The Glutamatergic Neurons in the Spinal Cord of the Sea Lamprey: An In Situ Hybridization and Immunohistochemical Study

Abstract

Glutamate is the main excitatory neurotransmitter involved in spinal cord circuits in vertebrates, but in most groups the distribution of glutamatergic spinal neurons is still unknown. Lampreys have been extensively used as a model to investigate the neuronal circuits underlying locomotion. Glutamatergic circuits have been characterized on the basis of the excitatory responses elicited in postsynaptic neurons. However, the presence of glutamatergic neurochemical markers in spinal neurons has not been investigated. In this study, we report for the first time the expression of a vesicular glutamate transporter (VGLUT) in the spinal cord of the sea lamprey. We also study the distribution of glutamate in perikarya and fibers. The largest glutamatergic neurons found were the dorsal cells and caudal giant cells. Two additional VGLUT-positive gray matter populations, one dorsomedial consisting of small cells and another one lateral consisting of small and large cells were observed. Some cerebrospinal fluid-contacting cells also expressed VGLUT. In the white matter, some edge cells and some cells associated with giant axons (Müller and Mauthner axons) and the dorsolateral funiculus expressed VGLUT. Large lateral cells and the cells associated with reticulospinal axons are in a key position to receive descending inputs involved in the control of locomotion. We also compared the distribution of glutamate immunoreactivity with that of γ-aminobutyric acid (GABA) and glycine. Colocalization of glutamate and GABA or glycine was observed in some small spinal cells. These results confirm the glutamatergic nature of various neuronal populations, and reveal new small-celled glutamatergic populations, predicting that some glutamatergic neurons would exert complex actions on postsynaptic neurons.

Anatomical recovery of the spinal glutamatergic system following a complete spinal cord injury in lampreys

“…The antibody has been characterized with respect to cross reactivity by antibody dilution experiments as well as by absorption experiments53. The same staining pattern was obtained with both monoclonal and polyclonal anti-glutamate antibodies in this study and in sections of the brain and retina (unpublished observations), and it does not differ from the pattern of expression of VGLUT as shown by in situ hybridization304151.…”

“…A detailed description of the spinal glutamatergic system of the sea lamprey has been previously reported30. Briefly, in control un-lesioned animals, glutamate immunoreactive (-ir) neuronal processes were observed in the dorsal, ventral and lateral columns of the white matter, being more abundant in the dorsal column (Fig.…”

“…On the other hand, the lateral population of glutamate-ir cells recovers faster in the caudal than in the rostral stump (2 vs 4 wpl, respectively). Cells of the lateral population are considered to be mainly excitatory interneurons (EINs) that participate in the central pattern generator (CPG)30, excite motor neurons and other interneurons and are involved in rhythm generation (for review see ref. 19,20).…”

“…19,20). Cells of the dorsal population probably participate in the sensory relay and have dendrites that reach the dorsal column30. In electrophysiological studies, muscle locomotor activity has been observed at 2 wpl just below the lesion1640.…”

“…These include the standard 20 amino acids found in proteins, the non-protein amino acids D-serine, D-alanine and D-aspartate, GABA and the glycine containing tripeptide glutathione, which did not yield significant cross reactivity. This antibody was developed by Dr David V. Pow (University of Newcastle, New South Wales, Australia) and used in previous studies of the lamprey brain and spinal cord2530415051. This antibody did not label any native protein band from the sea lamprey brain51.…”