A solvent-free matrix application method for matrix-assisted laser desorption/ionization imaging of small molecules.

Matrix application continues to be a critical step in sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). Imaging of small molecules such as drugs and metabolites is particularly problematic because the commonly used washing steps to remove salts are usually omitted as they may also remove the analyte, and analyte spreading is more likely with conventional wet matrix application methods. We have developed a method which uses the application of matrix as a dry, finely divided powder, here referred to as dry matrix application, for the imaging of drug compounds. This appears to offer a complementary method to wet matrix application for the MALDI-MSI of small molecules, with the alternative matrix application techniques producing different ion profiles, and allows the visualization of compounds not observed using wet matrix application methods. We demonstrate its value in imaging clozapine from rat kidney and 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylic acid from rat brain. In addition, exposure of the dry matrix coated sample to a saturated moist atmosphere appears to enhance the visualization of a different set of molecules.

Responses of magnocellular neurons to osmotic stimulation involves coactivation of excitatory and inhibitory input: an experimental and theoretical analysis.

How does a neuron, challenged by an increase in synaptic input, display a response that is independent of the initial level of activity? Here we show that both oxytocin and vasopressin cells in the supraoptic nucleus of normal rats respond to intravenous infusions of hypertonic saline with gradual, linear increases in discharge rate. In hyponatremic rats, oxytocin and vasopressin cells also responded linearly to intravenous infusions of hypertonic saline but with much lower slopes. The linearity of response was surprising, given both the expected nonlinearity of neuronal behavior and the nonlinearity of the oxytocin secretory response to such infusions. We show that a simple computational model can reproduce these responses well, but only if it is assumed that hypertonic infusions coactivate excitatory and inhibitory synaptic inputs. This hypothesis was tested first by applying the GABA(A) antagonist bicuculline to the dendritic zone of the supraoptic nucleus by microdialysis. During local blockade of GABA inputs, the response of oxytocin cells to hypertonic infusion was greatly enhanced. We then went on to directly measure GABA release in the supraoptic nucleus during hypertonic infusion, confirming the predicted rise. Together, the results suggest that hypertonic infusions lead to coactivation of excitatory and inhibitory inputs and that this coactivation may confer appropriate characteristics on the output behavior of oxytocin cells. The nonlinearity of oxytocin secretion that accompanies the linear increase in oxytocin cell firing rate reflects frequency-facilitation of stimulus-secretion coupling at the neurohypophysis.

Uterine contractile activity stimulates supraoptic neurons in term pregnant rats via a noradrenergic pathway.

Oxytocin secretion is important for the normal progress of parturition in the rat. We tested the hypotheses that contractions of the uterus before pup delivery activate oxytocin neurons, and that they do so via a noradrenergic projection. In anesthetized 22-day (term) pregnant rats, i.v. oxytocin pulses enhanced both uterine contractile activity and the firing rate of oxytocin and vasopressin neurons in the supraoptic nucleus, and these were significantly correlated. The same oxytocin treatment also increased the expression of Fos in both the supraoptic nucleus and the nucleus of the tractus solitarius, but not in 21-day pregnant or virgin rats. In five of eight rats on the day of expected parturition, noradrenaline release in the supraoptic nucleus (sampled by microdialysis) exhibited sudden peaks during oxytocin administration, seen in only one of nine rats given vehicle pulses. Noradrenaline release was significantly greater in rats that went into labor or gave birth to a pup than in rats not in labor. In rats infused with the alpha(1)-noradrenergic receptor antagonist, benoxathian, into the supraoptic nucleus before and during iv oxytocin administration, Fos expression in supraoptic neurons was significantly less than that in vehicle controls. Thus, at term pregnancy, uterine contractions activate both oxytocin and vasopressin neurons in the SON, and this activation involves a noradrenergic pathway.

Modelling the pituitary response to luteinizing hormone-releasing hormone.

The pituitary response to luteinizing hormone-releasing hormone (LHRH) is steroid-dependent and varies throughout the reproductive cycle, but the rapid rise in pituitary sensitivity on the day of the ovulation-inducing LH surge is due to a 'self-priming' effect of exposure to LHRH that results in a potentiation of pituitary responsiveness 35-40 min later. The expression of this effect is itself steroid-dependent, and is most marked on pro-oestrus. Here, a model of LHRH-induced LH release was developed to incorporate the changes in pituitary sensitivity observed throughout the reproductive cycle. LH release is based on the Law of Mass Action, and a component related to self-priming is included in the model, incorporating the delay between initial exposure and potentiation of responsiveness and an upper maximum to the achievable level of priming. Where possible, model parameters were obtained from biological values, otherwise they were optimized to fit an experiment performed in vivo. These parameters were then used to test the model against other experimental data obtained both in vivo and in vitro. The model provided a good fit to the in vivo data but the in vitro experimental data required a change in one parameter, the upper limit of priming. We conclude that this model of the pituitary release mechanism can simulate the changes in pituitary responsiveness throughout the reproductive cycle. We suggest that substitution of this model in a previous model of the LHRH pulse generator could allow more appropriate tests of the LHRH pulse generator model.

Enhancement of homocysteine toxicity to insulin-secreting BRIN-BD11 cells in combination with alloxan.

Previous studies have shown that homocysteine (HC) has a detrimental impact on insulin secretion and pancreatic beta cell function. The aim of the present study was to determine the role of reactive oxygen species (ROS) in the in vitro toxic effects of HC on insulin secretion and function of BRIN-BD11 insulin-secreting cells. In this study, insulin secretion from BRIN-BD11 cells was determined radioimmunologically, cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and glucokinase activity by a glucose phosphorylation assay following culture with HC plus alloxan (Alx). Treatment with HC resulted in concentration-dependent inhibition of insulin secretion induced by glucose and other insulinotropic agents. HC in combination with Alx resulted in a more pronounced decline in insulin secretion, including that induced by 20  mM alanine, by 43% (P<0.001) and 30  mM KCl by 60% (P<0.001), compared with control culture. The glucokinase phosphorylating capacity in cells cultured with HC plus Alx was significantly lower, compared with control cells. The cells also displayed a significant 84% (P<0.001) decline in cell viability. Prolonged, 72-h culture of insulin-secreting cells with HC followed by 18-h culture without HC did not result in full restoration of beta cell responses to insulinotropic agents. In vitro oxygen consumption was enhanced by a combination of Alx with HC. The study arrived at the conclusion that HC generates ROS in a redox-cycling reaction with Alx that explains the decline in viability of insulin-secreting cells, leading to reduced glucokinase phosphorylating ability, diminished insulin secretory responsiveness and cell death.