Glutamate and AMPA receptor immunoreactivity in Ia synapses with motoneurons and neurons of the central cervical nucleus

Abstract

Axonal tracing and high resolution immunocytochemistry were used to identify transmitter content and postsynaptic receptors in synapses between Ia primary afferents and motoneurons and in neurons of the central cervical nucleus (CCN), respectively, in the rat. The terminals, as well as the target neurons, were identified by postembedding immunogold detection of transganglionically or retrogradely, respectively, transported cholera toxin B subunit (CTB), and in adjacent sections postembedding immunogold was employed to demonstrate glutamate and AMPA receptors in the same synapses. A total of 390 CTB-labelled Ia boutons in apposition to CTB-labelled motoneurons, CCN neurons or unlabelled dendrites in the surrounding neuropil were traced in section series from two animals. A third animal was used as a control. In the motor nucleus, a majority of the synapses were with medium-sized dendrites, whereas in the CCN the distribution was skewed towards fine-calibre dendrites. In both nuclei, somatic and juxtasomatic synapses were quite infrequent (<10%). All of the CTB-labelled Ia boutons recovered in the sections incubated for glutamate (n=323) were enriched with glutamate immunoreactivity. One hundred and fifty of these disclosed synaptic contact in at least two ultrathin sections. In this sample, 50% (33-59%) appeared immunoreactive to receptor sub-units GluR1-4 in at least two ultrathin sections, whereas 35% were labelled in one section only. Distribution of gold particles relative to presynaptic and postsynaptic membrane profiles (n=23) revealed a close correlation between AMPA immunoreactivity and the postsynaptic membrane of the synapse. Finally, immunogold particles signalling GluR1 were observed much less frequently than particles signalling GluR2/3 or GluR4. Our results provide additional strong evidence that chemical transmission at Ia synapses is mediated by glutamate and identify GluR2/3 and GluR4 as important postsynaptic receptors.

Distribution of vesicular glutamate transporters 1 and 2 in the rat spinal cord, with a note on the spinocervical tract

“…There is now abundant evidence that VGLUT1 is present in large diameter low threshold skin and muscle primary afferent terminals in the spinal cord, corroborating extensive electrophysiological/pharmacological and glutamate immunogold labeling data supporting a transmitter role of glutamate in such terminals (e.g. Salt and Hill, 1983;Jahr and Yoshioka, 1986;Schouenborg and Sjölund, 1986;Gerber and Randic, 1989;Walmsley and Nicol, 1991;Maxwell et al, 1990a;Broman et al, 1993;Maxwell et al, 1993;Broman and Ådahl, 1994;Valtschanoff et al, 1994;Örnung et al, 1995;Larsson et al, 2001;Ragnarson et al, 2003). Dorsal rhizotomy results in significant depletion of large VGLUT1 immunolabeled varicosities in the ventral horn and deep dorsal horn (Li et al, 2003;Oliveira et al, 2003;Alvarez et al, 2004;Wu et al, 2004), and essentially all primary afferent terminals in these VGLUT1 and VGLUT2 co-localize in a proportion of varicosities in laminae III-IV and lamina IX, further support the notion that some primary afferent terminals in both the deep dorsal horn and the ventral horn express VGLUT2 in addition to VGLUT1.…”

Numbers, Densities, and Colocalization of AMPA- and NMDA-Type Glutamate Receptors at Individual Synapses in the Superficial Spinal Dorsal Horn of Rats

“…Popratiloff et al, 1996S. A. Popratiloff et al, 1998;Ragnarson et al, 2003), the pattern of AMPA and NMDA receptor expression at individual synapses remains largely elusive.…”

Widespread Expression of the AMPA Receptor GluR2 Subunit at Glutamatergic Synapses in the Rat Spinal Cord and Phosphorylation of GluR1 in Response to Noxious Stimulation Revealed with an Antigen-Unmasking Method

“…Although conventional immunocytochemical studies have been performed to detect AMPA subunits (Tachibana et al, 1994;Jakowec et al, 1995a;Popratiloff et al, 1996Popratiloff et al, , 1998Morrison et al, 1998;Spike et al, 1998), it is unlikely that these revealed receptors at glutamatergic synapses, because these are thought to be inaccessible due to extensive cross-linking of the protein meshwork of the synaptic cleft and postsynaptic density that results from aldehyde fixation (Baude et al, 1995;Ottersen and Landsend, 1997;Fritschy et al, 1998;Watanabe et al, 1998). Postembedding immunogold-labeling has been used to investigate synaptic AMPA receptors in the spinal cord (Popratiloff et al, 1996(Popratiloff et al, , 1998Morrison et al, 1998;Ragnarson et al, 2003); however, despite this, the pattern of AMPA subunit expression at individual synapses remains largely elusive.…”