Isolation of dendrodendritic synapses from swine olfactory bulbs

Kornguth, Steven; Juhl, U.; Knobeloch, Lynda; Sunderland, E.; Johnson, Timothy J. A.; Scott, Grayson

doi:10.1016/0006-8993(76)90591-6

Cited by 12 publications

(9 citation statements)

References 18 publications

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“…In the course of an immunohistochemical screening of a number of mAbs raised against dendrodendritic synaptosomes (3) of the rabbit olfactory bulb, we noticed that one of them, mAb 271A6, strongly labeled the gray matter of all regions belonging to the telencephalic segment, whereas it did not stain other brain segments. In the present study, we have examined the spatial distribution of antigen 271A6 [i.e., molecule(s) recognized by mAb 271A6] in various regions of the central and peripheral nervous systems of the rabbit, using immunohistochemical staining methods and the dot-immunobinding assay.…”

mentioning

confidence: 99%

Telencephalon-specific antigen identified by monoclonal antibody.

Mori¹,

Fujita²,

Watanabe³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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Immunohistochemical screening of monoclonal antibodies (mAbs) raised against fractions rich in the dendrodendritic synaptosomes of the rabbit olfactory bulb revealed that one of the mAbs (mAb 271A6) recognized a telencephalon-specific antigen or antigens. Thus, the stain with mAb 271A6 was observed throughout the gray matter of all regions of the neocortex, piriform cortex, hippocampus, striatum, septum, and the amygdaloid nucleus, in addition to the main and accessory olfactory bulbs. The mAb 271A6, however, labeled neither nontelencephalic regions of the central nervous system nor the peripheral nervous system so far examined. Dot-immunobinding assays of homogenates of various brain regions also showed the telencephalon-specific distribution of the antigen designated as 271A6. Antigen 271A6 is developmentally regulated. At birth, the antigen was expressed in a small quantity only in phylogenetically older telencephalic regions such as the olfactory bulb, piriform cortex, striatum, cingulate cortex, and hippocampus. It was hardly detectable in most areas of the neocortex. The densities and areas of 271A6-positive structures increased during the early postnatal period. These results demonstrate a molecular specificity of the most rostral brain segment, the telencephalon. mAb 271A6 may be a good tool for obtaining a better understanding of the molecular basis of the segmental organization or the segment-specific functions of the brain.During prenatal development, the vertebrate brain forms five segmental enlargements (vesicles)-telencephalon, diencephalon, mesencephalon (or midbrain), metencephalon (pons and cerebellum), and myelencephalon (or medulla oblongata) (1, 2). Each vesicle gives rise to specific regions of the adult brain. This segmental organization is therefore assumed to be the basic plan for the development of the central nervous system, and it may have great significance in ordering the structure and function of the brain. Among these vesicles or segments, the most rostral one (the telencephalon) expands most conspicuously in mammals and forms the complex telencephalic structures of the adult brain such as the cerebral neocortex, piriform cortex, hippocampus, striatum, septum, amygdaloid nucleus, and olfactory bulb.Although the molecular basis of the brain's segmental organization is unknown, it can be assumed that the vertebrate nervous system expresses certain molecules that are specific to one or more of the brain segments. In the course of an immunohistochemical screening of a number of mAbs raised against dendrodendritic synaptosomes (3) of the rabbit olfactory bulb, we noticed that one of them, mAb 271A6, strongly labeled the gray matter of all regions belonging to the telencephalic segment, whereas it did not stain other brain segments. In the present study, we have examined the spatial distribution of antigen 271A6 [i.e., molecule(s) recognized by mAb 271A6] in various regions of the central and peripheral nervous systems of the rabbit, using immunohistochemical staining methods and...

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mentioning

confidence: 99%

Telencephalon-specific antigen identified by monoclonal antibody.

Mori¹,

Fujita²,

Watanabe³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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“…Charles River C D male rats, weighing between 200 and 300 g, were killed by decapitation. The olfactory bulbs from 15-20 rats were rapidly removed and placed in icecold 0.4 mM potassium phosphate buffer (pH 7.0) containing 0.32 M sucrose and 1 mM MgCI, (Sucrose A; Kornguth et al, 1976). The pooled olfactory bulbs were homogenized in 10 volumes of Sucrose A with a Dounce homogenizer and fractionated as previously described (Quinn and Cagan, 1980) to obtain a crude fraction of dendrodendritic synaptosomes (DDS) called fraction Ps' , and a nuclear pellet P,.…”

Section: Tissue Fractionationmentioning

confidence: 99%

“…Fractions PB' and P2 were each suspended in the original volume of Sucrose A, and the synaptosomes were further purified by centrifuging in a discontinuous sucrose density gradient (Kornguth et al, 1976). Each band of material was collected, sedimented, washed twice in Sucrose A, and used later to prepare membranes.…”

Section: Subcellular Fractionationmentioning

confidence: 99%

“…The binding of both ligands Subcellular distribution of binding activity The distribution of specific binding activity for ["HIGABA and [3HH]muscimol following subcellular fractionation is shown in Table 3. Fraction PB', which contains the dendrodendritic synaptosomes (Kornguth e t al., 1976;Quinn and Cagan, 1980), accounted for 86% of the binding activity recovered from the homogenate. Following centrifugation in the sucrose density gradient, the highest specific binding activity for both ligands was recovered in band 4, at the 1.U1.4 M sucrose interface (Table 3).…”

Section: [3h]gaba and [3h]muscimol Bindingmentioning

confidence: 99%

“…It is believed to act at dendrodendritic synapses between granule cell dendrites and secondary dendrites of mitral cells. A synaptosomal preparation that is highly enriched in dendrodendritic synapses was morphologically characterized after being isolated from the olfactory bulbs of porcine and bovine (Kornguth et al, 1976;. We recently isolated dendrodendritic synaptosomes (DDS) from rat olfactory bulbs and demonstrated that they contain a considerable amount of glutamate decarboxylase (GAD) activity (Quinn and Cagan, 1980), lending neurochemical support to the hypothesis that GABA functions as a neurotransmitter at the dendrodendritic synapses.…”

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confidence: 92%

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High Specific Binding of [³H]GABA and [³H]Muscimol to Membranes from Dendrodendritic Synaptosomes of the Rat Olfactory Bulb

Quinn

Çağan

1982

Journal of Neurochemistry

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Olfactory bulbs contain dendrodendritic synapses, which occur between granule cells and mitral cells, and gamma-aminobutyric acid (GABA) is thought to act as an inhibitory neurotransmitter at these synapses. Synaptosomes derived from the dendrodendritic synapses of the olfactory bulb were shown previously to contain considerable L-glutamate decarboxylase activity. The subcellular distribution and binding parameters of [3H]GABA and [3H]muscimol binding sites have now been determined in the rat olfactory bulb. Of all fractions examined, crude synaptic membranes (CSM) prepared from the dendrodendritic synaptosomes were shown to have the highest specific binding activity and accounted for nearly all of the total binding activity for both ligands. The specific binding activities for [3H]GABA and for [3H]muscimol were greatly increased after treating the CSM with 0.05% Triton X-100. Binding was shown to be Na+-independent, reversible, pharmacologically specific, and saturable. High- and low-affinity sites were detected for both ligands, and both classes of sites had appreciably lower KD values for muscimol (KD1 = 3.1 nM, KD2 = 25.1 nM) than for GABA (KD1 = 8.6 nM; KD2 = 63.7 nM). The amounts of the high-affinity binding sites for muscimol and GABA were similar (Bmax = 1.7 and 1.5 pmol/mg protein, respectively). The results of the present experiments indicate that the GABA and muscimol binding sites represent the GABA postsynaptic receptor, presumably on mitral cell dendrites, and provide further support for the hypothesis that GABA functions as a neurotransmitter at the dendrodendritic synapses in the olfactory bulb.

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Chloride‐dependent binding sites for L‐[³H]glutamate on dendrodendritic synaptosomal membranes of rat olfactory bulb

Quinn

Spraguer

1986

J of Neuroscience Research

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Dendrodendritic synapses occur between granule cell dendrites and secondary dendrites of mitral cells within the olfactory bulb and are attainable in a subcellular fraction (DDS). Since the mitral cells are thought to utilize an excitatory amino acid as a neurotransmitter, we determined the pharmacologic specificity of Na+-independent L-[3H]glutamate binding to fresh membranes of DDS in 50 mM Tris-HCl, pH 7.1. Binding of L-glutamate to membranes of DDS was specific, Cl(-)-dependent, and saturable. Scatchard plots were analyzed by nonlinear regression analyses using the computer program LIGAND, and the data was best-fitted to a one-site model with KD of 0.56 +/- 0.04 microM and an apparent Bmax of 48 +/- 5 pmol/mg protein. Hill plots also indicated the presence of one site and no cooperativity (nH = 0.99 +/- 0.03). However, the relative effectiveness of several compounds in inhibiting L-glutamate binding to membranes of DDS clearly demonstrated the presence of more than one site. Electrophysiological studies suggest that 2-amino-4-phosphonobutyrate (APB) is a potent antagonist of evoked responses elicited by stimulation of mitral cell axons and that quisqualate is a potent agonist; both of these compounds were highly effective inhibitors of L-glutamate binding to DDS membranes. APB displaced about 70% of the sites labeled with 200 nM L-glutamate with a KI of 1.6 microM, whereas quisqualate inhibition of L-glutamate binding yielded a line that was curvilinear in the Scatchard plot and was resolved into two sites of relatively high affinity (KI values of 0.02 and 0.65 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

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Isolation of dendrodendritic synapses from swine olfactory bulbs

Cited by 12 publications

References 18 publications

Telencephalon-specific antigen identified by monoclonal antibody.

Telencephalon-specific antigen identified by monoclonal antibody.

High Specific Binding of [³H]GABA and [³H]Muscimol to Membranes from Dendrodendritic Synaptosomes of the Rat Olfactory Bulb

Chloride‐dependent binding sites for L‐[³H]glutamate on dendrodendritic synaptosomal membranes of rat olfactory bulb

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Isolation of dendrodendritic synapses from swine olfactory bulbs

Cited by 12 publications

References 18 publications

Telencephalon-specific antigen identified by monoclonal antibody.

Telencephalon-specific antigen identified by monoclonal antibody.

High Specific Binding of [3H]GABA and [3H]Muscimol to Membranes from Dendrodendritic Synaptosomes of the Rat Olfactory Bulb

Chloride‐dependent binding sites for L‐[3H]glutamate on dendrodendritic synaptosomal membranes of rat olfactory bulb

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High Specific Binding of [³H]GABA and [³H]Muscimol to Membranes from Dendrodendritic Synaptosomes of the Rat Olfactory Bulb

Chloride‐dependent binding sites for L‐[³H]glutamate on dendrodendritic synaptosomal membranes of rat olfactory bulb