Gudrun Seeger

Cortical areas are revealed by distribution patterns of proteoglycan components and parvalbumin in the Mongolian gerbil and rat

Cortical areas in rodents have been basically characterized by its cytoarchitecture, connectivity or by physiological parameters. In this study we show that they are revealed by distribution patterns of proteoglycans and parvalbumin-immunoreactivity. Brains of young adult Mongolian gerbils (Meriones unguiculatus) and Wistar rats were cut into series of transversal sections. Proteoglycan components were detected using the N-acetylgalactosamine binding Wisteria floribunda agglutinin (WFA) and antibodies against chondroitin sulphate proteoglycan (CSPG). Differences between cortical areas were found to exist with regard to the occurrence and the density of perineuronal nets, but were also expressed in varying staining intensities for WFA and CSPG of the neuropil. Primary neocortical areas (somatosensory, auditory, visual cortex) were characterized by an intense neuropil staining in layer IV and the upper part of layer VI. Using the same methods strong labelling was also typical of the neuropil in the retrosplenial cortex, of layer Ia in the prepiriform cortex and the hippocampal CA3 field. In tangential sections cut from gerbil cortical hemispheres, some of the heavily lectin-stained cortical areas were sharply delineated from adjacent faintly labelled regions, others showed more diffuse borders. In the rat, the area-specific staining for WFA was less clearly expressed than in the gerbil. Immunocytochemistry of the calcium-binding protein parvalbumin in alternate sections showed labelling patterns of neuropil which resembled those of WFA-binding and CSPG-immunoreactivity in the entire neocortex and hippocampus. From these results it can be concluded that functional peculiarities of cortical fields may not only be determined by neuronal network parameters but also by the spatial arrangement of extracellular matrix proteoglycans.

Mapping of perineuronal nets in the rat brain stained by colloidal iron hydroxide histochemistry and lectin cytochemistry.

Net-like structures, visualized with the Golgi technique and several histochemical and immunocytochemical methods, have been described to ensheath somata, parts of dendrites and axon initial segments of various types of neurons. The origin and function of these perineuronal nets have been controversially discussed. Recently, it was confirmed that they are glia-associated. In the present study such perineuronal nets were demonstrated by using colloidal iron hydroxide staining for detection of polyanionic components and the plant lectins Vicia villosa agglutinin and Wisteria floribunda agglutinin with affinity for N-acetylgalactosamine. This paper shows their distribution patterns and the occurrence of regional specialization of these nets which might provide a basis to suggest functional implications of these structures. Perineuronal nets were found in more than 100 brain regions, such as neocortex, hippocampus, piriform cortex, basal forebrain complex, dorsal lateral septal nucleus, lateral hypothalamic area, reticular thalamic nucleus, zona incerta, deep parts of superior and inferior colliculus, red nucleus, substantia nigra, some tegmental nuclei, cerebellar nuclei, dorsal raphe and cuneiform nuclei, central gray, trochlear nucleus, pontine and medullar reticular nuclei, superior olivary nucleus and vestibular nuclei. Neurons enwrapped by perineuronal nets not only differ in morphology but also in transmitter content. In neocortical and hippocampal regions there occurs a much higher number of perineuronal nets ensheathing non-pyramidal cells than in paleocortical structures. Most subcortical regions containing perineuronal nets were found to be integrated in motor functions. The findings are discussed with respect to known electrophysiological data of cell types described in our investigation as net-associated. There are some indications that such cells may represent fast firing types.

Neurons associated with aggrecan-based perineuronal nets are protected against tau pathology in subcortical regions in Alzheimer's disease

The biological basis for the selective vulnerability of neurons in Alzheimers disease (AD) is elusive. Aggrecan-based perineuronal nets (PNs) of the extracellular matrix have been considered to contribute to neuroprotection in the cerebral cortex. In the present study, we investigated the organization of the aggrecan-based extracellular matrix in subcortical regions known to be preferentially affected by tau pathology in AD. Immunocytochemistry of aggrecan core protein was combined with detection of neurofibrillary degeneration. The results show that many regions affected by tau pathology in AD, such as the basal nucleus of Meynert, the dorsal thalamus, hypothalamic nuclei, raphe nuclei, and the locus coeruleus were devoid of a characteristic aggrecan-based extracellular matrix. Regions composed of nuclei with clearly different intensity of tau pathology, such as the amygdala, the thalamus and the oculomotor complex, showed largely complementary distribution patterns of neurofibrillary tangles and PNs. Quantification in the rostral interstitial nucleus of the longitudinal fascicle potentially affected by tau pathology in AD revealed that tau pathology was not accompanied by loss of aggrecan-based PNs. Neuro-fibrillary tangles in net-associated neurons extremely rarely occurred in the pontine reticular formation. We conclude that the low vulnerability of neurons ensheathed by PNs previously described for cortical areas in AD represents a more general phenomenon that also applies to subcortical regions. The aggrecan-based extracellular matrix of PNs may thus, be involved in neuroprotection.

Involvement of Perineuronal and Perisynaptic Extracellular Matrix in Alzheimer’s Disease Neuropathology

Brain extracellular matrix (ECM) is organized in specific patterns assumed to mirror local features of neuronal activity and synaptic plasticity. Aggrecan‐based perineuronal nets (PNs) and brevican‐based perisynaptic axonal coats (ACs) form major structural phenotypes of ECM contributing to the laminar characteristics of cortical areas. In Alzheimers disease (AD), the deposition of amyloid proteins and processes related to neurofibrillary degeneration may affect the integrity of the ECM scaffold. In this study we investigate ECM organization in primary sensory, secondary and associative areas of the temporal and occipital lobe. By detecting all major PN components we show that the distribution, structure and molecular properties of PNs remain unchanged in AD. Intact PNs occurred in close proximity to amyloid plaques and were even located within their territory. Counting of PNs revealed no significant alteration in AD. Moreover, neurofibrillary tangles never occurred in neurons associated with PNs. ACs were only lost in the core of amyloid plaques in parallel with the loss of synaptic profiles. In contrast, hyaluronan was enriched in the majority of plaques. We conclude that the loss of brevican is associated with the loss of synapses, whereas PNs and related matrix components resist disintegration and may protect neurons from degeneration.

Neuronal activation of Ras regulates synaptic connectivity

A synRas mouse model was used expressing constitutively activated Ha‐Ras (Val12 mutation) in neurons to investigate the role of Ras‐MAPkinase signalling for neuronal connectivity in adult brain. Expression of the transgene in the cortex of these mice starts after neuronal differentiation is completed and allows to directly investigate the effects of enhanced Ras activity in differentiated neurons. Activation of Ha‐Ras induced an increase in soma size which was sensitive to MEK inhibitor in postnatal organotypic cultures. Adult cortical pyramidal neurons showed complex structural rearrangements associated with an increased size and ramification of dendritic arborization. Dendritic spine density was elevated and correlated with a twofold increase in number of synapses. In acute brain slices of the somatosensory and of the visual cortex, extracellular field potentials were recorded from layer II/III neurons. The input–output relation of synaptically evoked field potentials revealed a significantly higher basal excitability of the transgenic mice cortex compared to wild‐type animals. In whole cell patch clamp preparations, the frequency of AMPA receptor‐mediated spontaneous excitatory postsynaptic currents was increased while the ratio between NMDA and AMPA‐receptor mediated signal amplitude was unchanged. A pronounced depression of paired pulse facilitation indicated that Ras contributes to changes at the presynaptic site. Furthermore, synRas mice showed an increased synaptic long‐term potentiation, which was sensitive to blockers of NMDA‐receptors and of MEK. We conclude that neuronal Ras is a common switch of plasticity in adult mammalian brain sculpturing neuronal architecture and synaptic connectivity in concert with tuning synaptic efficacy.

Different dendrite and dendritic spine alterations in basal and apical arbors in mutant human amyloid precursor protein transgenic mice.

The extracellular deposition of amyloid-beta peptide (Abeta) in brain parenchyma is one of the characteristic features of Alzheimers disease and is suggested to induce reactive and degenerative changes in neuronal cell bodies, axons and dendritic processes. In particular, within and in close proximity to amyloid plaques, distinctive morphological alterations have been observed, including changes in neurite trajectory and decreases in dendritic diameter and in spine density. Apart from these plaque-associated focal aberrations, little is known regarding modifications of the global dendritic morphology including the detailed and comparative quantitative analysis of apical and basal arbors. The objective of the present study was to investigate the effects of amyloid plaque deposition and elevated soluble Abeta on neuronal morphology in mutant human amyloid precursor protein (hAPP) transgenic mice (line Tg2576; [K. Hsiao, P. Chapman, S. Nilsen, C. Eckman, Y. Harigaya, S. Younkin, F. Yang, G. Cole, Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice, Science 274 (1996) 99-102]). Retrogradelly labeled callosal-projecting pyramidal cells in the primary somatosensory cortex were three-dimensionally analyzed. Although basal dendrites remained unaffected, analysis of apical trees revealed a number of unambiguous morphological changes. Thus, in TG2576 mice, the apical arbors were shortened in total length and less branched. Furthermore, the diameter of proximal dendritic segments was increased whereas that of distal segments was reduced. Analysis of spine numbers and distribution on basal and apical trees demonstrated a significant reduction in spine densities along the whole course of dendrites. The findings suggest that Abeta-related pathology induces morphological aberrations in basal and apical arbors to different degrees which are unrelated to direct plaque-associated changes.

Inducible neuronal expression of transgenic TGF-β1 in vivo: dissection of short-term and long-term effects

Various chronic neurological diseases are associated with increased expression of transforming growth factor‐beta1 (TGF‐β1) in the brain. TGF‐β1 has both neuroprotective and neurodegenerative functions, depending on conditions such as duration and the local and temporal pattern of its expression. Previous transgenic approaches did not enable control for these dynamic aspects. To overcome these limitations, we established a transgenic mouse model with inducible neuron‐specific expression of TGF‐β1 based on the tetracycline‐regulated gene expression system. TGF‐β1 expression was restricted to the brain where it was particularly pronounced in the neocortex, hippocampus and striatum. Transgene expression was highly sensitive to the presence of doxycycline and completely silenced within 6 days after doxycycline application. After long‐term expression, perivascular thioflavin‐positive depositions, formed by amyloid fibrils, developed. These depositions persisted even after prolonged silencing of the transgene, indicating an irreversible process. Similarly, strong perivascular apolipoprotein E (ApoE) depositions were found after TGF‐β1 expression and these remained despite TGF‐β1 removal. These in vivo observations suggests that the continuous presence of TGF‐β1 as intitial trigger is not necessary for the persistence and development of chronic lesions. Neuroprotective effects were observed after short‐term expression of TGF‐β1. Death of striatal neurons induced by 3‐nitropropionic acid was markedly reduced after induced TGF‐β1 expression.

ELECTRON MICROSCOPIC EVIDENCE FOR A CHOLINERGIC INNERVATION OF GABAERGIC PARVALBUMIN-IMMUNOREACTIVE NEURONS IN THE RAT MEDIAL SEPTUM

The presence of interconnections between cholinergic and parvalbumin (PARV)‐containing gamma aminobutyric acid (GABA)ergic septohippocampal projection neurons is still a matter of debate. To search for contacts of cholinergic collateral axon terminals in the septal‐diagonal band region the immunotoxin 192IgG‐saporin was applied, which was proved to selectively destroy cholinergic basal forebrain neurons. Seven and 10 days after administration of the immunotoxin, choline acetyltransferase immunoreactivity had disappeared, and numerous neuronal somata and dendrites as well as axonal terminals revealed characteristics of electron‐lucent degeneration. Electron‐dense degeneration was never observed in dendrites and synaptic boutons. Degenerating terminals were found in contact with PARV‐immunopositive and PARV‐negative neurons. Because only cholinergic cells were degenerating, the terminals should be collaterals from cholinergic neurons. In addition to such contacts, PARV‐immunoreactive boutons were seen in contact with PARV‐positive and PARV‐negative cells, but were not identified at degenerating postsynaptic profiles. As suggested in other studies, cholinergic boutons contacting GABAergic PARV‐containing septal projection cells may influence hippocampal theta activity. Furthermore, multiple synaptic connections of both neuronal populations forming the septohippocampal pathway may contribute to their high rate of survival after fimbria‐fornix transection. J. Neurosci. Res. 54:248–253, 1998.

Unique features of extracellular matrix in the mouse medial nucleus of trapezoid body--implications for physiological functions.

The medial nucleus of the trapezoid body (MNTB) is a vital structure of sound localization circuits in the auditory brainstem. Each principal cell of MNTB is contacted by a very large presynaptic glutamatergic terminal, the calyx of Held. The MNTB principal cells themselves are surrounded by extracellular matrix components forming prominent perineuronal nets (PNs). Throughout the CNS, PNs, which form lattice-like structures around the somata and proximal dendrites, are associated with distinct types of neurons. PNs are highly enriched in hyaluronan and chondroitin sulfate proteoglycans therefore providing a charged surface structure surrounding the cell body and proximal neurites of these neurons. The localization and composition of PNs have lead investigators to a number of hypotheses about their functions including: creating a specific extracellular ionic milieu around these neurons, stabilizing synapses, and influencing the outgrowth of axons. However, presently the precise functions of PNs are still quite unclear primarily due to the lack of an ideal experimental model system that is highly enriched in PNs and in which the synaptic transmission properties can be precisely measured. The MNTB principal cells could offer such a model, since they have been extensively characterized electrophysiologically. However, extracellular matrix (ECM) in these neurons has not yet been precisely detailed. The present study gives a detailed examination of the ECM organization and structural differences in PNs of the mouse MNTB. The different PN components and their distribution pattern are scrutinized throughout the MNTB. The data are complemented by electron microscopic investigations of the unique ultrastructural localization of PN-components and their interrelation with distinct pre- and postsynaptic MNTB cell structures. Therefore, we believe this work identifies the MNTB as an ideal system for studying PN function.

Perineuronal and perisynaptic extracellular matrix in the human spinal cord.

Extracellular matrix (ECM) forms an active interface around neurons of the central nervous system (CNS). Whilst the components, chemical heterogeneity and cellular recruitment of this intercellular assembly in various parts of the brain have been discussed in detail, the spinal cord received limited attention in this context. This is in sharp contrast to its clinical relevance since the overall role of ECM especially that of its chondroitin sulphate-based proteoglycan components (CSPGs) was repeatedly addressed in neuropathology, regeneration, CNS repair and therapy models. Based on two post-mortem human specimen, this study gives the first and detailed description of major ECM components of the human spinal cord. Immunohistochemical investigations were restricted to the systematic mapping of aggrecan, brevican, proteoglycan link-protein as well as tenascin-R and hyaluronan containing matrices in the whole cranio-caudal dimension of the human spinal cord. Other proteoglycans like versican, neurocan and NG2 were exemplarily investigated in restricted areas. We show the overall presence of tenascin-R and hyaluronan in both white and grey matters whereas aggrecan, proteoglycan link-protein and brevican were restricted to the grey matter. In the grey matter, the ECM formed aggrecan-based perineuronal nets in the ventral and lateral horns but established single perisynaptic assemblies, axonal coats (ACs), containing link-protein and brevican in all regions except of the Lissauers zone. Intersegmental differences were reflected in the appearance of segment-specific nuclei but not in overall matrix distribution pattern or chemical heterogeneity. Perineuronal nets were typically associated with long-range projection neurons including cholinergic ventral horn motorneurons or dorsal spinocerebellar tract neurons of the Clarke-Stilling nuclei. Multiple immunolabelling revealed that nociceptive afferents were devoid of individual matrix assemblies unlike glycinergic or GABAergic synapses. The detailed description of ECM distribution in the human spinal cord shall support clinical approaches in injury and regenerative therapy.