Distribution of 28 kDa Calbindin-Immunopositive Neurons in the Cat Spinal Cord

Abstract

The distribution of vitamin D-dependent calcium-binding protein (28 kDa calbindin) was investigated in cat lumbar and sacral spinal cord segments (L1-S3). We observed specific multi-dimensional distributions over the spinal segments for small immunopositive cells in Rexed laminae II-III and medium-to-large cells of varying morphology in lamina I and laminae V-VIII. The small neurons in laminae II-III were clustered into the columns along the dorsal horn curvature. The medium-to-large cells were grouped into four assemblages that were located in (1) the most lateral region of lamina VII at the L1-L4 level; (2) the laminae IV-V boundary at the L5-L7 level; (3) the lamina VII dorsal border at the L5-L7 level; and (4) the lamina VIII at the L5-S3 level. The data obtained suggest that the morphological and physiological heterogeneity of calbindin immunolabeling cells formed morpho-functional clusters over the gray matter. A significant portion of the lumbosacral enlargement had immunopositive neurons within all Rexed laminae, suggesting an important functional role within and among the spinal networks that control hindlimb movements.

Keywords: Ca2+-binding proteins; calbindin-28 kDa; gray matter; interneurons; spinal cord.

Figure 1

Labeled cells analysis. (A) Scheme for frontal and horizontal slice preparation; (B) Gray matter at the frontal slice; small labeled cells are marked by small dots, medium-to-large cells are marked by gross dots; shaded rectangle—the part of the dorsal horn containing laminae II-III. (C) A labeled area assessment in shaded rectangle at (B); Upper image—an initial 2-D pattern of labeled cells in laminae II/III, low image—a blurred and contrasted 2-D pattern of labeled cells in laminae II/III. (D) Diagram of labeled cell distributions in different laminae (summed by all segments). X axis—laminae, Y axis—number of cells. R, C, D, V—rostral, caudal, dorsal and ventral directions.

Figure 2

Calbindin-immunopositive neuron distribution in different segments of the dorsal horn. (A–L) Dorsal horn frontal slices; the laminae I-II boundary is marked by a black dashed line. (E) Enlarged area of labeling (in rectangle at D). Gross labeled cells in d are indicated by thick arrows. (I) Enlarged area of labeling (in rectangle at H). Lamina I immunopositive cells are marked by thin arrows. D, V, M and L—dorsal, ventral, medial and lateral directions. Calibration marker is 100 μm.

Figure 3

Calbindin-immunopositive neuronal distribution in lamina I of gray matter. (A,B) Dorsal horn frontal slices at segment S1 (A) and segment L2. (B) Labeled cells are indicated by arrows; laminae I-II boundary is marked by black dashed line. Inserts in bottom—enlarged cells (in squares). (C) A schematic view of horizontal slice preparations; gray—gray matter, dark gray—laminae II-III. (D–F) Labeled neurons at horizontal slices (depth 1.0–1.1 mm). Arrows mark neurons described in text. D, V, R, C, M and L—dorsal, ventral, rostral, caudal, medial and lateral directions. Calibration marker is 100 μm.

Figure 4

Calbindin-labeled neuronal distribution in different Rexed laminae of the L1-S3 segments. (A) Diagrams of the distribution of medium-to-large cells in lamina I; (B) Diagrams of the distribution of small cells in laminae II-IV; (C) Diagrams of the distribution of medium-to-large cells in laminae V-VIII. (A–C) left most, upper—frontal slices, regions of interest are shaded. (A–C), left most, bottom—averaged from all cat data (TOT); (A–C) Medium and rightmost figures—diagrams for individual cats (K7, K8, K12 and K15). X axis—segments, Y axis—number of labeled cells. (B) Light gray—lamina IV, dark gray—laminae II-III; (C) Light gray—laminae V-VI, dark gray—laminae VII-VIII.

Figure 5

Calbindin-immunopositive neuronal distribution in the frontal (A) and horizontal (C,D) sections of the gray matter of the dorsal horn. (A–D) Clusters of calbindin-immunopositive cells in segment L4, laminae II-IV; clusters are indicated by arrows. (A) Frontal slice, (B) Schematic view of horizontal slice preparations, depths of 1.4, 1.5 and 1.6 mm from the pial surface. (C,D) Horizontal slices; clusters are indicated by arrows. (E–G) Columns of calbindin-immunopositive cells in laminae II-III. (F,G) Enlarged regions in rectangles. D, V, R, C, M and L—dorsal, ventral, rostral, caudal, medial and lateral directions. Calibration marker is 100 μm.

Figure 6

Calbindin-immunopositive neuronal distribution in the gray matter of L1-L4 segments. (A–P) Contoured images of frontal slices in segments L1 (A–D), L2 (E–H), L3 (I–L) and L4 (M–P) of cat K7 (A,E,I,M), K8 (B,F,J,N), K12 (C,G,K,O) and K15 (D,H,L,P). Immunopositive neurons in laminae II-IV are marked by small dots; those in laminae V-VIII—by gross dots; laminae VI-VII boundary is marked by a dashed line. Rexed laminae are indicated by Roman numerals. Lateral groups of labeled cells are shaded in blue. (Q–T) Labeled cells groups in lamina VII, lateral image. (Q,S) Frontal slice (segment L3), (R,T) horizontal slice (segment L4, depth 2.2 mm, only left hemisphere is shown). The laminae III/IV, IV/V and VI-VII boundaries are marked by dashed lines. Rexed laminae are indicated by Roman numerals. D, V, M and L—dorsal, ventral, medial and lateral directions. Calibration marker is 100 μm.

Figure 7

Calbindin-immunopositive neuronal distribution in the gray matter on L5-L7 segments. (A–L) Contoured images of frontal slices in segments L5 (A–D), L6 (E–H) and L7 (I–L) of cat K7 (A,E,I), K8 (B,F,J), K12 (C,G,K) and K15 (D,H,L). Immunopositive neurons in laminae II-IV are marked by small dots; those in laminae V-VIII—by gross dots; laminae VI-VII boundary is marked by a dashed line. Rexed laminae are indicated by Roman numerals. Groups of labeled cells are shaded in blue. (M–S) Labeled cells in laminae V-VIII. (M) Dorsal horn frontal slice (segment L7); (N–R) Enlarged cells (in dashed squares). (S) Bubble histogram of labeled cells located at the Laminae VI-VII boundary (as a sum of three frontal slices). Slices were centered apart from the central canal (CC, white circle). Cell area is marked by bubble size. Cell clusters are indicated by arrows (thick arrows—clusters of larger cells, thin arrows—clusters of smaller cells). D, V, M and L—dorsal, ventral, medial and lateral directions. Rexed laminae are indicated by Roman numerals. Calibration marker is 100 μm.

Figure 8

Calbindin-immunopositive neuronal distribution in the gray matter on the S1-S3 segments. (A–L) Contoured images of frontal slices in segments S1 (A–D), S2 (E–H) and S3 (I–L) of cat K7 (A,E,I), K8 (B,F,J), K12 (C,G,K) and K15 (D,H,L). Immunopositive neurons are marked by dots; laminae VI-VII and VII-VIII boundaries are marked by dashed lines. Rexed laminae are indicated by Roman numerals. (M,N) Frontal slices of segments S2 (M) and S3 (N). Labeled cells are indicated by dark arrows; motor neurons are indicated by light gray arrows. (O–R) Enlarged cells (in dashed squares). Rexed laminae are indicated by Roman numerals. Calibration marker is 100 μm.