Synaptic connections of GABA-containing boutons in the lateral cervical nucleus of the cat: An ultrastructural study employing pre- and post-embedding immunocytochemical methods

Maxwell, David; Christie, W.M.; Somogyi, Péter

doi:10.1016/0306-4522(89)90319-9

Cited by 24 publications

(11 citation statements)

References 42 publications

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“…Moreover, our quantitative results support the general observation of Maxwell et al (1989) that GAD-positive nerve terminals were more heavily labeled for GABA than GAD-negative terminals.…”

Section: Discussionsupporting

confidence: 88%

“…Only one previous study has directly examined GABA immunogold labeling over GAD-positive and GAD-negative nerve terminals (Maxwell et al 1989). …”

Section: Discussionmentioning

confidence: 99%

“…A general presumption is that all nerve terminals containing GAD 67 , the active form of the enzyme, contain high levels of GABA, but this relationship has not been analyzed quantitatively. Maxwell et al (1989) showed that, in the lateral cervical nucleus of the cat, GAD-immunoreactive terminals exhibited high densities of gold labeling for GABA but did not show quantitatively whether the density of GABA labeling was high over all GAD-positive terminals and low over all GAD-negative terminals. We have previously used tissue fixed with 2.5% glutaraldehyde to localize GAD 67 immunoreactivity and showed that it was suitable for immunogold localization of the amino acid neurotransmitter glutamate (Murphy et al 1996).…”

mentioning

confidence: 92%

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Pre-embedding Staining for GAD₆₇ Versus Postembedding Staining for GABA as Markers for Central GABAergic Terminals

Murphy

Pilowsky

Llewellyn‐Smith

1998

J Histochem Cytochem.

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SUMMARY Pre-embedding immunocytochemistry for the active form of glutamate decarboxylase (GAD 67 ) and postembedding staining for ␥-aminobutyric acid (GABA) were compared as markers for central GABAergic terminals in the phrenic motor nucleus, in which phrenic motor neurons had been retrogradely labeled with cholera toxin B-horseradish peroxidase. Nerve terminals with or without GAD 67 immunoreactivity were identified in one ultrathin section. GABA was localized with immunogold in an adjacent section after etching and bleaching. GABA labeling density was assessed over 519 GAD 67 -positive and GAD 67 -negative nerve terminals in the phrenic motor nucleus. Frequency histograms showed that statistically higher densities of gold particles occurred over most GAD 67 -positive terminals. However, some GAD 67 -negative terminals also showed high densities of gold particles, and some GAD 67 -positive terminals showed low densities. Preabsorption of the anti-GABA antibody with a GABA-protein conjugate, but not with other amino acid-protein conjugates, significantly reduced gold labeling over both GAD 67 -positive and GAD 67 -negative terminals. These results show that the presence of GAD 67 immunoreactivity correlates strongly with high densities of immunogold labeling for GABA in nerve terminals in the phrenic motor nucleus. Preabsorption controls indicate that authentic GABA was localized in the postembedding labeling procedure. Only a small proportion of intensely GABAimmunoreactive terminals lack GAD 67 , suggesting that both GAD 67 and GABA are reliable markers of GABAergic nerve terminals. (J Histochem Cytochem 46:1261-1268, 1998) GABAergic nerve terminals throughout the central nervous system have been identified using either preor postembedding immunogold methods to localize ␥-aminobutyric acid (GABA), or pre-embedding immunocytochemistry to visualize glutamate decarboxylase (GAD), the enzyme required for synthesis of GABA from l-glutamate (McLaughlin et al. 1975;Gabbott et al. 1986;Babb et al. 1988;Zhan et al. 1989; Helfert et al. 1992;Tai and Goshgarian 1996). A general presumption is that all nerve terminals containing GAD 67 , the active form of the enzyme, contain high levels of GABA, but this relationship has not been analyzed quantitatively. Maxwell et al. (1989) showed that, in the lateral cervical nucleus of the cat, GAD-immunoreactive terminals exhibited high densities of gold labeling for GABA but did not show quantitatively whether the density of GABA labeling was high over all GAD-positive terminals and low over all GAD-negative terminals. We have previously used tissue fixed with 2.5% glutaraldehyde to localize GAD 67 immunoreactivity and showed that it was suitable for immunogold localization of the amino acid neurotransmitter glutamate (Murphy et al. 1996).Using similarly processed tissue in this study, we determined whether GAD 67 -containing nerve terminals labeled by pre-embedding immunocytochemistry showed high densities of gold particles after postembedding im-

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Section: Discussionsupporting

confidence: 88%

“…Only one previous study has directly examined GABA immunogold labeling over GAD-positive and GAD-negative nerve terminals (Maxwell et al 1989). …”

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 92%

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Pre-embedding Staining for GAD₆₇ Versus Postembedding Staining for GABA as Markers for Central GABAergic Terminals

Murphy

Pilowsky

Llewellyn‐Smith

1998

J Histochem Cytochem.

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“…For example, there is a dense GABAergic regulation of neurons of the lateral cervical nucleus (21). Furthermore, many gray matter neurons extend dendrites into the adjacent white matter, notably in the lateral funiculus (22), where they can be contacted by axons that ascend or descend in the white matter.…”

Section: Discussionmentioning

confidence: 99%

Neurons in the dorsal column white matter of the spinal cord: Complex neuropil in an unexpected location

Abbadie

Skinner

Mitrovic

et al. 1999

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

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It is common to think of gray matter as the site of integration in neural circuits and white matter as the wires that connect different groups of neurons. The dorsal column (DC) white matter, for example, is the spinal cord axonal pathway through which a topographic map of the body is conveyed to the somatosensory cortex. We now describe a network of neurons located along the midline of the DCs. The neurons are present in several mammals, including primates and birds, and have a profuse dendritic arbor that expresses both the neuron-specific marker, microtubule-associated protein-2, and the neurokinin-1 receptor, a target of the neuropeptide, substance P. Electron microscopy and double immunostaining for synaptophysin and a marker of ␥-aminobutyric acid-ergic terminals documented a rich synaptic input to these neurons. Finally, injection of a ␥-aminobutyric acid type A receptor antagonist or of substance P into the cerebrospinal f luid of the rat spinal cord induced Fos expression and internalization of the neurokinin-1 receptor in these neurons, respectively, indicating that the DC neurons are under tonic inhibitory control and can respond to neurotransmitters that circulate in the cerebrospinal f luid.Although neuronal cell bodies are concentrated in the gray matter of the spinal cord and brain, there are several exceptions. Most notable are two neuronal groups in the white matter of the lateral columns of the spinal cord, the lateral cervical nucleus (1) and the lateral spinal nucleus (2). The former is located at the first and second cervical segments and is found in all species. The latter is prominent in the rat and is present at all levels of the spinal cord. Importantly, electrophysiological and anatomical studies have established that neurons within these two groups can be readily distinguished from neurons in the neighboring dorsal horn gray matter.To date, distinct neuronal cell groups have not been identified in the dorsal column (DC) white matter, which contains large-diameter primary afferent and second-order axons en route to the medulla (3). Here we provide evidence that the DCs, in fact, contain a population of neuronal cell bodies and a distinct neuropil. This finding was uncovered in the course of our studies of the organization of spinal cord neurons that express the neurokinin-1 (NK-1) receptor, which is targeted by the peptide substance P (SP). The cell bodies are concentrated near the midline of the DC white matter. In the present paper we establish that these cells are, in fact, neurons, that they receive a dense synaptic input and that they are under a profound tonic ␥-aminobutyric acid (GABA)-ergic inhibitory control.

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“…reactive substances in the LCN (Blomqvist et al, 1985a; Giesler ondarv antibodies were used for evaluating the distribution of and Elde, 1985;Broman and Westman, 1988;Blomqvist, 1989a,b, 1990;Maxwell et al, 1989) including an enrichment of Glu-LI in SCT terminals ). However, the findings in these studies do not give any evidence regarding the neurotransmitter used by the CTT neurons.…”

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confidence: 99%

Cervicothalamic tract terminals are enriched in glutamate-like immunoreactivity: an electron microscopic double-labeling study in the cat

Broman

Ottersen

1992

J. Neurosci.

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The distribution of glutamate-like immunoreactivity (Glu-LI) in the thalamic ventral posterolateral nucleus (VPL) of cats was studied with the EM immunogold technique in order to identify nerve terminal populations that may use glutamate as a neurotransmitter. The investigation was focused on cervicothalamic tract (CTT) terminals, which were labeled by WGA-HRP transported anterogradely from injection sites in the lateral cervical nucleus (LCN). The amount of Glu-LI in different profiles was evaluated quantitatively by counting the number of gold particles and then calculating the areal density of gold particles over different profile types. The highest density of gold particles was found over terminals with morphologic characteristics of terminals of cortical origin (RS terminals), a finding that further supports the glutamatergic nature of these terminals suggested by previous studies. Enrichment of Glu-LI was also found in CTT terminals and in non-peroxidase-labeled terminals with the same morphologic characteristics as CTT terminals (RL terminals). The labeling density over these terminals was about twice the average tissue density of gold particles. The labeling density over large VPL neuronal cell bodies was on average 127%, and that over vesicle-containing dendritic appendages and truncs (presynaptic dendrites) about 80%, of the average tissue density of gold particles. Immunogold labeling with antiserum against glutamine (Gln) indicated low levels of Gln-like immunoreactivity in CTT terminals and a high Glu:Gln ratio as compared to astrocytes and the average Glu:Gln ratio in the VPL. The present findings provide further support for a transmitter role of glutamate in terminals of ascending somatosensory afferents to the VPL, including the CTT. Taken together with previous findings of an enrichment of Glu-LI in terminals of the spinocervical tract (Broman et al., 1990), our results suggest that synaptic transmission in the spinocervicothalamic pathway is dependent on the release of glutamate both at the levels of the LCN and the VPL.

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Synaptic connections of GABA-containing boutons in the lateral cervical nucleus of the cat: An ultrastructural study employing pre- and post-embedding immunocytochemical methods

Cited by 24 publications

References 42 publications

Pre-embedding Staining for GAD₆₇ Versus Postembedding Staining for GABA as Markers for Central GABAergic Terminals

Pre-embedding Staining for GAD₆₇ Versus Postembedding Staining for GABA as Markers for Central GABAergic Terminals

Neurons in the dorsal column white matter of the spinal cord: Complex neuropil in an unexpected location

Cervicothalamic tract terminals are enriched in glutamate-like immunoreactivity: an electron microscopic double-labeling study in the cat

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Synaptic connections of GABA-containing boutons in the lateral cervical nucleus of the cat: An ultrastructural study employing pre- and post-embedding immunocytochemical methods

Cited by 24 publications

References 42 publications

Pre-embedding Staining for GAD67 Versus Postembedding Staining for GABA as Markers for Central GABAergic Terminals

Pre-embedding Staining for GAD67 Versus Postembedding Staining for GABA as Markers for Central GABAergic Terminals

Neurons in the dorsal column white matter of the spinal cord: Complex neuropil in an unexpected location

Cervicothalamic tract terminals are enriched in glutamate-like immunoreactivity: an electron microscopic double-labeling study in the cat

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Pre-embedding Staining for GAD₆₇ Versus Postembedding Staining for GABA as Markers for Central GABAergic Terminals

Pre-embedding Staining for GAD₆₇ Versus Postembedding Staining for GABA as Markers for Central GABAergic Terminals