocortex is due to increased adenosine concentrations that vary between synapses. Given the central role demonstrated for A1 receptor activation in determining synaptic amplitude and the statistics of transmission between mature layer 5 pyramidal cells, the emplacement of adenosine sources and sinks near the synaptic terminal could constitute a novel form of long-term synaptic plasticity

The sodium-potassium pump controls the intrinsic firing of the cerebellar Purkinje neuron

In vitro, cerebellar Purkinje cells can intrinsically fire action potentials in a repeating trimodal or bimodal pattern. The trimodal pattern consists of tonic spiking, bursting, and quiescence. The bimodal pattern consists of tonic spiking and quiescence. It is unclear how these firing patterns are generated and what determines which firing pattern is selected. We have constructed a realistic biophysical Purkinje cell model that can replicate these patterns. In this model, Na+/K+ pump activity sets the Purkinje cell's operating mode. From rat cerebellar slices we present Purkinje whole cell recordings in the presence of ouabain, which irreversibly blocks the Na+/K+ pump. The model can replicate these recordings. We propose that Na+/K+ pump activity controls the intrinsic firing mode of cerbellar Purkinje cells

Visfatin reduces gap junction mediated cell-to-cell communication in proximal tubule-derived epithelial cells

Background/Aims: In the current study we examined if the adipocytokine, visfatin, alters connexin-mediated intercellular communication in proximal tubule-derived epithelial cells. Methods: The effects of visfatin (10-200ng/mL) on cell viability and cytotoxicity in HK2-cells were assessed by MTT, crystal violet and lactate dehydrogenase assays. Western blot analysis was used to confirm expression of Cx26, Cx40 and Cx43. The effect of visfatin (10-200ng/mL) on TGF-β1 secretion was confirmed by ELISA, and the effects of both TGF-β1 (2-10ng/mL) and visfatin (10-200ng/mL) on connexin expression were assessed by western blot. Functional intercellular communication was determined using transfer of Lucifer Yellow and paired-whole cell patch clamp electrophysiology. Results: In low glucose (5mM), visfatin (10-200ng/mL) did not affect membrane integrity, cytotoxicity or cell viability at 48hrs, but did evoke a concentration-dependent reduction in Cx26 and Cx43 expression. The expression of Cx40 was unaffected. At 48hrs, visfatin (10-200ng/mL) increased the secretion of TGF-β1 and the visfatin-evoked changes in connexin expression were mimicked by exogenous application of the pro-fibrotic cytokine (2-10ng/ml). Visfatin reduced dye transfer between coupled cells and decreased functional conductance, with levels falling by 63% as compared to control. Although input resistance was increased following visfatin treatment by 166%, the change was not significant as compared to control. The effects of visfatin on Cx-expression and cell-coupling were blocked in the presence of a TGF-β1 specific neutralizing antibody. Conclusions: The adipocytokine visfatin selectively evoked a non-toxic reduction in connexin expression in HK2-cells. The loss in gap-junction associated proteins was mirrored by a loss in functional conductance between coupled cells. Visfatin increased TGF-β secretion and the pattern of change for connexins expression was mimicked by exogenous application of TGF-β1. The effect of visfatin on Cx-expression and dye transfer were negated in the presence of a TGF-β1 neutralising antibody. These data suggest that visfatin reduces connexin-mediated intercellular communication in proximal tubule-derived epithelial cells via a TGF-β dependent pathway. © 2013 S. Karger AG, Base

Combined electrophysiological and biosensor approaches to study purinergic regulation of epileptiform activity in cortical tissue

Background: Cortical brain slices offer a readily accessible experimental model of a region of the brain commonly affected by epilepsy. The diversity of recording techniques, seizure-promoting protocols and mutant mouse models provides a rich diversity of avenues of investigation, which is facilitated by the regular arrangement of distinct neuronal populations and afferent fibre pathways, particularly in the hippocampus. New method and results: We have been interested in the regulation of seizure activity in hippocampal and neocortical slices by the purines, adenosine and ATP. Via the use of microelectrode biosensors we have been able to measure the release of these important neuroactive compounds simultaneously with on-going epileptiform activity, even of brief durations. In addition, detailed numerical analysis and computational modelling has produced new insights into the kinetics and spatial distribution of elevations in purine concentration that occur during seizure activity. Comparison and conclusions: Such an approach allows the spatio-temporal characteristics of neurotransmitter/neuromodulator release to be directly correlated with electrophysiological measures of synaptic and seizure activity, and can provide greater insight into the role of purines in epilepsy

Detecting CO2-sensitive hemichannels in neurons in acute brain slices

This protocol provides two independent methods to functionally detect the neuronal expression of CO2-sensitive hemichannels. These hemichannels (consisting of connexins 26 or 30) are directly gated by CO2, independent of pH changes and until recently were thought to be only expressed by glia. This protocol outlines a method to change the concentration of CO2 without changing pH, using isohydric solutions and then utilizing this to detect opening and closing of functional hemichannels using whole-cell patch clamp recording and dye loading

Moderate changes in CO2 modulate the firing of neurons in the VTA and substantia nigra

The substantia nigra (SN) and ventral tegmental area (VTA) are vital for the control of movement, goal-directed behavior, and encoding reward. Here we show that the firing of specific neuronal subtypes in these nuclei can be modulated by physiological changes in the partial pressure of carbon dioxide (PCO2). The resting conductance of substantia nigra dopaminergic neurons in young animals (postnatal days 7–10) and GABAergic neurons in the VTA is modulated by changes in the level of CO2. We provide several lines of evidence that this CO2-sensitive conductance results from connexin 26 (Cx26) hemichannel expression. Since the levels of PCO2 in the blood will vary depending on physiological activity and pathology, this suggests that changes in PCO2 could potentially modulate motor activity, reward behavior, and wakefulness

Modelling microelectrode biosensors : free-flow calibration can substantially underestimate tissue concentrations

Microelectrode amperometric biosensors are widely used to measure concentrations of analytes in solution and tissue including acetylcholine, adenosine, glucose and glutamate. A great deal of experimental and modelling effort has been directed at quantifying the response of the biosensors themselves; however, the influence that the macroscopic tissue environment has on biosensor response has not been subjected to the same level of scrutiny. Here we identify an important issue in the way microelectrode biosensors are calibrated that is likely to have led to underestimations of analyte tissue concentrations. Concentration in tissue is typically determined by comparing the biosensor signal to that measured in free-flow calibration conditions. In a free-flow environment the concentration of the analyte at the outer surface of the biosensor can be considered constant. However, in tissue the analyte reaches the biosensor surface by diffusion through the extracellular space. Because the enzymes in the biosensor break down the analyte, a density gradient is set up resulting in a significantly lower concentration of analyte near the biosensor surface. This effect is compounded by the diminished volume fraction (porosity) and reduction in the diffusion coefficient due to obstructions (tortuosity) in tissue. We demonstrate this effect through modelling and experimentally verify our predictions in diffusive environments