Enrichment of Glutamate‐like Immunoreactivity in Primary Afferent Terminals Throughout the Spinal Cord Dorsal Horn

Abstract

Although several lines of evidence indicate that glutamate is a neurotransmitter in primary afferent terminals, controversies exist on the proportion and types of such terminals that release glutamate. In the present study quantitative analysis of immunogold labelling was used to assess the presence of glutamate-like immunoreactivity in primary afferent terminals in laminae I-V of the rat spinal cord dorsal horn. Anterograde transport of choleragenoid-horseradish peroxidase from a spinal ganglion and tetramethyl benzidine histochemistry were used to identify primary afferent terminals in laminae I and III-V. Presumed C-fibre terminals in lamina II were identified on morphological criteria (dense sinusoid axon terminals). Primary afferent terminals in all dorsal horn laminae displayed significantly higher levels of glutamate-like immunoreactivity than pleomorphic vesicle-containing profiles in laminae III-IV and large neuronal cell bodies in laminae III-V. The density of gold particles over primary afferent terminals also significantly exceeded the average density of gold particles over laminae II and III-IV. The highest densities of gold particles were present over dense sinusoid axon terminals in lamina II. These findings suggest that glutamate, alone or in combination with other neuroactive compounds, is involved in the transfer of all sensory modalities from primary afferent fibres to dorsal horn neurons.

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.…”

“…This presumably explains why their disappearance is usually undetected following dorsal rhizotomy. Primary afferent terminals in lamina II contain high levels of glutamate (DeBiasi and Rustioni, 1988;Maxwell et al, 1990b;Broman et al, 1993;Broman and Ådahl, 1994;Valtschanoff et al, 1994;Larsson et al, 2001) and there is abundant physiological/pharmacological evidence that support glutamate as a fast primary afferent neurotransmitter in such terminals (e.g. Weinberg, 1989, Aanonsen et al, 1990;Broman et al, 2000).…”

Synaptic Vesicular Localization and Exocytosis ofl-Aspartate in Excitatory Nerve Terminals: A Quantitative Immunogold Analysis in Rat Hippocampus

“…286, Amiry-Moghaddam et al, 1994;no. 607, Ericson et al, 1995 andBroman et al, 1993;no. 34, Laake et al, 1986).…”

3-Hydroxyanthranilic acid oxygenase-containing astrocytic processes surround glutamate-containing axon terminals in the rat striatum

“…The sections, collected on nickel mesh grids and dried overnight at 50°C were immersed sequentially in the following solutions: (1) 1% periodic acid (HIO,) in doubledistilled water (ddW) for 7 min; (2) 8% sodium m-periodate (NaIO,) in ddW for 15 min; (3) 1% human serum albumin (HSA) in ddW for 10 min; (4) 1% HSA in Tris phosphate-buffered saline (TPBS, 0.4 gm KCl, 0.2 gm NaN,, 7.0 gm NaCl per liter of 0.01 M PB + 0.01 M Tris/ HCl) for 10 min; (5) glutamate antiserum (#607; diluted 1: lOO-1:900 in TPBS containing 2% HSA and preadsorbed with 100 pM aspartateglutaraldehyde complex, 200 FM glutamine-glutaraldehyde complex, and 200 PM P-alanine-glutaraldehyde complex) for 2 hr Broman et al, 1993); (6) 1% HSA in TPBS for 10 min; (7) 0.05 M Tris and 0.5 mg/liter polyethyleneglycol in ddW ("Tris buffer") for 3 min; and (8) colloidal gold (20 nm) coupled to anti-rabbit IgG (diluted 1:20 in Tris buffer) for 2 hr. All steps were followed with thorough rinses in either ddW or TPBS.…”