Synaptophysin immunoreactivity and distributions of calcium-binding proteins highlight the functional organization of the rat's dorsal column nuclei.

The mammalian dorsal column nuclei (DCN) are principally composed of the cuneate (CN) and gracile (GN) nuclei. Data presented here support previously published anatomical and functional evidence that the longitudinal organization of the CN and GN reflect the complex role of the DCN in somatosensory processing. The CN is organized longitudinally into three parts. Within the middle portion of this nucleus, primary afferent projections and cuneothalamic cells are concentrated. Although traditional cytoarchitectonic analyses had failed to detect this tripartite organization in rats, we found evidence for it, with a functional middle region, extending approximately 0.2-0.9 mm caudal to the obex, characterized by precise somatotopy of primary afferent terminations and corresponding somatotopy of cytochrome oxidase (CO) blotches. Additional evidence is presented here consistent with a functionally distinct middle region within the rat's CN: (1) patches of dense synaptophysin (a synaptic-vesical-associated protein)-immunoreactivity (SYN-IR) are limited to the middle CN region, coincident with the dense CO blotches; (2) neurons immunoreactive for the calcium-binding proteins calbindin-D28 (CB), calretinin (CR) and parvalbumin (PV) are concentrated in the middle CN region. Furthermore, in adult rats subjected to perinatal forepaw removal, (1) the patterns of SYN-IR in the middle region of the CN are disrupted, as had previously been shown for the patterns of CO blotches; (2) in contrast, however, distributions of CN cells with PV-, CB- and CR-IR are unaffected. Evidence for a tripartite division in the GN is also presented, based on the distributions of cells with PV-, CB- and CR-IR.

Enhanced cytochrome-oxidase staining of the cuneate nucleus in the rat reveals a modifiable somatotopic map.

Existing cytochrome oxidase (CO)-staining techniques were modified to enhance sensitivity and contrast in order to examine patterns of CO-activity in the dorsal column nuclei (DCN) of adult Long-Evans rats. Within a rostrocaudally limited region in the middle of the cuneate nucleus (CN) distinctive blotches of intense CO-activity were observed. The CO-staining was maximally differentiated approximately 0.3-0.7 mm caudal to the obex. No CO-blotches were observed anywhere else in the DCN. Transganglionic labelling (WGA-HRP) demonstrated that some of the CO-blotches in the rat CN are related to the terminal projection fields of primary afferents from the skin of the forepaws. The corresponding location of primary afferent termination fields and CO-staining patterns supports a tripartite rostrocaudal division in the rat CN, similar to that described by other investigators in cats, monkeys and raccoons. Comparing the patterns of CO-staining to (1) the cytoarchitecture (Nissl-stained sections), or to (2) the dendritoarchitecture (distribution of microtubule-associated protein 2 (MAP2) or to (3) the organization of retrogradely labelled (WGA-HRP/HRP) cuneothalamic cells, revealed no topographical organization corresponding to the CO-blotches. Postnatal (at least up to 11 days postpartum) forepaw deafferentation or removal disrupted the CO-staining pattern in the CN.

The cuneate nucleus in the rat does have an anatomically distinct middle region.

Recently obtained anatomical evidence supports the division of the rat cuneate nucleus (CN) into three rostrocaudal regions, with the middle region receiving a disproportionately greater share of the primary sensory input. The CN in the rat conforms to the basic rostrocaudal CN pattern described in other mammals, including cat, monkey and raccoon.

Organization of cutaneous primary afferent fibers projecting to the dorsal horn in the rat: WGA-HRP versus B-HRP.

Primary afferent projections from cutaneous afferents in the forelimb and hindlimb digits to the dorsal horn (DH) were examined using 4 tracers: (1) 25% free horseradish peroxidase (HRP), (2) 2.5% wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), (3) a mixture of 25% free HRP and 2.5% WGA-HRP (WGA-HRP/HRP) or (4) 0.1% HRP conjugated to cholera toxin (B-HRP). The tracer was injected intracutaneously into the digits. Three to 4 days later, the rats were perfused transcardially, transverse sections (60-microns thick) were cut and the HRP was reacted using the tetramethyl benzidine (TMB) method. The location of the label was reconstructed by camera lucida drawings. In rats which received an injection of HRP alone, no label was detected in the DH. Rats injected with WGA-HRP had projection patterns similar to those injected with WGA-HRP/HRP. Patterns of labelling with WGA-HRP differed markedly from those with B-HRP. WGA-HRP labelled cutaneous afferents projecting to Rexed's laminae I-III, with the densest label in lamina II; in contrast, B-HRP labelled cutaneous afferents projecting to laminae II-V, with the densest label in laminae III-IV. These results indicate that, for cutaneous primary afferents projecting to the DH, WGA-HRP and B-HRP labelled different subpopulations of fibers, with the B-HRP-labelled subpopulation biased toward afferents of larger diameter. Rostrocaudally, the extent of the densest fiber projections, whether labelled by WGA-HRP or by B-HRP, was essentially the same, but the extent of the less densely labelled projections was much greater with B-HRP than with WGA-HRP. Comparisons of the projection maps from each of the five digits, using either WGA-HRP or B-HRP, indicated that, as seen in transverse sections through the DH, there was extensive overlapping among the labelled cutaneous afferent fibers from adjacent, or even non-adjacent digits.

Somatotopic organization of the dorsal column nuclei in the rat: transganglionic labelling with B-HRP and WGA-HRP.

To analyze the patterns of cutaneous primary afferent fibers projecting to the dorsal column nuclei in the rat, horseradish peroxidase (HRP)-based tracers were injected intracutaneously into a number of discrete regions of the forelimbs and hindlimbs. Three-4 days following the HRP injections, the rats were perfused transcardially; 60 microns transverse sections were cut, and the HRP was reacted using the tetramethyl benzidine method. Comparisons were made of projections following injections with choleragenoid-conjugated horseradish peroxidase (B-HRP) or with wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). B-HRP and WGA-HRP produced similar patterns of labelling, but B-HRP produced greater intensity of labelling and slightly larger projection areas. In the cuneate nucleus (CN), HRP labelling of primary afferents from small, delimited regions, e.g., from a portion of the skin of a single digit, appeared to be precisely restricted in rostrocaudally oriented columns, with little or no overlap (in the mediolateral and dorsoventral plane) into adjacent regions. With respect to rostrocaudal organization, a region in the CN containing a dense population of cutaneous primary afferent fibers appeared to be similar to the middle, or cluster, region in cats and in raccoons and the pars rotunda in primates. Projection patterns were very consistent from rat to rat, but their somatotopic organization differed from that suggested by electrophysiological studies: cutaneous afferents from forelimb digit 1 projected near the ventral border of the CN; those from digit 5 projected dorsomedially to those from digit 1; the projections from the remaining digits formed a crescent between the projections from digits 1 and 5. In the gracile nucleus, the organization of cutaneous afferent projections from hindlimb digits was more variable and complex than that found in the CN.

Somatotopic organization of the cuneate nucleus in the rat: transganglionic labelling with WGA-HRP.

A novel somatotopic map of primary cutaneous afferents projecting to the cuneate nucleus in the rat was determined by transganglionic transport of wheat-germ agglutinin conjugated to horseradish peroxidase and free horseradish peroxidase. Intracutaneous injections of tracer into different limited regions of the forelimb resulted in discrete areas of label for each injection site, with little or no overlap into other projection areas. The map of cutaneous projections onto the cuneate nucleus revealed by our anatomical tracing provided much more detail than any previous study in the rat, and demonstrated some significant differences from earlier maps based on electrophysiological recordings.