Immunolesion-induced loss of cholinergic projection neurones promotes ß-amyloidosis and tau hyperphosphorylation in the hippocampus of triple-transgenic mice.

AIMS: Currently available animal models incompletely capture the complex pathophysiology of Alzheimer's disease (AD), typically involving ß-amyloidosis, neurofibrillary tangle formation and loss of basal forebrain cholinergic projection neurones (CPN). While age-dependent ß-amyloidosis and tau hyperphosphorylation are mimicked in triple-transgenic mice (3xTg), experimental induction of CPN loss in these mice is still lacking. Here, we introduce a more-complex animal model of AD by inducing cellular loss of CPN in an already existing transgenic background aiming to elucidate subsequent changes of hippocampal ß-amyloid (Aß) and tau pathology. METHODS: Twelve-month-old 3xTg mice intracerebroventricularly received the rabbit-anti-low affinity neurotrophin receptor p75-saporin, an immunotoxin specifically targeting forebrain CPN. After histochemical verification of immunolesion in immersion-fixed forebrains, markers of Aß and tau metabolism were analysed using quantitative Western blot analyses of hippocampi from these mice. In parallel, these markers and glial activation were investigated by multiple immunofluorescence labelling of perfusion-fixed hippocampi and confocal laser-scanning microscopy. RESULTS: Four months after immunolesion, the selective lesion of CPN was verified by disappearance of choline acetyltransferase and p75 immunolabelling. Biochemical analysis of hippocampi from immunolesioned mice revealed enhanced levels of Aß, amyloid precursor protein (APP) and its fragment C99. Furthermore, immunolesion-induced increase in levels of phospho-tau and tau with AD-like conformation were seen in 16-month-old mice. Immunofluorescence staining confirmed an age-dependent occurrence of hippocampal Aß-deposits and phospho-tau, and demonstrated drastic gliosis around Aß-plaques after immunolesion. CONCLUSION: Overall, this extended model promises further insights into the complexity of AD and contributes to novel treatment strategies also targeting the cholinergic system.

Microdomains for neuron-glia interaction: parallel fiber signaling to Bergmann glial cells.

Astrocytes are considered a reticulate network of cells, through which calcium signals can spread easily. In Bergmann glia, astrocytic cells of the cerebellum, we identified subcellular compartments termed 'glial microdomains'. These elements have a complex surface consisting of thin membrane sheets, contain few mitochondria and wrap around synapses. To test for neuronal interaction with these structures, we electrically stimulated parallel fibers. This stimulation increased intracellular calcium concentration ([Ca2+]i) in small compartments within Bergmann glial cell processes similar in size to glial microdomains. Thus, a Bergmann glial cell may consist of hundreds of independent compartments capable of autonomous interactions with the particular group of synapses that they ensheath.

P2 receptor expression in the dopaminergic system of the rat brain during development.

Extracellular ATP facilitates the release of dopamine via P2 receptor activation in parts of the mesolimbic system. To characterize P2X/Y receptor subtypes in the developing dopaminergic system, their expression in organotypic slice co-cultures including the ventral tegmental area/substantia nigra (VTA/SN) complex and the prefrontal cortex (PFC) was studied in comparison to the receptor expression in 3-5 day-old and adult rats. Reverse transcriptase-polymerase chain reaction (RT-PCR) with specific primers for the P2X(1,2,3,4,6,7) and P2Y(1) receptors in the tissue extracts of organotypic co-cultures revealed the presence of the P2X and P2Y receptor mRNAs investigated. Multiple immunofluorescence labeling of the P2X/Y receptor protein indicated differences in the regional expression in the organotypic co-cultures after 10 days of cultivation (VTA/SN, P2X(1,2,3,4,6,7), P2Y(1,6,12); PFC, P2X(1,3,4,6,7), P2Y(1,2,4,6,12)). At postnatal days 3-5, an immunofluorescence mostly comparable to that of adult rats was observed (VTA/SN and PFC: P2X(1,2,3,4,6,7), P2Y(1,2,4,6,12)). There was one important exception: the P2X(7) receptor immunocytochemistry was not found in adult tissue, suggesting a potential role of this receptor in the development. Only few P2 receptors (e.g. P2X(1), P2Y(1)) were expressed at fibers interconnecting the dopaminergic VTA/SN with the PFC in the organotypic co-cultures. The treatment of the cultures with the ATP analogues 2-methylthio-ATP and alpha,beta-methylene-ATP induced an increase in axonal outgrowth and fiber density, which could be inhibited by pre-treatment with the P2X/Y receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid. The co-localization of the dopamine-(D1) receptor with the P2X(1) receptor in organotypic slice cultures was evident. In the PFC of the co-cultures, and that of young but not adult rats, a number of tyrosine hydroxylase (TH)-positive cells also possessed P2Y(1)-immunoreactivity (IR). Additionally, a strong P2Y(1)-IR was observed on astrocytes. The present results show a time-, region- and cell type-dependent in vitro and in vivo expression pattern of different P2 receptor subtypes in the dopaminergic system indicating the involvement of ATP and its receptors in neuronal development and growth.

Axon initial segment ensheathed by extracellular matrix in perineuronal nets.

Perineuronal nets of extracellular matrix are associated with distinct types of neurons in the cerebral cortex and many subcortical regions. Large complexes of aggregating proteoglycans form a chemically specified microenvironment around the somata, proximal dendrites and the axon initial segment, including the presynaptic boutons attached to these domains. The subcellular distribution and the temporal course of postnatal formation suggest that perineuronal nets may be involved in the regulation of synaptic plasticity. Here we investigate structural and cytochemical characteristics of the extracellular matrix around axon initial segments virtually devoid of synaptic contacts. Wisteria floribunda agglutinin staining, the immunocytochemical detection of aggrecan and tenascin-R, as well as affinity-labeling of hyaluronan were used to analyze perineuronal nets associated with large motoneurons in the mouse superior colliculus. The molecular composition of perineuronal nets was divergent between neurons but was identical around the different cellular domains of the individual neurons. The axon initial segments largely devoid of synapses were covered by a continuous matrix sheath infiltrating the adjacent neuropil. The periaxonal zone penetrated by matrix components often increased in diameter along the initial segment from the axon hillock toward the myelinated part of the axon. The axonal and somatodendritic domains of perineuronal nets were concomitantly formed during the first three weeks of postnatal development. The common molecular properties and major structural features of subcellular perineuronal net domains were retained in organotypic midbrain slice cultures. The results support the hypothesis that the aggrecan-related extracellular matrix of perineuronal nets provides a continuous micromilieu for different subcellular domains performing integration and generation of the electrical activity of neurons.

Expression of ADAM15 in lung carcinomas.

ADAM15, a member of the ADAM (a disintegrin and metalloprotease) family, is a membrane protein containing both protease and adhesion domains and may, thus, be involved in tumor invasion and metastasis. The aim of this study was to analyze the expression of ADAM15 and its potential ligand, integrin alpha(v)beta3 (CD51/CD61), in lung carcinoma cell lines and tissues. Most small cell lung carcinomas (SCLCs) and non-SCLC cell lines were ADAM15, alpha(v) and beta3 integrin mRNA positive. Half of the cell lines expressed ADAM15, and three expressed the alpha(v)beta3 heterodimer at the cell surface as shown using flow cytometry. Paraffin sections of pulmonary epithelial tumors, including SCLCs (n=26), squamous cell cancer (SCCs, n=27) and adenocarcinomas (ACs, n=17) were stained with antibodies to the ectosolic and cytosolic domain of ADAM15 and alpha(v)beta3 integrin complex. The results were scored (0-12, according to Remmele's score). Normal epithelial cells of the lung were negative or slightly positive for ADAM15 (score<2). The score was always significantly higher for tumor cells. ACs showed the strongest staining (tumor center; ADAM15ecto; mean+/-SEM; 5.47+/-1.04), whereas SCLCs only showed weak ADAM15 expression (2.67+/-0.42; SCCs: 3.62+/-0.62). Frequently, significantly stronger ADAM15 expression has been shown in tumor cells located at the front of invasion compared with those within solid formations. Overall analysis of all tumor specimens and each tumor type revealed no significant correlation between tumor stage or degree of differentiation and ADAM15 ectosolic or cytosolic domain expression in tumor cells. Both molecules are often co-localized in the same tumor cells in ADAM15- and alpha(v)beta3 integrin-positive carcinomas. In summary, lung carcinoma cell lines and tissues were frequently ADAM15 positive.

Postnatal development of perineuronal nets in wild-type mice and in a mutant deficient in tenascin-R.

The extracellular matrix glycoprotein tenascin-R (TN-R), colocalizing with hyaluronan, phosphacan, and aggregating chondroitin sulphate proteoglycans in the white and grey matter, is accumulated in perineuronal nets that surround different types of neurons in many brain regions. To characterize the role of TN-R in the formation of perineuronal nets, we studied their postnatal development in wild-type mice and in a TN-R knock-out mutant by using the lectin Wisteria floribunda agglutinin and an antibody to nonspecified chondroitin sulphate proteoglycans as established cytochemical markers. We detected the matrix components TN-R, hyaluronan, phosphacan, neurocan, and brevican in the perineuronal nets of cortical and subcortical regions. In wild-type mice, lectin-stained, immature perineuronal nets were first seen on postnatal day 4 in the brainstem and on day 14 in the cerebral cortex. The staining intensity of these nets for TN-R, hyaluronan, phosphacan, neurocan, and brevican was extremely weak or not distinguishable from that of the surrounding neuropil. However, all markers showed an increase in staining intensity of perineuronal nets reaching maximal levels between postnatal days 21 and 40. In TN-R-deficient animals, the perineuronal nets tended to show a granular component within their lattice-like structure at early stages of development. Additionally, the staining intensity in perineuronal nets was reduced for brevican, extremely low for hyaluronan and neurocan, and virtually no immunoreactivity was detectable for phosphacan. The granular configuration of perineuronal nets became more predominant with advancing age of the mutant animals, indicating the continued abnormal aggregation of chondroitin sulphate proteoglycans complexed with hyaluronan. As shown by electron microscopy in the cerebral cortex, the disruption of perineuronal nets was not accompanied by apparent changes in the synaptic structure on net-bearing neurons. The regional distribution patterns and the temporal course of development of perineuronal nets were not obviously changed in the mutant. We conclude that the lack of TN-R initially and continuously disturbs the molecular scaffolding of extracellular matrix components in perineuronal nets. This may interfere with the development of the specific micromilieu of the ensheathed neurons and adjacent glial cells and may also permanently change their functional properties.

P2Y receptor expression on astrocytes in the nucleus accumbens of rats.

The expression of purinoceptor (P2)Y-subtypes on astrocytes in vivo under physiological conditions and after stab wound injury was investigated. Reverse transcriptase-polymerase chain reaction with specific primers for the receptor-subtypes P2Y1,2,4,6,12 in tissue extracts of the nucleus accumbens of untreated rats revealed the presence of all P2Y receptor mRNAs investigated. Double immunofluorescence visualized with laser scanning microscopy indicated the expression of the P2Y1,4 receptors on glial fibrillary acidic protein (GFAP)-labeled astrocytes under physiological conditions. After stab wound injury the additional expression of the P2Y2 and P2Y6 receptors, and an up-regulation of the P2Y1,4 receptor-labeling on astrocytic cell bodies and/or processes was observed. Astrocytes of cortical, in contrast to accumbal areas exhibited P2Y1,2,4,6 receptor-immunoreactivity (IR) under control conditions, which was up-regulated after stab would injury. Labeling for the P2Y12 receptor was not observed on GFAP-positive cortical and accumbal astrocytes under any of the conditions used. For the first time, the co-localization of different P2 receptor-subtypes (e.g. P2Y1 and P2X3) on the same astrocyte was shown immunocytochemically. The up-regulation of P2Y1 receptor-IR on astrocytes and non-glial cells after mechanical injury could be facilitated by microinfusion of the P2Y1,12,13 receptor agonist adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS). Proliferative changes after ADPbetaS-microinjection were characterized by means of double-staining with antibodies against GFAP and 5-bromo-2'-deoxyuridine. The non-selective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, the P2Y1 receptor antagonist N6-methyl-2'-deoxyadenosine 3',5'-bisphosphate and the P2Y1 receptor-antibody itself inhibited the agonist-induced effects. The data indicate the region-specific presence of P2Y receptors on astrocytes in vivo and their up-regulation after injury as well as the co-localization of P2X and P2Y receptor-subtypes on the same astrocyte. The dominant role of P2Y1 receptors in proliferation and the additional stimulation of non-P2Y1 receptors has been demonstrated in vivo suggesting the involvement of this receptor-type in the gliotic response under physiological and pathological conditions.

P2X receptor expression on astrocytes in the nucleus accumbens of rats.

Astrocytes express a variety of neurotransmitter receptors which render them capable of responding to extracellular stimuli, like ATP. Release of ATP, e.g. after brain injury, may initiate reactive gliosis via stimulation of purinergic P2X and P2Y receptors. In the present study, the expression and cellular localization of P2X receptor subtypes on astrocytes in the nucleus accumbens of rats under normal physiological conditions and after stab wound were investigated. Reverse transcription-polymerase chain reaction (RT-PCR) with specific P2X(1-7) primers, and double immunofluorescence with antibodies to glial fibrillary acidic protein (GFAP, a specific marker of fibrous astrocytes) and to different P2X receptor subtypes (P2X(1-4), P2X(7)) were used. The RT-PCR of tissue extracts of the nucleus accumbens of untreated rats revealed the presence of all seven currently known P2X receptor subtype mRNAs indicating the presence of these receptors in this region. A double immunofluorescence approach with confocal laser scanning microscopy showed the localization of P2X(2-4) receptor subtypes on GFAP-labelled astrocytes in untreated rats. Labelling for P2X(1) and P2X(7) receptor subtypes was not found. After mechanical damage all P2X receptor subtypes studied (P2X(1-4), P2X(7)) were observed on the GFAP-labelled reactive astrocytes. A characteristic distribution of the P2X receptors on astrocytic processes and cell bodies as well as an up-regulation of the P2X-immunofluorescence was found. In conclusion, the data show the presence of P2X receptors on rat nucleus accumbens astrocytes and suggest that astrogliosis in vivo is associated with an up-regulation of distinct P2X receptor subtypes.

Short-term consequences of N-methyl-D-aspartate excitotoxicity in rat magnocellular nucleus basalis: effects on in vivo labelling of cholinergic neurons.

Cholinergic neurons of the basal forebrain form one of the neuron populations that are susceptible to excitotoxic injury. Whereas neuropharmacological studies have aimed at rescuing cholinergic neurons from acute excitotoxic attacks, the short-term temporal profile of excitotoxic damage to cholinergic nerve cells remains largely elusive. The effects of N-methyl-D-aspartate (NMDA) infusion on cytochemical markers of cholinergic neurons in rat magnocellular nucleus basalis were therefore determined 4, 24 and 48 h post-lesion. Additionally, the influence of excitotoxic damage on the efficacy of in vivo labelling of cholinergic neurons with carbocyanine 3-192IgG was investigated. Carbocyanine 3-192IgG was unilaterally injected in the lateral ventricle. Twenty-four hours later, NMDA (60 nM/microl) was infused in the right magnocellular nucleus basalis, while control lesions were performed contralaterally. Triple immunofluorescence labelling for carbocyanine 3-192IgG, NMDA receptor 2A and B subunits and choline-acetyltransferase (ChAT) was employed to determine temporal changes in NMDA receptor immunoreactivity on cholinergic neurons. The extent of neuronal degeneration was studied by staining with Fluoro-Jade. Moreover, changes in the numbers of ChAT or p75 low-affinity neurotrophin receptor immunoreactive neurons, and the degree of their co-labelling with carbocyanine 3-192IgG were determined in basal forebrain nuclei. The effects of NMDA-induced lesions on cortical projections of cholinergic nucleus basalis neurons were studied by acetylcholinesterase (AChE) histochemistry. Characteristic signs of cellular damage, as indicated by decreased immunoreactivity for NMDA receptors, ChAT and p75 low-affinity neurotrophin receptors, were already detected at the shortest post-lesion interval investigated. Fluoro-Jade at 4 h post-lesion only labelled the core of the excitotoxic lesion. Longer survival led to enhanced Fluoro-Jade staining, and to the decline of ChAT immunoreactivity reaching a maximum 24 h post-surgery. Significant loss of p75 low-affinity neurotrophin receptor immunoreactivity and of cortical AChE-positive projections only became apparent 48 h post-lesion. Carbocyanine 3-192IgG labelling in the ipsilateral basal forebrain exceeded that of the contralateral hemisphere at all time points investigated and progressively declined in the damaged magnocellular nucleus basalis up to 48 h after NMDA infusion. The present study indicates that excitotoxic lesion-induced alteration of cholinergic neuronal markers is a rapid and gradual process reaching its maximum 24 h post-surgery. Furthermore, in vivo labelling of cholinergic neurons may be applied to indicate neuronal survival under pathological conditions, and enable to follow their degeneration process under a variety of experimental conditions.

P2 receptor-types involved in astrogliosis in vivo.

1. In the nucleus accumbens (NAc) of rats, the involvement of P2X and P2Y receptors in the generation of astrogliosis in vivo, was investigated by local application of their respective ligands. The agonists used had selectivities for P2X1,3 (alpha,beta-methylene adenosine 5'-triphosphate; alpha,beta-meATP), P2Y1,12 (adenosine 5'-O-(2-thiodiphosphate; ADP-beta-S) and P2Y2,4,6 receptors (uridine 5'-O-(3-thiotriphosphate; UTP-gamma-S). Pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (PPADS) was used as a non-selective antagonist. The astroglial reaction was studied by means of immunocytochemical double-labelling with antibodies to glial fibrillary acidic protein (GFAP) and 5-bromo-2'-deoxyuridine (BrdU). 2. The agonist-induced changes in comparison to the artificial cerebrospinal fluid (aCSF)-treated control side reveal a strong mitogenic potency of ADP-beta-S and alpha,beta-meATP, whereas UTP-gamma-S was ineffective. The P2 receptor antagonist PPADS decreased the injury-induced proliferation when given alone and in addition inhibited all agonist effects. 3. The observed morphogenic changes included hypertrophy of astrocytes, elongation of astrocytic processes and up-regulation of GFAP. A significant increase of both GFAP-immunoreactivity (IR) and GFA-protein content (by using Western blotting) was found after microinfusion of alpha,beta-meATP or ADP-beta-S. In contrast, UTP-gamma-S failed to increase the GFAP-IR. The morphogenic effects were also inhibited by pre-treatment with PPADS. 4. A double immunofluorescence approach with confocal laser scanning microscopy showed the localisation of P2X3 and P2Y1 receptors on the GFAP-labelled astrocytes. 5. In conclusion, the data suggest that P2Y (P2Y1 or P2Y12) receptor subtypes are involved in the generation of astrogliosis in the NAc of rats, with a possible minor contribution of P2X receptor subtypes.

Immunocytochemical demonstration of alpha 2-M-R/LRP on Müller (glial) cells isolated from rabbit and human retina.

The alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha 2-M-R/LRP) is a multifunctional receptor which has been implicated in lipoprotein metabolism, clearance of proteinase-proteinase inhibitor complexes and regulation of growth factor/cytokine metabolism. This receptor is abundantly present in numerous tissues and organs such as liver, lung, placenta and brain. In brain it is expressed in neurones but not in normal macroglia. Using immunocytochemistry and monoclonal antibodies against the large extracellular receptor subunit we have detected alpha 2-M-R/LRP on enzymatically isolated retinal Müller (glial) cells. This receptor may be involved in vital functions of Müller cells.

NMDA-activated currents in Bergmann glial cells.

NMDA receptors play a crucial role in synaptic plasticity of the central nervous system and were thought to be exclusive to neurones. In this study we provide evidence that Bergmann glial cells from mouse cerebellar slices show intrinsic responses to NMDA. As in neurones, NMDA increased membrane conductance and the responses were blocked by the NMDA antagonist ketamine, but not by the non-NMDA glutamate receptor antagonist CNQX. In contrast to responses in neurones, the current voltage relation of the glial NMDA-induced current was linear, reversed at -40 mV, currents were not blocked by Mg2+ or enhanced by glycine and NMDA did not induce an increase in cytosolic Ca2+ as recorded with a fura-2 imaging system. These data imply the presence of distinct NMDA receptors on Bergmann glial cells; these glial receptors could be the substitute for complex neurone-glia interactions in the cerebellum.

Mammalian Müller (glial) cells express functional D2 dopamine receptors.

Dopamine plays important functional roles in the vertebrate retina. Here we show that functional D2 dopamine receptors are present on mammalian retinal Müller (glial) cells. Using an antiserum directed to two oligopeptides predicted from rat D2 receptor DNA, patchy label was demonstrated immunocytochemically on virtually all Müller cells enzymatically isolated from guinea-pig and rat retinae. Application of exogeneous dopamine to voltage-clamped isolated living guinea-pig Müller cells caused either a decrease (40%), an increase (32%), or no change (28%) of the input resistance of the membrane. The D2 receptor agonist quinelorane caused an increase of the membrane's input resistance in 100% of the cells. This effect was completely blocked by the D2 receptor antagonist S(-)-sulpiride. When all voltage-activated K+ channels except the delayed rectifiers were blocked by Ba2+, quinelorane had no effect. Further, the reversal potentials of the responses were near the potassium equilibrium potential. We conclude that the activation of Müller cell D2 receptors closes (inwardly rectifying) K+ channels. The presence of functional dopamine receptors on mammalian Müller cells may have important consequences for retinal K+ clearance, and thus, for information processing in the retina.

Antibody for human p75 LNTR identifies cholinergic basal forebrain of non-primate species.

192-IgG is an antibody directed against the p75 low affinity nerve growth factor receptor in rats, whereas ME 20.4 was raised against the analogous protein in humans. Coupled to saporin, 192-IgG and ME 20.4 have been used to lesion basal forebrain neurons in rats and primates, respectively. We compared the cross-reactivity of 192-IgG and ME 20.4 in the basal forebrain of rat, human, dog, cat, raccoon, pig, and rabbit. We found excellent species cross-reactivity of ME 20.4 in dog, raccoon, cat, pig and rabbit. In contrast, 192-IgG did not label neurons in any species other than rat. Our findings suggest that ME 20.4-saporin could be used to produce cholinergic basal forebrain lesions in several non-primate species.

Co-localization of calretinin and calbindin in distinct cells in the hippocampal formation of the rat.

We studied the distribution of the calcium binding proteins calretinin and calbindin in the hippocampal formation of the rat brain by means of double-label immunofluorescence - confocal laser scanning microscopy. Colocalization of calretinin and calbindin occurred mostly in large neurons located in the alveus and stratum oriens of field CA1. Some double-labeled cells were observed in the transition area between field CA1 and the subiculum. Finally, double-labeled cells were present in the deep layer of the ventral subiculum. The cells in field CA1 co-expressing both proteins resemble neurons which in neurophysiological experiments by others have been identified as O-LM cells, and we believe that these co-expressing cells should be considered a distinct subpopulation of the calretinin and calbindin populations of GABAergic hippocampal interneurons.

Laser scanning and electron microscopic evidence for rapid and specific in vivo labelling of cholinergic neurons in the rat basal forebrain with fluorochromated antibodies.

Recently developed methods for the selective labelling of cholinergic basal forebrain neurons containing the low-affinity neurotrophin receptor p75 (p75(NTR)) in vivo and in vitro are based on carbocyanine 3 (Cy3)-tagged antibodies directed against p75(NTR). The present study focuses on the maintenance of this neuronal label after injection of such fluorescent antibodies into the cerebral ventricle. One, 3, and 10 days after injection this marker exclusively stains neurons immunoreactive for the cholinergic markers choline acetyltransferase and vesicular acetylcholine transporter in the rat medial septum, diagonal band and nucleus basalis. Thirty days after injection the in vivo labelling was nearly abolished. Predominant labelling of lysosomes was shown by electron microscopic analysis following photoconversion of the Cy3-label to an electron-dense reaction product. The pre-labelling of cholinergic neurons might facilitate pharmacological and electrophysiological approaches in living slices and cell culture systems as well as detailed investigations focused on the transport of neurotrophins in vivo and in animals with experimentally altered p75(NTR) expression.

Perineuronal nets in the rat medial nucleus of the trapezoid body surround neurons immunoreactive for various amino acids, calcium-binding proteins and the potassium channel subunit Kv3.1b.

Perineuronal nets (PNs) are known as chondroitin sulfate-rich, lattice-like coatings of the extracellular matrix ensheathing mainly GABAergic, parvalbumin-containing neurons especially in the cerebral cortex. PNs have also been detected around GABA-immunonegative cells which were shown to be not aminergic, cholinergic, nitrinergic or peptidergic in various brain regions of some mammalian species. To find out whether glycine and aspartate may occur in net-bearing neurons the present study was focused on the rat medial nucleus of the trapezoid body (MNTB) which contains a large portion of cells immunoreactive for these amino acids, but appears to be devoid of GABA-immunoreactive cell bodies. PNs were detected around many glycine- and aspartate-immunopositive neurons in the MNTB by carbocyanine double labeling and confocal laser scanning microscopy. An additional finding was that the lectin-cytochemically stained extracellular matrix surrounds the calretinin-immunoreactive calyces of Held known as giant glutamatergic endbulbs which cover glycinergic principal cells in the MNTB. As elucidated by triple fluorescence labeling, the vast majority of somata co-expressed the calcium-binding proteins parvalbumin and calbindin, but not calretinin. The observed co-localization of PNs and immunoreactivity for the voltage-dependent potassium channel Kv3.1b - as an established marker of fast-firing parvalbumin-containing neurons - supports the assumed function of PNs as a cation exchanger ensuring rapid ion transport as required by highly active nerve cells.

Axonal expression sites of tyrosine hydroxylase, calretinin- and calbindin-immunoreactivity in striato-pallidal and septal nuclei of the rat brain: a double-immunolabelling study.

Besides the dopaminergic afferent projection system, calbindin (CALB)- and calretinin (CR)-immunoreactive fibres of intrinsic and extrinsic origin represent the most abundant axonal categories in the rat striatal and lateral septal areas. The question arises whether or not they may represent separate populations, or whether they form subgroups which co-express more than one of these antigens. Therefore, the present study is focused on the distribution patterns of the axons single-immunolabelled by the catecholaminergic marker tyrosine hydroxylase (TH), and on TH-immunoreactive axons displaying also CR- and/or CALB-immunoreactivity in double-immunostained sections. Striking differences were found between the patch and matrix compartments of the caudate-putamen (CP). Whereas the vast majority of TH-immunoreactive fibres in the patches and a patch-associated subcallosal layer co-expressed CR but not CALB, fibres mono-labelled by the TH-immunoreactivity were predominant in the matrix. The matrix-like regions of the core of nucleus accumbens (CACC), fundus striati (FS), the striatal cell bridges (CB) and the striatal part of olfactory tubercle (OTU) coincided in this respect with the matrix in CP. The absence of CR-immunoreactivity was also characteristic of the TH-immunoreactive fibres in the patch-like areas of the accumbal core, although a high number of separate CR-immunoreactive axons were present. In the shell of nucleus accumbens (SACC) which receives a rich catecholaminergic innervation, fibres co-expressing either one of the calcium-binding proteins were absent. The islands of Calleja (CJI) displaying a strongly TH-immunoreactive centre and a periphery of lower staining intensity, showed only a low number of TH-immunoreactive fibres co-expressing CR or CALB. The broad shell-like band of TH-immunoreactive axons between medial and lateral part of the septum was single-stained with the TH-immunoreactivity. In contrast, the TH-positive fibres forming basket-like arrangements around some neurons in the dorsal lateral septal nucleus co-expressed also CR, but not CALB. The results are discussed in view of the recent concepts of basal forebrain organization and the cytochemical characteristics of mesencephalic dopaminergic nuclei giving rise to the vast majority of the striatal and septal TH-immunoreactive fibre supply, in order to correlate the known projection patterns with the content of calcium-binding proteins in TH-immunolabelled fibres and presumed cells of origin. The TH-immunoreactive fibres in the striatal patches displaying CR- but not CALB-immunoreactivity may originate mainly from neurons in the ventral tier of pars compacta (SNC) and from the pars reticulata of substantia nigra (SNR) which show identical cytochemical properties. Axons in the matrix of CP and the accumbal core as well as in the islands of Calleja single-labelled by the TH-immunoreactivity or additionally containing CALB and CR may originate from neurons in the dorsal tier of mesencephalic nuclei like SN, pars compacta and ventral tegmental area. CR-containing TH-immunoreactive basket-like axon terminations in the dorsal lateral septal nucleus are likely to originate either from mesencephalic nuclei or from the supramammillary region.

Cortical neurons immunoreactive for the potassium channel Kv3.1b subunit are predominantly surrounded by perineuronal nets presumed as a buffering system for cations.

Perineuronal nets (PNs) are known as chondroitin sulphate-rich, lattice-like coatings of the extracellular matrix. In the cortex of mammalian species investigated so far, they were mainly found around GABAergic neurons, but to a lesser degree also around pyramidal cells. Previous investigations in the rat revealed similar distribution patterns of fast-firing neurons expressing both the Kv3.1b subunit of voltage-gated potassium channels and the calcium-binding protein parvalbumin. In the present study, triple fluorescence labelling was applied for the simultaneous demonstration of PNs with the N-acetylgalactosamine-specific Wisteria floribunda agglutinin (WFA), parvalbumin-immunoreactivity (ir) with a monoclonal antibody and of Kv3.1b-ir with several rabbit antibodies. Subsets of non-pyramidal neurons - enwrapped by PNs and expressing parvalbumin and Kv3.1b - were detected in the rat and monkey neocortex and hippocampus. In the rat, faintly stained PNs were additionally found around several layer II/III and V pyramidal cells immunonegative for Kv3.1b, but contacted by Kv3.1b-containing boutons. In the monkey, more intensely labelled PNs frequently occurred around pyramidal cells which themselves appeared to be Kv3. 1b-immunopositive. We also observed minor Kv3.1b-ir and parvalbumin-ir cortical cell populations which were devoid of PNs; occasionally, nets were detected around neurons lacking both immunoreactivities. By confocal laser scanning microscopy, Kv3.1b-ir and WFA-binding sites were found adjoining at the soma and proximal dendritic surface, while lectin-binding sites usually extended on more distal dendritic segments and the axon initial segments which failed to express detectable Kv3.1b-ir. This spatial relationship of both markers was also confirmed by combined WFA-gold labelling and Kv3.1b-immunoperoxidase staining at the electron microscopic level. The data are used for a critical examination of current hypotheses concerning the functional role of PNs. We conclude that PNs may serve as rapid local buffers of excess cation changes in the extracellular space. Somatic membranes of fast-spiking neurons seem to be a main, but not the only source of such changes.

Expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), and Bcl-2 protooncogene protein by Müller (glial) cells in retinal light damage of rats.

In retinal light damage, degeneration of photoreceptors may cause alterations of glial (Müller) cells. We performed immunocytochemical studies on Müller cells isolated from retinae of rats exposed to enhanced illumination for 24 months, a procedure which leads to complete loss of photoreceptor cells. One group of rats was fed daily with Ginkgo biloba extract (EGb 761, an established free radical-scavenger) during the last 8 months of life when the remaining photoreceptors (about 50%) die. We found that (1) Müller cells respond to photoreceptor damage by increased expression of glial fibrillary acidic protein, (2) Müller cells reduce expression of glutamine synthetase when the major glutamate-releasing neurons are lost, and (3) the application of exogenous free radical scavengers prevents the expression by Müller cells of the protooncogene protein Bcl-2, a molecule assumed to activate endogenous free radical-scavenging activities.