Organization of Projection-Specific Interneurons in the Spinal Cord of the Red-Eared Turtle

Abstract

Using differential retrograde axonal tracing, we identified motoneurons (MNs) and projection-specific interneuron (IN) classes in lumbar segment D9 of the adult red-eared turtle spinal cord. We characterized the distribution of these neurons in the transverse plane, and estimated their numbers and proportions. Different labeling paradigms allowed us to distinguish ipsilateral INs (IINs) from commissural INs (CINs), and to identify IINs and CINs with either ascending (a) axons, descending (d) axons, or axons that bifurcate to both ascend and descend (ad). Local interneurons with axons shorter than 1 segment in length were not studied. We show that most retrogradely labeled INs are located dorsal to the MNs, in the ventral horn, the intermediate zone and the dorsal horn. IINs predominate in the dorsal horn. CINs are located on average more medially than the IINs in the ventral horn and intermediate zone. Within the IIN and CIN populations, aINs and dINs overlap extensively. The adIINs and adCINs make up only a small fraction of the total number of INs and are scattered throughout much of the respective IIN and CIN domains. The proportions of IINs and CINs are about equal, as are the proportions of aIINs versus dIINs, of aCINs versus dCINs, and of adIINs versus adCINs. The findings are compared to the organization of lumbar spinal INs in other vertebrate species.

Stereological Estimate of the Total Number of Neurons in Spinal Segment D9 of the Red-Eared Turtle

“…In contrast, the number of lumbar motoneurons is Ͻ2000 in adult bull frogs (Farel et al, 1992) and only 3500 in rodents (Oppenheim, 1986). Our finding that interneurons in segment D9 outnumber motoneurons by a factor of eight is surprisingly close to the ratio of 5:1 between projecting interneurons and motoneurons in the same segment (Nissen et al, 2008). The estimated number of projecting interneurons is conservative because fluorescent dextran may be a less effective tracer when crystals are applied at the cut surface of the spinal cord rather than the ventral root (Nissen et al, 2008).…”

“…The stereological counting procedure was verified by comparing the estimated average number of MN (2049) in segment D9 to the number of myelinated fibers in the segmental ventral roots (2114). The discrepancy in total number of motoneurons in a previous study (Nissen et al, 2008) may be due in part to the difference in definition of the boarders of segment D9 and in cell counting methods, since the former study was not based on purely unbiased counting methods. Based on the number of myelinated fibers in the ventral roots, each segment in D8 -D10 contains 2000 -3000 motoneurons (Nissen et al, 2008).…”

“…The discrepancy in total number of motoneurons in a previous study (Nissen et al, 2008) may be due in part to the difference in definition of the boarders of segment D9 and in cell counting methods, since the former study was not based on purely unbiased counting methods. Based on the number of myelinated fibers in the ventral roots, each segment in D8 -D10 contains 2000 -3000 motoneurons (Nissen et al, 2008). Assuming a similar number of segmental motoneurons in segments S1 and S2, we estimate the total number of hindlimb motoneurons in the lumbar enlargement of the turtle to be at least 10,000.…”

“…Motoneurons were provisionally identified from their morphology, large size, distinct slender and long shape, and location in the ventral half of the ventral horn (Fig. 2 B)-a distinction also used by Nissen et al (2008). The validity of the motoneurons count was verified by the counts of myelinated axons in D9 ventral roots.…”

“…Insight in the anatomy of the scratch network was provided by studies of the propriospinal projection patterns within the lumbar enlargement (Berkowitz, 2004) and from rostral segments serving the scratch receptive fields (Currie and Stein, 1990;Berkowitz and Stein, 1994). A recent study initiated attempts to quantify the intersegmental projections in the hindlimb enlargement (Nissen et al, 2008). The number of neurons in D9 with intersegmental projections was estimated to be five times higher than the number of motoneurons in the segment.…”