Hironobu Katsumaru

GABAergic neurons containing the Ca2+-binding protein parvalbumin in the rat hippocampus and dentate gyrus

The distribution of Ca2+-binding protein, parvalbumin (PV), containing neurons and their colocalization with glutamic acid decarboxylase (GAD) were studied in the rat hippocampus and dentate gyrus using immunohistochemistry. PV immunoreactive (PV-I) perikarya were concentrated in the granule cell layer and hilus in the dentate gyrus and in the stratum pyramidale and stratum oriens in the CA3 and CA1 regions of the hippocampus. They were rare in the molecular layer of the dentate gyrus, in the stratum radiatum and in the stratum lacunosum-moleculare of the hippocampus. PV-I axon terminals were restricted to the granule cell layer, the stratum pyramidale and the immediately adjoining zones of these layers. Almost all PV-I neurons were also GAD immunoreactive (GAD-I), whereas only about 20% of GAD-I neurons also contained PV. The percentages of GAD-I neurons which were also immunoreactive for PV were dependent on the layer in which they were found; i.e. 40-50% in the stratum pyramidale, 20-30% in the dentate granule cell layer and in the stratum oriens of the CA3 and CA1 regions, 15-20% in the hilus and in the stratum lucidum of CA3 region and only 1-4% in the dentate molecular layer and in the stratum radiatum and the stratum lacunosum-moleculare of the CA3 and CA1 regions. PV-I neurons are a particular subpopulation of GABAergic neurons in the hippocampal formation. Based on their morphology and laminar distribution, they probably include basket cells and axo-axonic cells.

Fast-spiking cells in rat hippocampus (CA1-region) contain the calcium-binding protein parvalbumin

Fast spiking cells in the CA1 region of the rat hippocampus were revealed as gamma-aminobutyric acid (GABA)ergic non-pyramidal cells containing the calcium-binding protein parvalbumin by intracellular injection of Lucifer yellow in vitro in combination with postembedding parvalbumin immunohistochemistry.

Nuclear actin bundles in Amoeba, dictyostelium and human HeLa cells induced by dimethyl sulfoxide

In a previous study we demonstrated that dimethyl sulfoxide (DMSO) induces the formation of microfilament bundles in the interphase nucleus of a cellular slime mold, Dictyostelium mucoroides [12], in which the microfilaments bound rabbit skeletal muscle heavy meromyosin, forming an ‘arrowhead’ structure, and that this binding could be reversed by Mg2+ and ATP. In the present study, we show electron microscopic data demonstrating the occurrence of such microfilament bundles in the nucleus of Amoeba proteus and human HeLa cells, as well as in D. mucoroides. The similarities in the morphology and dimension of the microfilanets, as well as the specific conditions by which they are induced, suggested that these microfilaments are actin. We present evidence that actin is involved in interphase nucleus of a variety of organisms, and that DMSO acts on the molecules to induce microfilament bundles specifically in the nucleus.

A quantitative study of synaptic reorganization in red nucleus neurons after lesion of the nucleus interpositus of the cat: an electron microscopic study involving intracellular injection of horseradish peroxidase

A quantitative electron microscopic analysis of the corticorubral projection was performed in the red nucleus (RN) of adult cats to determine morphological correlates of synaptic reorganization that occur following a lesion of the interpositus nucleus (IP). Corticorubral synaptic endings were identified by lesioning the sensorimotor cortex 2-6 days before electrophysiological experiments. Horseradish peroxidase (HRP) was injected into electrophysiologically identified RN neurons. Sagittal sections 100 micrometers thick were cut and reacted by diaminobenzidine. Sections containing HRP-positive neurons were selected and embedded in Epon. In normal cats, degenerating corticorubral terminals in the RN region frequently made contact with dendritic profiles, having small cross-sections, while a few made contact with somatic profiles. Similar results were obtained when degenerating terminals making contact with HRP-filled dendrites were analyzed. In the experimental animals, the cortical lesion was performed more than 8 weeks after lesion of the IP. In these animals, degenerating corticorubral terminals were frequently found on proximal dendrites and somata in RN region and HRP-positive neurons in contrast to the findings in normal cats. The results indicate that new corticorubral synapses were formed on proximal dendrites and somata of RN neurons as a consequence of IP lesions.

Plasticity of neuronal connections in developing brains of mammals

Although mature nervous systems show substantial malleability following various surgical or environmental manipulations, developing brains show far more prominent plasticity, particularly in terms of morphological features. Neuronal circuits, for example, can be dramatically rewired following neonatal but not adult brain lesions. It remains unknown why neuronal circuits in developing brains show such remarkable plasticity. A number of anatomical and physiological studies suggest that there are transient projections in developing brains and they are eliminated by cell death and/or collateral elimination as development proceeds. This raises a possibility that aberrant projections observed following various surgical or environmental manipulations such as partial denervation, results from retention or stabilization of transient projections. However, evidence suggests that cell death does not play an important role in developmental fine-tuning of neuronal projections. Furthermore, although the elimination of axon collaterals takes place, individual neurons appear to elaborate axonal arbors in appropriate target areas, resulting in a net increase in the size of axonal arbor emerging from individual neurons. In accord with these observations, the number of synapses appear to increase during the period when axonal elimination proceeds. Taken together, reinforcement of appropriate projections rather than elimination of excessive connections plays a major role in developmental specification of neuronal connections. Appearance of aberrant projections after partial denervation may not be a consequence of disordered axonal growth, since they form topographic maps which precisely mirrors those for normal projections. They may be induced due to reinforcement of pre-existing neuronal connections rather than to construction of novel pathways. Observations of axonal morphology in denervated areas indicate that lesion-induced enlargement of projections is due to transformation of axonal morphology, from simple and poorly branched to multiply branched. Perhaps such simple and poorly branched axons in inappropriate target areas may represent ones in the course of elimination but they may serve as a source of sprouting when denervated. In other words, after total elimination of axons any surgical or environmental manipulation cannot induce enlargement of projections. The mechanisms underlying such modifiability of neuronal connections remains unclarified but possible participation of an activity-dependent competitive mechanism is discussed.

Developing corticorubral axons of the cat form synapses on filopodial dendritic protrusions

Developing neurons transiently grow numerous spine- or filopodium-like dendritic protrusions (SLDPs). Electron microscopy on identified input and intracellular staining of postsynaptic cells were performed to gain insight into their significance. Newborn kitten-corticorubral axons, labelled with biocytin, commonly made synapses on SLDP, often multiply invaginated by the SLDPs. Correspondingly, intracellularly labelled kitten rubrospinal cells had numerous SLDPs. Taking into account that corticorubral synapses are largely formed on dendritic shafts in adult cats, it is likely that the SLDPs play some important role in the development of corticorubral synapses. We hypothesize that rubrospinal cells elongate SLDPs searching for corticorubral axons to form synapses.

Immunocytochemical demonstration of GABAergic synapses on identified rubrospinal neurons

GABAergic synapses on rubrospinal neurons were demonstrated with immunocytochemistry combined with intracellular injection of horseradish peroxidase. Sections containing red nucleus neurons were processed for glutamic acid decarboxylase (GAD) immunohistochemistry. GAD-immunoreactive synaptic endings formed synaptic contacts with somata and dendrites of red nucleus neurons and identified rubrospinal neurons. Our observation provides further evidence that GABA acts as an inhibitory transmitter mediating cortically evoked inhibitory postsynaptic potentials in red nucleus neurons.

GABAergic intrinsic interneurons in the red nucleus of the cat demonstrated with combined immunocytochemistry and anterograde degeneration methods

The presence of glutamic acid decarboxylase (GAD), the enzyme synthesizing gamma-aminobutyric acid (GABA), was investigated in the red nucleus by an immunocytochemical method. The ipsilateral sensorimotor cortex was ablated prior to the immunocytochemical procedures to examine whether cortical neurons make synaptic contacts with GAD-immunoreactive neurons. Small GAD-immunoreactive neurons with a major diameter of 16.1 +/- 3.2 micron (mean +/- S.D.) were observed in the red nucleus under both light and electron microscopy. They were uniformly distributed throughout the nucleus. Degenerating axon terminals were found making synaptic contact with GAD-immunoreactive neurons in the red nucleus, which suggests that there is an input from the ipsilateral sensorimotor cortex to these neurons. This observation, along with our previous findings that GABAergic axon terminals make synaptic contact with the rubrospinal neurons, provides anatomical evidence for the presence of intrinsic GABAergic interneurons which mediate cortical inhibition in cat rubrospinal neurons.

Dendritic and somatic appendages of identified rubrospinal neurons of the cat.

Giant neurons of the red nucleus of the cat were stained intracellularly with horseradish peroxidase and examined using light microscopy, electron microscopy of thin sections, and high voltage electron microscopy of thick sections (2-5 microns). Special attention was paid to the arrangement of dendritic spines and other appendages relative to the distribution of synaptic contacts from known sources. In the region of the neuron known to receive synaptic contacts from the nucleus interpositus of the cerebellum (soma and proximal 200-300 microns of dendrites), the dendrites were relatively unbranched, and free of long spines or complex appendages. The surface of the neurons in this region was covered with a dense layer of short thin appendages that invaginated or penetrated between the synaptic terminals that cover this part of the cells. The small spines received synapses of the types associated both with the cerebellar afferent fibers and with the local inhibitory interneurons. These same terminals made synaptic contacts directly onto the surface of the neurons and onto the lateral surfaces of the spines, suggesting that the spines may serve primarily to increase the available synaptic surface area. The more distal portion of the dendritic field, where cerebellar afferents do not make synaptic contacts, exhibited a dramatically different appearance. The dendrites were much more branched, and exhibited many and varied dendritic appendages. The appendages were of three general types. One was a large protrusion with a cup-shaped head that formed the principal postsynaptic component of a glomerular arrangement also involving an axon terminal and usually a presynaptic dendrite. A second was a long thin filiform process that usually occurred around the glomeruli. This appendage was occasionally postsynaptic. The third was a spherical appendage containing many lysosomal organelles resembling residual bodies. The glomerular dendritic protrusions were very common in the distal portion of the dendritic field, numbering at least 1000 per cell. At least some of the glomeruli are specialized for receipt of synaptic input from the corticorubral pathway, since lesions of sensorimotor cortex resulted in degeneration of the central synaptic terminal in some glomeruli on horseradish peroxidase-injected rubrospinal neurons. These specializations of dendritic structure may contribute to the differences in excitatory postsynaptic potential wave shape between cortical and cerebellar inputs, and they may play a role in the changes in the cortical excitatory postsynaptic potential that develop after lesions of cerebellar inputs.(ABSTRACT TRUNCATED AT 250 WORDS)

ULTRASTRUCTURAL LOCALIZATION OF TELENCEPHALIN, A TELENCEPHALON-SPECIFIC MEMBRANE GLYCOPROTEIN IN RABBIT OLFACTORY BULB

Localization of a telencephalon-specific glycoprotein, telencephalin (TCLN), in the olfactory bulb of the rabbit was studied with an electron microscope. Anti-TCLN antisera appeared to stain plasma membrane, Golgi apparatus and multivesicular bodies of granule cells which are local circuit interneurons in the bulb. Principal neurons, mitral and tufted cells, were not immunoreactive. No glial cells showed immunoreactivity. Thus, expression of telencephalin is specific not only to the telencephalic segment of the brain, but also to the neuronal types.