Inflammatory cell recruitment after experimental thromboembolic stroke in rats.

Inflammatory mechanisms were recently identified as contributors to delayed neuronal damage after ischemic stroke. However, therapeutic strategies are still lacking, probably related to the outstanding standardization on inflammatory cell recruitment emerging from predominantly artificial stroke models, and the uncertainty on functional properties of distinct subpopulations. Using a rodent model of stroke that closely reflects human embolic ischemia, this study was focused on the local recruitment of immunoreactive cells as well as their functional and regional characterization. Wistar rats underwent thromboembolic middle cerebral artery occlusion, followed by intravenous injection of the blood-brain barrier permeability marker fluorescein-conjugated albumin at 24h. One hour later, brain tissue was subjected to multi-parameter flow cytometry and Pappenheim staining to characterize cells invaded into the ischemia-affected hemisphere, compared to the contralateral side. Immunofluorescence labeling was applied to explore the distribution patterns of recruited cells and their spatial relationships with the vasculature. One day after ischemia onset, a 6.12-fold increase of neutrophils and a 5.43-fold increase of monocytes/macrophages was found in affected hemispheres, while these cells exhibited enhanced major histocompatibility complex class II expression and allocation with vessels exhibiting impaired blood-brain barrier integrity. Microglia remained numerically unaltered in ischemic hemispheres, but shifted to an activated phenotype indicated by CD45/CD86 expression and morphological changes toward an ameboid appearance in the bordering zone. Ischemia caused an increase of lymphoid cells in close vicinity to the affected vasculature, while further analyses allowed separation into natural killer cells, natural killer T cells, T cells (added by an unconventional CD11b(+)/CD3(+) population) and two subpopulations of B cells. Taken together, our study provides novel data on the local inflammatory response to experimental thromboembolic stroke. As concomitantly present neutrophils, monocytes/macrophages and lymphoid cells in the early stage after ischemia induction correspond to changes seen in human stroke, future stroke research should preferably use animal models with relevance for clinical translation.

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.

Spatio-temporal course of macrophage-like cell accumulation after experimental embolic stroke depending on treatment with tissue plasminogen activator and its combination with hyperbaric oxygenation.

Inflammation following ischaemic stroke attracts high priority in current research, particularly using human-like models and long-term observation periods considering translational aspects. The present study aimed on the spatio-temporal course of macrophage-like cell accumulation after experimental thromboembolic stroke and addressed microglial and astroglial reactions in the ischaemic border zone. Further, effects of tissue plasminogen activator (tPA) as currently best treatment for stroke and the potentially neuroprotective co-administration of hyperbaric oxygen (HBO) were investigated. Rats underwent middle cerebral artery occlusion and were assigned to control, tPA or tPA+HBO. Twenty-four hours, 7, 14 and 28 days were determined as observation time points. The accumulation of macrophage-like cells was semiquantitatively assessed by CD68 staining in the ischaemic area and ischaemic border zone, and linked to the clinical course. CD11b, ionized calcium binding adaptor molecule 1 (Iba), glial fibrillary acidic protein (GFAP) and Neuronal Nuclei (NeuN) were applied to reveal delayed glial and neuronal alterations. In all groups, the accumulation of macrophage-like cells increased distinctly from 24 hours to 7 days post ischaemia. tPA+HBO tended to decrease macrophage-like cell accumulation at day 14 and 28. Overall, a trend towards an association of increased accumulation and pronounced reduction of the neurological deficit was found. Concerning delayed inflammatory reactions, an activation of microglia and astrocytes with co-occurring neuronal loss was observed on day 28. Thereby, astrogliosis was found circularly in contrast to microglial activation directly in the ischaemic area. This study supports previous data on long-lasting inflammatory processes following experimental stroke, and additionally provides region-specific details on glial reactions. The tendency towards a decreasing macrophage-like cell accumulation after tPA+HBO needs to be discussed critically since neuroprotective properties were recently ascribed to long-term inflammatory processes.

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.

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.

P2X(2), P2X(2-2) and P2X(5) receptor subunit expression and function in rat thoracolumbar sympathetic neurons.

The present study investigated the pharmacological properties of excitatory P2X receptors and P2X(2) and P2X(5) receptor subunit expression in rat-cultured thoracolumbar sympathetic neurons. In patch-clamp recordings, ATP (3-1000 microM; applied for 1 s) induced inward currents in a concentration-dependent manner. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 30 microM) counteracted the ATP response. In contrast to ATP, alpha,beta-meATP (30 microM; for 1 s) was virtually ineffective. Prolonged application of ATP (100 microM; 10 s) induced receptor desensitization in a significant proportion of sympathetic neurons in a manner typical for P2X(2-2) splice variant-mediated responses. Using single-cell RT-PCR, P2X(2), P2X(2-2) and P2X(5) mRNA expression was detectable in individual tyrosine hydroxylase-positive neurons; coexpression of both P2X(2) isoforms was not observed. Laser scanning microscopy revealed both P2X(2) and P2X(5) immunoreactivity in virtually every TH-positive neuron. P2X(2) immunoreactivity was largely distributed over the cell body, whereas P2X(5) immunoreactivity was most distinctly located close to the nucleus. In summary, the present study demonstrates the expression of P2X(2), P2X(2-2) and P2X(5) receptor subunits in rat thoracolumbar neurons. The functional data in conjunction with a preferential membranous localization of P2X(2)/P2X(2-2) compared with P2X(5) suggest that the excitatory P2X responses are mediated by P2X(2) and P2X(2-2) receptors. Apparently there exist two types of P2X(2) receptor-bearing sympathetic neurons: one major population expressing the unspliced isoform and another minor population expressing the P2X(2-2) splice variant.

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.

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.

Perineuronal nets show intrinsic patterns of extracellular matrix differentiation in organotypic slice cultures.

Perineuronal nets (PNs), consisting of extracellular matrix proteoglycans, complexed with hyaluronan and colocalized with tenascins, are associated with distinct neuronal populations in mature mammalian brain. PNs have been shown to appear postnatally during the period of synaptic refinement and myelination, indicating the commencement of mature physiological properties of neurons. Here we show that the developmental patterns of formation of PNs are well preserved in organotypic slice cultures prepared from rats on postnatal day 3-5 and maintained in vitro for 3-10 weeks. Staining of cultures with Wisteria floribunda agglutinin and immunocytochemical detection of chondroitin sulfate proteoglycans revealed developing PNs in the basal forebrain, mesencephalic regions, and the cerebellum after 2 weeks in vitro, and later in the neocortical areas and hippocampus. In contrast, neurons known to be devoid of PNs in the adult rat brain such as cholinergic basal forebrain neurons and catecholaminergic tegmental neurons differentiate without any formation of PNs in slice cultures. We show further that environmental factors influence the development of PNs around the net-associated types of neurons. Notably, chronic depolarization of the cultures, imposed by an elevated concentration of external potassium ions, enhanced the development of PNs. Blocking of calcium channels with magnesium chloride or with the L-type calcium channel blocker nifedipine, suppressed the development of PNs, while a block of voltage-gated sodium channels by tetrodotoxin had no obvious effects. The results show that extracellular matrix components specifically contribute to the organotypic patterns that develop in brain slice cultures. Evidence is provided that the differentiation of PNs is regulated by calcium-dependent signaling.

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.

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.

Mitochondria of retinal Müller (glial) cells: the effects of aging and of application of free radical scavengers.

Age-related changes of mitochondria were studied in Müller (retinal glial) cells from guinea pigs fed with or without externally applied Ginkgo biloba extract EGb 761, an established radical scavenger. When Müller cell mitochondria from aged animals were compared with those from young adults, they displayed (1) a diminished number of well-defined cristae at the ultrastructural level, (2) a reduced membrane potential, as revealed by fluorimetry using the voltage-sensitive dye tetramethyl rhodamine methylester, and (3) a slightly reduced index of vitality assayed by tetrazolium salt colorimetry. Müller cell mitochondria were also studied in aged guinea pigs which had been fed daily by EGb 761 during the last 2 months before they were sacrificed. Such mitochondria displayed (1) many well-defined cristae at the ultrastructural level, and, compared with mitochondria from untreated aged animals, (2) a significantly enhanced membrane potential and (3) a significantly enhanced index of vitality. No age- or drug-related changes were observed in the mitochondrial content of GABA transaminase, as revealed by immunocytochemistry/densitometry. These results suggest that many but not all structural and functional parameters of aging Müller cell mitochondria are impaired by accumulating oxidative damage, and that externally applied radical scavengers may protect the organelles from the damaging actions of free radicals. As it has been shown earlier that EGb 761 treatment enhances the intrinsic glutathione content of aged guinea pig Müller cells, the protective radical-scavenging effect of the drug may be mediated both directly and indirectly.

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.

P2X7 receptors in Müller glial cells from the human retina.

ATP has been shown to be an important extracellular signaling molecule. There are two subgroups of receptors for ATP (and other purines and pyrimidines): the ionotropic P2X and the G-protein-coupled P2Y receptors. Different subtypes of these receptors have been identified by molecular biology, but little is known about their functional properties in the nervous system. Here we present data for the existence of P2 receptors in Müller (glial) cells of the human retina. The cells were studied by immunocytochemistry, electrophysiology, Ca(2+)-microfluorimetry, and molecular biology. They displayed both P2Y and P2X receptors. Freshly enzymatically isolated cells were used throughout the study. Although the [Ca(2+)](i) response to ATP was dominated by release from intracellular stores, there is multiple evidence that the ATP-induced membrane currents were caused by an activation of P2X(7) receptors. Immunocytochemistry and single-cell RT-PCR revealed the expression of P2X(7) receptors by Müller cells. In patch-clamp studies, we found that (1) benzoyl-benzoyl ATP (BzATP) was the most effective agonist to evoke large inward currents and (2) the currents were abolished by P2X antagonists; however, (3) long-lasting application of BzATP did not cause an opening of large pores in addition to the cationic channels. By microfluorimetry it was shown that the P2X receptors mediated a Ca(2+) influx that contributed a small component to the total [Ca(2+)](i) response. Activation of P2X receptors may modulate the uptake of neurotransmitters from the extracellular space by Müller cells in the retina.

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.

Patch-clamp study of neurons and glial cells in isolated myenteric ganglia.

Most of the physiological information on the enteric nervous system has been obtained from studies on preparations of the myenteric ganglia attached to the longitudinal muscle layer. This preparation has a number of disadvantages, e.g., the inability to make patch-clamp recordings and the occurrence of muscle movements. To overcome these limitations we used isolated myenteric ganglia from the guinea pig small intestine. In this preparation movement was eliminated because muscle was completely absent, gigaseals were obtained, and whole cell recordings were made from neurons and glial cells. The morphological identity of cells was verified by injecting a fluorescent dye by micropipette. Neurons displayed voltage-gated inactivating inward Na(+) and Ca(2+) currents as well as delayed-rectifier K(+) currents. Immunohistochemical staining confirmed that most neurons have Na(+) channels. Neurons responded to GABA, indicating that membrane receptors were retained. Glial cells displayed hyperpolarization-induced K(+) inward currents and depolarization-induced K(+) outward currents. Glia showed large "passive" currents that were suppressed by octanol, consistent with coupling by gap junctions among these cells. These results demonstrate the advantages of isolated ganglia for studying myenteric neurons and glial cells.