Kainate-activated cobalt uptake in the primary gustatory nucleus in goldfish: Visualization of the morphology and distribution of cells expressing AMPA/kainate receptors in the vagal lobe

Abstract

Gustatory afferent fibers of the vagus nerve that innervate taste buds of the oropharynx of the goldfish, Carassius auratus, project to the vagal lobe, which is a laminated gustatory nucleus in the dorsal medulla. As in the mammalian gustatory system, responses by second-order cells in the goldfish medulla are mediated by N-methyl-D-aspartate (NMDA) and non-NMDA ionotropic glutamate receptors. We utilized a cobalt uptake technique to label vagal lobe neurons that possess cobalt-permeable ionotropic glutamate receptors. Vagal lobe slices were bathed in kainate (40 microM) or glutamate (0.5 or 1 mM) in the presence of CoCl(2), which can pass into cells through the ligand-gated cation channels of non-NMDA receptors made up of certain subunit combinations. Cobalt-filled cells and dendrites were observed in slices that were activated by kainate or glutamate, but not in control slices that were bathed in CoCl(2) alone, nor in slices that were bathed with the non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (10 microM) in addition to an agonist. Likewise, simple depolarization of the cells with KCl failed to induce cobalt loading. Cobalt-filled round unipolar cells, elongate or globular bipolar cells, and multipolar cells with elongate or polygonal perikarya were distributed throughout the cell layers in the sensory zone of the vagal lobe. Numerous labeled neurons had dendrites spanning layers IV and VI, the two principal layers of primary afferent input. Apical and basal dendrites often extended radially through neighboring laminae, but many cells also extended dendrites tangential to the lamination of the sensory zone. In the motor layer, cell bodies and proximal dendrites of small, multipolar neurons, and large motoneurons were regularly loaded with cobalt.

Co-occurrence of calcium-binding proteins and calcium-permeable glutamate receptors in the primary gustatory nucleus of goldfish

“…To cobalt-label cells with Ca ϩϩ -permeable glutamate receptors, vagal lobe slices were prepared similar to previous studies (Smeraski et al, 2001;Sharp and Finger, 2002). In brief, goldfish were cold-anesthetized and the brain was removed quickly.…”

“…In contrast, Korada and Schwartz (2000) show little correlation between CaBPs expression and the absence of GluR2 in the cochlear nucleus. The purpose of our study was to directly assess the degree of colocalization of Ca ϩϩ -permeable AMPA/kainate receptors and CaBPs in the primary gustatory nucleus in goldfish relying on direct visualization of the Ca ϩϩ -permeable neurons by kainatestimulated Co ϩϩ uptake as used previously by us (Smeraski et al, 2001) and other investigators (Pruss et al, 1991;Toomin and Millington, 1998;Yin et al, 1999;Van Den Bosch et al, 2000;Ogoshi and Weiss, 2003;Ogoshi et al, 2005).…”

“…Neurons containing Ca ϩϩ -permeable AMPA/kainate receptors can be visualized by incubating brain sections in Co ϩϩ -containing Ringer's solutions and precipitating the intracellular Co ϩϩ with sulfide (Pruss et al, 1991;Toomin and Millington, 1998;Yin et al, 1999;Van Den Bosch et al, 2000;Smeraski et al, 2001;Ogoshi and Weiss, 2003;Ogoshi et al, 2005). Smeraski et al (2001) applied this technique and showed that many neurons in the primary gustatory nucleus of goldfish have Ca ϩϩ -permeable non-NMDA ionotropic glutamate receptors.…”

“…Smeraski et al (2001) applied this technique and showed that many neurons in the primary gustatory nucleus of goldfish have Ca ϩϩ -permeable non-NMDA ionotropic glutamate receptors. In the present study, we combined the Co ϩϩ -filling technique with immunocytochemistry for calretinin, calbindin, and parvalbumin to assess the degree of co-occurrence of these traits in individual neurons.…”

Glutamatergic neurotransmission in the nucleus tractus solitarii: Structural and functional characteristics

“…However, these results obtained in rat contrast with observations made in the goldfish. It was found that numerous neurons in the sensory layers of the vagal lobe (fish equivalent of the rostral gustatory portion of the mammalian NTS) were cobalt-labeled by kainate application and are therefore likely to possess calciumpermeable AMPA/kainate receptors (Smeraski et al, 2001;Ikenaga et al, 2006). Whether this apparent discrepancy originates from species differences or the investigated regions (mid-caudal general visceral part versus rostral gustatory part) remained to be determined.…”