Neuron,

Vol. 8, 1191-1204,

June,

1992, Copyright

0 1992 by Cell Press

CNS Glial Cells Support In Vitro Survivial, Division, and Differentiation of Dissociated Olfactory Neuronal Progenitor Cells Sarah K. Pixley Department of Anatomy and Cell Biology University of Cincinnati College of Medicine Cincinnati, Ohio 45267-0521

Summary Olfactory receptor neurons (ORNs) are replaced and differentiate in adult animals, but differentiation in dissociated cell culture has not been demonstrated. To test whether contact with the CNS regulates maturation, neonatal rat olfactory cells were grown on a culture substrate or on CNS astrocytes. Mature ORNs, immunopositive for olfactory marker protein (OMP), disappeared rapidly from both systems. Neurons positive for neuron-specific tubulin (immature and mature) disappeared from substrate-only cultures, but remained abundant in the cocultures. OMP-positive neurons reappeared after 10 days in vitro. Pulse labeling with 13H]thymidine showed extensive neurogenesis of both immature and mature olfactory neurons. This demonstrates, in vitro, both division and differentiation of olfactory progenitor cells. Introduction In the olfactory neuroepithelium, which forms part of the lining epithelium of the nasal cavity, olfactory receptor neurons (ORNs) are continuously generated from dividing neuronal progenitor cells in most normal adult animals, including mammals (Hinds et al., 1984; Mackay-Sim and Kittel, 1991; Miragall and Monti Graziadei, 1982; Graziadei and Monti Graziadei, 1978, 1979; Schwartz Levey et al., 1991). Continual neuronal turnover is very rare elsewhere in the mammalian nervous system. More extensive regeneration is seen in the olfactory epithelium after experimental manipulations that lead to death of significant numbers of mature ORNs (Camara and Harding, 1984; Harding et al., 1978; Monti Graziadei and Graziadei, 1979; Samanen and Forbes, 1984; Verhaagen et al., 1990). The newly generated ORNs differentiate, as shown by initiation of expression of the olfactory marker protein (OMP), a soluble protein of unknown function (Margolis, 1985) found almost exclusively in mature ORNs (Baker et al., 1989; Farbman and Margolis, 1980; Monti Graziadei et al., 1977,198O). Differentiation of the newly generated ORNs also includes formation of appropriate, functionally active central connections with the olfactory bulb (Costanzo, 1985; Harding et al., 1977; Hurrt et al., 1988; Simmons and Getchell, 1981; Wright and Harding, 1982). Because both neurogenic and differentiation events occur in the adult, there has been intense interest in using the peripheral olfactory epithelium as a model system for studies of the regulation of these processes.

Regulatory factors for ORN generation and differentiation have not been definitively identified, despite numerous studies. One proposal is that contact between the axons of ORNs and the olfactory bulb initiates final differentiation of the ORN. This hypothesis is supported by data from explant culture studies (Chuah and Au, 1988; Chuah and Farbman, 1983; Chuah et al., 1985), bulbectomy and nerve section studies (Costanzo and Graziadei, 1983; Harding et al., 1977; Miragall and Monti Graziadei, 1982; Monti Graziadei, 1983; Samanen and Forbes, 1984; Schwartz Levey et al., 1991), and developmental studies (Farbman and Margolis, 1980; Monti Graziadei et al,, 1980). However, other studies have shown that OMP expression can be stimulated in ORNs without specific bulb contact, for example, by contact between ORNs and nonolfactory bulb CNS tissues, or by placement of olfactory tissues in the anterior chamber of the eye (Barber and Jensen, 1988, Graziadei and Monti Graziadei, 1986; Monti Graziadei, 1983). Olfactory progenitor cell proliferation appears to be regulated by death of ORNs (Hinds et al., 1984; Monti Graziadei and Graziadei, 1979; Schwartz Levey et al., 1991) and/or spatial restrictions at the surface of the epithelium (Chuah and Au, 1988; Chuah et al., 1985). To identify more definitively the regulatory mechanisms controlling ORN generation and differentiation, it would be useful to have dissociated cell cultures of ORNs. ORNs have been difficult to identify and maintain in monolayer cultures, as shown by the fact that comparatively few primary systems had been described prior to 1991 (Calof and Chikaraishi, 1989; Coon et al., 1989; Gonzalez et al., 1985; Noble et al., 1984; Pixley and Pun, 1990; Schubert et al., 1985). In the systems in which ORNs were examined, OMPpositive neurons were not present, or OMP staining was possibly artifactual (Coon et al., 1989; Gonzalez et al., 1985; Pixleyand Pun, 1990). More recent reports suggest that the lack of OMP-positive neurons may have been due to a lack of trophic factors, because OMP-positive neurons can survive after plating onto CNS astrocytes (Chuah et al., 1991; Trombley and Westbrook, 1991), or after plating in medium containing nerve growth factor (Ronnett et al., 1991). No reports to date have shown differentiation of ORNs after plating. The latter situation would allow study and manipulation of the regulation of differentiation. Because astrocytes are one of the components of the CNS that ORNs contact after they enter the glomeruli of the olfactory bulb (Doucette, 1990) and becauseastrocytes had been shown to support neuronal survival in olfactory (Chuah et al., 1991; Trombley and Westbrook, 1991) and non-olfactory (Banker, 1980; Temple, 1989) cultures, disaggregated nasal epithelial cells from newborn rat pups were plated on monolayers of CNS astrocytes (cocultures) and compared with the same cells plated on the culture substrate

NWUXl 1192

alone (single cultures). Neither of these conditions supported survival of freshly disaggregated, OMPpositive, mature ORNs for more than a few days. However, in the cocultures, olfactory neurons immunopositive for neuron-specific tubulin, which stains both immature and mature neurons in newborn rat tissue sections, survived for more than 35 days. Moreover, differentiation of the tubulin-positive neurons occurred because OMP-positive neurons reappeared after IO days in culture. This effect was not olfactory bulb specific, as OMP-positive neurons also reappeared after plating nasal cells on glia from two other non-bulb CNS areas. [3H]thymidine pulse labeling demonstrated that the reappearance of OMP-positive neurons was due to division and differentiation of neuronal progenitor cells after plating. While division of olfactory neuronal progenitor cells has been described previously in cell cultures (Calof and Chikaraishi, 1989; Coon et al., 1989), this work demonstrates both division and differentiation of olfactory neuronal progenitor cells in a primary, dissociated cell culture system. Results Olfactory

Precursor

Cells

and

Mature

Olfactory

Neurons Died Rapidly in Cultures Plated on the Substrate Only Inapreviousstudy,cellsthatwereimmunostainedfor OMPwere not found in dissociated nasal cell cultures grown in a serum-containing medium on polylysinecoated glass coverslips, although neurons responsive to odors were abundant (Pixley and Pun, 1990). To determine whether or not OMP-positive neurons survived the disaggregation and entered the cultures, similar cultures were examined at earlier times after plating. A serum-free medium was used because it will be advantageous in future studies of regulatory mechanisms. A general neuronal marker antibody, the monoclonal antibody TUJI, which binds specificallyto a neuron-specific form of tubulin (the class Ill J3 isoform; Geisert and Frankfurter, 1989), was used to identify immature as well as mature neurons. In cryostat-cut, paraformaldehyde-fixed, tissue sections of the newborn rat olfactory epithelium, TUJI stained globose basal cells (olfactory neuron progenitor cells), immature olfactory neurons, and all mature, OMPpositive neurons (data not shown). In 1 day nasal cell culturesgrown on polylysine-coated glass (Figure IA), TUJI-positive neurons were abundant (5862 k 509 neurons per cm2, n = 4 experiments; Figure 2A; Table 1). However, the number of TUJI-positive cells dropped rapidly to only 5% of these values at day 4 after plating (293 + 98 neurons per cm2, mean + SEM, n = 5 experiments; Figure IB; Figure2A).TUJl-positive neurons were rare at day 6 (Figure IC; Figure 2A) and day 10 (Figure 2A) and were undetectable at day 15 (Figure 2A). Mature, OMP-positive, olfactory neurons were present at 1 day after plating, but at very low numbers

(258 k 56 neurons per cm2, n = 5 experiments; Figure ID; Figure 2A) compared with the number of TUJIpositive neurons. Because OMP-positive neurons disappeared at the same rate as TUJI-positive neurons: the number present at 4 days was barely detectable (14 k 5 neurons per cm2, n = 6 experiments; Figure IE; Figure 2B), which explains our earlier failure to detect them (Pixleyand Pun, 1990). OMP-positive neurons were rarely seen after 6 days (Figure IF; Figure 28). OMP-positive neurons were primarily non-process bearing (Figure ID), while the majority of TUJIpositive neurons had processes (Figure ‘iA). Cells not immunostained (predominantly glial cells and fibroblasts) also decreased in number with time in culture (Figures 16 and IF). The decrease in immunopositive cell numbers appeared to be due to cell death, based on changes in the patterns of immunostained debris and cell morphologywith time. The amount of TUJI-stained debris increased in reverse proportion to the number of live cells during days I-4, with most seen around days 3 and 4 (Figure IB). Much less debris was seen by and after day 6 (Figure IC). OMP-stained debris was most abundant duringthe first l-3 days after plating (Figure ID) and was far less evident after 3 days (Figure IE), which would be consistent with the numbers of ceils that were available to die. In addition, from day 2 on, the almost complete lack of processes and the small size of the few intact OMP-positive cells seen in culture suggested that the cells were in poor health. Double immunofluorescence labeling in 1 day cuitures showed that virtually all (97.6%) OMP-positive cells inthesubstrate-onlycultureswereaJsoTUJ1 positive (n = 338 OMP-positive neurons examined, multiple experiments; see Figures 5A and SB). The lower number of processes on OMP-positive cells was not duetoa lackof staining in the processes becauseTUJ1 staining showed the same pattern (see Figure 5). The few OMP-positive neurons (2.4%) that wereTUJ1 negative appeared to be stained nuclei of dying OMPpositive cells that had lost all other cytoplasm, because OMP staining appeared to be both nuclear and cytoplasmic, while TUJI staining was exclusively cytoplasm?. Olfactory Neuronal Precursor Cells Survived and Differentiated in Nasal-Glial Cell Cocultures ORN axons contact their target neurons only after they enter the glomerular area of the olfactory bulb. ThisisalsothefirstplaceinwhichtheyencounterCNS astrocytes. Prior to that point, they are ensheathed by the unique glial cells of the olfactory nerves (Doucette, 1990). Because the stimuli for ORN support and differentiation may come from the olfactory bulb and because astrocytes have survival-promoting and differentiating effects on neurons in cu!ture (Banker, 1980; Chuah et al., 1991; Temple, 1989; Trombley and Westbrook, IVVI), the hypothesis that contact with CNS glial cells might promote survival and possibly differentiation of ORNs was tested. Nasal epithelial

In Vitro 1193

Neurogenesis

and Differentiation

Figure Grown tures)

1. ORNs in Cultures of Nasal Cells on the Substrate Only (Single Cul-

(A-C) TUJI immunostaining; (D-F) OMP immunostaining. (A and D) 1 day cultures; (B and E) 4 day cultures; (C and F) 6 day cultures. Cells positive for TUJI (A) were much more abundant than OMP-positive neurons(D) in 1 day cultures. TUJI-positive cells in 4 day cultures (B) were still very healthy, although far fewer in numbers. Abundant TUJl-positive debris was present. OMP-positive neurons and debris were very infrequent at 4 days (E). At 6 days after plating, TUJI-positive (C) or OMPpositive (F) neurons were rarely seen. Bar, 71 pm.

., C8

F

cells were plated on monolayers of CNS (for source see below), which were prepared dard purification methods (Levison and 1991) and contained predominantlyastrocytes

Table

Culture

1. Estimated

Type

Single Cultures f-nrllltllrPs - - - -. .-. - -

Total

Yield

of Neurons

Ave. Number Neurons/cm2 5,900 26.000

‘astrocytes by stanMcCarthy, 085%)

at 1 Day after

of

at the mined (GFAP) Survival

start of the coculture by staining for glial (data not shown). of TUJI-positive

experiments, fibrillary acidic neurons

to 1 day

as deterprotein after

plat-

Plating

Total Yield of Neurons/Experiment

Yield of Neurons as % of Total Cells Plated

7 x 105 3 x 106

6% 26%

Colume 2: numbers of TUJI-positive neurons per cm* were determined at 1 day after plating for single cultures and cocultures. Column 3: the average total yield of neurons per experiment was estimated by multiplying the column 2 numbers by the approximate area of culture surface plated in an average experiment (120 cm2; 120 wells, 2.5 plates of 48 wells, each well having a surface area of 0.96 cm?. Column 4: the yield of neurons, as a percentage of all cells plated the day before, was determined by dividing the number of neurons seen at 1 day by the number of cells originally plated (1 x IO5 cells per cm2).

Neuron 1194

Figure 2. Quantification ing In Vitro

of Immunostain-

(Aand B) Quantification of immunostained ceils in single-cell cultures. The number of TUJI-positive neurons per cm* (A: was at all times much higher than the number of OMP-positive neurons per cm2 (B), but both cultures showed rapid exponential drops in cell number between days 1 and 4. TUJI-positive cell numbers were underDA”S IN CO-CULTL!?E estimates at days 1 and 2 because of overlap I ot cells In occasionally seen small clumps, -^ N 10 presumably remnants of undissociated epithelium. (C) OMP-positive neurons in nasal-glial cell cocultures were more numerous at 1 day than in single cultures, but the numbers dropped just as rapidly in the first 4 days after plating. At 10 days after plating, the numbersof OMP-positive neurons had begun to rise and reached a plateau by day 14 that remained high through day 20. Bars give SEMs from 3-17 experiments. (D) Measurements were made of the outer diameter ot the sphencal, central areas ot clumps contalnlng TUJI-positive neurons (open circles) and clumps containing only nonneuronal cells (closed circles). Clumps with neurons increased in size with time in culture, while nonimmunostained clumps did not. Linear regression lines and r2 values were computed with the Sigmaplot software.

ing was approximately 4 times greater in the cocultures than in the single cultures (Figure 3A; Table ‘I). This number (a slight underestimate because of cell clumping)was roughlyone-fourth (26%)ofall thecells plated 1 day earlier (1 x IO5 cells per cm2; Table 1). At 4 days after plating, TUJI-positive neurons were still very abundant (Figure 3B), although counting was no longer possible because almost all neurons were found in clusters or clumps, with considerable cell overlap (Figure 36). By IO days, TUJI-positive neurons were very abundant and formed densely packed, large, circular clumps, with neuronal processes forming radiating bundles (Figure 3C). The size of neuronal, but not nonneuronal, clumps increased with time in culture (outer diameters were measured at the central portion of the clumps; Figure 2D). TUJIstained debris was abundant before and at 4days after plating (Figure 3B), but was rare at 10 days or later (Figure 3C; Figure 4A). By 15 days postplating, multicellular clumps were large enough to be seen by eye. TUJI neurons were either densely packed in the large clumps (Figure 4A, right side) or more loosely associated in flattened clusters (left side). TUJI-stained cells were asymmetrically bipolar, like ORNs in vivo. Dendrite-like processes were thicker and shorter, with terminal, expanded endings reminiscent of the dendritic knobs of ORNs (Figure 4A; Figure 5C). Axon-like processes were thinner and longer. Most TUJI-positive cell bodies and processes were tightly associated with other TUJIpositive cells or processes. The stained processes formed bundles (Figure 4A) with nonneuronal cells. These bundles either interconnected clumps, ended spread out on the glial monolayer, or joined other process bundles to end in tangles in nonneuronal clumps (data not shown).

OMP-positive neurons were much more numerous in the cocultures at 1 day after plating (732 neurons per cm*; Figure 2C) than in cultures grown on the substrate. More cells had processes (Figure 3D). By day 4,OMP-positive cell numbers had decreased to a low of 47 f 8 neurons per cm2 (Figure 3E; Figure 2C), and the numbers remained at this level through 6 and 7days (Figure 26). Surprisingly, at 10 days postplating, some experiments (5111) showed the reappearance of OMP-positive neurons (Figure 3F; Figure Figure 2C). At 14-15 days after plating, the cocultures were dramatically different because they contained numerous, densely OMP-stained neurons (Figure 4B). OMPpositive neurons were found exclusively within the multicellular clumps and the interconnecting fiber bundles (Figure 4B; Figure 50). OMP staining appeared to be both nuclear and cytopiasmic, and the density of staining was variable in both places (Figure 4A). Similar nuclear staining and variable densities have been reported in tissue section staining (Samanen and Forbes, 1984). Because these were wholemount-like preparations we cannoi say whether the staining was intranuclear or perinuclear. OMP-positive neurons in general differed from the majority of TUJI-positive cells by having shorter processes and fewer cells with processes and by being equidistantly spaced within the clumps. They were never tightly packed, as seen with TUJI staining. This suggests that, in addition to OMP expression, other cellular characteristics change during maturation of ORNs. Identical staining patterns were seen with rabbit anti-OMP serum (data not shown). Only diffuse background staining was seen when the primary (goat) anti-OMP serum was replaced with normal goat serum, at the same dilution (Figure 4F). Double immunostaining with TUJl and anti-OMP

In Vitro 1195

Neurogenesis

and Differentiation

Figure 3. Nasal Cells Cocultured with cal Astrocytes (O-IO Days in Culture)

Corti-

(A-C) TUJI immunostaining; (D-F) OMP immunostaining. (A and D) 1 day cocultures; (B and E) 4 day cultures; (C and F) 10 day cultures. TUJI-positive cells in 1 day cultures (A) were more numerous and healthier in appearance than cells in single cultures. The numberofTUJl-positivecells in 4 day cultures (B) remained high, unlike the number in single cultures. TUJl-positive cells clustered together ([B] right side), resulting in cellular overlap that impeded counting. Abundant TUJI-positive debris was also seen at l-4 days after plating ([B] left side). TUJI-positive neurons in 10 day cultures (C) formed large, multicellular clumps, with neuritic processes emerging from the clumps in tightly packed bundles. OMP-positive neurons were more common in 1 day cultures (D) than in single cultures, but there were very few compared with the number of TUJI-positive cells. Some OMP-positive cells in clumps had processes. OMP-positive neurons at 4 days (E) were rare. Greater numbers of OMP-positive neurons, with lower staining densities in general, were seen in 10 day cultures (F). Bar, 71 Wm.

serum (Figures 5C and 5D) in the 15 day cocultures showed that all OMP-positive neurons that could be clearly examined were TUJI positive. Evaluation of individual cells in the center of the clumps was not possible because of the intense fluorescence with TUJI. After 15 days in culture, signs of degeneration were seen in the clumps (Figures 4C and 4D show cultures at 25 days), even though the number of OMP-positive neurons remained constant through day 20 (Figure 2C). Fewer OMP-positive neurons were process bearing (Figure 4C). With TUJI staining, visible breaks wereseeninthepackingdensityintheclumps(Figure 4D), and an increase in TUJI-stained cellular debris (data not shown) suggested the loss of cells. Disorganized areas of either tangled process endings or cellular debris were seen in the clumps (Figure 4D, arrow).

It became more difficult to maintain the cultures after 15 days because the clumps and/or the whole cellular sheet showed a tendency to detach during feeding, fixation, or antibody staining. Previously, Chuah and Farbman (1983) showed that epithelial explants plated for 10 days in vitro, in contact with olfactory bulb explants, contained twice as many OMP-positive neurons as epithelial explants plated alone. This effect was not seen when epithelial explants were grown with other CNS or PNS tissues. To examine the specificity of the astrocytes in the cocultures, nasal cells were plated on glial cells derived from the newborn rat brain olfactory bulb, cortex, or cerebellum.Table2showsthatglialcellsfromallthree sources supported the appearance of very similar numbers of OMP-positive neurons in 15 day cocultures.

NelJrCJn 1196

Figure trols

4. Cocultures

(>I5

Days)

and

Con-

(AandB)15daycocultures;(CandD)25day cocultures;(E and F) controls. TUJI-positive cells in cocultures fixed at 15 days were either densely packed in clumps ([A] right side) or found in looser groups spread out more on the monolayer ([A] left side). Neuritic process bundles emerged from ihe clumps (A). OMP-positive neurons in 15 day cocultures (6) were numerous in large, multicellularclumps,withvariablestaining densities, although numbers were lower than those with TUJI staining. Few OMPpositive neurons had processes, and those processes were shorter than the average TUJI-positive cell. OMP-positive neurons were as numerous at 25 days after plating (C) as at 15 days, but far fewer cells had stained processes. TUJI-positive cells at 25 days after plating (D) no longer formed complete packing layers in the clumps. Also, areas of dense staining that were either tangles of processes or cellular debris ([D] arrow) were seen. No OMP-positive cells were seen in nasal cell cultures fed with fresh 50% astrocyte-conditioned medium at every medium change, fixed at 15 days,and immunostainedforOMP(E).Normat goat serum substituted for OMP primary antiserum showed high background staining in 15 day cocultures, but no densely stained cells (F). Bar, 71 pm.

Cell-cell communication can occur via soluble factors or physical contact. Chuah and Farbman (1983) showed that physical contact, and not a soluble factor, was responsible for the olfactory bulb-stimulated increase in OMP-positive neurons in epithelial explants. To test these alternatives in the current system, single cultures of nasal cells were fed with freshly collected DSNI medium, conditioned by the CNS astrocytes, at concentrations of 50% and 25%. After 15 days, cell numbers in general were higher than those without conditioned medium, but noOMP-positive(Figure4E) or tau-positive neurons were seen. (Tau staining resembled TUJI staining; this was another measure of general neuronal staining.) The stimulus for support of neuronal survival and reappearance of OMP-positive neurons might have been simply the higher cell numbers used in the cocultures. To test this, nasal cells were plated at twice

the normal plating density on the substrate only, to roughly approximate the cell numbers plated in the cocultures. At 15 days postplating, cell density in general had increased, but the cultures were not confluent, and no OMPor TUJI-positive neurons were detected (data not shown; culture appearance was very similar to Figure 4E). The DSNl culture medium contained supplements not commonly used in brain cultures. To determine how this medium would affect the glial cells, glial cultures were maintained in DSNl for 15 days, fixed, and immunostained with TUJI and anti-OMP serum. No OMP staining was seen, but low numbers of TUJIpositive cells were present in the glial cultures. The latterwere multipolar and showed very littletendency to associate with other TUJI-positive cells (data not shown). Similar multipolar, TUJI-positive cells were found as isolated cells outside the clumps and ridges

In Vitro 1197

Neurogenesis

and Differentiation

Figure 5. Double TUJI and Anti-OMP

lmmunolabeling Serum

with

(A and B) 1 day single cultures; (C and D) 15 day cocultures. (A and C) TUJI labeling; (B and D) OMP labeling. In 1 day cultures of nasal cells grown on the substrate only, 97.6% of all OMP-positive neurons (B) were TUJI positive (A). Double-labeled cells rarely had processes. In 15 day cocultures, all OMP-positive neurons (D) that could be examined were TUJI positive (C) (arrows show double-immunolabeled cells). TUJl staining was too intense in clumps to distinguish individual cells. At all times, OMP staining was in both the cytoplasm and the nucleus, while TUJI staining was only cytoplasmic. OMP was visualized with rhodamine optics and TUJI with flourescein. Bars, 35 urn. Bar in (B) applies to (A) and (B); bar in (D) applies to (C) and (D).

in the cocultures. Because of their suspected glial origin, they were not included in the pulse-labeling counts (see below). Mature Olfactory Neurons Were Generated from Precursor Cells in Nasal-Glial Cell Cocultures Reappearance of OMP-positive neurons in the cocultures indicated induction of differentiation in progenitor cells that were either rescued from dying by plating on astrocytes or newly generated after plating.

Table 2. Olfactory Neuron Astrocytes from Different

Astrocvte

Source

Olfactory Cortex Cerebellum

Bulb

Differentiation Brain Regions

after

Growth

OMP-Positive Neurons/cm*

SEM (n)

2270 2974 2384

541 VI 897 (4) 390 (3)

on

Nasal cells were plated on purified astrocytes derived from newborn rat olfactory bulb, cortical, or cerebellar tissues. The numbers of OMP-positive neurons per cm2 were determined for cultures fixed at 15 days after plating. Average and SEM values are given for at least 3 experiments per cell type. No significant differences were seen (p > 0.05, Student’s t test).

To determine whether OMP-positive neurons were generated by division of progenitor cells after plating, the cocultured cells were pulse labeled with [3H]thymidine. Neurons that had divided during the pulse, and therefore had taken up isotope, were detected by combined immunocytochemistry and liquid emulsion autoradiography. Double-labeled cells (immunostained cells with silver grains deposited over the nuclei) were seen with both OMP (Figures 6A and 6B) and TUJI (Figures 6C and 6D) staining. This demonstrates that both immatureand matureORNsweregenerated by division of progenitor cells after plating in culture. Both immunoperoxidase (Figures 6A and 68) and immunofluorescence (Figures 6C and 6D) staining techniques gave successful double labeling. The variability of silver grains per stained cell (Figure 6) is consistent with an unsynchronized dividing cell population (Simpson-Herren, 1987; Bayer and Altman, 1991). Because some neurons (Figures 6C and 6D, closed arrows) contained approximately as many silver grains as the most intensely labeled nonneuronal cells (Figures 6C and 6D, open arrows), differentiation of these neurons might have occurred after only one cell division. To ensure detection of double-labeled cells and to

Neuron 1198

figure

6. Neurogenesis

in the Cocuitures

(A and B) Cells immunostained with antiOMP serum using immunoperoxidase; (C and D) cells immunostained with TUJI using immunoflourescence (rhodamine). OMP-stained neurons (A and B) showed deposition of significant numbers of silver grains in the emulsion iayer centered over the cell nucleus, after a pulse of [3H]thymidine was delivered to the cells between days 1 and 4 (A) or8 and 10 (B). Cells immunostained for TUJI (C and D) also showed silver grain deposition after a pulse at 8-11 days after plating. (5) is a combined brightfield and fluorescence exposure. Arrows in (C) and (D) indicate a double-labeled cell, and open arrows show grain deposition over an unstained, presumably nonneuronal ceil. Bars, 35 Km. Bar in (B) applies to (A) and (B); bar in (D) applies to (C) and (IX

determine the timing patterns of progenitor cell division during the standard 15 day culture period, a pulse labeling protocol that covered all time periods in culturewasused,andthenumberof double-labeledcells generated was quantified (see Experimental Procedures; Figure 7). Immediate uptake of t3H]thymidine into OMP-positive neurons in culture was not expected because this does not occur in the intact animal (Graziadei and Metcalf, 1971; Graziadei and Monti Graziadei, 1978,1979; Hinds et al., 1984; Mackay-Sim and Kittel, 1991; Miragall and Monti Craziadei, 1982; Moulton, 1974; Schwartz Levey et al., 1991). In fact, it takes 7 days after injection of isotope into a normal animal before isotopic label can be detected in OMPpositive neurons (Miragall and Monti Craziadei, 1982). Because we did not know whether this timing would apply to the cultures and because cell division might not have been occurring at all times after plating, a pulse-labeling paradigm that allowed coverage of all I5 days of the standard culture time was chosen. The paradigm used was similar to that used to determine neuronal birthdates in the whole animal (Bayer and Altman, 1991). As diagramed in Figure 7A, PH]thymidine (in culture medium) was added to a separate set of wells at each medium change (dotted lines). Nonra-

dioactive medium was used for all other medium changes (Figure 7A, solid lines). All cells were fixed at day 15, an arbitrary time chosen as representative of a mature culture. Figure 7A shows that the OMPpositive cells that were double labeled after fixation at 15 days were generated almost exclusively between 6 and 11 days after plating. Double-labeled cells positive for TUJl staining (Figure 7C) were generated almost exclusively between 8 and 13 days after plating. The lack of overlap between the two antibody peaks at 6 and 7 days is not at odds with the doubleimmunolabeling data for OMP and TUJI because the actual numbersof OMP-positive, double-labeled cells were very low compared with the numbers of TUJIpositive cells. This technique does not allow determination of whether neurogenesis occurred at other time points during the culture. The “age” of double-labeled neurons was the time between “birth” (midpoint of pulse that resulted in labeling) and “death” (time of fixation). Using the upper scales in Figure 7, the average age of the doublelabeled, OMP-positive neurons was 6.5 days, while the average age of theTUJl-positive cells was 4.5 days, the midpoints of the respective peaks of doublelabeling. Because all OMP-positive neurons were

In Vitro 1199

Neurogenesis

and Differentiation

tioned medium or a higher density of plating, and it did not depend on the source of CNS glia. Furthermore, we have shown that both immature and differentiated olfactory neurons were generated in culture by restricted bursts of neurogenesis.

3H THYMIDINE ADMINISTRATION (IN DAYS PRIOR TO FIXATION)

fyyy*

0

1

2

3

14

4

5

12

6

7

10

8

9

8

10

11

6

12

13

4 0

40 ~-

z

30

15

2

,

16 06

OMP

0

s

14

0

x 9

0

20.-

0

1

2

3

4

5

6

10

7

8

8

9

10

6

11

12

4 0

0

10

11

13

14

15

2

yc

TUJ 1

10 0 0

1

2

3

4

5

6

7

8

9

12

13

14

15

3H THYMIDINE ADMINISTRATION (IN DAYS AFTER PLATING) Figure 7. Timing with pH]thymidine

of Neurogenesis:

Analysis

of Pulse

Labeling

A sample experimental protocol is shown in (A). pH]thymidine was added to one set of culture wells at each feeding (dotted lines) and removed at the next. All feedings were covered, but different wells received each pulse. Except at the pulse time, cells were maintained in nonradioactive medium (solid lines). Two coverslips each were stained with TUJI or anti-OMP serum, and the number of stained cells with silver grains was expressed as a percentage. The average value was assigned to the midpoint time of the pulse (closed diamonds in [A]; open circles in [B] and [Cl). Linear regression curves (solid lines) were generated using the Sigmaplot software with an order of 8 for OMP (r2 = 0.65) and an order of 6 for TUJI (rZ = 0.87). Only one subset of all OMP-positive and TUJI-positive cells is considered by fixing and analyzing the cells at day 15. Given this, the data in (B) and (C) show that the cell division that produced the subsets occurred only during restricted times after plating.

TUJI positive, this means that the OMP-positive neurons, as a group, were an older subset of all TUJIpositive neurons.Thedatado not provide information on whether all “older” TUJI-positive cells were OMP positive. Discussion Mature, OMP-positive neurons disappeared rapidly after plating nasal cells in dissociated cell culture. However, if nasal cells were plated onto a CNS glial cell monolayer, continued survival of immature neurons was seen, followed by differentiation into olfactory neurons. This effect was not duplicated by condi-

Clial Cell Support of Neurogenesis and Differentiation Glial cell support of survival of non-olfactory CNS neurons or neuroblasts has been described previously (Banker, 1980; Temple, 1989). In our cultures, the glial monolayers were not providing an abundant or robust soluble factor, because conditioned medium was not as effective as the live monolayer. This is in agreement with previous explant culture studies, in which physical contact and not soluble factors regulated ORN cell numbers (Chuah and Farbman, 1983). In our cultures, a soluble factor cannot be completely ruled out by the conditioned medium experiments because a soluble factor(s) could have been either labile (rapidly degraded in the medium) or required in higher concentrations (i.e., those occurring when cells are closely apposed). While the CNS cultures were composed of predominantlyGFAP-positiveastrocytes, purified according to standard techniques (Levison and McCarthy, 1991), the small numbers of non-astrocytic cells present may have provided the survival support. Cocultures of nasal cells with other types of cells, notably Schwann cells or fibroblasts, were not tested because both types of cells were present in the nasal cell cultures (which contain all of the cell types of the lamina propria), and support of neurons was not seen even after plating the nasal cells at twice the normal density. Despite this control, it is still possible that if the other nasal cell types were purified and present in larger numbers, a survival-promoting effect might be seen. Such an effect should exist because, in vivo, immature ORNs must survive prior to establishment of contacts between ORN axons and the olfactory bulb. Our laboratory has previously demonstrated that there is a small subpopulation of densely GFAP-positive, astrocyte-like cells in the nasal cell cultures (approximately 10% of all cultured nasal cells) and in the olfactory nerves (Pixley, 1992). It is tempting to speculate, although no data exist to support this at present, that the astrocyte-like subpopulation of the olfactory nerve glia might provide the needed minimal level of ORN support, perhaps amplified by enclosure within the olfactory nerve spaces. The unexpected appearance of small numbers of TUJI-positive cells in the glial monolayers, found with all three sources of glia, introduces the possibility that the neurons seen in cocultures were actually from the glial monolayer.Argumentsagainstthis possibility are that OMP staining was never seen in glial monolayers grown alone; OMP-positive cells were never multipolar; separate counts of double-labeled, multipolar, TUJI-positive cells after addition of [3H]thymidine gave a constant level of 20%-30% labeling (data not

NCWlWl 1200

shown), not the restricted pattern of labeling seen for bipolar, TUJI-positive cells; and astrocytes grown in nasal cell-conditioned medium did not show production of OMP-positive neurons (Pixley, unpublished data). Despite this, it is still conceivable that contact with the nasal cells could convert glial, TUJI-positive cells into bipolar, OMP-positive cells. More involved experimentation is needed to address this issue. Interestingly, the TUJI-positive cells do not appear to be type 2 astrocytes, because they do not show double immunolabeling with anti-GFAP serum (Pixley, unpublished data). Regional specificity of glial support of ORN progenitor cell division and expression of OMP was not seen. A definite region-specific effect of the olfactory bulb on olfactory neurons has been demonstrated only in explant culture studies (Chuah and Farbman, 1983). Other studies have shown that ORNs can express OMP without making any contact with the CNS (Monti Craziadei, 1983), after making contact with cortical tissue during regeneration (Graziadei and Kaplan, 1980; Graziadei and Monti Graziadei, 1986), or after transplantation of epithelial tissues into nonolfactory CNS areas, even into the anterior chamber of the eye (reviewed in Barber and Jensen, 1988). In addition, partial recovery of OMP protein levels, mRNA levels, and OMP-positive cell numbers has been observed in vivo after unilateral bulbectomies (Costanzo and Craziadei, 1983; Margolis et al., 1974; Monti Graziadei, 1983; Verhaagen et al., 1990). Finally, in the explant studies, many OMP-positive neurons survived for 10 days in olfactory epithelial explants grown alone. Coculture with olfactory bulb explants resulted only in a2-fold increase in OMP-positive cells in the epithelial explants (Chuah and Au, 1988; Chuah and Farbman, 1983). The coculture study shown here suggests that the cue for regulation of ORN survival and maturation was produced equally by astrocytes from three different brain regions (or by another type of CNS nonneuronal cell equally distributed in all the glial monolayers). ORN Genesis and Differentiation in Culture Division of olfactory neuron progenitor cells in culture has been demonstrated previously. Calof and Chikaraishi (1989) showed genesis of N-CAM-positive olfactory neurons in a monolayer culture system derived from epithelial explants from embryonic mice. Coon and colleagues (Coon et al., 1989) showed genesis of olfactory neuroblasts in cell lines with neuronal characteristics. The study described here demonstrates both generation and differentiation of OMPpositive olfactory neurons in dissociated cell culture. The glial monolayer has been identified as the source of support for neurogenesis and differentiation. L3Hlthymidine uptake is generally accepted as evidence of cell division. However, other possibilities exist. To rule out uptake of [xH]thymidine by mitochondrial DNA synthesis, or breakdown of thymidine and use of the 3H in other compounds, only cells with

silver grains deposited in a nuclear pattern (most commonly seen) were included in analysis. Uptake into DNA as a repair mechanism did not seem likely, because some neurons were as heavily labeled as nonneuronal cells. Also, the timing of labeling was not correlated with accumulation of immunostained cellular debris (a measure of cell distress), and the TUJI and OMP labeling peaks occurred at different times. The latter would not happen if neuronal repair were occurring either continually, or in response to a specific stimulus. Neurogenesis occurred at specific, restricted times after plating for a specific set of TUJ’l-positive and OMP-positive neurons that was identified by fixation at day 15. The interpretation of the pulse-labeling data must be conservative because all cells could not be counted and there was some loss of data sets due to technical problems (see Experimental Procedures). The linear regression curves shown in Figure 7 are for illustration only, and they are not intended to imply a correlation with a biologically significant model. However, it is interesting that the fits are significant. Becauseonlyone staticwindowoftime in culturewas analyzed by fixing all cells at day 15, the status of neurogenesis at earlier or later times in culture is not known. The data suggest that the olfactory neuronal progenitor cell was not OMP positive. This is shown by the low levels of double labeling on the right side of the OMP curve seen in Figure 7B. Using the upper scale in the figure, ORNs that were less than 4-5 days old (labeled after day IO-II in culture) were not OMP positive at day 15. This suggests that it takes about 4-5 days for a progenitor cell to finish its division and then differentiate into an OMP-positive neuron. Similarly, the low levels of dou ble labeling of TUJI-positive cells immediately before fixation (Figure 7B) appear to suggest that the dividing progenitor cell was not TUJI positive. However, these data are less definitive than the OMP data because, if the cell cycle time (unknown) was greater than 24 hr, then insufficient numbers of cells might have been labeled in the shorter pulses used just before fixation to give an accurate reading. In support of the idea of a TUJl-negative progenitor cell is the fact that the strictly bipolar, processbearing, cultured TUJI-positive cells were very different from the “cloud-like,” N-CAM-positive, neuronal progenitor cells described by Calof and Chikaraishi (1989) in their olfactory cell cultures and from the globose basal cells seen in our staining of newborn rat tissue sections. The differences between our cocultures and the Calof cultures could be a consequence of the age of the host tissue (we used newborn rats and they used embryonic mouse tissues), or differences in the substrate and medium conditions. However, it is also possible that there is a TUJI-negative progenitor cell in both the epithelium and our cocultures that we have not yet detected. The lack of significant labeling after early pulse times (the left side of the curves in Figure 7) could

In Vitro 1201

Neurogenesis

and Differentiation

be explained if neuronal progenitor cells died shortly after being generated, if there was low cell division during the first few days after plating, or if the neuronal progenitors at early times in the cultures divided sufficient numbers of times that the isotopic label was too dilute to detect. Because OMP-positive neurons were not seen until around 10 days after plating, the first two explanations are favored since either might occur if the conditions in early cultures were not adequate. The[3H]thymidine pulse-labeling data indicated that OMP-positive neurons were a longer lived subset of all TUJI-positive cells. This raises an interesting question. Are culture conditions actually inducing differentiation and OMP expression, or are they simply allowing longer survival of immature neurons, which then are intrinsically programmed to express OMP after a certain length of time? This question is interesting because, even after coculture with astrocytes, the culture conditions did not appear to be optimal. Relatively few ORNs completed maturation compared with the numbers of TUJI-positive cells. Also, the estimated average ORN lifespans (4.5 and 6.5 day medians for TUJIand OMP-positive cells, respectively) were relatively short, compared with estimates in vivo of either around 30days (Graziadei and Monti Graziadei, 1979; Mackay-Sim and Kittel, 1991), or up to or past 90 days (Hinds et al., 1984; Mackay-Sim and Kittel, 1991). Addition of some factor that might be bulb specific, i.e., contact with olfactory bulb neurons, might be needed to improve ORN survival. Alternately, better survival, or complete maturation, of ORNs might require formation of a three-dimensional organization similar to the intact epithelium. This is suggested by two observations: OMP expression in the cocultures occurred only after the formation of complex multicellular clumps, and OMP-positive neurons were found almost exclusively in the clumps or cellular ridges. In summary, improvements in the culture conditions to allow longer survival of immature neurons may be all that is necessary to provide greater numbers of OMP-positive neurons that live longer in culture. The liming of In Vitro Phenomena: Comparisons with the Situation In Vivo Despite the differences in survival times between cultured ORNs and ORNs in vivo, other variables were similar between the in vitro and in vivo situations. First, in both single cultures and cocultures, OMPpositive neurons disappeared by around 6 days after disaggregation, apparently as the result of cell death. This is very similar to the situation in vivo, where nerve section or bulbectomy results in maximal decreases in OMP protein levels or number of OMP-positive neurons by days 3-8 after injury (Harding et al., 1977; Margolis et al., 1974; Monti Graziadei, 1983; Samanen and Forbes, 1984; Schwartz Levey et al., 1991; Verhaagen et al., 1990). Second, reappearance of OMPpositive cells in the cocultures occurred by around 10

days after plating. This is similar to the reappearance of OMP-positive cells after nerve section or bulbectomy in the animal, which occurs around 8-12 days after injury (Craziadei and Monti Graziadei, 1980; Samanen and Forbes, 1984). Third, the time needed for differentiation of ORNs into OMP-positive cells was estimated at around 4-5 days, or a median time of 6.5 days, in the more established, more stable 15 day cocultures. This situation can be compared to that in the intact animal, in which 7 days is the time needed for injected isotope to appear in mature ORNs (Graziadei and Monti Graziadei, 1978; Miragall and Monti Graziadei, 1982). Not only are the times in culture very similar to those in vivo, both situations show a slightly longer time to differentiation after injury, which is perhaps necessary for clearing of debris and repair of damage before initiation of neurogenesis and differentiation. In summary, we have found novel dissociated cell culture conditions that provide support of olfactory neuronal progenitor cell survival, division, and differentiation into olfactory receptor neurons. The timing of events in culture was very similar to that of events in vivo, which suggests that these cultures can serve as a model system for studies of the regulation of olfactory neurogenesis and neuronal differentiation. These cultures might also be useful for investigation of the developmental regulation of, for example, the recently discovered multigene families of putative olfactory receptor proteins (Buck and Axel, 1991) and putative odorant-binding proteins (Dear et al., 1991). Experimental

Procedures

Newborn Rat Nasal Cell Cultures Details of the culture methods for the newborn olfactory epithelial cells are given in Pixley and Pun (1990) and Pixley (1992). Briefly, the cartilage and overlying soft nasal tissues of newborn to 2-day-old rat pups were cut into small pieces, rinsed in sterile suspension minimum essential Eagle’s medium (CIBCO, Long Island, NY) with 1.1 g/l sodium bicarbonate, 13 mM HEPES (pH 7.3), 1 g/l bovine serum albumin (BSA) (Sigma, St. Louis, MO), 2.5 g/l nystatin, 2 mM glutamine, 100 U/ml penicillin, and 0.1 mglml streptomycin and incubated in the same with 0.3% BSA, 0.125% trypsin, and 0.175 mg/ml collagenase (Sigma #C-0130) (2 ml for tissues from 10 rat pups) for 1 hr. The tissues were mechanically disaggregated by passage through a plastic pipette, and passed through a 210 Pm nylon mesh (Tetco, Elmsford, NY) to remove cartilage. The resultant dissociated cells were counted with trypan blue (by hemocytometer) and plated at 1 x IO5 cells per cm* on glasscoverslips (IO mm diameter rounds; Dynalab, Rochester, NY) that had been detergent washed, etched with 10 M NaOH, and coated with poly-r-lysine (#P7890, Sigma), at 0.05 mg/ml in 0.15 M sodium borate buffer (pH E.O), overnight. Cells were grown in a serum-free medium, DSNI: Dulbecco’s modified Eagle’s medium (Whittaker, Walkersville, MD) supplemented with 4.5 g/l glucose, 3.7 g/l sodium bicarbonate, 18 mM HEPES, 1 mM sodium pyruvate, 1 x nonessential amino acids, 4 mM glutamine, 30 pM hypoxanthine, 3 ftM thymidine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 25 PM 2-mercaptoethanol, and a modified Nl supplement mix (Nl with 20 pglml transferrin; Bottenstein, 1984). Unless specified, all materials were from Sigma or CIBCO. CNS Astrocyte Cultures Olfactory bulb, cortex,

and

cerebellum

tissues

from

newborn

NWKX? 1202

animals were disaggregated in 5% trypsin (CIBCO) for 15 min at room temperature with mechanical dissociation. Cells were maintained in a serum-containing medium (DMEMIF-lZ[l:I]with 15 mM HEPES, 1.2gll sodium bicarbonate, 100 U/ml penicillin,O.l mglml streptomycin, 2.5 ml/l nystatin, and 10% fetal calf serum [Hyclone, Logan, UT]) for 7 days, then shaken to remove oligodendrocytes and neurons (Levison and McCarthy, 1991). The attached cells were removed with trypsin and EDTA and replated at 1 x IO* cells per cm2 on NaOH-etched, uncoated glass coverslips in 48 well plates in the astrocyte medium. Before adding nasal cells, the medium was changed to serum-free DSNI. To prepare the nasal-glial cell cocultures, nasal cells in DSNI were seeded onto CNS nonneuronal cells at 1 x IO5 cells per cm2 and maintained for 4 days before the first medium change, with medium changes every 2-3 days thereafter. lmmunocytochemistry on Cultured Cells Cells were fixed without rinsing (to minimize cell loss) at 37OC in 4% paraformaldehyde (Fisher, Cincinnati, OH) in 0.1 M phosphate buffer (pH 7.4) for 15 min at room temperature. Fixed cells were incubated for 2 hr in Hank’s balanced salt solution with 0.2% Triton X-100 and 10% horse serum, overnight at room temperature in the primary antibody, for2 hr in biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, CA), and for 1.5 hr in the Elite ABC reagent (avidin DH-biotinylated horseradish peroxidase, 1:400; Vector Laboratories). The chromogen was diaminobenzidine (0.5 mg/ml in 0.1 M phosphate buffer [pH 7.31) with excess glucose oxidase to generate H,O, (ltoh et al., 1979). All reagent dilutions and rinses were in phosphatebuffered saline (0.01 M phosphate, 0.15 M NaCl [pH 7.31) with 0.2% Triton X-100, except the ABC and color development steps. Antibodies used were TUJI, a monoclonal antibody specific for the class III B isoform of tubulin (1:SOOO; gift of A. Frankfurter; Geisert and Frankfurter, 1989), anti-OMP serum (goat antiserum, 1:3000; rabbit antiserum, 1:lOOO; both gifts of F. Margolis), antitau serum (1:250, monoclonal antibody; gift of L. Binder), and normal goat serum used as a control in place of the goat antiOMP serum (equivalent sources were Vector Laboratories and Sigma). For double immunofluorescence staining, primary goat and mouse antibodies were combined, secondary antibodies were a mixture of unlabeled rabbit anti-goat (1:200; Southern Biotech. Assoc., Inc., Birmingham, AL) and biotinylated horse anti-mouse (1:200; Vector Laboratories) sera. The tertiary antibodies were FITC-labeled streptavidin (1:150; Vector Laboratories) and TRITC-labeled donkey anti-rabbit IgG (1:200; Chemicon, Temecula, CA). After immunostaining, coverslips were mounted cell side down on microscope slides with Gelvatol (Harlow and Lane, 1988). Cells were counted in 10 microscope fields per coverslip at 100 x magnification (total of 0.25 cm2, 32% of the IO mm coverslip). Routinely, two coverslips were counted per condition per experiment. Clump diameterwas determined with an eyepiece micrometer. Pulsing labeling with [3H]thymidine and Combined lmmunocytochemistry and Autoradiography For pulse labeling, [3H]thymidine (specific activity 6.7 Ci/mmol; DuPont, Bannockburn, IL) was diluted in DSNI and added to a different set of 4 culture wells at each feeding time, providing consecutive coverage of 15 days in culture (see Figure 7A for sample experimental design). At all other feedings, cells received nonradioactive DSNI. The first medium change was not done until day 4 to reduce loss of cells, then at l-3 day time periods. All cells were fixed at 15 days and immunostained with TUJI or anti-OMP serum, as described above. Cells on coverslips were postfixed in95% ethanol,airdried, mounted cell sideuponglass slides with Permount (Fisher), and dried for 24 hr. For liquid emulsion autoradiography, the slides were dipped in Kodak NTB-2 emulsion (I:1 with water, 37”C, in the dark), dried horizontally, and exposed in desiccated, light-tight boxes at 4°C for 2 weeks (for 0.2 and 0.1 uCi/ml [‘Hlthymidine) or 4 weeks (for 0.05, 0.02, and 0.01 LrCi/ml). Slides were developed in Dektol-19 (I:1 in water, 6 min, 15OC), fixed in Kodak Rapid Fix, dehydrated in alcohol, cleared in Hemo-de (Fisher), and coverslipped with Per-

mount (Fisher). Coverslips from one experiment were immunostained and processed together. Double-labeled cellswere those immunostained cells that had significantnumbersofsilvergrains(over background)deposited in the emulsion over the cell nucleus. Cells with grains only over the cytoplasm (rare) were not counted. Two coverslips per pulse were stained with anti-OMP serum and two with TUJI. One hundred immunopositive cells were examined per coverslip, and the average number of stained cells per pulse per experiment, expressed as a percentage, was assigned to the midpoint of the puIsetime(Figure7A, closed diamonds). Only cells in monolayer areas or on the uppermost surface of the clumps were counted, as the emulsion did not penetrate the clumps. TUJI-positive, isolated, stellate cells, presumably derived from the astrocyte layer and easily distinguished from bipolar, aggregaiing neurons, were not included in the analysis. A separate count (data not shown) of these cells gave a fairly constant rate of 20%-30% double-labeled cells in each pulse period. Pulse times were variable, in part because the feeding schedule varied with the health of cells and density of cultures and in part because this allowed generation of more time points without increasing the frequency of rinsing. Increased number of rinses resulted in lower numbers of OMP-positive neurons at 15 days (data not shown). Also, initially, removal of isotope was followed by rinsing three times. This appeared to be the cause of selective loss of all or almost all OMP-positive (but not TUJIpositive) neurons at pulse times that later were determined to correspond roughly to times of high OMP-positive neurogenesis. Rinsing was changed to simple removal and addition of medium (a reduction by l/IO of the medium cpm). Toxicity of pH]thymidine (0.2 LrCilml) was the other possible variable, so different concentrations of pH]thymidine were tested. Despite differences in thymidine concentration, pulse length, and completeness of pulse coverage, all data points from 6 experiments showed tight clustering without normalization for peak values (Figures 7B and 7C, open circles). Peak widths and timing did not vary if the data were normalized for length of pulse time (by dividing the percentage of labeled cells by the days of pulse and plotting a portion on each day of that pulse). The values plotted at the pulse midpoints are shown in Figure 7, as this involved the least amount of data manipulation. Best fit curves were generated by Sigmaplot (Jandel Scientific, Corte Madera, CA). Acknowledgments I would like to acknowledge the excellent technical support of Mei Shi and Raymond Grill. I would also like to acknowledge the excellent support and constructive criticism of the members of the Program Project Grant on Development and Regeneration in Vertebrate Chemoreception. This work was supported by American Paralysis Association Grant #PBl-8803-I and National Institutes of Health grant #DC00347. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

March

6, 1992; revised

April

16, 1992.

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