NADPH diaphorase staining within the developing olfactory bulbs of normal and unilaterally odor-deprived rats

Brain Research, 460 (1988) 323-328 Elsevier

323

BRE 13932

NADPH diaphorase staining within the developing olfactory bulbs of normal and unilaterally odor-deprived rats Carolyn E. Croul-Ottman and Peter C. Brunjes Department of Psychology, University of Virginia, Charlottesville. VA 22903 (U.S.A.) (Accepted 12 April 1988)

Key words: Olfactory bulb development; Nicotinamide adenine dinucleotide phosphate diaphorase histochemistry; Short-axon cell: Sensory deprivation

Littermate rat pups underwent either unilateral surgical occlusion of the right external naris or sham surgery on postnatal day 1. At 10, 21) or 30 days postpartum olfactory bulbs were sectioned and stained using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) bistochemistry. Two types of staining were observed and analyzed. The reaction produced a Golgi-like filling of short-axon cells in both deep and superficial bulb areas. No differences in the number, morphology or distribution of these cells were found either across ages or treatment conditions, indicating that the cells are resistant to the effects of the deprivation paradigm. Large regional variations in glomerular and olfactory nerve layer staining density were also observed at each age, reinforcing notions of functional or structural differences between glomeruli at very early ages.

INTRODUCTION While the intrinsic circuitry and development of. the major neuron classes in the olfactory bulb has been extensively studied 7, little is known about the development or function of a varied population of interneurons found throughout the granule cell (GCL), external plexiform (EPL) and glomerular (GLM) layers known as short-axon cells 142°'21"2s'29. Bayer-' concluded from thymidine studies that most shortaxon cells found in the E P L are born during the first 3 postnatal days. Mair et al. is indicated that cells within both the EPL and G C L do not stain reliably with Golgi techniques until approximately day 14. Attempts to further study short-axon cells have been foiled by their relative scarcity and morphological diversity. However, Scott et al. 29 have recently observed that at least a subpopulation of the cells stain with nicotinamide adenine dinucleotide phosphate ( N A D P H ) diaphorase histochemistry ( N A D P H - d + cells). The technique, which has been used to mark discrete populations of cells in a number of brain re-

gions 12"15'17'22-26'33-36,produces a Golgi-like filling of cells as well as patterns of neuropil staining resembling those seen after treatments used to visualize relative levels of metabolic enzyme activity and 2deoxyglucose uptake ~-~,25.29. Although the enzyme is a widely employed cofactor, the actual function of the N A D P H - d detected histochemically remains to be determined 12.16.22-25,27,29.33. The present study was designed to examine the development of short-axon cells using N A D P H - d staining as a marker. Tissue from both normal and unilaterally deprived rat pups was examined. Unilateral odor deprivation, accomplished via surgical occlusion of the external naris, has profound consequences on the development of the bulb; occlusion on day 1 results in a 25% reduction in the size of the ipsilateral olfactory bulb by day 30 (refs. 6, 7). Earlier effects of occlusion on day 1 include: decreased succinate dehydrogenase (SDH) and cytochrome oxidase staining by postnatal day 4 (ref. 10), decreased dopamine content by day 8 (ref. 8) and decreased numbers of tufted cells and granule cells and

Correspondence: P.C. Brunjes, 102 Gilmer Hall, University of Virginia, Charlottesville, VA 22903, U.S.A. 0006-8993/88/$03,50 © 1988Elsevier Science Publishers B.V. (Biomedical Division)

324 glia after day 20 (refs. 4, 13, 19, 30, 31). The widespread changes seen in the bulb suggest that many cell populations, including perhaps short-axon cells, are affected.

HC1 buffer, pH 8.0) and removed when sufficient staining had occurred (50-120 min). Sections were mounted onto 1% gel-subbed slides, air-dried overnight, and coverslipped with Eukitt.

MATERIALS AND METHODS

Data analysis

Subjects The offspring of L o n g - E v a n s hooded rats (Charles River, Wilmington, MD) were housed in polypropylene cages (48 x 25 x 16 cm) with water and Purina Rat Chow available ad libitum. The breeding colony was maintained on a 16/8 h light/dark cycle and all animals appeared healthy and vigorous throughout the study. On the day after the day of birth (postnatal day 1) litters were culled to 10 pups. All pups were anesthetized with hypothermia, half underwent right external nares occlusion via cautery, and the remainder (controls) experienced cautery application to the dorsal nose surface 5'19. Tissue from 4 - 5 occluded and control littermate pairs was examined at each test age (days 10, 20 and 30).

The number, location, orientation and morphology of all superficial and internal N A D P H - d reacted cells was recorded in each bulb. Cells which surround the accessory olfactory bulb ( A O B ) and the anterior olfactory nucleus ( A O N ) were not counted, nor were the large numbers of neurons characteristically preceding these structures 27'>. A cell was counted only if its entire soma was within the plane of section and/ or processes could be readily identified emanating from the cell body. Due to the intense cell staining (Fig. 1), count/recount reliability was more than 95%. In order to compare the rostral/caudal distribution of cells each bulb was divided into 6 areas each containing equal numbers of sections. RESULTS

Normal patterns of staining in developing rats Tissue preparation Animals were anesthetized with urethane and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde (both in 0.1 M phosphate buffer, pH 7.2). The amount of perfusate was proportional to body weight (approx. 500-600 ml/kg) as the degree of perfusion was found to affect cellular, but not glomerular staining intensity >. After postfixing for 32 h, bulbs were extracted from the cranium and placed overnight in a 20% phosphate-buffered sucrose solution. Serial 80/~m coronal sections were stained (procedure modified from ref. 17) at 40 °C in individual wells containing 0.4-0.5 ml of incubation medium (100 mg% nitroblue tetrazolium, 50 rag% N A D P H (Sigma; reduced, type 1), 125 mg% monosodium malate, 0.8% Triton X-100 in 50 mM Tris-

Analyses of variance (2 x 3, Condition x Age) of total cell number (Age: F = 0.95, df = 2, P = 0.40; Condition: F = 0.42, df = 1, P = 0.52; Age x Condition: F = 0.32, df = 2, P = 0.73), total number of G C L cells (Age: F = 0.03, df = 2, P = 0.97; Condition: F = 1.57. df = 1, P = 0.23; Age x Condition: F = 1.04, df = 2, P = 0.37), and differences in the total number of cells between left and right bulbs (Age: F = 0.16, df = 2, P = 0.85; Condition: F = 2.35; Age × Condition: F = 1.86, df = 2, P = 0.18) yielded uniformly negative findings, suggesting no effect of either age or the treatment procedure (see Table I). Although adult-like cell morphology was seen in all age groups (compare Fig. 1 with those in ref. 29), processes of many of the stained cells in the 10-dayold animals appeared to be shorter and more 'swol-

Fig. 1. (a) NADPH-d+ cell with irregular soma found in 10-day-old pup. (b) NADPH-d+ cell with round soma, found in 10-day-old animal. (c) NADPH-d+ cell with fusiform cell body, found in 30-day-old animal. (d) Two darkly stained NADPH-d+ (positive) cells lining the subependymal layer (delineated by stars) of the bulb. Note lightly staining granule cells (arrows). (e) NADPH-d+ cell in EPL of 30-day-old subject. Note process (arrow) traversing into the GCL. (f) NADPH-d+ cell at base of glomeruli (curved arrows). Note lightly staining periglomerular cells (arrows). (g) Staining in olfactory nerve layer and glomeruli, (h) Coronal section through bulb of 30-day-old subject demonstrating differential staining of olfactory glomeruli and the olfactory nerve layer. Scale bars: a-f, 25 urn: g. lO0,um: h, 0.5 mm.

325

32~

TABI.E 1 .lh,m~ total number c)/' N A D P H - d cell.~ (S. E. M. ) it~ l<12 and right o!facto D' bMbs o.t"both control and occluded animals al I 0 . 2 0 a~ut 3:) dav~ G L M and E P L le[~ Control

Experimental

GCL right *

Total

lef?

right *

10

5.11(I).82)

6.1111.8)

21/ 30

20,8 (5.2) 15,214.91

18.8 13.11) 16,612.6)

99.3(111.8) 108,3 (15.9) 106,8(13.7)

119.3(9.31 105.5 (28.5) 113.4115.91

114.8(6. l) 127.9 (24,2) 126.1/116.11

10 20 30

5.8 (2.1/) 15,8(1.61 11.513,51

5.8 (1.6) 19.313.31 14.013.4)

109.3 (7.2) 119.3117,7) 97.5111.4)

115.5 (8.9) 148.3(21.71 127.3(25.31

118.1 (8.6) 152.5(21/.61 125.1(19.11

* Occluded side in Experimental subjects.

len" than in those of older ages. Furthermore, in 10day-old animals a number of small, irregular dark blebs were observed within the inner and superficial layers. These did not appear to be cells as they were, small and had no identifiable processes. While the data in Table I suggest that there was an increase in the number of superficial cells from day 10 to day 20, the small numbers of cells observed suggested that statistical comparisons were not appropriate. In each of the age groups examined large intensely stained cells were identified throughout the GCL. The distribution of observed cells did not appear to change with age; nearly all were located in the inner third of the GCL, with approximately one third of the deep cells bordering the subependymal layer (SUB). Cell morphology was quite variable. N A D P H - d + cells had large irregular, round or fusiform somata (Fig. la-c). The latter cell type was typically seen bordering the medial and lateral aspects of the SUB, with processes running parallel to the bulb lamina. Other cells contained processes radial, perpendicular and/or parallel to the bulb layering. A few had a thin process extending into the SUB. Nevertheless, it was not always possible to follow processes to their termination, a finding common with the procedure 22'29and one which obviated attempts to use the technique to quantify ontogenetic changes in the length of processes. Characteristic varicosities along the dendritic b r a n c h e s 22"25"34 and lack of spines on N A D P H - d + cells were also apparent 29 (Fig. 1). The rostral-caudal distribution of stained cells was relatively uniform. However, as previously noted in adult rats, increased numbers of cells were observed near the rostral border of the AOB and AON :v>. In

addition, lightly stained somata resembling those of granule cells in both morphology and distribution were apparent in some sections from all age groups (Fig. ld). Within the GLM most N A D P H - d + cells were located at the base of glomeruli, but a few were also observed between glomeruli or near the pial side. In all ages, the N A D P H - d + cells were large, darkly staining, and aspiny with round or irregular somata. Dendrites extended outward from the soma parallel to the EPL with some spanning 4 - 6 glomeruli. Occasionally, many lightly stained cells were seen clustered around and in between glomeruli in sections from all age groups (Fig. lf). They possessed small, rounded somata and a prominent process which branched into the neighboring glomeruli and thus appeared to be periglomerular cells. A few N A D P H - d + cells were observed in the EPL. Some in the inner EPL had a thin process extending into the GCL (Fig. le). Dendrites of deep cells arborized within the middle EPL while those more externally located had processes encircling the bases of neighboring glomeruli. Differential staining of the glomeruli was quite obvious at every age (Fig. lg,h). NADPH-d staining appeared to be associated with primary afferents as labelled processes could be seen entering glomeruli from the olfactory nerve layer (ONL) and the amount of ONL staining was proportional to that of corresponding glomeruli. In general, at all ages glomeruli on the medial and dorsolateral aspects of the bulb stained more heavily than glomeruli in other regions, although isolated heavily-labelled areas were found in many locations.

327

The effects of deprivation The overall morphology and distribution (see Table 1) of N A D P H - d + cells within the bulbs of both control and occluded animals appeared similar at all ages, and, as mentioned above, cell counts revealed no differences between occluded and control animals. Deprivation also did not seem to affect staining patterns within glomeruli as deprived bulbs exhibited a range of staining intensity similar to that of controis. However, since left and right bulbs were stained separately, quantitative comparisons of differences in glomerular staining could not be made. DISCUSSION The results of this study show that: (a) N A D P H d+ cells are apparent as early as day 10; (b) the relative number and distribution of N A D P H - d + cells changes little during early life; (c) unilateral odor deprivation does not appear to affect the number or distribution of positively labelled cells; and (d) similar patterns of glomerular staining are found to occur at all ages and in both conditions. N A D P H - d + cells appeared to be quite well formed in the 10-day-old bulb. Most were similar to those previously described in the adult 14'29, and included cells with processes extending considerable distances, including across laminar borders. Unlike previous reports, N A D P H - d + cells were observed within the middle and external GCL in each age group. The rarity of cells within these areas (1-3% of the total in the GCL) as well as strain and age differences may account for the observation. Approximately 130 total cells were observed per bulb in pups at each age and in each condition (with approximately 90% of these found in the GCL), suggesting remarkable stability of N A D P H - d cell number during a period of rapid change within the bulb 13. Scott et al. 29 reported that there were approximately 200 NADPH-d-stained neurons per bulb within the adult rat GCL, a figure approximately 60% higher than that reported above. While a number of factors could account for the difference (including variations in staining protocols (but see ref, 25), recognition criteria, strain and age differences), equal numbers of N A D P H - d + cells were observed in superficial layers in both studies, suggesting these cells may be more constant across various conditions. Nevertheless, the

numerical estimates reported above probably underestimate total numbers as all short axon cells may not be N A D P H - d + . While unilateral nares occlusion has been shown to decrease dramatically the number of cells of several types, it had little effect on number of N A D P H - d + cells. The deprivation procedure appears to affect primarily cells which are quite immature at birth (cf. ref. 13). The resistance of the N A D P H - d + cells to the procedure, along with their precocious morphology, suggests that they are well established early during life. Interestingly, N A D P H - d + cells have been shown to be quite resistant to a number of chemical and surgical insults in several brain regions 32. Furthermore, some N A D P H - d + neurons have been reported to be selectively resistant to the endogenous N-methyl-D-aspartate (NMDA) agonist, quinolinate 16. N M D A receptors are thought to play an important role in regulating neu/onal plasticity TM. Their absence perhaps buffers the cells against the effects of alterations in the early developmental milieu. Although deprivation reduces the amount of SDH and CO staining within glomeruli l°, no gross differences were seen with the NADPH-d procedure. Nevertheless, dramatic regional variations in the amount of glomerular staining were observed. Similar observations have been reported with several techniques used to visualize metabolic activity (e.g. SDH and cytochrome oxidasel°; 2-DG1"7'9). Staining with the metabolic markers tends to be localized to the glomeruli and the EPL, while NADPH-d staining was confined to the glomeruli and ONL, suggesting the techniques mark different systems, and offering convergent evidence that there are regional variations in glomerular function or organization which are evident in quite young rats. These observations reinforce the suggestion made by Cullinan and Brunjes l° regarding the importance of studying patterns and gradients of glomerular activity as potential organizers during early life. ACKNOWLEDGEMENTS Thanks to K. Ottman, D. Korol, Dr. P. Gold and Dr. D. Hill for their assistance and discussions. The work was supported by grants from the NINCDS, (NS23154), the Office of Naval Research (N0001486-K-0342) and the Whitehall Foundation.

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