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. Author manuscript; available in PMC: 2016 Apr 6.
Published in final edited form as: Eur J Neurosci. 2001 Jan;13(1):48–56.

Effect of Nicotine on Cerebellar Granule Neuron Development

Lisa A Opanashuk 1, James R Pauly 2, Kurt F Hauser 1,3
PMCID: PMC4823140  NIHMSID: NIHMS773419  PMID: 11135003

Abstract

To assess the role of nicotinic cholinergic receptors (nAChR) on neuronal maturation, nAChR expression and the direct effects of nAChR activation were examined in cerebellar external granular layer (EGL) precursors isolated in vitro. Treatment of EGL neuroblasts with nicotine elicited a concentration-dependent increase in DNA content and synthesis, implying an increase in cell numbers. Pretreatment of cultures with the nAChR antagonist dihydro-β-erythroidine (DHBE) attenuated nicotine-induced changes in DNA abundance and synthesis. Furthermore, chronic nicotine treatment for 4–7 days promoted EGL cell survival. Epibatidine but not cytisine stimulated granule neuroblast DNA synthesis and survival. Survival effects mediated by nicotine and epibatidine were attenuated by pre-treating cultures with DHBE. Immunocytochemical analysis revealed that EGL neurons possessed α3, but not α4, nAChR immunoreactivity. Quantitative autoradiography was used to determine which nAChRs are present during the period of granule cell neurogenesis in vivo. On postnatal day 5, the EGL was intensely labeled by [3H]-epibatidine but virtually devoid of [3H]-A85380 binding suggesting that a high concentration of α3 nAChRs is present in granule neuroblasts. The pharmacology of [3H]-epibatidine displacement from EGL neurons also suggested an interaction with the α3-nAChR subunits. Together these data provide novel evidence that the activation of nAChRs directly affect the development of primary cerebellar neuroblasts and further suggest that the effects are mediated through the α3-nAChR subtype.

Keywords: neuroblast proliferation, cerebellar development, neurogenesis, programmed cell death, nicotinic receptors, α3 nicotinic receptor subunits

Introduction

Accumulating evidence suggests that the cholinergic system is important during cerebellar development. Levels of choline acetyltransferase, the enzyme that catalyzes acetylcholine synthesis, are particularly high in cerebellum during the early postnatal period in humans (Brooksbank et al., 1978; Court et al., 1993) and rats (Gould & Butcher, 1987; Clos et al., 1989). During this period of development, choline acetyltransferase levels are relatively higher than levels of the degradative enzyme for acetylcholine, acetylcholinesterase (Boegman et al., 1988; Clos et al., 1989; Court et al., 1993). A developmental role for cholinergic agonists is further strengthened by several reports indicating that both muscarinic and nicotinic (nAChR) cholinergic receptor subtypes are present in perinatal periods in rats (Zoli et al., 1995; Tice et al., 1996; Winzer-Serhan & Leslie, 1997) and humans (Kinney et al., 1993; Court et al., 1995). In particular, previous studies have demonstrated nAChRs in granule and Purkinje neurons in rats (Zoli et al., 1995; Dominguez et al., 1997; Morley, 1997) and in the external granular layer (EGL), the location of granule neuron precursors, in humans (Court et al., 1995). Multiple nAChR subtypes, including α3, α4, α5, α7, β2, and β4, have been detected in cultured rat granule neurons (Didier et al., 1995). Granule neurons are the targets for cholinergic mossy fiber innervation from the dorsal pontine brainstem nuclei occurring later in development (Jaarsma et al., 1997), suggesting that acetylcholine may participate in synaptogenesis and neuromodulation (Role & Berg, 1996). Moreover, recent evidence suggests that choline itself, which is abundant in developing brain, as well as other ligands, may act as an agonist for α7 nAChRs and as a partial agonist for α3 nAChRs (Alkondon et al., 1997; Albuquerque et al., 1997a; 1997b). Together, these findings suggest that the cholinergic system may play an important role in the mediation of trophic events throughout cerebellar development, particularly in the maturation of granule neurons.

The cerebellum is an ideal system to explore potential developmental roles for nAChR because a considerable amount of cerebellar development, in both rats and mice, occurs during the first three postnatal weeks (Miale & Sidman, 1961; Altman, 1972a). In addition, cerebellar cytoarchitecture has been extensively characterized in both rats (Altman, 1972a) and mice (Miale & Sidman, 1961). Neuroblasts migrate from the rhombic lip and assume a position on the cerebellar surface to form the EGL (Altman & Bayer, 1978), which exclusively contains granule neuron progenitors (Zhang et al., 1996). In the rodent cerebellum, the EGL consists of an outer proliferative zone, with peak rates of neuroblast division occurring between postnatal days 5–8, and an inner post-mitotic premigratory zone (Altman, 1972a; Miale & Sidman, 1961). By postnatal day 21, the EGL has disappeared with cells having migrated into the internal granule cell layer (Altman, 1972b). This developmental program corresponds to the cerebellar changes seen during the late fetal period though the first 1.5 years in humans (Bayer et al., 1993). The presence of nAChRs in the EGL suggests that nicotinic agonists directly affect granule neuron precursors (Court et al., 1995).

Although previous evidence implies that nicotine can alter cerebellar development (McFarland et al., 1991; Slotkin et al., 1993; Slotkin, 1998), the direct effects of nicotinic agonists on the genesis of isolated granule neuron precursors have not been evaluated. Furthermore, it remains unclear which nAChR subtypes reside in the mouse EGL. In the present study, the effects of nicotine treatment on granule neuroblast cell turnover were examined in an in vitro model of the mouse EGL. The findings of this study suggest that nAChR activation influences granule neuron maturation during the early postnatal period.

Materials and Methods

Materials

Tissue culture media, sera, and the supplements B27 and N2 were purchased from Gibco (Life Technologies, Grand Island, NY, USA). Percoll was obtained from Pharmacia (Piscataway, NJ, USA). Cytisine, epibatidine, and dihydro-β-erythroidine (DHBE) were purchased from RBI (Natick, MA, USA). Alpha 3 and 4 nAChR subtype-specific antibodies were bought from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Secondary antibodies were purchased from Vector Laboratories (Burlingame, CA, USA). The CyQuant and Live/Dead assay kits were purchased from Molecular Probes (Eugene, OR, USA). Nicotine and all other chemicals were bought from Sigma Chemical Co. (St. Louis, MO, USA).

Granule neuroblast culture

Granule neuronal precursors were purified from the EGL of 5 day-old ICR mouse cerebella according to procedures described by Hatten and coworkers (Hatten, 1985; Gao et al., 1991). Cerebella were dissected and digested with a mixture of trypsin and DNAse in Ca2+-Mg2+-free Tyrode’s solution to produce a cell suspension. The dissociated cells were layered and centrifuged over a 35%: 60% Percoll step gradient. The granule cell fraction was preplated onto poly-D-lysine (0.1 mg/ml) substrate three times for 30 minutes each to remove contaminating glia. Nonadherent cells (granule neuroblasts) were resuspended in basal minimal essential medium containing glutamine (292 μg/ml), glucose (9 mg/ml), donor horse serum (10%) and fetal bovine (5%) serum and allowed to reaggregate into clusters in 96-well plates (3×105 cells/well) for 24 hours (34–35°C in 5% CO2/95% air). The characterization of these cultures by morphological and immunocytochemical criteria, as previously described (Gao et al., 1991), indicated that >95% of the cells were granule neurons and/or precursors. In all experiments, the effects of nicotinic agents were compared in cultures containing identical cell densities (3 × 105/well). Immediately after plating, cells were continuously exposed to nicotinic agonists and/or the antagonist DHBE for ~1 day (21 or 24 hours), or 2, 5, or 7 days.

Thymidine incorporation

Thymidine incorporation into DNA was measured by procedures similar to those previously described (Gao et al., 1991; Tao et al., 1997; DiCicco-Bloom et al., 1993; Opanashuk & Hauser, 1998). Reaggregate cell clusters were plated in serum-free medium at 3 × 105 cells/well in 96-well plates. After 24 hours in serum-free medium, [3H]-thymidine (2 μCi/ml) was added continuously for 21 hours in the presence or absence of nAChR agonists and/or DHBE, a competitive nAChR-antagonist. DHBE was added for 15 minutes prior to the addition of nicotine. Cell clusters were harvested onto filter paper with trichloroacetic acid washes and repeated water elution using a Skatron cell harvester as previously described (Opanashuk & Hauser, 1998). After the filters were dried, thymidine labeling was measured by liquid scintillation counting. At least four independent determinations from separate cell preparations were evaluated for each treatment.

Measurement of DNA content

Cells were grown as described for the [3H]-thymidine incorporation studies and treated with drugs for 48 hours. Total DNA content of EGL neurons was assessed using the CyQuant DNA assay according to the procedure provided by the manufacturer as previously described (Opanashuk & Hauser, 1998). Plates were frozen at −80°C until assayed. For determination of total DNA content, cells were thawed and resuspended in a detergent-based lysis buffer containing 1 mM EDTA and 1 μg/ml DNAse-free RNAse. After 1 hour, the CyQuant reagent, a green fluorescent dye that binds to nucleic acids, was added for 5 minutes and fluorescence measured using a Cytofluor 2300 microplate reader (Millipore, Bedford, MA, USA) with 480 nm excitation and 520 nm emission filters. The amount of DNA in each culture was extrapolated by using linear regression with bacteriophage DNA as a standard. At least 4 independent determinations from separate cell preparations were made for each treatment.

Cell Survival Assay

Reaggregate cell clusters were plated onto poly-L-lysine-coated glass coverslips (3 × 105 cells/coverslip/culture) for 2 hours to permit cell adhesion to the substrate. Cells were subsequently grown in serum free medium for 24 hours before continuous exposure to nAChR agonists and/or DHBE for an additional 1–7 days. Cultures were rinsed and incubated in Dulbecco’s phosphate buffered saline containing 4.0 μM ethidium homodimer and 3.5 μM calcein-AM at 34°C in 5% CO2/95% air. After 30 minutes, coverslips with attached cells and cell clusters were removed from culture plates, mounted on microscope slides using Prolong Antifade (Molecular Probes, Eugene, OR, USA), and counted within 24 hours. Cells were counted using a Nikon Diaphot fluorescence microscope with a long working-distance 100x-oil immersion objective and a 485 nm excitation filter. The emission filters were 530 nm for calcein and 590 nm for the ethidium ion. Live cells were distinguished by their intracellular esterase activity, determined by the enzymatic conversion of the non-fluorescent calcein-AM to calcein, which is fluorescent green in live cells. Ethidium is excluded by an intact plasma membrane, and enters cells with damaged membranes, binds to nucleic acids and fluoresces bright red. To arbitrarily sample cells, the microscope stage controller was moved along a line through the center of the coverslip and all cell clusters within the field were sampled. For each value, a random sample of 1000 cells was counted. At least 4 independent determinations from separate cell preparations were evaluated for each treatment.

Immunocytochemistry

After a 24-hour reaggregation period, EGL neuroblasts were plated onto poly-L-lysine-coated glass coverslips (3 × 105 cells/coverslip/culture) and given 2 hours to adhere. The media was subsequently removed and cells were rinsed 3 times with Dulbecco’s minimal essential medium then maintained in serum free medium, comprised of Dulbecco’s minimal essential medium with glutamine (292 μg/ml), glucose (9 mg/ml), B27, and N2. After 24 hours in serum free medium, EGL neuronal clusters were fixed in 100% ethanol containing 5% acetic acid for 15 minutes at 4°C then rinsed 3 times with phosphate buffered saline, pH 7.2. Fixed cells were incubated in phosphate buffered saline (pH 7.2) containing 1.0 % bovine serum albumin, 0.1% Triton X-100, and 10% rabbit serum for 30 minutes at room temperature. Cell clusters were then incubated with the appropriate dilution of primary antibodies (2 μg/ml) in phosphate buffered saline (pH 7.2) containing BSA and Triton-X-100 for 24 hours at 4°C for 24 hours on an orbital shaker. After 3 rinses with phosphate buffered saline (pH 7.2) cells were incubated with the rhodamine-conjugated rabbit anti-goat antibodies for one hour. After several rinses, coverslips were mounted on to glass slides in Prolong Antifade mountant (Molecular Probes). For peptide neutralization experiments, the primary antibodies were incubated with a five-fold excess of peptide antigen for 2 hours at room temperature prior to accomplishing the remainder of the immunocytochemical procedure. Controls lacking primary antiserum were used to determine the specificity of the reaction.

Receptor autoradiography

Receptor autoradiography was used to evaluate the expression of cholinergic receptor subtypes in the developing mouse cerebellum. Nicotinic binding was compared on adjacent tissue sections from P5 mice. Brain sections (16 μM thick) were sliced using a Leica CM1850 cryostat and thaw-mounted on sections coated with gelatin, chromium potassium sulfate and poly-L-lysine to increase tissue adherence. All binding assays were performed in parallel. [3H]-epibatidine autoradiography was performed as described by Perry and Kellar (1995), with minor modifications. Sections were incubated for 40 minutes in buffer containing 50 mM Tris HCl, 120 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 2.5 mM CaCl2 (pH 7.0) and 200 pM [3H]-epibatidine (DuPont/New England Nuclear, Boston, USA; specific activity = 59.7 Ci/mmol). The sections were then washed twice in ice-cold buffer (5 minutes each), once in diluted ice-cold buffer (10 sec) and once in ice-cold deionized water (10 sec). Identical binding conditions were used for 3H-A85380 autoradiography (specific activity =69.5 Ci/mmol). The sections were then washed 3 times in 50 mM Tris-HCl (4 min each; 4°C), once in diluted Tris-HCl (10 sec; 4°C) and once in deionized water (10 sec; 4°C).

Following binding, the sections were gently dried with ambient airflow from a desktop fan, desiccated overnight under a vacuum at room temperature and then exposed to Amersham Hyperfilm (Amersham Pharmacia Biotech, Piscataway, NJ, USA) for 5 weeks. Films were processed in Kodak (Kodak, Rochester, NY, USA) D-19 developer (5 min), stop bath (30 sec) and Kodak rapid fixer (5 min). After the films were developed, the sections were stained with thionin so that the autoradiographic images could be directly compared to the corresponding histological section.

Results

Nicotine increases granule neuroblast DNA synthesis and content

Nicotine caused significant increases in DNA content in EGL cultures at 48 hours (Fig. 1A, B) that were preceded by increased rates of [3H]-thymidine incorporation at 21 hours (Fig. 1C, D). As noted, [3H]-thymidine incorporation was used to estimate the proportion of dividing neuroblasts, while DNA content was used to estimate EGL cell numbers (all non-dividing diploid cells contain the same amount of DNA). DNA content was assessed at 48 h following nicotine treatment to allow sufficient time for changes in DNA synthesis (determined at 24 h) to be manifest as actual changes in cell numbers, as previously determined using other drugs (Hauser et al., 2000). The effect of nicotine on DNA synthesis was concentration-dependent (Fig. 1C) and markedly suppressed by pretreating cultures with DHBE at (Fig. 1D). Similarly, the effect of nicotine on DNA content was concentration-dependent (Fig. 1A) and significantly attenuated by DHBE (Fig. 1B) at the later (48-hour) time point. Maximal increases in thymidine incorporation and/or DNA content were observed following 1 μM nicotine and were approximately 1.5-fold higher than untreated controls. Considering that the number of cells was identical in each culture at the start of each experiment and that nicotinic agonists did not significantly affect the rate of cell death at 24–48 hours, the drug-induced increases in granule neuroblast DNA synthesis and content could result from increases in cell proliferation.

Figure 1.

Figure 1

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Effect of nicotine on DNA content (A and B) and [3H]-thymidine incorporation into DNA (C and D) in external granule layer (EGL) neuroblasts. (A) Nicotine treatment stimulated a significant increase in DNA levels after 48 hours. The effect of nicotine on DNA levels was (A) concentration-dependent and (B) antagonized by the nicotinic acetylcholine receptor (nAChR) antagonist DHBE (10 μM). Results are expressed as percent of control where DNA content in control cultures was 945 ± 242 ng/well. (C) Treatment of EGL cells with nicotine (nic) for 21 hours resulted in a modest but significant elevation in [3H]-thymidine incorporation into DNA. Increases in [3H]-thymidine incorporation were (C) concentration-dependent and (D) attenuated by pretreatment with the nAChR antagonist DHBE (10 μM). Results are expressed as percent of control defined as thymidine incorporation in serum-free media alone. In controls, there were 2.3 × 103 ± 162 c.p.m. Data represent the mean ± SEM from at least 4 separate experiments and were analyzed by ANOVA with Newman-Keuls post hoc analysis, *P < 0.05 vs. untreated cultures, bP < 0.025 vs. untreated cultures, #P < 0.05 vs. nicotine -treated and untreated cultures, aP < 0.05 vs. nicotine -treated cultures.

Epibatidine but not cytisine stimulates DNA synthesis in cultured EGL neuroblasts

To elucidate the selectivity of the effect of nicotine on DNA synthesis, thymidine incorporation was evaluated in EGL cells treated with the nicotinic agonists epibatidine or cytisine. Treatment of EGL cells with epibatidine stimulated a concentration-dependent increase in DNA synthesis (Fig. 2A). Significant increases in thymidine incorporation were observed following 1 pM epibatidine. Furthermore, the nAChR antagonist DHBE (Fig. 2B) attenuated the increases in DNA synthesis following epibatidine treatment. In contrast, treatment with cytisine failed to elicit significant changes in DNA synthesis (Fig 2A) even at 100 nM concentrations (data not shown). Baseline [3H]-thymidine counts were 2.9×103 ± 878 cpm and 1.6×103 ± 364 (for epibatidine A and B, respectively) and 2.4×103 ± 1103 (for cytisine) in untreated controls.

Figure 2.

Figure 2

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Effect of nicotinic acetylcholine receptor (nAChR) agonists on EGL neuroblast DNA synthesis. (A) Treatment of EGL cells with epibatidine (epi) but not cytisine for 21 hours resulted in elevated rates of [3H]-thymidine incorporation into DNA. Increases in [3H]-thymidine incorporation were (A) concentration-dependent and (B) attenuated by pretreatment with DHBE (10 μM). Results are expressed as percent of control defined as thymidine incorporation in serum-free media alone. Data represent the mean ± SEM from at least 4 separate experiments and were analyzed by ANOVA with Newman-Keuls post hoc analysis, *P < 0.02 vs. untreated cultures, #P < 0.005 vs. nicotine -treated cultures.

Nicotine promotes granule neuroblast cell survival

To evaluate whether the alterations in DNA synthesis and content could be attributed to altered rates of cell survival, EGL cell viability was assessed following nicotine treatment. As a control for the viability assay, EGL cells were lysed with 0.1% saponin, resulting in 100% cell death (data not shown). The live/dead markers did not overlap in the same cell and all cells were labeled as either living or dead. In general, there was a low percentage of EGL cell death 24 hours after nicotine was added, between 5–8%, in control cultures (Fig. 3). After 4 days, approximately 20 % of untreated EGL cells were dead; cell survival was slightly enhanced in response to continuous nicotine treatment (Fig. 3). Larger and more significant effects on EGL neuroblast survival were observed following 7 days of nicotine exposure. Because the greatest nicotine response on cell survival was observed 7 days following treatment, all subsequent experiments were assessed on day 7. Nicotine effects on granule neuroblast survival at 7 days were completely reversed by DHBE (Fig 3B). Furthermore, DHBE alone had no effect on cell survival.

Figure 3.

Figure 3

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Effect of nicotine on EGL cell survival. (A) Chronic nicotine (nic) (1 μM) treatment for 4–7 days results in enhanced granule neuroblast survival. (B) Nicotine -mediated survival effects at 7 days were reversed by pretreatment with DHBE. Data represent the mean ± SEM from at least 4 independent experiments and were analyzed by ANOVA with Duncan’s post hoc analysis, * P <0.05 vs. untreated cultures, #P < 0.05 vs. nicotine.

Epibatidine but not cytisine is a survival factor for EGL neuroblasts in vitro

Continuous treatment with epibatidine simulated a concentration-dependent increase in EGL neuroblast survival, compared to untreated controls, at 7 days in vitro (Fig. 4 A). The epibatidine-mediated increases in granule neuroblast survival were attenuated by DHBE (Fig 4B). In contrast, treatment with cytisine had no effect on EGL cell survival (Fig. 4A) even at concentrations up to 100 nM (data not shown).

Figure 4.

Figure 4

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Effect of nicotinic acetylcholine receptor (nAChR) agonists on EGL cell survival. (A) Epibatidine, but not cytisine, treatment for 7 days displays a concentration-dependent increase in granule neuroblast survival. (B) Epibatidine (10 pM) -mediated survival effects at 7 days were reversed by pretreatment with DHBE. Data represent the mean ± SEM from at least 4 independent experiments and were analyzed by ANOVA with Duncan’s post hoc analysis, *P <0.05 vs. untreated cultures, #P < 0.05 vs. epibatidine treatment.

EGL neuronal precursors display α3 nAChR subunit immunoreactivity and binding

EGL neuronal precursors displayed α3 nAChR subunit immunoreactivity (Figs. 5A, C), which was not evident in controls lacking primary antiserum (Fig. 5B) or in preabsorbed controls (5D). In contrast, α4 nAChR immunoreactivity was not seen in the reaggregate cultures. Alpha 3 immunoreactivity was typically diffuse and associated with neuronal cell bodies and neurites, but not the nucleus.

Figure 5.

Figure 5

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EGL neuronal precursors display α3 nicotinic acetylcholine receptor (nAChR) subunit immunoreactivity and radioligand binding. (A–D) EGL neuronal precursors form neurosphere clusters in vitro. EGL neuronal precursors isolated from 5-day-old mice displayed α3, but not α4, nAChR subunit immunoreactivity. α3 immunoproduct was associated with the cell body and neurites as visualized using (A) nickel -intensified DAB or (C) using immunofluorescence. Specific immunoreactivity was not evident in (B) controls lacking primary antiserum or in (D) preabsorbed controls. In contrast, α4-nAChR immunoreactivity was not seen in the neuronal cultures. (F–I) Autoradiographs showing nicotinic acetylcholine receptor (nAChR) binding using radioligands selective for nAChR subtypes. Radioligands were bound to coronal sections from 5-day-old mice. Note the external granule layer (arrows) at the cerebellar surface is prominently labeled with [3H]-epibatidine (F and H), and not labeled with [3H]-A85380 (G), suggesting the presence of α3 nAChR subunits in cerebellar neuroblasts. Specific [3H]-epibatidine binding was evident in the presence of unlabeled cytisine, suggesting the presence of α3 sites. Note sections F and G are more rostral than H and I; superior colliculus (SC); inferior colliculus (IC); medial vestibular nucleus (MVN). Scale bars, 20 μm (A and B); 25 μm (C–E).

Pseudocolor enhanced photomicrographs depicting nicotinic acetylcholine receptors in the mouse brainstem labeled by [3H]-epibatidine (200 pM) and [3H]-A85380 binding (200 pM) are shown in Figures 5F and 5G, respectively. In most regions of the mouse brainstem, the binding of these two radioligands was nearly identical. For example, prominent binding of both radioligands was identified in the superior colliculus (SC), inferior colliculus (IC), medial vestibular nucleus (MVN) and the deep nuclei of the cerebellum. Epibatidine binding was also present in the EGL of the cerebellum (Figure 5F, arrows); in contrast, there was no detectable binding of [3H]-A85380 in the EGL (Figure 5G). The discrepancy between [3H]-epibatidine and [3H]-A85380 binding in the EGL may indicate that epibatidine binds to multiple nAChR subtypes in developing mouse brain, similar to what has been reported in adult rodent brain. The pharmacological profile of [3H]-epibatidine binding in the mouse cerebellum was also evaluated in the present study. Sets of adjacent sections were incubated in either 200 pM [3H]-epibatidine alone (Figure 5H) or with 200 pM [3H]-epibatidine plus 50 nM unlabeled cytisine (Figure 5I). The addition of cytisine to the incubation buffer reduced [3H]-epibatidine binding to the level of film background in all brain regions except the inferior colliculus, the medial vestibular nucleus and the EGL of the cerebellum. This result supports the notion the [3H]-epibatidine binds to multiple nicotinic receptor subtypes in the developing mouse brain and that the EGL expresses a receptor with a unique pharmacological profile.

Discussion

Neurotransmitters can act as signals to influence neuronal maturation and can differentially regulate cellular proliferation, migration, survival and differentiation (Lipton & Kater, 1989; Schwartz, 1992; Hauser & Mangoura, 1998; Hauser et al., 2000). The abundance of cholinergic synthetic enzymes and receptors in the postnatal cerebellum (Brooksbank et al., 1978; Court et al., 1993; 1995) is consistent with the notion that acetylcholine is important during postnatal cerebellar development. Granule neurons contain both muscarinic (Aloso et al., 1990) and nicotinic receptors (Didier et al., 1995), and receive cholinergic innervation from mossy fiber subpopulations (Jaarsma et al., 1997). Periods of peak developmental change in granule neurons (Miale & Sidman, 1961; Altman, 1972a) coincide with the maturation of cholinergic systems during the first 3 postnatal weeks in rodents (Brooksbank et al., 1978; Court et al., 1993; 1995; Jaarsma et al., 1997).

The results of cell growth, immunohistochemical, receptor autoradiography and pharmacological displacement experiments in the present study provide some information concerning the nAChR phenotype expressed in EGL neurons. We speculate that α3 nAChR subunits are involved with nicotine-induced proliferation and survival of EGL neuronal precursors. This is based imunohistochemical evidence that α3, but not α4 nAChR’s are expressed in cultured EGL cells (Fig. 5A–E) and radioligand binding showing robust and specific binding of [3H]-epibatidine in the EGL, but negligible binding of [3H]-A85380 in the EGL in vivo. Previous autoradiographic binding studies have concluded that [3H]-epibatidine binds with high affinity to both α3 and α4 receptor subunits, whereas other radioligands such as [3H]-nicotine and [3H]-cytisine only identify receptors that express α4 subunits (Marks et al., 1998; Perry & Kellar, 1995; Flores et al., 1996). Alternatively, A-85380 reportedly has high affinity for the α4 nAChR, but not α3 receptors (Pauly et al., 1996). Findings that epibatidine, but not cytisine, affect neuronal growth and survival further support this notion.

α3 nicotinic receptor binding in the developing cerebellum has not been reported in the mouse. In the developing rat cerebellum, α3 messenger RNA is expressed in the early postnatal EGL using in situ hybridization techniques (Zoli et al., 1995; Morley, 1997). In the adult rat cerebellum, α3 mRNA expression is largely restricted to the Purkinje cell layer (Wada et al., 1989). Our findings indicate that α3 nAChR subtypes are expressed by immature EGL neurons in the mouse and by isolated EGL neurons in vitro. Interestingly, in addition to the α3 variant, granule neurons appear to express other type nAChR subtypes. In rats, α7 nAChR mRNA is expressed by Purkinje cells and the deep cerebellar nuclei (Dominguez et al., 1997). In adult granule neurons, α7-subunit immunoreactivity has a limited pattern of expression and is associated with dendrites but not the cell body (Caruncho et al., 1997). We saw little α7 binding ([125I]-BTX) binding associated with the EGL on postnatal day 5 (data not shown). Thus, although the present study strongly implicates the α3 nAChR in EGL neuroblast proliferation, other nAChR subtypes may also be present and influence development.

Heterologous expression studies demonstrate that α3 nAChRs exhibit some unique pharmacological properties that may influence development. Chronic exposure to nicotinic agonists causes a dose dependent increase in the density of brain α4 nAChRs, possibly as a result of desensitization or permanent inactivation (Pauly et al., 1991; Schwartz & Kellar, 1983; Collins & Marks, 1996; Changeux et al., 1998). However, α3 receptors respond differently to chronic agonist treatment in terms of receptor number compared to other nAChR isoforms (Olale, et al., 1997). Alpha 3 nAChRs are also more resistant that α4 subunits in terms of upregulation following agonist exposure (Flores et al., 1997; Peng et al., 1997). Why α3 receptors fail to desensitize like other subtypes is not known but repeated exposure to mainstream (or side-stream) cigarette smoke may have unique properties in neuronal populations expressing the α3 phenotype.

The effects of nicotine on development are complex, as nAChR activation appears to either stimulate or inhibit cell proliferation depending on the target cell type and experimental paradigm used. For instance, in many in vitro studies, nAChR activation can increase cell proliferation, e.g., in a thymic cell line, lung cell carcinoma, subsets of normal and neoplastic cervical cells, neuroendocrine and vascular smooth muscle cells (Tominaga et al., 1989; Maneckjee & Minna, 1990; Quik et al., 1994; Strohschneider et al; 1994; Waggoner & Wang, 1994; Carty et al., 1997). In contrast, several studies report that nicotine administration decreases rat brain DNA synthesis in several regions including the cerebellum (Navarro et al., 1989; McFarland et al., 1991; Slotkin et al., 1994), although one recent report suggests that increased numbers of mitotic neural cells are present in rat embryos exposed to nicotine in vitro (Roy et al., 1998). Studies in vivo have shown that lesions of forebrain cholinergic neurons reduce the thickness of cortical lamina, and decrease neuronal cell body size and dendritic field size in the developing neocortex (Hohmann et al., 1988; Robertson et al., 1998), suggesting that cholinergic input is trophic. Furthermore, nAChRs reportedly regulate the migration and differentiation of developing retinal ganglion neurons (Wong, 1995).

The complexity of nAChR subtypes and rich diversity of developmental actions among different species, ages, and brain regions indicates that nicotinic agonists convey highly selective and individualized information depending on the particular cell type and brain region that is targeted. In addition, the pharmacodynamics of nicotine administration and particular experimental paradigms used (e.g., in vivo versus in vitro) may affect outcome. Moreover, nAChR activation has potent systemic effects on the cardiovascular, respiratory, and endocrine systems that are likely to indirectly influence neurogenesis. For these reasons, it was important to define the intrinsic effects of nicotine on granule neuron maturation. Alternatively, because granule neurons do not normally develop in isolation, other factors (e.g., interactions with other cell types or non-nicotinic trophic factors) are likely to modify the intrinsic response of granule neurons to nAChR activation. For example, isolated EGL neurons stop proliferating when astroglia are present (Gao et al., 1991) and no longer respond to morphine when epidermal growth factor-like ligands are present (Opanashuk & Hauser, 1998). Thus, we feel that caution should be used when comparing our results to other studies because of significant differences in the developmental models utilized. It is premature to speculate whether granule neurons might respond similarly to nAChR activation in vivo, or whether the untimely exposure to nicotine during maturation might influence cerebellar development. Lastly, our data indicate that EGL cells predominantly express an α3 nAChR phenotype. The effects of nicotine on cell proliferation and survival may be exclusive to cells that express this receptor subtype. The mixed populations of cells in vivo may express alternative nAChR subunit types (α4 and α7) that may mask/overshadow the α3 mediated-decreases in DNA synthesis seen in an isolated population of neurons.

Granule neuron death occurs during normal cerebellar development in vivo (Wood et al., 1993) and in EGL cell cultures (Gao et al., 1991). It has previously been reported that nicotine prevents apoptosis of immune cells (Aoshiba et al., 1996) and lung cancer cells (Maneckjee & Minna, 1994; Heusch & Maneckjee, 1998), and is a survival factor for spinal motor neurons (Messi et al., 1997). Alternatively, in other systems nicotine can promote cell death, e.g., in embryonic rat brains (Roy et al., 1998) and at high concentrations in vascular cells (Villablanca, 1998). Like its effects on cell replication, it is likely that nicotine’s apoptotic actions are highly individualized based on cell type and developmental stage. We found that nicotine did not alter EGL neuroblast survival at early time-points in vitro, when the range of percentages of dying cells in untreated control cultures varied between 5–8%. However, as the rate of cell death increased in more mature cultures, significant effects of nicotine on survival became evident. Together the cell proliferation and survival data suggest that nicotine is initially mitogenic and later serves as a survival factor when EGL cells are dying at a higher rate. Interestingly, Yan and coworkers (1995) reported that acetylcholine prevented apoptosis in cultured granule neurons via an interaction with muscarinic AChRs. It is conceivable that activation of both nAChR and muscarinic AChRs regulate the maturation of cerebellar granule neurons, as has been suggested in retina (Wong, 1995). Last, there are numerous examples in which nicotine is neurotoxic. For example, at high concentrations (1, 10 or 100 μM), nicotine is neurotoxic to the brains of whole rat embryos (Roy et al., 1998) and causes apoptosis in cultured hippocampal neurons (Berger et al., 1998). Nicotine neurotoxicity may differ depending on the particular nAChR subtype affected. Hippocampal neurotoxicity appears to be preferentially associated with the presence of α7 nAChR subtypes, which permit large inward Ca2+ currents (Berger et al., 1998).

The mechanisms by which nAChR activation modulates granule neuron maturation are not understood. Recent reports suggest that nicotine can regulate the synthesis and/or degradation of trophic factors, including PDGF, TNFα, and TGFβ, each of which can affect cellular growth (Rakowicz-Szulczynska, et al., 1994; 1996). Nicotine stimulates the production and secretion of basic fibroblast growth factor in smooth muscle cells (Carty et al., 1996; Cucina et al., 1999). Although similar scenarios involving trophic factors could be proposed for cholinergic systems during cerebellar development see Tao et al., 1997), our findings suggest that nicotinic agonists intrinsically affect the development of isolated neuroblasts in the absence of many of the extracellular signals normally present. For example, the proliferative effects of nicotine could be related to the high Ca2+ permeability of nAChRs (Wong, 1995; Role & Berg, 1996; Berger et al., 1998). Intracellular Ca2+ signals regulate cell entry into and exit from mitosis (Reddy, 1994). Cells deprived of Ca2+ cannot progress through the G1/S phase of the cell cycle (Means & Rasmussen, 1988; Reddy, 1994). Although the specific mechanisms underlying trophic events following nAChR activation require further evaluation, this study provides evidence for a direct cholinergic role in neurogenesis.

Acknowledgments

We thank Todd Meyerrose for technical assistance. Supported by the Kentucky Tobacco and Health Research Institute, NIH NRSA NS 10007 (LAO), DA 08443 (JRP), and DA 06204 (KFH).

Abbreviations

AChR

acetylcholinergic receptor

BrdU

5′-bromodeoxyuridine

DAB

diaminobenzidine

DHBE

dihydro-β-erythroidine

EGL

external granular layer

G1

pre-DNA synthesis phase of the cell cycle

IC

inferior colliculus

MVN

medial vestibular nucleus

nAChR

nicotinic cholinergic receptor

PDGF

platelet-derived growth factor

PBS

phosphate buffered saline

S-phase

DNA synthesis phase of the cell cycle

SC

superior colliculus

TGFβ

transforming growth factor β

TNFα

tumor necrosis factor α

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