A comparative study of the calcium-binding proteins calbindin-D28K, calretinin, calmodulin and parvalbumin in the rat spinal cord

BRAIN RESEARCH RlEVlEWS ELSEVIER

Brain Research Reviews 19 (1994) 163-179

Full-length Review

A comparative study of the calcium-binding proteins calbindin-D28K, calretinin, calmodulin and parvalbumin in the rat spinal cord K. Ren * and M.A. Ruda Neuro&&gy and Anesthesiology Branch, National institute of Dental Research, National Institutes of Health, Building 49, Room IAll, 9000 Recur Pike, Rethesda, MD 20892, USA (Accepted 13 July 1993)

contents 163

Summary............................................................................................. Introduction

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Materials and Methods. .................... 2.1. Tissue preparation .................... 2.2. Antibodies .......................... 2.3. Immunocytochemistry .................. 2.4. Data analysis ........................

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Resuits ............................................................................. 3.1. Comparison of immunohistochemicai localization of the calcium-binding proteins .................... 3.2. Effect of unilateral multiple dorsal root ganglionectomies ...................................... Discussion ............. 4.1. General ........... 4.2. Superficial dorsal horn 4.3. Dorsal gray commissure 4.4. Ventral horn ........ 4.5. Fiber tracts ......... 4.6. Cell nucteus ........ Acknowledgements References

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Abstract Comparison of the immunocytochemical localizations revealed distinct patterns of differential distribution and overlapping of calbindin-D28K (CB-D28K), calretinin (CR), calmodulin (CM) and parvalbumin (PV) in the rat spinal cord. In some areas, one of the four calcium-binding proteins (CBPs) appears to be predominant, for example, CB-D28K in lamina I and ependymal cells, PV at the inner part of laminae II, CR in Iaminae V and VI and CM in motoneurons of lamina IX. In other regions of the spinal cord, more than one CBPs was abundant. CB-D28K and CR were similarly distributed in lamina II and the lateral spinal and cervical nucleus; CM and PV were similarly abundant in the ventromedial dorsal horn, internal basilar and central cervical nucleus; CR and PV were similarly heterogeneous in the gracile fasciculus from caudal to rostra1 spinal cord. In the sacral dorsal gray ~rn~~ure, the dist~bution patterns of CR and PV were clearly ~mplernent~. The unilateral g~~ionectomies resulted

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in a substantial reduction of CBP-like immunoreactivity (CBP-LI) in the dorsal columns and a reduction of CM- and PV-LI in the ventromedial dorsal horn. In the motor system, only CM labeled large motoneurons in lamina IX and CB-D28K lightly stained pyramidal tract. The apparent absence of CM-L1 in the superficial dorsal horn is contradictory to the presence of a CM-dependent nitric oxide synthase in that region. These data indicate that most CBP-LI in the dorsal column pathway had primary afferent origin, while the superficial dorsal horn exhibited intrinsic CBP immunoreactivity. The differential and selective localizations of CBPs in the spinal cord suggest a role for these proteins in spinal nociceptive processing, visceral regulation and dorsal column sensory pathways. Key words: Immunocytochemistry; Superficial dorsal horn; Dorsal gray commissure; Dorsal column; Ventral horn; Canglionectomy

1. Introduction

in the spinal cord, whereas CR-like immunoreactivity (LI) was found in dorsal horn neurons rather than As one of the important intracellular signaling molemotoneurons and glia ce11s32~58,59. The differential discules, calcium ions (Ca*‘) are involved in fundamental tribution of CBPs in certain regions of the CNS suggest physiological processes, such as signal transduction. It that each CBP may perform distinct functions. For has become clear that the effects of Ca2” are closely instance, CB-D28K and PV are located in distinct related to a superfamily of the structurally related compartments in the thalamus where they may be calcium-binding proteins (CBPs), which bind calcium involved in relaying sensory information of different with high affinity. modalities, respectively56. The prototype CBP is calmodulin (CM). CM was There has been an increased interest in CBPs duridentified in 1967 (see ref. 16 for a review) and has ing recent years. Alterations in CBPs may result in been intensively studied since then. The activity of perturbation of Ca 2+ homeostasis; and abnormal more than 20 enzymes are regulated by Ca2+-CM comCa2+-concentrations could lead to cytotoxicity 28*47.In plexes (see ref. 39 for a review). A number of CBPs, fact, alterations in CBPs have been found in neurodeincluding calbindin-D28K (CB-D28K). pa~albumin generative disorders such as ~zheimer’s disease, Par(PV) and calretinin (CR), have been found widely kinson’s disease and epilepsy”. On the other hand, the distributed in the central nervous system (CNS) in presence of CBPs may protect neurons from chemicalrecent years (see ref. 6 for a review). CB-D28K was or ischemia-induced neurotoxicity3198*48. In the rat hippocampus, neurons containing either CB-D28K or PV first found in chick intestine as a vitamin D-dependent are insensitive to seizure activity”. Resembling neuCBP74. It was later localized in mammalian brain33*67. rodegenerative disorders, calcium-mediated excitotoxiPV was first found in muscular tissue3’j and then also city in the spinal cord may also be related to abnormal in the brain15. The EF-hand structure, a consensus pain sensations such as allod~ia and hyperalgesia*$. amino acid sequence involved in calcium-binding activThus, studying spinal CBPs may provide insight into ity for over 160 CBPs, was first identified in PV (see the mechanisms of nociception and hyperalgesia. ref. 54 for a review). CR was discovered recently60,61,75. The spinal cord is an important site for processing It is closely related to CB-D28K but appears to be a of nociceptive input and integration of sensorimotor neuronal specific CBP51@. functions. The immunohistochemical distribution of The significance of the binding of Ca2” by CBPs CB-D28K, PV and CR in the spinal cord have been may be related to either the triggering of a physiologistudied recently 1s8*59*77-79 (also see ref. 3 for a review). cal process or modulation of intra~llular Ca*+ conA detailed i~unohist~hemical mapping of CM is centrations. The precise function of most CBPs, howstill missing in spinal cord. Like other regions of the ever, is still largely unknown6. Using primarily the nervous system, CBPs also appear to exhibit differenimmunohistochemical technique, interesting distributial and complementary distribution patterns in the tion patterns of CBPs have been described in the CNS. spinal cord. For instance, CR is similarly, but not CB-D28K and PV are present largely in different equally, distributed as its close relative, CB-D28K; and groups of neurons in the cerebral cortex29’73, hippocampus7’, superior colliculus43, thalamic nuclei 34Z55*56, PV is distributed quite differently from CR and CBD28K. To further reveal the anatomy and physiology of medullary dorsal horn’ and spinal cord1,77,78.CR, when the CBPs and its possible involvement in spinal nocicompared to CB-D28K and PV, also is present in ceptive processing, this study was conducted to give a different sets of neurons, although the three CBPs do direct and detailed comparison of immunohistochemioverlap to some extent’3,57v62,63. CM has been found in cal localizations of four CBPs CM, CB-D28K, PV and glia cells in the cerebellum’* and large motoneurons69’76

Fig. 1. The distribution of ~~cium-binding protein-like immunoreactivi~ in the lumbar W spinal cord. A: calbindin-D28K (CB); B: calretinin (CR); C: calmodufin (CM); D: parvalbumin (PV). Note the differential staining patterns in the superficial laminae of the dorsal horn and the lateral spinal nucleus (LSN). Also note the dense calmodulin- and parvalbumin-like immunoreactivity in the ventromedial dorsal horn (medial laminae V/VI, asterisks). GR, gracile fasciculus; PT, pyramidal tract. Roman numerals I-X indicate spinal cord laminae. DAR reaction. Scale bar (same for A-D) = 200 pm,

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CR in the rat spinal cord. The response of these CBPs to unilateral multiple ganglionectomies was also examined.

2. Materials and methods 2.1. Tissue preparation

Ten male Sprague-Dawley rats (200-300 gl were used in the present study. Five of the rats received unilateral multiple dorsal root ganglianectomies under sterile and anesthetic conditions and were allowed to survive 1 (n = 2) to 2 fn = 3) weeks after the surgery. All rats were deeply anesthetized with sodium pentobarbital and perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The extent of the ganglioaectomies was verified after the perfusion. The cervical, thoracic, lumbar and sacral spinal cord were removed, immersed in the same fixative overnight at 4°C and transferred to 30% sucrose (w/v) in phosphate buffer for several days for cryoprotection. Thirty I.tm-thick sections were cut with a cryostat at -20°C. The adjacent sections were collected in phosphate buffered saline (PBS) and processed simultaneously for CB-D28K, CR, PV and CM ~~un~ochemist~, respectively. Using the same material, CR-L1 has been reported previously”. Observations on CR-L1 is presented here for the purpose of comparison.

2.2. Antibodies Mouse anti-chicken gut CB-D28K (monoclonal IgGl, Clone No. CL-300; Lot 06OH4816) and mouse anti-carp muscle PV (monoclonal IgGl, Clone No. PA-235; Lot 49F4824) were purchased from Sigma Chemical (St. Louis, MO). The specificity of CB-D28K and PV antibodies has been examined (Sigma). The anti-CB-D28K reacts specifically with CB-D28K in brain tissue from rat and stains the 45Ca binding spot of CB-D28K (M.W. = 28,000, pI = 4.8) in a two-dimensional immunoblot. The anti-PV reacts specifically with PV of cultured nerve cells from rat and stains specifically the 45Ca-binding spot of PV (M.W. = 12,000 and pl = 4.9) by immunoblotting. Mouse antj-bovine CM (mon~lonal IgGI) was purchased from Upstate Biotechnology (Lot 10905l. The specificity of this CM antibody has been characterized@. The CM antibody identifies both Ca”-bound and Ca2+-free rat CM. The primary antiserum against guinea pig CR was raised in rabbits as described”. The specificity of the CR antiserum has been detailed previously2,32. The CR antibody does not cross-react with CB-D28K. On a protein blot of the guinea pig brain sample, the anti-CR only detected CR but not CB-D28K75. 2.3. Immunocytochemistry Free floating tissue sections were rinsed in PBS with 0.75% Triton X-100 for 1 h, 3% normal goat serum (NGS) or 5% normal horse serum (NHS) in PBS for 30 min, then incubated with anti-CBP antibodies (diluted in PBS; 1: 25-50 K for CB-D28K 1: 200 K for

Fii. 2, Comparison of ~lcium-b~d~g protein-like immunoreactivi~ in the superficial dorsal horn of the I.5 spinal cord. A: calbindin-D~K (CI3); B: calretinin (CR); C: caimodulin (CM). D: parvalbumin (PV). Some Iamina I calbindin-D28K- and calretinin-immunoreactive neurons are marked (arrows in A and B). Arrowheads in B indicate the dendrites of a Waldeyer type lamina I neuron. Note the absence of CM-IR cell bodies and fibers in laminae I and II, while a few varicose fibers and labeled cells (arrow) can be identified in lamina III (0. Parvalbumin primarily labeled fiber networks in the inner lamina II (D). Two parvaibumin-IR cell bodies were indicated (arrows in D). DAB reaction. Scale bar (same for A-D) = 50 pm.

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Ruda /Brain Research Reviews 19 (1994) 163-l 79

PV, 1: 10-20 K for CM and 1: 7.5-10 K for CR) with l-3% NGS of NHS for 48-72 h with gentle agitation. After two washes in PBS (10 min each) and a 20-mitt rinse in PBS with 3% NGS or 5% NHS, the sections were incubated with biotinylated horse anti-mouse or goat anti-rabbit IgG (Vector, 1:200 in PBS) for 30-60 min. Following rinses in PBS (10 min X 2) and PBS with NGS or NHS (20 mitt), the sections were then incubated with avidin and biotinylated horseradish peroxidase complex (Vector, 1:50 in PBS) for 30-60 min. After

167

washes in PBS or Tris HCLbuffered saline (TBS), the tissue sections were reacted with 0.05% diaminobenzidine dihydrochloride (DAB, Sigma) in 0.1 M phosphate buffer containing 0.003% hydrogen peroxide for 3-6 min. In some experiments, the sections were pre-incubated in 0.2% nickel ammonium sulfate in 0.1 M phosphate buffer for 10 min before incubation with DAB. Both procedures produced the same immunostaining pattern in the tissue although the nickel ammonium sulfate incubation resulted in increased con-

Fig. 3. Comparison of calcium-binding protein-like immunoreactivity in the neck of the dorsal horn (laminae V and VI) of the L4 spinal cord. Some labeled cell bodies are indicated by arrows. A: calbindinD28K (CBl. Arrowheads indicate an axon-like process issued from a multipolar neurons with densely labeled nucleus. Note fewer cells and fibers in lamina VI are labeled; B: calretinin (CR). Calretinin-immunoreactive cell bodies and fibers are abundant in laminae V and VI. Note fewer cells and fibers in lateral lamina V exhibited calretinin-like immunoreactivity; C: calmodulin (CM). Fibers in the ventromedial portion of laminae V and VI are densely labeled with calmodulin (asterisk). A bipolar neuron with a densely stained nucleus in lamina V is indicated (arrow); D: parvalbumfn (PV). Mixed fiber and cell bodies exhibit strong immunoreactivity to parvalbumin in the ventromedial portion of laminae V and VI (asterisk). Many rostrocaudal running small fiber bundles in lateral lamina V show parvalbumin-like immunoreactivity (arrowheads). Approximate border between laminae V and VI is indicated by a short dotted line. Directions: top, dorsal; right, lateral. DAR reaction. Scale bar (same for A-D) = 50 pm.

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trast. Some sections were later counterstained with Cresyl violet to allow visualization of the genera1 histology of the tissue. Control sections were processed with the same method except that the primary antisera were omitted. The sections were finally rinsed in PBS, mounted on gelatin-coated slides, dehydrated in alcohols, cleared in xylenes and coverslipped with Eukitt (Kindler, Germany). 2.4. Data analysis

Immunostaining intensity and packing density of CBP-immunoreactive (IR) cell bodies and fibers in specified spinal cord regions were qualitatively assessed (see2332s9).Rat spinal cord structures were identified and named according to the stereotaxic atlas of Paxinos and Watsons3 (see also refs. 44,45).

3. Results

3.1. Comparison of immunohistochemical localization of the calcium-binding proteins

Distinct patterns of irnmunohistochemical localization of CB-D28K, CR, CM and PV were demonstrated in the cervical through sacral spinal cord. The distribution of the four CBPs are generally consistent with previous reports in rats’,‘2,58,59,77,78. After comparing

Fig. 4. Comparison of calcium-binding protein-like immunoreactivity in the ventral horn of the L4 spinal cord. A: calbindin-D28K (CB); B: calretinin (CR); C: calmodulin (CM); D: parvalbumin (PV). Many laminae VII and VIII neurons are immunoreactive to calbindin-D28K and calretinin (A and B). Large motoneurons are preferentially labeled by calmodulin antibody (arrows in 0. Neurons in the ventromedial motor nucleus of lamina IX also exhibit strong calmodulin-like immunoreactivity (arrowheads in Cl. Some neurons with darkly stained nuclei are indicated by asterisks (C). Directions: top, dorsal (d); left, lateral (1). Nickel enhanced DAB reaction. Scale bar (same for A-D) = 100 km.

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Ruda /Brain Research Reviews 19 (1994) 163-179

CBP-LI in adjacent sections processed simultaneously but against CB-D28K, CR, CM or PV antibodies, respectively, a clear differential distribution of immunoreactivity for these CBPs was found in many structures of the spinal cord, including the superficial (laminae I and II) and ventromedial (medial portion of laminae V and VI> dorsal horn, the ventral horn (espeqially lamina IX), the dorsal lateral funiculus (DLF), the dorsal gray commissure (DGC) and the dorsal column. In the spinal gray matter, axons and dendrites were well stained by CB-D28K, CR and PV antibodies, while CM-IR staining appeared to be more restricted to cell bodies. All four CBPs exhibited immunoreactivity in the cell nucleus. The i~unostaining in the nucleus was often more intense than in the cytoplasm (e.g., see Figs. 3 and 4). Few cell nuclei were found not stained by CR and PV antibodies. The cell nucleolus was always not labeled. Extensive immunostaining was also demonstrated in the spinal white matter throughout

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the length of the spinal cord. The examples of CBD28K-, CR-, CM- and PV-LI in the spinal cord are shown in Fig, 1. In lamina I, many neurons exhibited intense CBD28K-LI (Figs. lA, 2A). Fewer neurons were labeled by CR antibody, including large Waldeyer type neurons (Fig. 2B, also see refs. 59,78). The staining for CM in lamina I was barely above the background and essentially no CM-IR cell bodies were found in lamina I (Figs. lC, 2C). No PV-LI was observed in lamina I (Figs. lD, 2D). A comparable number of cells showed immunoreactivity for CB-D28K and CR in lamina II (Fig. 2). Densely packed CB-D28K- and CR-IR cell bodies and fibers were distributed throughout lamina II, forming a dense IR band in the superficial dorsal horn (Fig. lA,B). In contrast, lamina II was not stained with CM antibody, leaving a blank band in the superficial dorsal horn (Fig. 10. An intense PV-IR band, primarily

Fig. 5. Comparison of calcium-binding protein-like immunoreactivity in the area around central canal from the Cl spinal cord. Note the ependymal cells in the central canal (asterisk) are preferentially labeled by calbindin-D28K (CB) antibody (A). Internal basilar (IBN) and central cervical (CCN) nuclei exhibit different staining intensities for cafretinin (CR)(B), calmodulin (CM) (0, and pawalbumin (PV) CD). PT, pyramidal tract. DAB reaction. Scale bar (same for A-D) = 100 ym.

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consisting of terminal like structures, was localized mainly in the inner part of lamna II (Figs. 1D and 2D). CR- and PV-LI in lamina II were not evenly distributed at the cervical and lumbar enlargement. The most medial portions of the CR- and PV-IR bands were usually less intensely stained than the lateral portions of lamina II (Fig. lB,D). In contrast, CB-D28K was evenly distributed in lamina II (Fig. 1A). In laminae III and IV, CM-L1 was found in some neurons (Fig. 20 A few varicose CM-IR fibers could also be seen. CB-D28K- and CR-L1 were in general not observed, but occasional CB-D28K and CR-IR cell bodies were found in laminae III and IV (Figs. lA,B and 2A,B). Laminae III and IV generally lacked PV-LI. All four CBPs exhibited immunoreactivity in laminae V and VI, although CR-IR cell bodies and fibers were the most abundant. Cell bodies and fibers labeled with CR intermingled in this region, primarily in the middle third of laminae V and VI (also see ref. 59). Very few CB-D28K-IR cell bodies and fibers were found in lamina VI (Fig. 3A); and fewer CR-IR cell bodies were found in lateral lamina V (Fig. 3Bl. In the medial half of laminae V and VI (ventromedial dorsal horn) at the level of lumbar enlargement, strong CMand PV-LI were noteworthy (Figs. lC,D and 3C,D). CM-L1 in this region consisted primarily of fiber networks and punctate staining, while PV-LI was present as mixed positive cell bodies and fibers (Figs. lC,D and 3C,D). PV-IR fibers were seen to enter from the dorsal column and radiated towards the ventral gray matter (Figs. 1D and 3D). In lateral reticular portion of lamina V, small rostrocaudal running bundles were frequently found with PV-IR (Fig. 3D). Many medium- to large-sized neurons were IR to each of the four CBPs in laminae VII and VIII. CR-IR

cell bodies and processes were the most abundant among the four CBPs (Figs. 1 and 4). The morphology of neurons was primarily multipolar although bipolar and pyramidal neurons were also found. Although all four CBPs demonstrated immunoreactive staining in lamina IX, large motoneuronal cell bodies were stained primarily, if not exclusively, by CM antibody (Fig. 4). A few small CB-D28K, CR or PV-IR cell bodies were occasionally seen in lamina IX. In lamina X, CR-IR cell bodies were mainly distributed in the area dorsal to the central canal (Figs. 1B and 5B). Antal et al.’ reported that CB-D28K and PV-LI were most prominent at the lateral and ventral aspects of the central canal. In the present study, densely packed CB-D28K-IR cell bodies also were found dorsal to the central canal (Fig. 5A). CM-IR cell bodies were rarely seen in lamina X. The ependymal cells surrounding the central canal were labeled densely by CB-D28K and lightly by CM, but not by CR and PV antibodies (Fig. 5). At the level of the sacral spinal cord, a sharp contrast of CBP-LI was found in the DGC, an area dorsal to the central canal and extending beyond the borders of lamina X. As illustrated in Fig. 6A, a cluster of medium to large densely packed CR-IR cell bodies and CR-IR fibers were located immediately dorsal to the central canal, forming a dense CR-IR network in the DGC. From this CRlabeled cell group, many fibers ran dorsally to reach the edge of the gray matter; other fibers traveled bilaterally and horizontally to connect the intermediolateral columns (Fig. 6A). PV-IR fiber networks encircled the DGC and appeared to define the borders of this region (Fig. 6B). Only a few varicose PV-IR fibers were seen in the center of the DGC. Thus, the pattern of PV-LI in the DGC was clearly complementary to

Fig. 6. Distinct complementarity of calretinin- (CR) (A) and parvalbumin(PV) (B) like immunoreactivity in the dorsal gray commissure (DGC) of the ~$2 spinal cord. The central canal is indicated by asterisks. Top: dorsal. Nickel enhanced DAB reaction. Scale bar (same for A and B) = 100 Km.

K. Ren, MA. Ruda /Brain Research Reviews 19 (1994) 163-179

CR (Fig. 5). The CB-D28K-LI was similar to CR, except that the cell bodies and fibers were less densely packed (data not shown). Scattered small CM-IR cell bodies were distributed in the sacral DGC. At the rostra1 cervical spinal cord, intensely labeled PV-IR cell bodies and fibers were densely packed in the internal basilar nucleus (IBN) and central cervical nucleus (EN), making the borders of the two nuclei clearly distinguishable (Fig. 5D). The IBN and CCN also were labeled by CM and CR antibodies. The IBN and CCN generally lacked CB-D28KLI (Fig. 5A). Thus, the relative intensity of immunoreactivity in the IBN and CCN was PV > CM > CR > CB-D28K = 0.

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Similar relative levels of immunoreactivity were exhibited in Clarke’s column (data not shown). The lateral spinal nucleus (LSN) and lateral cervical nucleus (IEN) were intensely labeled by CB-D28K and CR antibodies (Figs. lA,B and 7A, B). Cell bodies and fibers labeled with CB-D28K and CR were observed. The CB-D28K-LI appeared to spread more extensively than CR-LI. In the DLF, CM-L1 was mainly located in the ventrolateral part at the lumbar level (Fig. 1C). A nerve bundle at the dorsolateral extreme of the DLF was also densely stained by CM antibody at the rostra1 cervical level (Fig. 70 PV-LI was only detectable at the ventral portion of the DLF (Figs. ID and 7D). The

Fig. 7. Comparison of ~lcium-binding protein-like i~unoreactivi~ in the dorsal lateral funiculi (DLF) of the Cl spinal cord. A: calbindin-D~K (CB); B: calretinin (CR); C: calmodulin (CM& D: parvalbumin (PV). Arrows in A outline the area of DLF. I and II indicate the lateral end of spinal laminae I and II. LCN, lateral cervical nucleus; UN, lateral spinal nucleus. Top = dorsal cd), right = lateral (1). DAB reaction. Scale bar (same for A-D) = 100 Wm.

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differential distribution of the four CBPs in the region of DLF was most conspicuous at the rostra1 cervical level. At the Cl spinal cord, the dorsal portion of the DLF, where LSN and LCN could be identified, were predominantly stained by CB-D28K and CR antibodies (Fig. 7A,B). CM-L1 was most prominent in the dorsolateral edge of the DLF (Fig. 7C) and PV mainly labeled an area ventral to the LSN and LCN (Fig. 7Dl. In Lissaeur’s tract, labeled fibers for each CBP were found. However, they were typically few in number, with PV being found in the least number of fibers (Fig. 1). In the dorsal columns, only CM-L1 was uniformly detected in bath fasciculi gracilis and cuneatus and the fiber tract in the dorsal midline region (tail zone), from sacral through cervical spinal cords (Figs. 1C and 8C). CR and PV antibodies similarly labeled the gracile fasciculus at the caudal spinal cord (Fig. lB,Dl and labeled cuneate, but not gracile fasciculus at the rostra1 spinal cord (Fig. 8B,D). CB-D28K-LI was marginally

detectable in fasciculus gracilis from the sacral to cervical spinal cord. The fasciculus cuneatus was devoid of CB-D28K-LI. The pyramidal tract at the base of the dorsal column appeared to have a few fibers labeled with CB-D28K (Fig. 1Al. Using a different polyclonal antibody, the CB-D28K-LI was not detected in the pyramidal tract previously’. There appeared to be weak immunostaining for CM in the pyramidal tract. The pyramidal tract generally lacked immunoreactivity to CR and PV. At the lumbar and sacral levels, the dorsal portion of the pyramidal tract often stained lightly for PV antibody. Comparisons of immunoreactive cell bodies and fibers of CB-D28K, CR, CM and PV are summarized in Tables 1 and 2. 3.2. Effect of unilateral multiple dorsal root ganglionectomies

Five rats received unilateral multiple ganglionectomies primarily at the lumbar levels. The levels of the

Fig. 8. Comparisonof calcium-binding protein-like immunoreactivity in the dorsal column fasciculi in the Cl spinal cord. A: calbindin-D (CB); B: calretinin (CR); C: calmodulin (CM). D: parvalbumin (PV). CU, cuneate fasciculus; GR, gracile fasciculus; PT. pyramidal tract. spinal cord laminae I and II. DAB reaction. Scale bar (same for A-D) = 100 pm.

28K

I 1II,

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Table 1 c~~~~

of thed~t~ut~o~

of ~mmuno~a~tive

fibers of four pallid-binding

proteins in the rat spinal cord

+ + +, high density; + +, moderate density; +, low density; +, rarely found; -, not found. SpiMl cord region

Iamina I Lamina II Laminae III/ IV Lantina V/VI Lamina VII Lamina VIII Lamina IX Lamina X Internal basilar nucleus Central cervical nucleus Clarke’s column Lissaeur’s tract Lateral spinal nucleus Intermediolateral nucleus Intermediomedial nucleus Gracile fasciculus Lumbosacral Cervicothoracic Cuneate fasciculus Pyramidal tract

Staining in:ensity Calbindin-D28K

Calretinin

Calmodulin

+, ++ +++ -3 + + +, ++ +, ++ +, ++ + + f f + ++, +++ ++ 2 -

+ +++ -9 + ++ ++ ++ ++ + -9 + + ++ + +-i-+ ++ +

-> f

+ + -, * +

+++ It +f+

+++ +++ -t++ -9 +

Parualbumin

--,+++I + f, + ++ ++ + +, +, +, + +, rt +

++

++ i-f ++ f

+, ++ +, ++ f, ++ f, ++ -, + +++ +++ +++ + _ + ++,+++3 +++ f +++ -, ++4

’ There was no immunostaining

for patvalbumin in the outer portion of lamina II and most parts of lamina III. However, high density parvalbumin-LI was found primarily in the inner part of Iamina II. ’ Most obvious at the sacral levels. 3 Most obvious at the thoracic levels. 4 The pyramidal tract was generally lacking parvalbumin-11. However, parvalbumin-11 was found in the dorsal portion of the pyramidal tract at the lumbar and sacral levels.

Table 2 Comparison of the distributions of immunoreactive cell bodies of four calcium-binding proteins in the rat spinal cord + +, intense;

+ , light; f , rarely found; -, not found, h, high density; m, moderate density; 1, low density; r, rarely found; n, not found.

Spinal cord region

Lamina I Lamina II Laminae III/ IV Lamina V/VI Lamina VII Lamina VIII Lamina IX Lamina X Internal basilar nucleus Central cervical nucleus Clarke’s column Lateral spinal nucleus Inte~ediolaterai nucfeus Inte~ediomediai nucleus Ependymal cells Cell nucleus Cell nucleolus

Staining inter&& /packing

density

Ca~bi~~-~28K

Ca~ret~n~n

+, + +/1 +, + +/h +, + +/1 +, + +/1 +, + +/I +/I -, f +, + +/1 f f

f, ++/r ++/m,h f, + +/r +, + +/I, m +, ++/l,m 4, ++,A

+/I ++/m3

+, ++/1 + +/I, r f -

+++ +, -I-+

’ There were no parvalbumin - immunoreactive

-I-, ++/i,m

Calculi +/n +/I + /I +/m +/m rt

+, + +/1

+, ++

rt + f + + +, ++

Par~alb~min -, -

++/r

’

+, ++/n,m* +, + +/1 +/r + + +/I + +/I

* -,

+, ++

cell bodies in the outer portion of lamina II and most parts of lamina III. However, parvafbumin - ~mmunoreactive cell bodies were found along the border of laminae II and III. 2 Pa~albumin - immunoreact~e cell bodies with moderate packing density were found in the medial part of famina V (next to the dorsal column) at the lumbar levels. 3 Most obvious at the sacral levels.

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ganghonectomies were L2-5, Ll-5, L3-Sl and L2-6. One rat had Ll-2 ganglionectomies and L3 and L6 dorsal root axotomies. FolIowing the gangIionectomies, a common change for all four CBPs was a substantial loss of immunoreactivity in the dorsal column gracile fasciculus at related segments ipsilateral to the ganglionectomy. CM-L1 was almost depleted in the ipsilateral gracile fasciculus (Fig. 9A) and only scattered punctate staining re-

mained in the dorsal column for PV (Fig. 9Bl. In spinal gray matter, a great reduction of CM- and PV-LI, primarily from fiber network, was observed at the medial portion of laminae V and VI (ventromedial dorsal horn) ipsilateral to the ganglionectomy (Fig. 9). Ipsilateral to the surgery, the PV-IR band at the inner part of iamina II, especially the medial part, was less intensely stained when compared to the contralaterai side (Fig. 9B). CM-L1 was also reduced in ipsilateral

Fig. 9. The effects of multiple unilateral dorsal root ganglionectomies at the lumbar level (L2-5) on calmodulin- (CM) (A) and parvalbumin- (PV) (B) like immunoreactivi~ in the L.5 segment of the spinal cord. CONTRA, contralateral; GR, gracile fascicuius; IPSI, ipsiiateral. I,II, spinal cord laminae I and II. Note large reduction of immunoreactivi~ in the GR and ventromedial (asterisks) dorsal horn IPSl to the gan~lionectomy. The intensity of parvalbumin-IR band in the superficial dorsal horn was slightly reduced. Nickel enhanced DAR reaction. Scale bar (same for A and B) = 200 pm.

K Ren, MA. Ruda /Brain Research Reviews 19 (1994) 163-179

laminae III, IV and lamina IX. In some sections, CM-L1 was marginally increased at ipsilateral medial Iamina I and outer portion of lamina II, but this appeared to be inconsistent across all sections. Comparatively, changes in CB-D28K- and CR-L1 in spinal gray matter were less apparent after ganglionectomy. As described previouslys9, CR-L1 was reduced slightIy in Clarke’s column rostrai to the surgery levels and increased slightly in the medial third of ipsilateral Iamina II.

4. Discussion 4.1. General It is clear from this i,mmunohistochemical localization study that the distribution of CBPs CM, CB-D28K, PV and CR can be both differentia1 and overlapping in the rat spinal cord. In some areas, one of the four CBPs appears to be predominant, for example, CBD28K in lamina I, PV at the inner portion of laminae II, CR in laminae V, VI and VII, CM in motoneurons of lamina IX. In other regions of the spinai cord, more than one CBP was abundant. CB-D28K and CR were similarly distributed in lamina II and LSN, CM and PV were similarly abundant in the ventromedial dorsal horn, IBN and CCN; CR and PV were similarly heterogeneous in the fasciculus gracilis from the caudal to rostra1 segments of the spinal cord. Data from the ganglionectomy experiment indicated that most CBP-LI in the dorsal column pathway was of primary afferent origin, while the superficial dorsal horn exhibited intrinsic CBP-LI. 4.2. ~~pe~~ial dorsal horn Intense CB-D28K- and CR-L1 were demonstrated in Iaminae I and II. PV-LI was specifically abundant in the inner part of Iaminae II. These observations are consistent with published results1*58~59.77~78. The absence of CM-L1 in the superficial dorsal horn has not been reported previously. There have been quite a few discussions about the differential distribution of CBPs in the CNS. Most attention has been given to CB-D28K and PV’~7*‘4,77-79, It is generally found that CB-D28K and PV are located in different sets of neurons in the rat spinal cord”. In fact, in the superficial dorsal horn characteristic PV-IR terminal-like structures define a unique band which is indistinguishable by traditional histological methods44*45.CR is a close relative of CB-D28K1’y5’. The primary translation product of CR mRNA is a 29 KDa protein@ and CR and CB-D28K have 50-60% sequence homology 50751,M)Z7s. ~mparing PV and CR, they appear to be generally present in different subpopulations of neurons 63. In the rat hipp ocampus, CR is not

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co-localized with PV 42. Despite the close resemblance between CR and CB-D28K, the two CBPs have been shown to exist in different sets of neurons in the brain2,‘4*32Y42763. The distribution patterns for CR and CB-D28K in the superficial dorsal horn were very similar, although CB-D28K was more abundant than CR in lamina I. Rogers et a163has reported colocahzation of CR and CB-D28K in neurons in the substantia gefatinosa of rat spinal cord. Using double-sabering fluorescence immunohistochemistry, we found that most labeled neurons in the superficial dorsal horn are only immunoreactive to either CR or CB-D28K, although a small percentage of neurons exhibited both CR- and CB-D28K-LI (Ren and Ruda, unpublished observations). Thus, it appears that CB-D28K CR and PV are primarily located in different subpopulations of neurons in the superficial dorsal horn. The superficial dorsal horn is an important relay station for nociceptive input. Most nociceptive primary afferents terminate in laminae I and II. ~mparing immunoreactive localization, CR and CB-D28K are more likely to play a role in nociceptive processing. Many CR- and CB-D28K-IR neurons may be interneurons that are important for nociceptive transmission; some immunoreactive neurons, especiahy the large cells in lamina I, may project supraspinally4i,78. CM and PV appeared to be less important for functional activity in the superficial dorsal horn. The intense CR- and CBD28K-LI in the superficial dorsal horn, however, did not appear to originate from primary afferents, since foIlowing multipte gangIionectomies, i~unoreactive fiber plexus remained essentially unchanged. A surprising result of this study was the lack of CM-L1 in the superficial dorsal horn. This result is unexpected in that a brain nitric oxide synthase (NOS), which has been found to be CM-dependent”, has been localized to that region’. Reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase has been found largely co-localized with brain NOS9. The NOS in neurons may be responsible for NADPH-diaphorase staining2430. Not suprisingly, dense NADPHdiaphorase staining was found in the superficial dorsal horn’*, resembling the immunostaining pattern of NOS9. The apparent absence of CM-L1 in lamina II is very puzzling as far as the CM-dependency of NOS is concerned. It is unlikely that an isoform of CM may exist which was not detected by the antibody used. It has been found that although there are multipIe CM genes and mRNAs, they all encode a CM with identical amino acid sequence in rats49,66. It is interesting that other CBPs, CR, CB-D28K and PV are specifically distributed in the superficia1 dorsal horn where CM-L1 is lacking. It is not clear at this time if other CBP(s) could regufate NOS activity in a way similar to CM. However, the absence of CM and presence of CR, CB-D28K and PV suggest such a possibility. Further

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studies are necessary to elucidate the role of CBPs in the superficial dorsal horn. 4.3. Dorsal gray commissure A number of peptides, including dyno~hin, enkephalin, somatostatin, chole~stokinin, avian pancreatic polypeptide, vasoactive intestinal polypeptide, substance P and neurotensin, have been localized immunohistochemically to cell bodies and fibers of the sacral DGC35*65.NIPS-diaphorase positive cell bodies and fiber networks are also densely stained in the DGC72. The distribution pattern of CR- and CBD28K-LI in the sacral DGC is very similar to that of NADPH-diaphorase” and some peptides, especially dynorphin, enkephalin and somatostatin65. Neurons in the sacral DGC may be important for processing of visceral afferent information, since these neurons receive visceral primary afferents” and send axons to reach the intermediolateral columns6’ (also see the present results). Neurons surrounding the central canal are also nociceptive and project supraspinally46. CR and CB-D28K are likely involved in visceral regulation and/or nociceptive processing in the DGC. Interestingly, the distribution of PV-LI is clearly complementary to CR and does not appear to resemble other substances in the DGC25,65. It would be very interesting to know if CR and PV are differentially involved in functional activities of DGC neurons. Further studies are necessary to identify the significance of complementary distribution of PV and CR in the DGC. 4.4. Ventral horn The present results showed that large motoneurons in the spinal lateral and ventromedial motor nuclei (lamina IX) exhibited immunoreactivity to CM antibody. This is consistent with previous reports using polyclonal antibodies to CM69,76.Comparatively, these results suggest a predominant role of CM in spinal motoneuronal activity. CB-D28K, CR and PV, on the other hand, may be involved in control of spinal motoneuronal function. Supporting the idea are the facts that there are abundant CB-D28K-, CR- and PV-LI in laminae VII and VIII, and Renshaw cells, that mediate recurrent inhibition of motoneurons, may contain CBD28K4. The distinct immunostaining of spinal motoneurons by CM antibody forms a striking contrast to apparent lack of CM-L1 in the superficial dorsal horn and less extensive staining in laminae III and IV. 4.5. Fiber tracts Among four CBPs studied, only CM labeled dorsal column gracile and cuneate fasciculi throughout all spinal cord segments, CR- and PV-LI demonstrated a rostrocaudal difference in the dorsal c01umns~~~~(see also the present results). The heterogeneity of CR-L1 in the dorsal columns has been discussed previously59.

The previous and present data suggest that in the gracile fasciculus at the lumbosacral levels, CBPs CR and PV selectively labeled fibers that terminate below the level of the gracile nucleus. These CR- and PV-IR fibers appeared to be from medium to large dorsal root ganglion (DRG) neurons, since the PV- and CR-L1 in the dorsal columns were not sensitive to capsaicin treatment7’. The ascending projections in the dorsal columns to the dorsal column gracile and cuneate nuclei include primarily axons.of large DRG neurons”. Some small capsaicin-sensitive DRG neurons may also project directly to the dorsal column nuclei via a dorsal column pathway 19s5* . These neurons may not contain CR or PV. Different from a previous report’, fibers in the gracile fasciculus were lightly stained with CB-D28IS through the rostrocaudal extent of the spinal cord. CB-D28K also showed the least immunoreactivity in Clarke’s column when compared to CM, CR and PV. The comparison suggests that a subpopulation of DRG neurons that contain CB-D28K may project directly to the dorsal column nuclei. Thus, using CBPs will provide additional markers for the study of the dorsal column pathways. The results of the ganglionectomy experiment indicated that most CBP-LI in the dorsal column gracile fasciculus was of primary afferent origin. The CBP-LI in the gracile fasciculus was greatly reduced following muftiple ganglionectomies. Similar loss of CB-D28K and PV-LI in the dorsal column after dorsal root rhizotomy has been reported”. Neurons in the lumbar ventromedial dorsal horn are known to transmit proprioceptive input to the thalamus40. Distinct CM- and PV-IR fibers and cell bodies were localized in the lumbar ventromedial dorsal horn and the CM- and PV-LI in this region were also reduced by the ganglionectomy. Yamamoto et al.” reported an apparent increase in PV-LI in the lumbar ventromedial dorsal horn after L3-5 dorsal root rhizotomies. The difference may be due to different experimental procedures. In the present study, all DRG neurons were eliminated unilaterally from multiple spinal segments, while some DRG neurons could still have spinal connections after limited dorsaf root rhizotomy. These data again suggest that CBPs appear to be involved in proprioceptive pathways. The DLF in the rat contains two functionally and anatomically distinct nuclei, the LSN and LCN26. Both ascending (spinocervical, spinocerebeliar and spinomesencephalic) and descending (from raphe and dorsal column nuclei) fibers have been identified in the DLF (see ref. 71 for a review). In general, CB-D28KfCR and CM/PV exhibited two patterns of distributions in the DLF, although further trivial distinction could be made between CB-D28k and CR and CM and PV (see the present results). It is possible that each pathway in the DLF employs one or two types of CBPs for its

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distinct functional activity. For example, descending serotoninergic fibers travel within the dorsal portion of the DLF”. Comparing the distribution of the four CBPs, CR, CB-D28K and probably CM, but not PV, may be involved in descending modulation of pain sensation mediated by serotoninergic pathways. The dense staining of cell bodies and fibers by PV in the CCN and Clarke’s column suggests that PV may be closely related to the dorsal spinocerebeilar pathway which originates primarily from CCN and Clarke’s column and ascend in the DLF37,71. Thus, the CBPs examined here could be specific markers for studying pathways associated with the DLF. Rat pyramidal tract (corticospinal tract) is located at the base of the dorsal columns between dorsal column fasciculi and the spinal gray matter. Among the four CBPs studied, only CB-D28K appeared to stain lightly the pyramidal tract. However, the existence of CBD28K in the pyramidal tract is not definitive. Using different antisera, CB-D28K has not been detectable in the pyramidal tract of human and rat1yz3.At the origin of the pyramidal tract, pyramidal neurons in layer 5B of the primary somatosensory cortex are immunoreactive to CB-D28K73, but not to PV73 and CR32,61.CBD28K mRNA is also localized in neurons in layer 5 of the cortex6’. Since CB-D28K is a soluble protein, it is conceivable that the pyramidal tract at the spinal levels would be labeled by CB-D28K antibody.

Cell nuclei were found stained by all four CBPs studied. In most cases, the nuclear staining was more intense than that of the cytoplasm. Immunostaining in the nucleus has been reported for CM12z76.For the other three CBPs, the cell nucleus is often found lacking CBP-LI in some other regions of the CNS. For example, CB-D28K immunostaining is not found in the nuclei of Purkinje and Golgi cells in the cerebellum24; and CR is not found in the nuclei of granule cells in the olfactory bulb and bipolar cells in the cerebral cortex32. PV is also not detected in the neuronal nucleus in rats”, although Bliimcke et al.* reported nuclear staining for PV in the monkey striate cortex neurons. The neuronal intranuclear immunohistochemical localization of CBPs appears to be exclusive of the nucleolus. This is different from hepatocytes that have CM in the nucleolar regiona. The functional significance of intranuclear presence of CBPs has recently been reviewed’. Other than the four CBPs compared in the present study, several other CBPs, such as calcineurin, calpactin and calpains, are also present in the cell nucleus. Abundant CBPs in the cell nucleus suggest that Ca2+ concentrations in the cytoplasm and nucleus may be differentially or independently regulated. Nuclear CBPs may participate in many important functional activities in

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the nucleus. CM has been shown to be involved in nuclear functions such as DNA replication, gene transcription and DNA repair (see ref. 5 for a review).

Acknowledgements We thank Dr. D.M. Jacobowitz for providing CR antiserum, preparing ganglionectomized animals and reviewing the final version of the manuscript and Drs. D. Besse, R. Dubner and D.J. Messersmith for reviewing the preliminary version of the manuscript.

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