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T F Freund

Publications and source records attributed to T F Freund.

153 records · Page 9Linked to original sources
PubMed1983

Glutamate decarboxylase-immunoreactive terminals of Golgi-impregnated axoaxonic cells and of presumed basket cells in synaptic contact with pyramidal neurons of the cat's visual cortex.

Glutamate decarboxylase (GAD)-immunoreactive varicosities were found around cell bodies of nonimmunoreactive and immunoreactive neurons in the cat's visual cortex; they also occurred along apical dendrites and axon initial segments of pyramidal neurons. By examination in the electron microscope of structures first identified in the light microscope, it was established that the GAD-immunoreactive varicosities were boutons in symmetrical synaptic contact with pyramidal cells in layers II-IV. More than 90% of 142 boutons surrounding the cell bodies of 20 pyramidal neurons were immunoreactive for GAD. Since such a high proportion of the axosomatic boutons are GAD-immunoreactive, it is likely that the terminals of basket cells are included in this population and so the basket cell probably uses gamma-aminobutyrate as a transmitter, as suggested by previous authors. Almost all the 68 boutons in symmetrical contact with the axon initial segments of six pyramidal neurons could be shown to be GAD-immunoreactive, which makes it very likely that the boutons of axoaxonic cells contain GAD-immunoreactivity. This was established unequivocally for an individual Golgi-impregnated axoaxonic cell by combining Golgi impregnation and immunocytochemistry in the same sections: A Golgi-impregnated axoaxonic cell whose cell body was in layer II gave rise to numerous terminal segments, some of which were examined in the electron microscope after gold-toning. These boutons were in synaptic contact with axon initial segments and not only contained the Golgi precipitate but were also immunoreactive for GAD. It is concluded that the axoaxonic cell in the visual cortex uses gamma-aminobutyrate as a transmitter. An individual axoaxonic cell in layer II/III was filled with horseradish peroxidase by intracellular iontophoresis. The very extensive local axonal field was composed of 330 terminal bouton rows in layer II/III and a sparse descending collateral projection to infragranular layers. A computer-assisted reconstruction of the axonal field in three dimensions revealed the following: The main output of the cell is to pyramidal neurons that lie deeper than the soma; the axonal arborization occupies an area of 400 micron in the anteroposterior axis and extends 200 micron along the mediolateral axis; the terminal bouton rows in layer II/III form clusters about 50 micron wide running approximately at right angles to the border between areas 17 and 18, with an intercluster interval of about 100 micron. These findings suggest that the terminals of an individual axoaxonic cell could be contained within one ocular dominance column but that there may be inhomogeneities in the weighting of the axoaxonic input to pyramidal cells in the supragranular layers.

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PubMed1983

The section-Golgi impregnation procedure. 1. Description of the method and its combination with histochemistry after intracellular iontophoresis or retrograde transport of horseradish peroxidase.

A method is described for impregnating neurons by the Golgi procedure in sections of brain tissue that are of a thickness 80-100 microns, so allowing a variety of histochemical procedures to be carried out prior to Golgi-impregnation. The method has been applied to the retina of the adult primate and to brain sections incubated to reveal horseradish peroxidase activity with substrates that give electron-dense reaction products. Sections containing neurons that have been filled with horseradish peroxidase by intracellular iontophoresis following electrophysiological characterization, or containing neurons retrogradely labelled by horseradish peroxidase, can be impregnated by this Golgi procedure. Subsequent gold-toning of the Golgi-impregnated neurons makes it possible to study, in the electron-microscope, both Golgi-impregnated and peroxidase-containing neurons and their afferent and efferent synaptic contacts within the same section. The procedure also makes it possible to repeat Golgi impregnation on sections already Golgi-impregnated and gold-toned, or to repeat the Golgi impregnation if the first impregnation is not satisfactory. The procedure is illustrated by examples from the monkey retina, cat visual cortex and rat neostriatum.

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PubMed1983

The section-Golgi impregnation procedure. 2. Immunocytochemical demonstration of glutamate decarboxylase in Golgi-impregnated neurons and in their afferent synaptic boutons in the visual cortex of the cat.

Sections of the cat's visual cortex were stained by an antiserum to glutamate decarboxylase using the peroxidase-antiperoxidase method; they were then impregnated by the section Golgi procedure and finally the Golgi deposit was replaced by gold. Neurons containing glutamate decarboxylase immunoreactivity were found in all layers of the visual cortex, without any obvious pattern of distribution. Fifteen immunoreactive neurons were also Golgi-impregnated and gold-toned, which enabled us to study the morphology and synaptic input of identified GABAergic neurons. These neurons were found to be heterogeneous both with respect to the sizes and shapes of their perikarya and the branching patterns of their dendrites. All the immunoreactive, Golgi-impregnated neurons had smooth dendrites, with only occasional protrusions. The synaptic input of glutamate decarboxylase-immunoreactive neurons was studied in the electron microscope. Immunoreactive neurons received immunoreactive boutons forming symmetrical synapses on their cell bodies. The Golgi-impregnation made it possible to study the input along the dendrites of immunoreactive neurons. One of the large neurons in layer III whose soma was immunoreactive was also Golgi-impregnated: it received numerous non-immunoreactive asymmetrical synaptic contacts along its dendrites and occasional ones on its soma. The same neuron also received a few boutons forming symmetrical synaptic contacts along its Golgi-impregnated dendrites; most of these boutons were immunoreactive for glutamate decarboxylase. Glutamate decarboxylase-immunoreactive boutons were also found in symmetrical synaptic contact with non-immunoreactive neurons that were Golgi-impregnated. A small pyramidal cell in layer III was shown to receive several such boutons along its somatic membrane. It is concluded that the combination of immunoperoxidase staining and Golgi impregnation is technically feasible and that it can provide new information. The present study has shown that there are many morphologically distinct kinds of aspiny GABAergic neurons in the visual cortex; that the predominant type of synaptic input to the dendrites of such neurons is from boutons forming asymmetrical synapses, but that some of the GABAergic neurons also receive a dense symmetrical synaptic input on their cell bodies, and occasional synapses along their dendrites, from the boutons of other GABAergic neurons. These findings provide a morphological basis, firstly, for a presumed powerful excitatory input to GABAergic interneurons and, secondly, for the disinhibition which has been postulated from electrophysiological studies to occur in the cat's visual cortex.

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PubMed1983

Retrograde transport of gamma-amino[3H]butyric acid reveals specific interlaminar connections in the striate cortex of monkey.

Several lines of evidence suggest that gamma-aminobutyric acid is an inhibitory neurotransmitter in the cerebral cortex. To study the intracortical projection of neurons that selectively accumulate this amino acid, we injected radioactive gamma-aminobutyric acid into the upper layers of the striate cortex of monkeys along tracks at an oblique angle to the pia. Sections from the injected area were then processed by a combination of autoradiography and Golgi impregnation to reveal the distribution of labeled neurons and their morphological characteristics. Labeled neurons always occurred around the injection site in each layer. In addition, a consistent radial pattern of perikaryal labeling was observed in layers IVc-VI below the injection track in layers I-IVa. The closer the injection track was to the pia the deeper the peak density of labeled cells appeared. After injection in layers IVa and the lower part of III, the highest number of labeled neurons was in layer IVc; after injection in the upper part of layer III, most labeled neurons were in layer V; and, after injection in layers I and II, the proportion of labeled neurons increased in the lower part of layer V and in layer VI. All these neurons in the infragranular layers are presumably labeled by retrograde axonal transport via the labeled fiber bundles that extended from upper to lower layers. Thirty-four Golgi-stained neurons of various types were also examined for retrograde labeling. Two were labeled, and both were aspiny stellate cells in layer V. The arrangement of these putative GABAergic neurones, with axons that ascend from lower to upper layers in a regular pattern and arborize locally, would enable them to mediate inhibition within cortical columns and between neighboring columns.

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PubMed1982

The axo-axonic interneuron in the cerebral cortex of the rat, cat and monkey.

The synaptic connections of a specific type of identified cortical interneuron, the axo-axonic cell, were studied using Golgi methods. In the light-microscope axo-axonic cells were demonstrated in certain layers of the primary and secondary visual cortex of rat, cat and monkey, in the motor cortex of cat and in the subiculum and pyriform cortex of rat. The dendrites originating from the oval soma were oriented radially in a lower and upper spray within a cylinder about 100-150 microns wide. Electron-microscopy of Golgi impregnated, gold-toned axo-axonic cells showed predominantly but not exclusively asymmetrical synaptic contacts on their dendrites and spines, few synaptic contacts on the perikarya some of which were asymmetrical, and no synaptic contacts on the axon initial segment. The axon usually arborized within the vicinity of the cell's own dendritic field in an area 100-200 microns in diameter. In the kitten motor cortex the axon of a neuron in layer III descended to layer VI, providing a columnar arborization. The axon formed specialized, 10-50 microns long terminal segments invariably oriented parallel with the axon initial segment of pyramidal cells. All 85 identified symmetrical-type synaptic contacts, deriving from 31 specialized terminal segments, were found exclusively on the axon initial segment of pyramidal neurons. Rare, lone boutons of axo-axonic cells also made synaptic contact only with axon initial segments, confirming the exclusive target specificity of these cells. In identified gold-toned boutons, flattened pleomorphic vesicles were present. Electron-microscopy showed that axons ending in specialized terminal segments may originate from myelinated fibres, indicating that Golgi impregnation has revealed only part of the axon. Counting of axon terminal segments, each of which was in contact with the axon initial segment of a pyramidal neuron, revealed 166 pyramidal neurons receiving input from a partially reconstructed axo-axonic cell in the motor cortex of the kitten, and 67 from another cell in the visual cortex of the cat. The convergence of five axo-axonic cells onto one pyramidal cell was demonstrated in the striate cortex of the cat by counting all synaptic contacts on three initial segments. Cells from a one-month-old kitten were compared with those of the adult. The axon of the developing neurons was more diverse, having many growth cones and filopodia which made no specialized membrane contacts. However, the developing specific terminal segments formed synapses only with axon initial segments. It is concluded that the presence of axo-axonic cells in all the species and cortical areas we have examined suggests their association with the structural design of pyramidal cells, wherever the latter occur, and with their participation in the information processing of pyramidal cells. Axo-axonic cells are uniquely endowed with the means of simultaneously influencing the action potential at the site of origin in groups of pyramidal cells...

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PubMed1981

Selectivity of neuronal [3H]GABA accumulation in the visual cortex as revealed by Golgi staining of the labeled neurons.

[3H]GABA was injected into the visual cortex of rats in vivo. The labeled amino acid was demonstrated by autoradiography using semithin sections of Golgi material. Selective accumulation was seen in the perikarya of Golgi-stained, gold-toned, aspinous stellate neurons. Spine-laden pyramidal-like cells did not show labeling. This method gives direct information about the dendritic arborization of a neuron, and its putative transmitter, and allows the identification of its synaptic connections.

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PubMedDate not supplied

Construction of a recombinant herpesvirus expressing the jellyfish green fluorescent protein.

Here we report the insertion of a synthetic version of the cDNA encoding the jellyfish (Aequorea victoria) green fluorescent protein (gfph ) into the genome of pseudorabies (Aujeszky's disease) virus (PrV). A putative latency promoter (PLAT) located at the inverted repeat region of the PrV genome was chosen as the target site for the insertion. Recombinant viral DNA designated as vLAT-gfp was generated as a result of homologous recombination between the transfected viral DNA and a plasmid containing the GFP-expression cassette flanked by viral sequences homologous to the target region. Plaques containing recombinant virus were selected visually using a fluorescent microscope. We demonstrated a GFP-expression in infected neurons of rat brain which showed normal morphology at early stage of viral infection by monitoring fluorescent light emission.

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PubMedDate not supplied

Simultaneous anterograde labelling of two afferent pathways to the same target area with Phaseolus vulgaris leucoagglutinin and Phaseolus vulgaris leucoagglutinin conjugated to biotin or dinitrophenol.

Anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L) and PHA-L conjugated to either biotin or 2,4-dinitrophenol were used to simultaneously label two populations of axons converging onto the same target area. Using the rat hippocampus as a test system, the septohippocampal and contralateral hilar afferents were labelled with the tracers. Double immunohistochemical procedures and either nickel-enhanced 3,3'-diaminobenzidine (DAB) reaction (blue-black colour) or DAB alone (brown colour) were used to produce contrasting colours for the different tracers in the same section. Both the biotinylated PHA-L and PHA-L conjugated with dinitrophenol were used successfully as anterogradely transported axonal tracers. They produced extensive axonal labelling in the hippocampal formation. Excellent double labelling could be produced with the simultaneous application of PHA-L and biotinylated PHA-L. Biotinylated PHA-L was visualized by a sequence of avidin-biotinylated peroxidase complex (ABC), biotinylated goat antiavidin and ABC again using nickel-enhanced DAB as chromogen yielding a blue-black reaction endproduct. PHA-L alone was detected by the unlabelled antibody enzyme (PAP) method using goat antibodies to PHA-L, and DAB as chromogen for the peroxidase reaction, resulting in brown axons. The combination of biotinylated PHA-L and DNP-conjugated PHA-L gave similar results, although the sensitivity of detection by the latter procedure was inferior to that obtained with the other tracers. These protocols permitted visualization of axons of different origin, together with their terminals, either in a blue-black or brown colour, and also allowed the demonstration of overlapping inputs in strata radiatum and lacunosum moleculare of the hippocampus and stratum moleculare and hilus of the dentate gyrus.

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