Mark N. Wallace

Nitric oxide synthase in reactive astrocytes adjacent to β-amyloid plaques

This study provides the first evidence that nitric oxide is released by astrocytes surrounding β-amyloid plaques. Nitric oxide is involved in many neuropathological conditions and can have either a neuroprotective or a neurotoxic function depending on its concentration and the redox state of the tissue. It is produced by the enzyme nitric oxide synthase, which can be located by a simple histochemical technique for demonstrating NADPH diaphorase. Using this method we examined tissue from 10 brains where there were varying numbers of β-amyloid plaques in the cerebral cortex. In the 6 brains with moderate or high densities of plaques, primitive and cored plaques were associated with between 1 and 10 reactive astrocytes that contained NADPH diaphorase or were immunoreactive for the inducible form of nitric oxide synthase. In the 4 brains which had only low densities of plaques, the plaques were not associated with diaphorase-containing astrocytes. The percentage of plaques associated with 1 or more NADPH diaphorase-containing astrocyte varied between 1 and 21% and was correlated with the density of plaques. Astrocytes were the only form of NADPH diaphorase-positive glial cell associated with the plaques. There was no evidence of any nitric oxide synthase occurring in microglia.

Identification and localisation of auditory areas in guinea pig cortex

Abstract. The organisation of guinea pig auditory cortex was studied by combining histological methods with microelectrode mapping. This allowed the location of seven auditory areas to be determined in relation to the visual and primary somatosensory areas. The auditory areas were identified by single-unit recordings and their borders defined by evoked potential mapping. The visual areas were identified by their relatively high densities of myelinated fibres, while the primary somatosensory cortex was identified by its characteristic barrels of high cytochrome oxidase (CYO) activity in layer IV. The auditory region had moderate levels of CYO and myelin staining. When staining was optimal, there was a clear edge to the moderate CYO activity, which apparently corresponds to the dorsal border of the primary auditory area (AI) and the other core field that lies dorsocaudal to it (DC). Thus the primary somatosensory area and the visual and auditory regions were separated from each other by a region with lower levels of CYO and myelin staining. The ventral borders of AI and DC could not be determined histologically as there were no sharp transitions in the levels of CYO or myelin staining. The two core areas were partially surrounded by belt areas. The dorsorostral belt and most of the belt around DC responded more strongly to broad-band stimuli than pure tones, while the ventrorostral belt, small field and a belt zone ventral to the rostral part of DC responded better to pure tones. Units in the small field (S) typically had higher thresholds and broader tuning to pure tones than AI, while units in the ventrorostral belt typically had longer onset latencies and gave more sustained responses than units in AI.

Intrinsic inter- and intralaminar connections and their relationship to the tonotopic map in cat primary auditory cortex

SummarySmall iontophoretic injections of the lectin, Phaseolus vulgaris leucoagglutinin (PHA-L), were made into different layers of the primary auditory cortex (AI) of cats. Injections in layer I labeled two types of morphologically distinct fibers in layer I as well as a smaller number of axons in layers II and III. Layer II injections labeled descending axons that produced a dense plexus of terminal fibers in layers I–III of both AI and adjacent auditory fields. Injections in layer III also labeled a dense plexus of axon collaterals at the junction of layers V and VI and labeled patches of terminal fibers in both AI and adjacent auditory fields. These were densest in layers I–III but usually extended into layers IV and V as well. The patches were partly formed by axon collaterals of layer III pyramidal cells that traveled for over 4 mm in the gray matter. Injections confined to layer IV labeled axons in all layers of the cortex but none of these axons appeared to reach the white matter. The axons spread laterally in layer IV and up into the superficial layers and ramified especially layer I. Injections in layers V and VI labeled axons in all layers of the cortex but these were densest in the deep layers where labeling was fairly homogeneous. In the upper layers the labeling was arranged in semi-discrete patches. Large injections involving layers I–III were studied in tangential sections. Between 3 and 8 patches of terminal labeling were observed in AI and these were mainly arranged in a band with its long axis aligned approximately in the dorsoventral direction. However dense patches of terminal labeling also occurred both anterior and posterior to the injection site. In selected experiments portions of the tonotopic map in AI were mapped by single unit recording and subsequently the map was related to patches of anterogradely labeled fibers that surrounded injections of PHA-L. Rows of dorsoventrally oriented patches were among cells with a similar best frequency to those in the injection site. However patches located anterior or posterior to the injection site were among cells with higher or lower best frequencies. Two injections of PHA-L close together produce different patterns of labeling. One of the injections usually produces one or more patches that has no correlate among the patches of fibers labeled by the adjacent injection. This is clearest when one of the injections is made with biotinylated PHA-L that can be visualized directly without the use of primary antibodies. Thus the intrinsic connections of AI arising from nearby cylinders of neurons are not homogenous and clusters of cells can be identified by their unique pattern of connections within AI.

NADPH-diaphorase activity in activated astrocytes represents inducible nitric oxide synthase

In paraformaldehyde-fixed sections of healthy brain, glial cells at the light-microscope level do not contain measurable levels of NADPH-diaphorase. However, after a variety of lesions in the mouse brain, some reactive astrocytes express varying amounts of this enzyme. Following stab wounds, activated astrocytes or related glial cells surrounding the lesion, contained moderate to high levels of NADPH-diaphorase in the cerebellum, midbrain, thalamus, striatum, hippocampal formation and neocortex. Double-labelling experiments confirmed that this corresponds to an inducible form of nitric oxide synthase, similar to that found in activated macrophages. Within the lesion there were large numbers of macrophages which also contained NADPH-diaphorase. After 10 min of global hypoxic ischaemia, some reactive astrocytes also contained NADPH-diaphorase. These cells were confined to the dorsal part of the hippocampal formation (the dentate fascia and CA1 areas) and to the anterolateral striatum. More focal ischaemic damage, produced by dividing an arterial branch, also produced a rim of reactive astrocytes containing NADPH-diaphorase, that surrounded the area of necrosis. Low levels of NADPH-diaphorase were induced within one day of a stab wound and the enzyme activity reached near maximal levels by two days postlesion. Moderate NADPH-diaphorase activity was still present at 63 days postlesion, but only a small number of astrocytes were stained in the immediate vicinity of the lesion. These experiments confirm that NADPH-diaphorase activity represents inducible nitric oxide synthase in activated astrocytes and probably in inflammatory macrophages. We conclude that a high proportion of activated astrocytes and a small proportion of invading macrophages are induced to express moderate to high levels of nitric oxide synthase following neuronal damage. Our results indicate that following a variety of lesions reactive astrocytes are synthesizing significant levels of nitric oxide within 24 h. This nitric oxide may be involved in modulating the likelihood of epileptic seizures.

Activated astrocytes of the mouse hippocampus contain high levels of NADPH-diaphorase

NADPH-Diaphorase has recently been shown to be identical to nitric oxide synthase in brain neurones. In the intact brain, NADPH-diaphorase is only present in selected populations of neurones and is not detectable in glia. However following a lesion in the mouse retrosplenial cortex, activated astrocytes display intense NADPH-diaphorase activity throughout their cytoplasm. After a control saline injection, NADPH-diaphorase-positive activated glia are only observed in damaged tissue immediately surrounding the injection site, but when kainic acid is injected unilaterally, activated astrocytes occur in the hippocampal formation bilaterally. This indicates that astrocytes activated by intense neuronal activity, as well as by direct mechanical damage, express high levels of NADPH-diaphorase.

Histochemical demonstration of sensory maps in the rat and mouse cerebral cortex

The variations in metabolic activity of two mitochondrial enzymes (succinate dehydrogenase and cytochrome oxidase) have been mapped histochemically in rat and mouse brains prepared in such a manner as to reveal the whole surface of the neocortex in one plane. In each species the enzymes appear to outline the complete body representation map in the somatosensory cortex as well as the primary visual and auditory areas.

Onset Neurones in the Anteroventral Cochlear Nucleus Project to the Dorsal Cochlear Nucleus

Considerable circumstantial evidence suggests that cells in the ventral cochlear nucleus, that respond predominantly to the onset of pure tone bursts, have a stellate morphology and project, among other places, to the dorsal cochlear nucleus. The characteristics of such cells make them leading candidates for providing the so-called “wideband inhibitory input” which is an essential part of the processing machinery of the dorsal cochlear nucleus. Here we use juxtacellular labeling with biocytin to demonstrate directly that large stellate cells, with onset responses, terminate profusely in the dorsal cochlear nucleus. They also provide widespread local innervation of the anteroventral cochlear nucleus and a small innervation of the posteroventral cochlear nucleus. In addition, some onset cells project to the contralateral dorsal cochlear nucleus.

Organisation of binaural interactions in the primary and dorsocaudal fields of the guinea pig auditory cortex

This study investigated the nature and topography of binaural interactions in the primary auditory field (AI) and dorsocaudal field (DC) of the urethane anaesthetised guinea pig auditory cortex. Single and multi-units were classified by their responses to monaural and binaural stimulation. In both AI and DC, units displayed binaural facilitation, binaural inhibition, or a level dependent mixture of facilitation and inhibition. There was a significant difference in the distribution of binaural response types between the two fields. Facilitated units predominated in DC (facilitated: 58%; inhibited: 24%; mixed: 6%; non-interacting: 12%), while inhibited units were the most common class in AI (facilitated: 15%; inhibited: 44%; mixed: 18%; non-interacting: 22%). It has previously been suggested that inhibited and facilitated units are concerned with processing different areas of space suggesting a possible separation of function between the two core fields. Topographically, the binaural response properties in AI and DC varied along isofrequency bands, with neurones displaying similar interactions aggregating in clusters. These clusters were similar in size for the two fields and often overlapped neighbouring isofrequency bands. However, their shape and position varied between different animals. This clustered organisation of binaural interactions is similar to that reported in recent studies of AI in other mammals.

Chemoarchitectonic organization of the cat primary auditory cortex.

SummaryAcetylcholinesterase (AChE) activity, demonstrated histochemically, defines an area of cortex on the middle ectosylvian gyrus that appears to correspond to the cytoarchitectonically defined area 41 and the physiologically defined primary auditory area (AI). In this area there are high levels of AChE in layers III, IV and VI while in the surrounding areas there are comparatively low levels of enzyme in these layers. The monoclonal antibody CAT 301, which was raised against a cell surface proteoglycan, also defines this area. There are high levels of CAT 301 immunoreactivity in cell bodies and the neuropil of layer III and an absence of very large immunoreactive neurons in layer V. Furthermore there are higher levels of the calcium binding protein, parvalbumin and the metabolic enzyme, cytochrome oxidase, in layers III and IV of AI, than in most of the surrounding cortex. By contrast the distribution of the calcium binding protein, calbindin and the distribution of myelinated fibers are similar in area 41 and the surrounding areas.

Spatial relationship of NADPH-diaphorase and acetylcholinesterase lattices in the rat and mouse superior colliculus

In the intermediate layers of the rat and mouse colliculus there is a lattice-like pattern of high nicotinamide adenine dinucleotide phosphate diaphorase activity. This lattice is composed of dark bands that are 100-200 micron wide and enclose pale areas of irregular shape. A very similar lattice of high acetylcholinesterase activity is also found in the intermediate layers and this overlaps the diaphorase lattice almost completely. However, in deeper layers the enzymes have a complementary organization with high levels of one being associated with low levels of the other. It is concluded that the histochemical lattices will provide useful patterns with which to compare the terminal organization of afferent systems.