Anti-cholinergic drug burden in patients with dementia increases after hospital admission: a multicentre cross-sectional study.

BACKGROUND: Anticholinergic medications are drugs that block cholinergic transmission, either as their primary therapeutic action or as a secondary effect. Patients with dementia may be particularly sensitive to the central effects of anticholinergic drugs. Anticholinergics also antagonise the effects of the main dementia treatment, cholinesterase inhibitors. Our study aimed to investigate anticholinergic prescribing for dementia patients in UK acute hospitals before and after admission. METHODS: We included 352 patients with dementia from 17 UK hospital sites in 2019. They were all inpatients on surgical, medical or Care of the Elderly wards. Information about each patient's medications were collected using a standardised form, and the anticholinergic drug burden of each patient was calculated with an evidence-based online calculator. Wilcoxon's rank test was used to look at the correlation between two subgroups upon admission and discharge. RESULTS: On admission to hospital, 37.8% of patients had an anticholinergic burden score ≥ 1 and 5.68% ≥3. On discharge, 43.2% of patients with an anticholinergic burden score ≥ 1 and 9.1% ≥3. The increase in scores was statistically significant (p = 0.001). Psychotropics were the most common group of anticholinergic medications prescribed at discharge. Of those patients taking cholinesterase inhibitors, 44.9% were also prescribed anticholinergic medications. CONCLUSIONS: Our cross-sectional, multicentre study found that people with dementia are commonly prescribed anticholinergic medications, even if concurrently taking cholinesterase inhibitors, and are significantly more likely to be discharged from hospital with a higher anticholinergic burden than on admission.

Controlled release of cytokines using silk-biomaterials for macrophage polarization.

Polarization of macrophages into an inflammatory (M1) or anti-inflammatory (M2) phenotype is important for clearing pathogens and wound repair, however chronic activation of either type of macrophage has been implicated in several diseases. Methods to locally control the polarization of macrophages is of great interest for biomedical implants and tissue engineering. To that end, silk protein was used to form biopolymer films that release either IFN-γ or IL-4 to control the polarization of macrophages. Modulation of the solubility of the silk films through regulation of ß-sheet (crystalline) content enabled a short-term release (4-8 h) of either cytokine, with smaller amounts released out to 24 h. Altering the solubility of the films was accomplished by varying the time that the films were exposed to water vapor. The released IFN-γ or IL-4 induced polarization of THP-1 derived macrophages into the M1 or M2 phenotypes, respectively. The silk biomaterials were able to release enough IFN-γ or IL-4 to repolarize the macrophage from M1 to M2 and vice versa, demonstrating the well-established plasticity of macrophages. High ß-sheet content films that are not soluble and do not release the trapped cytokines were also able to polarize macrophages that adhered to the surface through degradation of the silk protein. Chemically conjugating IFN-γ to silk films through disulfide bonds allowed for longer-term release to 10 days. The release of covalently attached IFN-γ from the films was also able to polarize M1 macrophages in vitro. Thus, the strategy described here offers new approaches to utilizing biomaterials for directing the polarization of macrophages.

Potential of wind turbines to elicit seizures under various meteorological conditions.

PURPOSE: To determine the potential risk of epileptic seizures from wind turbine shadow flicker under various meteorologic conditions. METHODS: We extend a previous model to include attenuation of sunlight by the atmosphere using the libradtran radiative transfer code. RESULTS: Under conditions in which observers look toward the horizon with their eyes open we find that there is risk when the observer is closer than 1.2 times the total turbine height when on land, and 2.8 times the total turbine height in marine environments, the risk limited by the size of the image of the sun's disc on the retina. When looking at the ground, where the shadow of the blade is cast, observers are at risk only when at a distance <36 times the blade width, the risk limited by image contrast. If the observer views the horizon and closes their eyes, however, the stimulus size and contrast ratio are epileptogenic for solar elevation angles down to approximately 5 degrees. DISCUSSION: Large turbines rotate at a rate below that at which the flicker is likely to present a risk, although there is a risk from smaller turbines that interrupt sunlight more than three times per second. For the scenarios considered, we find the risk is negligible at a distance more than about nine times the maximum height reached by the turbine blade, a distance similar to that in guidance from the United Kingdom planning authorities.

Magnetic resonance imaging of neuronal and glial swelling as an indicator of function in cerebral tissue slices.

Here we demonstrate a new basis of signal change in magnetic resonance imaging (MRI) related to neuronal function, independent of blood oxygenation or flow. Time series MRI data acquired from living, superfused brain slices of adult rats revealed that the signal intensity reversibly increased with depolarization evoked by briefly elevating extracellular K(+). This was presumably a consequence of increased tissue water in the intracellular compartment. Reversible increases in light transmittance (LT) demonstrating a similar time course in response to K(+) elevation supported cellular swelling as generating the MRI signal intensity changes. This was confirmed by reversibly swelling cells in the slice under hypoosmotic challenge, which increased both MRI and LT signals with an identical time course. Conversely, shrinking cells under hyperosmotic challenge reversibly decreased the MRI and LT signals. We propose that specific MRI of neuronal function (fMRI) signals detected under identical parameters during predominantly proton-density-weighted fMRI of the spinal cord can now be explained by neuronal and glial swelling in activated central nervous system (CNS) regions. These observations demonstrate the biophysical basis of the fMRI contrast mechanism that has been termed "signal enhancement by extravascular water protons," or SEEP.

Elevated polyunsaturated fatty acids in blood serum obtained from children on the ketogenic diet.

The authors analyzed blood metabolites in nine children with epilepsy prior to starting the ketogenic diet (KD) and 3 to 4 weeks after KD therapy. Elevated beta-hydroxybutyrate and cortisol levels were observed in all children on the KD. Free fatty acids increased 2.2-fold on the KD, with significant elevations in most polyunsaturated fatty acids (PUFA; arachidonate increased 1.6- to 2.9-fold and docosahexaenoate increased 1.5- to 4.0-fold). The rise in total serum arachidonate correlated with improved seizure control. Elevated PUFA may represent a key anticonvulsant mechanism of the KD.

Anoxic depolarization mediates acute damage independent of glutamate in neocortical brain slices.

An important but poorly understood event associated with ischemia is anoxic depolarization (AD), a sudden and profound depolarization of neurons and glia in cortical and subcortical gray matter. Leao first measured the AD as a wave of electrical silence moving across the cerebral cortex in 1947 and noted its similarity to spreading depression (SD). SD is harmless when coursing through normoxic cortical tissue as during migraine aura. However for 3-4 h following focal ischemia, the additional metabolic stress arising from recurring SD in the penumbra expands the ischemic core, so SD blockade is potentially beneficial therapeutically. In the present study, we measured intrinsic optical signals (IOSs) to monitor anoxic depolarization in submerged rat neocortical slices during O2/glucose deprivation (OGD). After approximately 6 min of OGD, the AD was imaged as a focal increase in light transmittance which then propagated across neocortical gray at approximately 2 mm/min. Although the slice was globally stressed, the AD always initiated focally, sometimes at multiple sites. Its propagation was coincident with a transient negative shift in the extracellular potential, the electrical signature of AD. Acute damage to neocortex (measured as a delayed decrease in LT and as a loss of the evoked field potential) followed only where the AD had propagated, so it is the combined metabolic demands of AD and OGD that acutely damages all layers of the neocortex. Glutamate receptor antagonists (2 mM kynurenate or 25 microM AP-5/10 microM CNQX) did not block AD initiation, slow its propagation or prevent post-AD damage. This study shows that acute ischemic damage is greatly exacerberated by AD during metabolic stress and that glutamate receptor antagonists are not protective. Using this slice model, therapeutically tolerable drugs that block the AD and SD can be investigated.

Imaging anoxic depolarization during ischemia-like conditions in the mouse hemi-brain slice.

Focal ischemia evokes a sudden loss of membrane potential in neurons and glia of the ischemic core termed the anoxic depolarization (AD). In metabolically compromised regions with partial blood flow, peri-infarct depolarizations (PIDs) further drain energy reserves, promoting acute and delayed neuronal damage. Visualizing and quantifying the AD and PIDs and their acute deleterious effects are difficult in the intact animal. In the present study, we imaged intrinsic optical signals to measure changes in light transmittance in the mouse coronal hemi-brain slice during AD generation. The AD was induced by oxygen/glucose deprivation (OGD) or by ouabain exposure. Potential neuroprotective strategies using glutamate receptor antagonists or reduced temperature were tested. Eight minutes of OGD (n = 18 slices) or 4 min of 100 microM ouabain (n = 14) induced a focal increase of increased light transmittance (LT) in neocortical layers II/III that expanded concentrically to form a wave front coursing through neocortex and independently through striatum. The front was coincident with a negative voltage shift in extracellular potential. Wherever the LT front (denoting cell swelling) propagated, a decrease in LT (denoting dendritic beading) followed in its wake. In addition the evoked field potential was permanently lost, indicating neuronal damage. Glutamate receptor antagonists did not block the onset and propagation of AD or the extent of irreversible damage post-AD. Lowering temperature to 25-30 degrees C protected the tissue from OGD damage by inhibiting AD onset. This study shows that anoxic depolarization evoked by global ischemia-like conditions is a spreading process that is focally initiated at multiple sites in cortical and subcortical gray. The combined energy demands of O(2)/glucose deprivation and the AD greatly exacerbate neuronal damage. Glutamate receptor antagonists neither block the AD in the ischemic core nor, we propose, block recurrent PID arising close to the core.

Interferon-alpha inhibits long-term potentiation and unmasks a long-term depression in the rat hippocampus.

Interferons (IFN) appear to have various neuromodulatory actions. Here, we characterized the actions of IFN-alpha on the electrophysiological properties of CA1 hippocampal neurons using intracellular recordings. Superfusion of this cytokine did not alter the resting membrane potential, cell input resistance, action potentials, nor GABA-mediated fast synaptic potentials. IFN-alpha inhibited glutamate-mediated excitatory postsynaptic potentials (gEPSPs) and reversed or prevented long-term potentiation (LTP) induced by high-frequency tetanic stimulation. IFN-alpha reduced gEPSP amplitude far below its control value. Only a short-term potentiation (STP) was observed when either IFN-alpha or D-2-amino-5-phosphonovalerato (APV; NMDA receptor antagonist) were present during tetanic stimulation. After this STP in presence of APV, IFN-alpha had no effect on gEPSPs. APV had no effect on LTP when applied after tetanic stimulation and did also not prevent IFN-alpha effect on LTP. Genistein (a tyrosine kinase inhibitor) or heat inactivation prevented IFN-alpha effects. IFN-alpha also decreased the depolarization induced by local application of glutamate but did not modify those induced by NMDA. Similarly, IFN-alpha reversed the potentiation (induced by tetanic stimulation) of glutamate-induced depolarizations. IFN-alpha did not affect long-term depression (LTD) induced by low-frequency tetanic stimulation. In conclusion, IFN-alpha-induced inhibition of LTP is, at least in part, mediated by a postsynaptic effect, by tyrosine kinase activity, and by non-NMDA glutamate receptors. Inhibition of LTP by IFN-alpha unmasks LTD which is induced by the same high-frequency tetanic stimulation.

ATP inhibits glutamate synaptic release by acting at P2Y receptors in pyramidal neurons of hippocampal slices.

It has been proposed that extracellular ATP inhibits synaptic release of glutamate from hippocampal CA1 synapses after its catabolism to adenosine. We investigated the possibility that at least part of this effect is mediated by ATP itself acting on P2Y receptors. ATP and various analogs decreased the amplitude and duration of glutamate-mediated excitatory postsynaptic potentials in all tested neurons. This effect was reversible and concentration-dependent and had the following rank order of agonist potency: AMP = ATP = adenosine-5'-O-(3-thio)triphosphate > adenosine = ADP. alpha,beta-Methylene ATP, beta,gamma-methylene ATP, 2-methylthioadenosine 5'-triphosphate, GTP, and UTP induced only a partial response. The depolarization induced by exogenous glutamate was not affected by ATP, indicating that this nucleotide acts presynaptically to inhibit glutamate-mediated excitatory postsynaptic potentials. Neither inhibition of ectonucleotidase activity with alpha,beta-methylene ADP, suramin, or pyridaxalphosphate-6-azophenyl-2',4'-disulfonic acid 4-sodium nor removal of extracellular adenosine (with adenosine deaminase) altered ATP effects. 8-Cyclopentyltheophylline competitively inhibited ATP effects, whereas P2 receptor antagonists (pyridaxalphosphate-6-azophenyl-2',4'-disulfonic acid 4-sodium, suramin, and reactive blue 2) were ineffective. ATP effects were by far more sensitive to pertussis toxin (PTX) than those of adenosine. After PTX, adenosine-5'-O-(3-thio)triphosphate induced only a partial response, and ATP concentration-response curve was biphasic. The second phase of this curve was blocked by adenosine deaminase, implying that it is mediated by adenosine as a result of ATP catabolism. Under control conditions, however, catabolism of ATP is not required to explain its actions. In conclusion, ATP inhibits synaptic release of glutamate by direct activation of P2Y receptors that are PTX- and 8-cyclopentyltheophylline-sensitive.

Glutamate does not mediate acute neuronal damage after spreading depression induced by O2/glucose deprivation in the hippocampal slice.

This study argues that, in contrast to accepted excitotoxicity theory, O2/glucose deprivation damages neurons acutely by eliciting ischemic spreading depression (SD), a process not blocked by glutamate antagonists. In live rat hippocampal slices, the initiation, propagation, and resolution of SD can be imaged by monitoring wide-band changes in light transmittance (i.e., intrinsic optical signals). Oxygen/glucose deprivation for 10 minutes at 37.5 degrees C evokes a propagating wave of elevated light transmittance across the slice, representing the SD front. Within minutes, CA1 neurons in regions undergoing SD display irreversible damage in the form of field potential inactivation, swollen cell bodies, and extensively beaded dendrites, the latter revealed by single-cell injection of lucifer yellow. Importantly, glutamate receptor antagonists do not block SD induced by O2/glucose deprivation, nor do they prevent the resultant dendritic beading of CA1 neurons. However, CA1 neurons are spared if SD is suppressed by reducing the temperature to 35 degrees C during O2/glucose deprivation. This supports previous electrophysiologic evidence in vivo that SD during ischemia promotes acute neuronal damage and that glutamate antagonists are not protective of the metabolically stressed tissue. The authors propose that the inhibition of ischemic SD should be targeted as an important therapeutic strategy against stroke damage.

Interpretation of intrinsic optical signals and calcein fluorescence during acute excitotoxic insult in the hippocampal slice.

Immediate (acute) neuronal damage in response to overstimulation of glutamate receptors results from toxic exposure to food poisons acting as glutamate analogues. Glutamate agonist application evokes dramatic intrinsic optical signals (IOSs) in the rat hippocampal slice preparation, particularly in the CA1 region. Theoretically IOSs are generated by alterations to neuronal and glial structure that change light transmittance (LT) in live brain tissue. To better understand such signals, IOSs evoked by the glutamate agonist N-methyl-D-aspartate were imaged in the rat hippocampal slice. We correlated these excitotoxic signals with: (1) biophysical principles governing light transport, (2) tissue volume changes as measured using a free intracellular fluorophore (calcein), (3) dendritic morphology visualized by dye injection, and (4) standard histopathology. In theory LT elevation evoked during acute excitotoxic swelling is generated by change to subcellular structure that reduces light scattering during cell swelling. However, in responsive dendritic regions, initial LT elevation caused by cell swelling was overridden by the formation of dendritic beads, a conformation that increased light scattering (thereby reducing LT) even as the calcein signal demonstrated that the tissue continued to swell. Thus IOS imaging reveals acute somatic and dendritic damage during excitotoxic stress that can be monitored across slices of brain tissue in real time.

Potential sources of intrinsic optical signals imaged in live brain slices.

Changes in how light is absorbed or scattered in biological tissue are termed intrinsic optical signals (IOSs). Imaging IOSs in the submerged brain slice preparation provides insight into brain activity if it involves significant water movement between intracellular and extracellular compartments. This includes responses to osmotic imbalance, excitotoxic glutamate agonists, and oxygen/glucose deprivation, the latter leading to spreading depression. There are several misconceptions regarding these signals. (1) IOSs are not generated by glial swelling alone. Although neuronal and glia sources cannot yet be directly imaged, several lines of evidence indicate that neurons contribute significantly to the changes in light transmittance. (2) Excitotoxic swelling and osmotic swelling are physiologically different, as are their associated IOSs. Hyposmotic swelling involves no detectable neuronal depolarization of cortical pyramidal neurons, only the passive drawing in of water from a dilute medium across the cell membrane. In contrast excitotoxic swelling involves sustained membrane depolarization associated with inordinate amounts of Na+ and Cl- entry followed by water. IOSs demonstrate substantial damage in the latter case. (3) Osmotic perturbations do not induce volume regulatory mechanisms as measured by IOSs. The osmotic responses measured by IOSs in brain slices are passive, without the compensatory mechanisms that are assumed to be active on a scale suggested by studies of cultured brain cells under excessive osmotic stress. (4) Spreading depression (SD) can cause neuronal damage. Innocuous during migraine aura, SD induces acute neuronal damage in brain slices that are metabolically compromised by oxygen/glucose deprivation, as demonstrated by IOSs. Neighboring tissue where SD does not spread remains relatively healthy as judged by a minimal reduction in light transmittance. IOSs show that the metabolic stress of SD combined with the compromise of energy resources leads to acute neuronal damage that is resistant to glutamate antagonists. (5) While hyperosmotic conditions reduce LT by causing cells to shrink, excitotoxic conditions reduce LT by causing dendritic beading. This conformational change increases light scattering even as the tissue continues to swell.

Spreading depression determines acute cellular damage in the hippocampal slice during oxygen/glucose deprivation.

During ischaemia neurons depolarize and release the neurotransmitter L-glutamate, which accumulates extracellularly and binds to postsynaptic receptors. This initiates a sequence of events thought to culminate in immediate and delayed neuronal death. However, there is growing evidence that during ischaemia the development of spreading depression (SD) can be an important determinant of the degree and extent of ischaemic damage. In contrast, SD without metabolic compromise (as occurs in migraine aura) causes no discernible damage to brain tissue. SD is a profound depolarization of neurons and glia that propagates like a wave across brain tissue. Brain cell swelling, an early event of both the excitotoxic process and of SD, can be assessed by imaging associated intrinsic optical signals (IOSs). We demonstrate here that IOS imaging clearly demarcates the ignition site and migration of SD across the submerged hippocampal slice of the rat. If SD is induced by elevating [K+]O, the tissue fully recovers, but in slices that are metabolically compromised at 37.5 degrees C by oxygen/glucose deprivation (OGD) or by ouabain exposure, cellular damage develops only where SD has propagated. Specifically, the evoked CA1 field potential is permanently lost, the cell bodies of involved neurons swell and their dendritic regions increase in opacity. In contrast to OGD, bath application of L-glutamate (6-10 mM) at 37.5 degrees C evokes a non-propagating LT increase in CA1 that reverses without obvious cellular damage. Moreover, application of 2-20 mM glutamate or various glutamate agonists fail to evoke SD in the submerged hippocampal slice. We propose that SD and OGD together (but not alone) constitute a 'one-two punch', causing acute neuronal death in the slice that is not replicated by elevated glutamate. These findings support the proposal that SD generation during stroke promotes and extends acute ischaemic damage.

Imaging spreading depression and associated intracellular calcium waves in brain slices.

Spreading depression (SD) was analyzed in hippocampal and neocortical brain slices by imaging intrinsic optical signals in combination with either simultaneous electrophysiological recordings or imaging of intracellular calcium dynamics. The goal was to determine the roles of intracellular calcium (Ca2+int) waves in the generation and propagation of SD. Imaging of intrinsic optical signals in the hippocampus showed that ouabain consistently induced SD, which characteristically started in the CA1 region, propagated at 15-35 micrometer/sec, and traversed across the hippocampal fissure to the dentate gyrus. In the dendritic regions of both CA1 and the dentate gyrus, SD caused a transient increase in light transmittance, characterized by both a rapid onset and a rapid recovery. In contrast, in the cell body regions the transmittance increase was prolonged. Simultaneous imaging of intracellular calcium and intrinsic optical signals revealed that a slow Ca2+int increase preceded any change in transmittance. Additionally, a wave of increased Ca2+int typically propagated many seconds ahead of the change in transmittance. These calcium increases were also observed in individual astrocytes injected with calcium orange, indicating that Ca2+int waves were normally associated with SD. However, when hippocampal slices were incubated in calcium-free/EGTA external solutions, SD was still observed, although Ca2+int waves were completely abolished. Under these conditions SD had a comparable peak increase in transmittance but a slower onset and a faster recovery. These results demonstrate that although there are calcium dynamics associated with SD, these increases are not necessary for the initiation or propagation of spreading depression.

Intrinsic optical signaling denoting neuronal damage in response to acute excitotoxic insult by domoic acid in the hippocampal slice.

Using the seafood contaminant domoic acid (an AMPA/kainate receptor agonist), we demonstrate a distinct excitotoxic sequence of events leading to acute neuronal damage in the hippocampal slice as measured by (1) loss of the evoked CA1 field potential, (2) irreversible changes in light transmittance, (3) histopathology, and (4) lucifer yellow injection of single CA1 pyramidal neurons. Change in light transmittance (LT) through the submerged slice indirectly measures altered cell volume, both neuronal and glial. At 37 degrees C, a 1-min superfusion of 10 mu M domoate induced a prolonged reversible increase in LT, primarily in the dendritic regions of CA1 and dentate granule cells (GC), but not in the CA3 region. Spectral analysis (400-800 nm) revealed a wide-band transmittance increase, indicating cell swelling as a major source of the intrinsic signal. The evoked field potential recorded in the CA1 cell body region (PYR) was lost as LT peaked, but completely recovered upon return to the baseline LT level. Increasing domoate exposure to 10 min elicited a different and distinct LT sequence in CA1 and dentate regions. An initial LT increase in dendritic regions evolved in an irreversible decrease in LT. At the same time, LT irreversibly increased in cell body regions (CA1 PYR and GC) and the evoked field potential was irretrievably lost. Also, there was histological damage to cell body and dendritic regions of CA1 and granule cells. Injection of lucifer yellow into single CA1 neurons in slices displaying the irreversible LT sequence revealed extensive dendritic beading, whereas CA1 cells in control slices displayed a smoothly contoured arbor. Consistent with acute neuronal damage, the optical changes generated by domoate did not require extracellular Ca2+, and lowering the temperature protected the slice from irreversible damage to CA1 and GC regions. Although glial changes may also occur, we conclude that imaging light transmittance reveals dynamic and compartmentalized excitotoxic changes in neuronal volume. Beading of the dendritic arbor increases light scatter, thereby decreasing LT and highlighting damaged dendritic regions.

Evidence against volume regulation by cortical brain cells during acute osmotic stress.

The cell bodies of neurons and glia examined in culture respond to severe osmotic stress (100 to 200 mOsm) by passive volume change that is followed within several minutes by volume regulation, even in the face of maintained osmotic change. However, in clinical situations, the brain does not experience such precipitous and severe changes in brain hydration. In this study we examined if there is evidence from the hippocampal slice preparation supporting the type of volume regulation observed in cultured brain cells. Within the CA1 region we imaged changes in light transmittance (LT), recorded the evoked field potential, and monitored tissue resistance (all measures of cell volume change) during the first hour of osmotic stress to search for evidence of volume regulation. During superfusion of hypo-osmotic aCSF (-40 mOsm), LT increased 24 to 28% in the dendritic regions of CA1 neurons. The LT reached a plateau which was maintained throughout a 45-min application interval, more than enough time to reveal a regulatory volume decrease. Upon return to control saline, LT immediately returned to baseline and settled there. Hypo-osmolality reversibly increased the relative tissue resistance (RREL) measured across the CA1 region with a time course identical to the increase in LT. Conversely, hyperosmotic aCSF (mannitol, +40 mOsm) decreased both RREL by 8% and LT by 15.5% with no indication of a regulatory volume increase. The CA1 cell body layer showed only slight hypo-osmotic swelling whereas exposure to the glutamate agonist quinolinic acid caused pronounced swelling in this region. Even when osmolality was decreased by 120 mOsm for 20 min, dendritic regions responded passively with no regulatory volume decrease. However, when aCSF Cl- was substituted, the CA1 dendritic regions displayed immediate swelling followed by a dramatic volume reduction under normosmotic conditions, indicating that such behavior can be evoked by extreme aCSF dilution. We conclude that in the brain slice preparation, the cortical cells do not exhibit classic volume regulation in response to sudden physiological changes in osmolality. Moreover it is the dendritic region, not the cell body region, that displays dynamic volume change during osmotic challenge.

Imaging NMDA- and kainate-induced intrinsic optical signals from the hippocampal slice.

1. Brain ischemia causes excess release and accumulation of glutamate that binds to postsynaptic receptors. This opens ionotropic channels that mediate neuronal depolarization and ionic fluxes that can lead to neuronal death. 2. The CA1 pyramidal cell region of the hippocampus is particularly susceptible to this neurotoxic process. Brain cell swelling is considered an early excitotoxic event, but remains poorly under stood and documented. As cells swell, light transmittance (LT) increases through brain tissue, so we hypothesized that brief exposure to glutamate agonists would elicit cell swelling that could be imaged in real time in the hippocampal slice. 3. A 1-min bath application of 100 microM N-methyl-D-aspartate (NMDA) or 100 microM kainate at 22 degrees C greatly increased LT, particularly in the dendritic regions of CA1. The response peaked by 2-3 min and slowly reversed over the subsequent 20 min following exposure. Peak LT increases were > 50% in CA1 stratum radiatum and > 20% in both CA1 stratum oriens and the dendritic region of the dentate gyrus, all areas with a high concentration of NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors. The CA3 stratum radiatum, which contains fewer of these receptors, showed a comparatively small LT increase. 4. The NMDA receptor antagonist 2-amino-5-phosphonovalerate (AP-5) [but not 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)] blocked the CA1 response to NMDA, whereas the non-NMDA receptor antagonist CNQX (but not AP-5) blocked the response to kainate. The relative tissue resistance measured across CA1 stratum radiatum increased after NMDA or kainate exposure with a time course similar to the LT change described above. The increase in relative tissue resistance was blocked by kynurenate, a nonspecific glutamate antagonist. Increases in both LT and tissue resistance provide two independent lines of evidence that cell swelling rapidly developed in CA1 dendritic areas after activation of NMDA or AMPA receptors. 5. This swelling at 22 degrees C was accompanied by a temporary loss of the evoked CA1 field potential. However, at 37 degrees C the dendritic swelling rapidly progressed to an irreversible LT increase (swelling) of the CA1 cell bodies accompanied by a permanent loss of the evoked field. 6. We propose that dendritic swelling mediated by NMDA and AMPA receptors is an early excitotoxic event that can herald permanent damage to CA1 neurons, those cells most vulnerable to ischemic insult.

Real-time imaging of intrinsic optical signals during early excitotoxicity evoked by domoic acid in the rat hippocampal slice.

Excitotoxicity involves neuronal depolarization and eventual cell death primarily through excess activation of glutamate receptors. Neuronal cell swelling is considered an early excitotoxic event mediated by ionic influx (mainly Na+ and Cl-) followed by water. Changes in the intrinsic optical signals of nerve tissue correlate with neuronal activity such that light transmittance (LT) increases across the brain slice as cells swell. The present study examined the effects of domoic acid, a potent excitotoxic food contaminant and glutamate analogue, on intrinsic optical signals in the rat hippocampal slice. A brief 1-min exposure to 10 microM domoate at 22 degrees C elevated LT by 58% in the apical dendritic region of CA1 and to a lesser extent in the molecular layer of the upper dentate gyrus. The responses peaked by 5 min and slowly reversed during a 30-min wash. The same responses were evoked by a 1-min application of 10 microM alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) at 22 degrees C. Minor changes were observed in the CA3 region and the lower blade of the dentate gyrus. At 37 degrees C, exposure to 10 microM domoate for 10 min resulted in apparent irreversible neuronal damage in the CA1 and upper dentate regions. The Na+ channel blocker tetrodotoxin (1 microM) eliminated the evoked CA1 population spike but not the LT increase, indicating that the domoate signal is not associated with action potential discharge pre- or post-synaptically. However, the response to domoate at 22 degrees C was reversibly blocked by the nonspecific glutamate receptor antagonist kynurenate and the non-N-methyl-D-aspartate (non-NMDA) receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). The response was not blocked by the NMDA receptor antagonist 2-amino-5-phosphonovaleate (AP-5) nor the kainate receptor blocker gamma-D-glutamylaminomethyl sulfonate (GAMS). Relative tissue resistance (RREL) measured across the CA1 dendritic region increased rapidly in response to domoate and fell slowly over 30 min, which paralleled the LT response described above. The increase in RREL was blocked by kynurenate. We propose that domoate binding to AMPA receptors opens channels mediating ionic influx, presumably Na+ followed passively by Cl-. Water follows, producing prolonged postsynaptic swelling in the CA1 and dentate regions where AMPA receptors are most abundant. At higher temperature this swelling can progress to permanent neuronal injury. Imaging intrinsic optical signals allows a real-time view of early excitotoxic events and may prove useful in assessing potentially therapeutic agents that reduce damage induced by excitotoxic agents or ischemia.

Time-dependent decreases of atrial natriuretic peptide release from the isolated rat atrium: evidence for a readily releasable pool.

In vitro, atrial distension causes a rapid increase in atrial natriuretic peptide (ANP) release. This stretch-induced release, however, declines to baseline levels within minutes without significant depletion of the total hormone stores. It has been observed that the basal rate of ANP release from isolated atria also declines over time despite evidence that the tissue retains its viability. We examined this time-dependency of ANP release from isolated rat atria and some parameters that may explain the diminishing release. Mean ANP secretion was 60 pg/min for both spontaneously beating and electrically paced preparations. Although ANP secretion steadily declined over time, there was no time-dependent effect on the amplitude of intracellularly recorded action potentials. The total ANP content in atria obtained after dissection was 133 +/- 28.9 micrograms/g (n = 3) which was not significantly different from atria that were perfused for 3 h (137 +/- 21.2 micrograms/g; n = 3). Only the 28 amino acid circulating form of ANP was released. The ANP mRNA appeared to be partially degraded in atria after 30 min equilibration or after perfusion for 3 h. These results demonstrated that ANP release from isolated atrial preparations declines steadily despite the maintenance of normal electrophysiological activity. This decline was not due to significant depletion of the ANP stores suggesting that a readily releasable pool of ANP exists and represents only a small fraction of the total hormone stores. Finally, degradation of ANP mRNA implies a reduction of de novo synthesis in our preparation which suggests that the observed depletion of the releasable pool was related to a decline in newly synthesized ANP.

Effects of micromolar and nanomolar calcium concentrations on non-synaptic bursting in the hippocampal slice.

To determine how [Ca2+]0 affects non-synaptic epileptogenesis in the CAI area of hippocampal slices, we compared the extracellularly recorded hyperactivity induced by ACSF containing either micromolar ('low-Ca2+, LC-ACSF) or nanomolar concentrations of Ca2+ ('zero-Ca2+, ZC-ACSF). Both solutions effectively blocked chemical synaptic transmission but spontaneous bursts developed more quickly and consistently in ZC-ACSF and were longer in duration and more frequent than those recorded in LC-ACSF Antidromically evoked bursts were less epileptiform, i.e., they exhibited fewer population spikes (PSs), in ZC-ACSF. Increasing [Mg2+]0 or decreasing [K+]0 suppressed spontaneous LC-ACSF bursting but only decreased the intensity and frequency of bursting in ZC-ACSF. Either manipulation increased the epileptiform nature of the antidromically evoked field potential, thereby mimicking the effect of increasing [Ca2+]0 from nanomolar to micromolar levels. Bath application of 250-500 microM GABA commonly arrested spontaneous bursting in LC-ACSF. In ZC-ACSF, GABA decreased the burst frequency but paradoxically superimposed high amplitude PSs on each burst. These effects were reversed by the GABAA receptor antagonists bicuculline methiodide or picrotoxin (50-100 microM). These results indicate that simply lowering [Ca2+]0 from micromolar to nanomolar concentrations increases the burst propensity and intensity of the CA1 population and can dramatically alter responses to pharmacological agents.