P. Streit

Prion (PrPSc)-specific epitope defined by a monoclonal antibody

Prions are infectious particles causing transmissible spongiform encephalopathies (TSEs). They consist, at least in part, of an isoform (PrPSc) of the ubiquitous cellular prion protein (PrPC). Conformational differences between PrPCand PrPScare evident from increased β-sheet content and protease resistance in PrPSc(refs 1,2,3). Here we describe a monoclonal antibody, 15B3, that can discriminate between the normal and disease-specific forms of PrP. Such an antibody has been long sought as it should be invaluable for characterizing the infectious particle as well as for diagnosis of TSEs such as bovine spongiform encephalopathy (BSE) or Creutzfeldt–Jakob disease (CJD) in humans. 15B3 specifically precipitates bovine, murine or human PrPSc, but not PrPC, suggesting that it recognizes an epitope common to prions from different species. Using immobilized synthetic peptides, we mapped three polypeptide segments in PrP as the 15B3 epitope. In the NMR structure of recombinant mouse PrP, segments 2 and 3 of the 15B3 epitope are near neighbours in space, and segment 1 is located in a different part of the molecule. We discuss models forthe PrPSc-specific epitope that ensure close spatial proximity of all three 15B3 segments, either by intermolecular contacts in oligomeric forms of the prion protein or by intramolecular rearrangement.

Two classes of cortical GABA neurons defined by differential calcium binding protein immunoreactivities

SummaryCalcium ions play a key role in many aspects of neuronal behavior and certain calcium binding proteins that may influence this behavior are differentially distributed in the central nervous system. In this study it is shown that immunoreactivity for calbindin-28 and for parvalbumin is localized in separate populations of inhibitory GABA interneurons in all areas of the neocortex of Old World monkeys. Virtually all GABA neurosn show immunoreactivity for one or other calcium binding protein but, except for a few cells in layer IV, GABA cells do not show immunoreactivity for both proteins. Among the two cell populations, parvalbumin immunoreactivity characterizes basket neurons while calbindin immunoreactivity characterizes double bouquet neurons. These findings suggest that the two GABA cell types differ in their regulation of calcium homeostasis and may yield clues to their different roles in intracortical circuitry.

Subsynaptic segregation of metabotropic and ionotropic glutamate receptors as revealed by immunogold localization

Glutamate is a major neurotransmitter in the brain that acts both through fast ionotropic receptors and through slower metabotropic receptors coupled to G proteins. Both receptors are present throughout the somatodendritic domain of neurons as shown by immunohistochemical and patch clamp recording studies. Immunogold labelling revealed a concentration of metabotropic receptors at the edge, but not within the main body of anatomically defined synapses, raising the possibility that ionotropic and metabotropic receptors are segregated. We applied double immunogold labelling to study glutamatergic parallel and climbing fibre synapses in the cerebellar cortex. The ionotropic AMPA type receptors occupy the membrane opposite the release site in the main body of the synaptic junction, whereas the metabotropic receptors are located at the periphery of the same synapses. Furthermore, immunoreactivity for AMPA receptors is at least twice as high in the parallel fibre synapses as in glutamatergic mossy fibre synapses. We suggest that the spatial segregation of ionotropic and metabotropic glutamate receptors permits the differential activation of these receptors according to the amount of glutamate released presynaptically, whereas the different densities of the ionotropic receptor at distinct synapses could allow the same amount of glutamate to evoke fast responses of different magnitude.

A new and sensitive staining method for axonally transported horseradish peroxidase (HRP) in the pigeon visual system.

Since its introduction as intra-axonally transported marker substance t2-t4 horseradish peroxidase (HRP) has been mainly applied to the somatopetal* tracing of connections 1~ in the nervous system. However, it was reported from the beginning that H R P could also be used as a somatofugal* axonal rnarker7,15,17,zL It was proposed 5,19 that somatofugal tracing with HRP would be simpler and faster than with radioactively labeled proteins, especially at the electron microscope (EM) level, and it would have the additional advantage that the labeled elements are visualized directly and not only by an epiphenomenon overlying them. On the other hand, tracing unknown connections at the EM level would be much easier if the relevant areas could be recognized already at the light microscope (LM) level as is the case with LM autoradiography of radioactively labeled proteins. Since somatofugally transported H R P was demonstrated at the LM level mainly by means of methods other s,~7,22 than the usual diaminobenzidine staininge, negative results obtained with the latter technique3,14, 20 might be due to deficiencies in the method itself. In the present paper a modified diaminobenzidine method is presented for the demonstration, at the LM level, of HRP transported in somatofugal as well as somatopetal directions. The method was tested in the retinal projections of the adult pigeon. The efferent, non-reciprocal projection from the isthmo-optic nucleus (IO) to the retina 2,4,~4,~5 was used to test the staining method in a somatopetal system. Sixteen adult pigeons (Columba livia) anesthetized with Penthrane ® (Abbott) received injections of 10-25 mg HRP (Boehringer RZ 3) dissolved in 50 ttl Ringer solution into the vitreous humor of one eye. The animals were perfused under Penthrane anesthesia after survival times valylng between 0.5 and 3.5 days. Intracardial injections of 1 ml sodium nitrite and 1 ml Liquvmine ® (Roche) were followed by a flush with 6 % Macrodex® solution (Pharmacia) and then 250-500 ml of

NMDA Receptor Content of Synapses in Stratum Radiatum of the Hippocampal CA1 Area

Glutamate receptors activated by NMDA (NMDARs) or AMPA (AMPARs) are clustered on dendritic spines of pyramidal cells. Both the AMPAR-mediated postsynaptic responses and the synaptic AMPAR immunoreactivity show a large intersynapse variability. Postsynaptic responses mediated by NMDARs show less variability. To assess the variability in NMDAR content and the extent of their coexistence with AMPARs in Schaffer collateral–commissural synapses of adult rat CA1 pyramidal cells, electron microscopic immunogold localization of receptors has been used. Immunoreactivity of NMDARs was detected in virtually all synapses on spines, but AMPARs were undetectable, on average, in 12% of synapses. A proportion of synapses had a very high AMPAR content relative to the mean content, resulting in a distribution more skewed toward larger values than that of NMDARs. The variability of synaptic NMDAR content [coefficient of variation (CV), 0.64–0.70] was much lower than that of the AMPAR content (CV, 1.17–1.45). Unlike the AMPAR content, the NMDAR content showed only a weak correlation with synapse size. As reported previously for AMPARs, the immunoreactivity of NMDARs was also associated with the spine apparatus within spines. The results demonstrate that the majority of the synapses made by CA3 pyramidal cells onto spines of CA1 pyramids express both NMDARs and AMPARs, but with variable ratios. A less-variable NMDAR content is accompanied by a wide variability of AMPAR content, indicating that the regulation of expression of the two receptors is not closely linked. These findings support reports that fast excitatory transmission at some of these synapses is mediated by activation mainly of NMDARs.

In Vitro Release of Endogenous Excitatory Sulfur-Containing Amino Acids from Various Rat Brain Regions

Abstract: Efflux of various amino acids from rat brain slices was determined under resting or depolarizing conditions. Slices of neocortex, hippocampus, striatum, cerebellum, mesodiencephalon, pons‐medulla, and spinal cord were depolarized by K+ (50 mM) or veratrine (33 μg/ml). The 4‐N, N‐dimethylamino‐azobenzene‐4′‐isothiocyanate (DABITC) derivatization method of Chang [Biochem. J.199, 537–545 (1981)] for HPLC was adapted for analysis of amino acids and peptides in superfusion solutions. It allowed the separation and simultaneous detection of the sulfur‐containing amino acids cysteine sulfinic acid (CSA), cysteic acid (CA), homocysteine sulfinic acid (HCSA), and homocysteic acid (HCA) at the picomole level. All four were shown to be released on depolarization in a Ca2+‐dependent manner from brain slices. CSA and HCSA were released from cortex, hippocampus, mesodiencephalon, and, for HCSA only, striatum. HCA release, observed in all regions, was most prominent in cortex and hippocampus. CA was slightly increased by depolarization in hippocampus and mesodiencephalon. These sulfur‐containing amino acids have been shown to exert an excitatory action on CNS neurons. The fact that these sulfur‐containing amino acids are released as endogenous substances from nervous tissue supports the hypothesis that they play a role in CNS neurotransmission.

Monoclonal antibodies demonstrating GABA-like immunoreactivity

SummaryMouse monoclonal antibodies (mAb) to GABA were developed following immunization with GABA coupled to bovine serum albumin (GABA-BSA). The selection of hybridoma cell lines producing antibodies which reacted with GABA-BSA but not with glutamate-BSA conjugates as well as the characterization of chosen mAb was performed by enzyme linked immunosorbent assays (ELISA). The five mAb selected were all of the IgG class and displayed different patterns of crossreactivities with the amino acid- and dipeptide-BAS conjugates tested. MAb 3A12 reacted approximately 4,000 times better with GABA-BSA than with β-alanine-BSA conjugates according to serial dilution experiments of the antibody in ELISA. Immunoreactivity was even lower for other conjugates tested including glycine-, taurine-, glutamate-, and glutamine-BSA. Immunohistochemical results in rat and chicken brain indicated that the patterns of GABA-like immunoreactivity observed with these mAb were consistent with the available information on the distribution of GABA-containing neurons.

Characterization of the pigeon isthmo-tectal pathway by selective uptake and retrograde movement of radioactive compounds and by Golgi-like horseradish peroxidase labeling.

Abstract Autoradiographs of the pigeon optic lobe were prepared after injection of various tritiated amino acids or choline in the optic tectum or in the subtectal nucleus isthmi, pars parvocellularis (Ipc). Horseradish peroxidase (HRP) was also injected into Ipc. A topographically organized reciprocal connection was observed between the tectum and Ipc. The tectal-Ipc pathway had cell bodies lying in sublayer IIi. The acetylcholinesterase staining disappeared topographically in Ipc after tectal lesion. The Ipc-tectal fibers project mainly upon the superficial sublayers IId and IIb. These terminals, filled with HRP, were seen at the electron microscopic level to contain mainly round vesicles and primarily to contact horizontally running structures. In the lateral and caudal part of the tectum the application of [ 3 H]glycine, [ 3 H]serine, [ 3 H]α-alanine or [ 3 H]choline was followed by labelling of the cell bodies in Ipc, while the injection of all the other amino acids tested lead only to neuropil labeling. [ 3 H]Glycine application to the superficial sublayers of the optic tectum was a necessary condition for the appearance of label within Ipc neurons. Rostral tectal injections of [ 3 H]GABA, but not [ 3 H]glycine, resulted in a labeling of rostral-Ipc somata. Glycine injected directly into Ipc was accumulated by neurons while little uptake of GABA or DABA was seen except for a small number of profiles within rostral regions of the nucleus. It is suggested that glycine and GABA, as putative neurotransmitters, are picked up by the Ipc-tectal terminals and that the label is transported retrogradely to the cell bodies. It is further proposed that glycine or a glycine related transmitter is active within portions of the Ipc-tectal pathway while rostrally located neurons of the subtectal nucleus are chemically differentiated and use GABA as their neurotransmitter.

Assembly intracellular targeting and cell surface expression of the human N-methyl-d-aspartate receptor subunits NR1a and NR2A in transfected cells

The intracellular trafficking, assembly, and cell surface targeting of the human N-methyl-D-aspartate receptor subunits NR1a and NR2A has been studied using both transiently and permanently transfected mammalian cell lines. The expression of either NR1a or NR2A alone does not result in significant cell surface expression of either subunit as determined by cell surface biotinylation and immunofluorescence staining. When NR1a is expressed alone large intracellular accumulations of the subunit are formed which do not co-localize with the golgi apparatus markers protein p58 and wheat germ agglutinin, but do co-localize with the endoplasmic reticulum marker calreticulin. Co-expression of NR1a and NR2A results in a reduction of these intracellular accumulations and the appearance of both subunits on the cell surface. Immunoprecipitation of NR1a from in vitro translated subunit proteins showed that NR2A could only be immunoprecipitated with NR1a when both subunits were co-synthesized in the presence of microsomes. When cells expressing NR1a and NR2A were incubated with [35S]methionine in the presence of Brefeldin-A, a drug which prevents protein transport from the endoplasmic reticulum, NR2A could be immunoprecipitated by an antiserum specific for NR1a. Together these results suggest that the NMDA receptor subunits are assembled in the endoplasmic reticulum and that co-synthesis of the subunits is necessary for their association and their successful cell surface targeting.

Insect optic lobe neurons identifiable with monoclonal antibodies to GABA

SummaryFive monoclonal antibodies aginst GABA were tested on glutaraldehyde fixed sections of optic lobes of three insect species, blowflies, houseflies and worker bees. The specificity of these antibodies was analyzed in several tests and compared with commercially available anti-GABA antiserum.A very large number of GABA-like immunoreactive neurons inncrvate all the neuropil regions of these optic lobes. Immunoreactive processes are found in different layers of the neuropils. The immunoreactive neurons are amacrines and columnar or noncolumnar neurons connecting the optic lobe neuropils. In addition some large immunoreactive neurons connect the optic lobes with centers of the brain.Some neuron types could be matched with neurons previously identified with other methods. The connections of a few of these neuron types are partly known from electron microscopy or electrophysiology and a possible role of GABA in certain neural circuits can be discussed.