Search

Neonicotinoid as insecticide on oilseed rape can reduce bee colony density, but its effect at a large geographical scale is unclear. This study describes 18-year long wild bee tracking data in England and show neonicotinoid use is correlated with wild bee population declines at real landscape scales.

In the central nervous system, nitric oxide (NO) transmits signals from one neurone to another, or from neurones to astrocytes or blood vessels, but the possibility of oligodendrocytes being physiological NO targets has been largely ignored. By exploiting immunocytochemistry for cGMP, the second messenger generated on activation of NO receptors, oligodendrocytes were found to respond to both exogenous and endogenous NO in cerebellar slices from rats aged 8 days to adulthood. Atrial natriuretic peptide, which acts on membrane-associated guanylyl cyclase-coupled receptors, also raised oligodendrocyte cGMP in cerebellar slices. The main endogenous source of NO accessing oligodendrocytes appeared to be the neuronal NO synthase isoform, which was active even under basal conditions and in a manner that was independent of glutamate receptors. Oligodendrocytes in brainstem slices were also shown to be potential NO targets. In contrast, in the optic nerve, oligodendrocyte cGMP was raised by natriuretic peptides but not NO. When cultures of cerebral cortex were continuously exposed to low NO concentrations (estimated as 40-90 pM), oligodendrocytes responded with a striking increase in arborization. This stimulation of oligodendrocyte growth could be replicated by low concentrations of 8-bromo-cGMP (maximum effect at 1 µM). It is concluded that oligodendrocytes are probably widespread targets for physiological NO (or natriuretic peptide) signals, with the resulting rise in cGMP serving to enhance their growth and maturation. NO might help coordinate the myelination of axons to the ongoing level of neuronal activity during development and could potentially contribute to adaptive changes in myelination in the adult., (© 2014 The Authors. Glia Published by Wiley Periodicals, Inc.)

Nitric oxide (NO) is a widespread signaling molecule with potentially multifarious actions of relevance to health and disease. A fundamental determinant of how it acts is its concentration, but there remains a lack of coherent information on the patterns of NO release from its sources, such as neurons or endothelial cells, in either normal or pathological conditions. We have used detector cells having the highest recorded NO sensitivity to monitor NO release from brain tissue quantitatively and in real time. Stimulation of NMDA receptors, which are coupled to activation of neuronal NO synthase, routinely generated NO signals from neurons in cerebellar slices. The average computed peak NO concentrations varied across the anatomical layers of the cerebellum, from 12 to 130 pm. The mean value found in the hippocampus was 200 pm. Much variation in the amplitudes recorded by individual detector cells was observed, this being attributable to their location at variable distances from the NO sources. From fits to the data, the NO concentrations at the source surfaces were 120 pm to 1.4 nm, and the underlying rates of NO generation were 36-350 nm/s, depending on area. Our measurements are 4-5 orders of magnitude lower than reported by some electrode recordings in cerebellum or hippocampus. In return, they establish coherence between the NO concentrations able to elicit physiological responses in target cells through guanylyl cyclase-linked NO receptors, the concentrations that neuronal NO synthase is predicted to generate locally, and the concentrations that neurons actually produce.

Brain function is usually perceived as being performed by neurons with the support of glial cells, the network of blood vessels situated nearby serving simply to provide nutrient and to dispose of metabolic waste. Revising this view, we find from experiments on a rodent central white matter tract (the optic nerve) in vitro that microvascular endothelial cells signal persistently to axons using nitric oxide (NO) derived from the endothelial NO synthase (eNOS). The endogenous NO acts to stimulate guanylyl cyclase-coupled NO receptors in the axons, leading to a raised cGMP level which then causes membrane depolarization, apparently by directly engaging hyperpolarization-activated cyclic nucleotide-gated ion channels. The tonic depolarization and associated endogenous NO-dependent cGMP generation was absent in optic nerves from mice lacking eNOS, although such nerves responded to exogenous NO, with raised cGMP generation in the axons and associated depolarization. In addition to the tonic activity, exposure of optic nerves to bradykinin, a classical stimulator of eNOS in endothelial cells, elicited reversible NO- and cGMP-dependent depolarization through activation of bradykinin B2 receptors, to which eNOS is physically complexed. No contribution of other NO synthase isoforms to either the action of bradykinin or the continuous ambient NO level could be detected. The results suggest that microvascular endothelial cells participate in signal processing in the brain and can do so by generating both tonic and phasic NO signals.

Excessive nitric oxide (NO) production from the inducible isoform of nitric oxide synthase (iNOS) has been invoked as a causative factor in many neurodegenerative disorders, including multiple sclerosis. This hypothesis has been supported by in vitro studies showing that glial iNOS expression results in toxic NO concentrations (near 1 microm). To investigate the relevance of such findings, experiments were carried out ex vivo on optic nerves from rats with exacerbated experimental allergic encephalomyelitis, a model of multiple sclerosis. The nerves displayed characteristic immunopathology and expression of iNOS in macrophages and/or microglia and there was overt axonal damage in localized regions of the optic chiasm. The resulting NO levels in the optic nerve were sufficient to cause activation of guanylyl cyclase-coupled NO receptors, resulting in marked cGMP accumulation in axons throughout the nerve. Nevertheless, calibration of cGMP levels against those evoked by exogenous NO indicated that the nerves were not compromised metabolically and that their ambient NO concentration was only approximately 1 nm. Consistent with this observation, electrophysiological tests indicated that there was no ongoing malfunctioning of the type that can be elicited by high exogenous NO concentrations. It is concluded that, with iNOS expressed in physiological locations and levels, the tissue levels of NO remain at concentrations far lower than those shown to have toxic effects, despite continuous NO synthesis. The fact that NO can rise to much higher levels in dispersed cultures in vitro may be attributable to a deficiency in NO inactivation in such preparations.

Nitric oxide (NO) may act as a toxin in several neuropathologies, including the brain damage resulting from cerebral ischaemia. Rat striatal slices were used to determine the mechanism of enhanced NO release following simulated ischaemia and, for estimating the NO concentrations, the activity of guanylyl cyclase served as a biosensor. Exposure of the slices for 10 min to an oxygen- and glucose-free medium caused a 70% fall in cGMP levels. On recovery, cGMP increased 2-fold above basal, where it remained for 40 min before declining. The pattern of changes matched those of cGMP or NO oxidation products measured during and after brain ischaemia in vivo. The increase observed during the recovery period was blocked by inhibition of NO synthase or NMDA receptors and was curtailed by tetrodotoxin, implying that it was caused by glutamate release leading to activation of the NMDA receptor-NO synthase pathway. Calibration of the cGMP levels against NO-stimulated guanylyl cyclase yielded a basal NO concentration of 0.6 nm. The peak NO concentration achieved on recovery from simulated ischaemia was estimated as 0.8 nm. These values are compatible with the low micromolar concentrations of NO oxidation products (chiefly nitrate) found by microdialysis in vivo, providing the NO inactivation rate (forming nitrate) is accounted for. NO at a concentration around 1 nm is unlikely to be toxic to cells. However, if the NO inactivation mechanism were to fail (as it can) the NO production rate normally providing only subnanomolar NO could readily generate toxic (microM) NO concentrations.

In the rat isolated optic nerve, nitric oxide (NO) activates soluble guanylyl cyclase (sGC), resulting in a selective accumulation of cGMP in the axons. The axons are also selectively vulnerable to NO toxicity. The experiments initially aimed to determine any causative link between these two effects. It was shown, using a NONOate donor, that NO-induced axonal damage occurred independently of cGMP. Unexpectedly, however, the compound YC-1, which is an allosteric activator of sGC, potently inhibited NO-induced axonopathy (IC(50) = 3 microM). This effect was not attributable to increased cGMP accumulation. YC-1 (30 microM) also protected the axons against damage by simulated ischemia, which (like NO toxicity) is sensitive to Na(+) channel inhibition. Although chemically unrelated to any known Na(+) channel inhibitor, YC-1 was effective in two biochemical assays for activity on Na(+) channels in synaptosomes. Electrophysiological recording from hippocampal neurons showed that YC-1 inhibited Na(+) currents in a voltage-dependent manner. At a concentration giving maximal protection of optic nerve axons from NO toxicity (30 microM), YC-1 did not affect normal axon conduction. It is concluded that the powerful axonoprotective action of YC-1 is unrelated to its activity on sGC but is explained by a novel action on voltage-dependent Na(+) channels. The unusual ability of YC-1 to protect axons so effectively without interfering with their normal function suggests that the molecule could serve as a prototype for the development of more selective Na(+) channel inhibitors with potential utility in neurological and neurodegenerative disorders.

Excessive nitric oxide formation may contribute to the pathology occurring in diseases affecting central white matter, such as multiple sclerosis. The rat isolated optic nerve preparation was used to investigate the potential toxicity of the molecule towards such tissue. The nerves were exposed to a range of concentrations of different classes of nitric oxide donor for up to 23 h, with or without a subsequent period of recovery, and the damage assessed by quantitative histological methods. Degeneration of axons and macroglia occurred in a time- and concentration-dependent manner, the order of susceptibility being: axons>oligodendrocytes>astrocytes. Use of NONOate donors differing in half-life indicated that nitric oxide delivered in an enduring manner at relatively low concentration was more toxic than the same amount supplied rapidly at high concentration. The mechanism by which nitric oxide affects axons was studied using a donor [3-(n-propylamino)propylamine/NO adduct, PAPA/NO] with an intermediate half-life that produced selective axonopathy after a 2-h exposure (plus 2 h recovery). Axon damage was abolished if, during the exposure, Na(+) or Ca(2+) was removed from the bathing medium or the sodium channel inhibitors tetrodotoxin or BW619C89 (sipatrigine) were added. In electrophysiological experiments, the donor elicited a biphasic depolarisation. The second, larger component (occurring after 7-10 min) was associated with a block of nerve conduction and could be inhibited by tetrodotoxin. Coincident with the secondary depolarisation was a reduction in ATP levels by about 50%, an effect that was also inhibited by tetrodotoxin. It is concluded that nitric oxide, in submicromolar concentrations, can kill axons and macroglia in white matter. The findings lend support to the hypothesis that nitric oxide may be of importance to white matter pathologies, particularly those in which inducible nitric oxide synthase is expressed. The axonopathy, at least when elicited over relatively short time intervals, is likely to be caused by metabolic inhibition. As in anoxia and anoxia/aglycaemia, nitric oxide-induced destruction of axons is likely to be caused by the Ca(2+) overload that follows a reduction in ATP levels in the face of continued influx of Na(+) through voltage-dependent channels.

A major transduction pathway for nitric oxide (NO) is stimulation of soluble guanylyl cyclase and the generation of cyclic GMP (cGMP). In the central nervous system, the NO-cGMP pathway has previously been associated primarily with synapses, particularly glutamatergic synapses. We report here that NO caused a large increase in the levels of cGMP in a central white matter tract devoid of synapses, namely in the rat isolated optic nerve. Cyclic GMP immunohistochemistry indicated that this response was confined to the axons. Accordingly, nerves previously subjected to 1 h of oxygen/glucose deprivation, which leads to irreversible axonal damage, displayed an 80% reduction in their subsequent capacity to generate cGMP in response to NO and a corresponding reduction in the numbers of cGMP-immunostained axons. Protection of the axon cGMP response against this insult was achieved by omission of Ca2 + or Na + from the incubation medium, and by the pharmacological agents tetrodotoxin, lamotrigine, BW619C89 and BW1003C87, all of which protect axonal structure from oxygen/glucose deprivation-induced damage. The results suggest that the NO-cGMP pathway has a hitherto unsuspected function in the optic nerve. Additionally, the expression of NO-stimulated guanylyl cyclase in optic nerve axons provides a simple, sensitive and specific marker of their functional integrity that is likely to be valuable in investigating the mechanisms responsible for axon degeneration in ischaemia and other conditions.

The mechanism of ischaemic injury to white matter axons was studied by transiently depriving rat optic nerves in vitro of oxygen and glucose. Light and electron microscopic analysis showed that increasing periods of oxygen/glucose deprivation (up to 1 h) caused, after a 90-min recovery period, the appearance of increasing numbers of swollen axons whose ultrastructure indicated that they were irreversibly damaged. This conclusion was supported by experiments showing that the damage persisted after a longer recovery period (3 h). To quantify the axonal pathology, an automated morphometric method, based on measurement of the density of swollen axons, was developed. Omission of Ca2+ from the incubation solution during 1 h of oxygen/glucose deprivation (and for 15 min either side) completely prevented the axonopathy (assessed following 90 min recovery). Omission of Na+ was also effective, though less so (70% protection). The classical Na+ channel blocker, tetrodotoxin (1 microM), provided 92% protection. In view of this evidence implicating Na+ channels in the pathogenesis of the axonal damage, the effects of three different Na+ channel inhibitors, with known neuroprotective properties towards gray matter in in vivo models of cerebral ischaemia, were tested. The compounds used were lamotrigine and the structurally-related molecules, BW619C89 and BW1003C87. All three compounds protected the axons to varying degrees, the maximal efficacies (observed at 30 to 100 microM) being in the order: BW619C89 (>95% protection) > BW1003C87 (70%) > lamotrigine (50%). At a concentration affording near complete protection (100 microM), BW619C89 had no significant effect on the optic nerve compound action potential. Experiments in which BW619C89 was added at different times indicated that its effects were exerted during two distinct phases, one (accounting for about 50% protection) was during the early stage of oxygen/glucose deprivation itself and the other (also about 50%) during the first 15 min of recovery in normal incubation solution. The results are consistent with a pathophysiological mechanism in which Na+ entry through tetrodotoxin-sensitive Na+ channels contributes to Na+ loading of the axoplasm which then results in a lethal Ca2+ overload through reversed Na(+)-Ca2+ exchange. The identification of BW619C89 as a compound able to prevent oxygen/glucose deprivation-induced injury to white matter axons without affecting normal nerve function opens the way to testing the importance of this pathway in white matter injury in vivo.

Nitric oxide (NO), generated upon glutamate receptor activation, elicits cyclic GMP accumulation through stimulation of guanylyl cyclase. NO is also a potential cytotoxin that has been suggested, on the basis of tissue culture experiments, to mediate neuronal damage associated with excessive activity of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. We have investigated the involvement of NO in the toxicity of glutamate receptor agonists in brain slice preparations. Slices of cerebellum and hippocampus from the developing rat exhibited neuronal necrosis following exposure (5-30 min) to NMDA (100 microM or 1 mM). When the exposures were carried out in the presence of NO synthase inhibitors, at concentrations suppressing NMDA-induced NO formation (as judged by measurements of cyclic GMP accumulation), the extent of injury was unaffected. To determine if exogenous NO is able to replicate NMDA toxicity, the slices were exposed to high concentrations of NO donating compounds for up to 2 hr. No damage was detectable. NO donors, moreover, neither reduced NMDA toxicity, nor potentiated the degeneration caused by just suprathreshold NMDA concentrations. The toxicities of non-NMDA agonists, or of glutamate itself, were also unaltered by NO synthase inhibitors or NO donors. Similar results were obtained using hippocampal slices from more mature animals. We conclude that the acute neurodegeneration mediated by NMDA or non-NMDA receptors in the slice preparations is not mediated by NO, nor is NO neuroprotective under these conditions.

Mobilisable intracellular Ca2+ stores are highly enriched in the cerebellum, particularly in Purkinje cells. We have detected, by light and electron microscopy, striking morphological changes in the presumed Ca2+ stores of Purkinje cells when slices of eight-day-old rat cerebellum were incubated in Ca(2+)-deficient media. After 30 min under these conditions, the endoplasmic reticulum became thinned and elongated. By 2 h, it was transformed into multilamellar, whorl-like inclusions with electron-dense cores. These changes were reversed on reintroduction of Ca2+. Analogous changes in other neurons were not observed. The results suggest that Ca2+ storage sites within Purkinje cells are capable of dramatic morphological change depending on the availability of Ca2+. The transformations may reflect, initially, depletion of Ca2+ from the stores and then homeostatic alterations in their capacity.

In slices of 8-day-old rat cerebellum, the lowest concentration of glutamate that induced toxicity (30 min exposure; 90 min recovery) was 100 microM, but the damage only occurred in the outermost regions. As the concentration was raised, the band of necrosis became progressively deeper until, at 3 mM, it was uniform across the slice thickness. At a test concentration of 300 microM, the width of the necrotic band did not change when either the exposure time or the recovery period was varied between 30 min and 3 h. These results are predicted by a theoretical model in which the diffusion of glutamate into brain tissue is countered by cellular uptake of the amino acid, and they argue against the idea that glutamate toxicity is inherently self-propagating. When slices were examined immediately after exposure (300 microM), a prominent swelling of glial cells was present at the slice surface. Swelling per se did not appear to compromise their uptake function, and the model predicts that cellular swelling, by reducing the rate of diffusion of glutamate, protects against glutamate toxicity. The damage produced by 3 mM glutamate, which was primarily exerted against granule cells, was prevented by N-methyl-d-aspartate (NMDA) receptor blockade, whereas antagonists acting at alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors were ineffective. Under conditions of energy deprivation, the neurotoxic potency of glutamate was markedly enhanced and a normally non-toxic concentration (30 microM) became maximally toxic towards granule cells. Dark vacuolar degeneration of Purkinje cells was also present, and this could be inhibited by blocking AMPA receptors. The results and theoretical analysis suggest that intact brain tissue is remarkably resistant to glutamate toxicity, chiefly because of the formidable properties of the uptake system. However, under special circumstances, glutamate can become a potent neurotoxin and its toxicity can then involve both NMDA and AMPA receptors.

Excitatory amino acid-induced death of central neurons may be mediated by at least two receptor types, the so-called NMDA (N-methyl-d-aspartate) and AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate) receptors. We have studied the neurodegenerative mechanisms set in motion by AMPA receptor activation using incubated slices of 8-day-old rat cerebellum and hippocampus. In both preparations, AMPA induced a pattern of degeneration that differed markedly from the one previously shown to be elicited by NMDA. In cerebellar slices, AMPA induced the degeneration of most Purkinje cells together with a population of Golgi cells; in hippocampal slices the neurons were affected in the order CA3 > CA1 > dentate granule cells. Three mechanisms could be discerned: an acute one in which neurons (e.g. cerebellar Golgi cells) underwent a rapid degeneration; a delayed one in which the neurons (Purkinje cells and hippocampal neurons) appeared to be only mildly affected immediately after a 30 min exposure but then underwent a protracted degeneration during the postincubation period (1.5 - 3 h); and finally a slow toxicity, which took place during long (2 h) exposures to AMPA (3 - 30 microM). Although Purkinje cells were vulnerable in both cases, the efficacy of AMPA was higher for the delayed mechanism than for the slow one. The pathology displayed by the acutely destroyed Golgi neurons was a classical oedematous necrosis, whereas most neurons vulnerable to the delayed and slow mechanisms displayed a 'dark cell degeneration', whose cytological features bore a close resemblance to those of neurons irreversibly damaged by ischaemia, hypoglycaemia or status epilepticus in vivo.

The mechanisms underlying the neurodegenerative effects of the glutamate receptor agonist, AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate), were studied using brain slice preparations of young rat (8 - 9 days old) cerebellum and hippocampus. Rapid AMPA toxicity (exerted on some cerebellar interneurons) was inhibited by including the appropriate receptor blocker, CNQX (6-cyano-7-nitroquinoxaline-2,3-dione, 10 microM), in the exposing solution. The degeneration of other neurons, including Purkinje cells and hippocampal pyramidal neurons, persisted. It could, however, be largely prevented if CNQX was included for 1.5 h during the post-incubation period, suggesting that an enduring 'rebound' AMPA receptor activation was responsible for this delayed type of degeneration, not the exposure itself. In cerebellar slices, independent evidence for the occurrence, postexposure, of persisting AMPA receptor stimulation was obtained electrophysiologically. Omission of Ca2+ during the exposure period (and for 10 min beforehand) markedly reduced rapid AMPA toxicity but was ineffective in protecting most of the Purkinje cells. However, if the slices were previously starved of Ca2+ for 1 h, then most of these neurons survived, even if the ion was reinstated during the recovery period. Slow AMPA toxicity, which takes place during long (2 h) exposures, could be inhibited either by CNQX or by omission of Ca2+ (30 min preincubation). The results indicate that the rapid oedematous necrosis induced by AMPA, like that caused by N-methyl-d-aspartate and kainate, is likely to involve excessive influx of Ca2+. In contrast, the induction of the delayed mechanisms, as well as its 'expression' during the postincubation period, probably depends on intracellular Ca2+, rather than Ca2+ influx.

In young rat cerebellar slices, histological methods showed that the neurotoxic potency of N-methyl-D-aspartate (NMDA) towards granule cells and intracerebellar nucleus neurons was increased 2- to 3-fold on removal of Mg ions, which have a blocking effect on NMDA-activated ion channels. The depolarizing potency of NMDA on granule cells, recorded using a gap method, was similarly enhanced whereas that of kainate, a non-NMDA receptor agonist, was unchanged. The neurotoxic potency of kainate (towards Golgi cells) was also unaltered by removal of Mg2+. In Mg2+-containing medium, neuronal depolarization induced either by kainate or by high K+ potentiated NMDA toxicity, apparently by reducing the channel block by Mg2+. The results strongly support the hypothesis that excessive Ca2+ influx through NMDA/Mg2+-gated ion channels mediates NMDA toxicity. They also have clear implications regarding the likely mechanism of toxicity of agonists, such as glutamate, able to activate both NMDA and non-NMDA receptors.

Exposure of slices of young (8 days old) rat hippocampus to 100 microM N-methyl-D-aspartate (NMDA) for 20 min followed by 90 min recovery, resulted in widespread, oedematous necrosis of all classes of neurones. The NMDA antagonist, D,L-2-amino-5-phosphonovalerate (APV) or omission of Ca2+ from the exposing solution prevented this cell death, but a large reduction in Cl- was ineffective. Quisqualate (100 microM, 20 min) led to a different pathological pattern characterised most strikingly by large numbers of cells undergoing 'dark cell degeneration'. Numerically, the neurones were affected in the order CA3 greater than CA1 greater than dentate granule cells. Quisqualate toxicity was not prevented by APV nor by reducing Ca2+ or Cl-. It is concluded that, as in cerebellar slices (but unlike in cultures of hippocampal neurones) NMDA toxicity in hippocampal slices is Ca2+-dependent and Cl- -independent. However, quisqualate exerts its pathological effects through a different mechanism. This mechanism may be primarily metabolic rather than ionic.

Depolarizing responses of Purkinje and granule cells to excitatory amino acid receptor agonists were recorded from rat cerebellar slices at various stages of postnatal maturation using a gap technique. No major developmental changes in relative potency or efficacy of kainate and quisqualate were observed. However, Purkinje and granule neurones both became less responsive to N-methyl-D-aspartate (NMDA) with age, most dramatically so between 14 and 21 days. This transient chemosensitivity to NMDA may reflect a special role of the NMDA receptor system in cerebellar development.

Incubated slices and freshly dissociated cells from 8-day-old rat cerebellum were used to try to identify the cells that participate in the large increases in cyclic GMP levels that follow activation of excitatory amino acid receptors in this tissue. In the slices, cyclic GMP responses to L-glutamate and related excitants were unaffected by tetrodotoxin and could be replicated by the guanylate cyclase activator nitroprusside. Nitroprusside and the receptor agonists appeared to activate the same pool of the enzyme. Prior destruction of neuroblasts, deep nuclei, or Golgi neurones did not cause loss of responses to L-glutamate. If granule cells were rendered necrotic, however, the cyclic GMP responses to all excitants tested were reduced by greater than or equal to 90%. Substantial losses of responses to veratridine and high K+ levels also occurred, but the nitroprusside-induced elevations were unaffected. In dissociated cell suspensions, the magnitude of responses to receptor agonists, but not those to nitroprusside, was markedly dependent on cell concentration. Responses to L-glutamate were the same in cell suspensions that were Purkinje cell depleted and Purkinje cell enriched. It is concluded that granule cells are primarily involved in the cyclic GMP responses to excitatory amino acids but that the cyclic GMP accumulations occur elsewhere, probably in glial cells.

Serum immunoglobulin concentrations in clinically healthy German shepherd dogs were compared with those in mongrel dogs and Irish setters. Serum IgA concentrations were significantly lower in the German shepherd dogs than in the other two groups, while there were no differences in the concentrations of IgM or IgG. These findings suggest that production of IgA by gut associated lymphoid tissue may be abnormally low in the German shepherd dog.

Incubation of slices of immature rat cerebellum for 30 min with quinolinate (QUIN), an endogenous neurotoxin, resulted in the selective necrosis of granule cells and intracerebellar nucleus neurones. Concentration of QUIN in the millimolar range were needed for these effects. The same neuronal populations were also selectively killed by N-methyl-D-aspartate (NMDA) but the toxic potency of NMDA was 40-fold higher than that of QUIN. Depolarizing responses of granule cells to brief applications of QUIN and NMDA were recorded using a gap method. Dose-response curves to the two compounds appeared parallel but NMDA was 30-fold more potent than QUIN. The depolarizing and toxic actions of QUIN and NMDA were inhibited by the NMDA antagonist, 2-amino-5-phosphonopentanoate. We conclude that the selective toxicity of QUIN in this tissue arises from its activity on NMDA receptors.

The serum proteins present at the skin surface in dogs were found to include the immunoglobulins (Ig) G, M and A, complement (C3) and albumin. Within the skin IgG and IgM were consistently found in the interstitial tissue throughout the dermis and were commonly present in the dermal blood vessels and hair papillae. IgA was undetectable or present in small amounts in the dermis in most of the 16 samples examined. It was found in the sweat glands, both in the lumen and the fundic epithelium, in some skin samples. C3 was demonstrated in the dermis and in the inter-cellular spaces of the stratum corneum. Elution of fresh skin specimens with phosphate buffered saline removed the majority of interstitial immunoglobulin and C3 from the dermis and some of the IgA from the sweat glands. IgM could still be demonstrated in the region of the epidermal basement membrane and C3 was not eluted from the stratum corneum. Removal of interstitial IgG and IgM facilitated the identification of immunoglobulin-bearing cells in the dermis. The distribution of IgG and IgM in dog skin is similar to that found in ruminants. Demonstration of IgA in the canine sweat gland fundus lends further support to the concept of IgA as a secretory immunoglobulin in the skin.

Slices of 400 microns thickness were prepared from various regions of the adult rat brain using a vibratome, and incubated in oxygenated Krebs-Henseleit medium for 3 h prior to processing for light and electron microscopy. Previous findings in cerebellar slices suggest that the structural recovery requires at least a 1 h period of incubation. In the present study, vibratome-cut slices of other brain areas such as the hippocampus, parietal cortex, striatum and median eminence were shown to display after this period a gross and fine structure comparable to in situ fixed tissues, and to retain it for at least 3 h of incubation. These areas are thought to be promising for further in vitro functional morphological experimentation.