Agonist-mediated regulation of presynaptic receptor function during development of rat septal neurons in culture

Authors

  • Andreas Ehret,

    1. Laboratory of Neuropharmacology, Institute for Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Hansastraße, Freiburg, Germany
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  • Anja Birthelmer,

    1. Laboratory of Neuropharmacology, Institute for Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Hansastraße, Freiburg, Germany
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  • Susanne Rutz,

    1. Laboratory of Neuropharmacology, Institute for Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Hansastraße, Freiburg, Germany
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  • Céline Riegert,

    1. Laboratory of Neuropharmacology, Institute for Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Hansastraße, Freiburg, Germany
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  • Anna Katharina Rothmaier,

    1. Laboratory of Neuropharmacology, Institute for Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Hansastraße, Freiburg, Germany
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  • Rolf Jackisch

    1. Laboratory of Neuropharmacology, Institute for Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Hansastraße, Freiburg, Germany
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Address correspondence and reprint requests to Dr Rolf Jackisch, Laboratory of Neuropharmacology, Institute for Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Hansastraße 9A, D-79104 Freiburg, Germany.
E-mail: rolf.jackisch@pharmakol.uni-freiburg.de

Abstract

Presynaptic receptors modulating the release of acetylcholine (ACh) were studied in fetal septal neurons cultured in a growth medium to which various drugs were added from day 3 in vitro (DIV 3) to DIV 14. The influence of these drugs on the function of the presynaptic muscarinic (M-) autoreceptor was determined at DIV 14 by measuring the inhibitory effect of the M-agonist oxotremorine on the electrically-evoked release of [³H]ACh from cultures pre-incubated with [³H]choline. The presence of the M-agonists oxotremorine (100 μmol/L) or carbachol (100 μmol/L) from DIV 3 to DIV 14, or from DIV 13 to DIV 14, abolished M-autoreceptor function at DIV 14, whereas the presence of the M-antagonist atropine (10 μmol/L from DIV 3 to DIV 14) during growth left M-autoreceptor function unaltered. Inhibition of ACh esterase by donepezil (1 μmol/L from DIV 3 to DIV 14) weakly decreased M-autoreceptor function at DIV 14; inhibition of neuronal firing by 0.1 tetrodotoxin (0.1 μmol/L from DIV 3 to DIV 14) did not tend to affect M-autoreceptor function at DIV 14. Co-cultivation of fetal septal and raphe neurons for 2 weeks yielded cell cultures containing both vesicular ACh transporter- and tryptophan hydroxylase-immunopositive cells. From these cultures, the release of both [³H]ACh and [³H]5-HT could be induced by electrical field stimulation. In co-cultured neurons versus septal-only ones the inhibitory effect of oxotremorine on the evoked release of [³H]ACh appeared almost normal, whereas that of the selective 5-HT1B agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrollo[3,2-b]pyrid-5-one (CP-93,129) was completely abolished. The effects of CP-93,129 were also absent on DIV 14 in septal mono-cultures grown in the presence of CP-93,129 (10 μmol/L) from DIV 3 to DIV 14. It is therefore concluded that the regulation of presynaptic receptor function strongly depends on the concentrations of endogenous transmitters in the neuronal environment.

Abbreviations used
ACh

acetylcholine

5-HT

serotonin

CP-93,129

3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrollo[3,2-b]pyrid-5-one

DIV

days in vitro

ED

embryonic day

GR-55,562

3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenyl]benzamide

GPCRs

G-protein coupled receptors

KH buffer

Krebs–Henseleit buffer

NS

not significant

NGF

nerve growth factor

TrpH

tryptophan hydroxylase

TTX

tetrodotoxin

VAChT

vesicular acetylcholine transporter.

The hippocampal release of acetylcholine (ACh), which originates from axon terminals of the septo-hippocampal cholinergic projection, is modulated by ACh itself via presynaptic muscarinic autoreceptors of the M2 subtype (Richards 1990; Wall et al. 1994) and, among other heteroreceptors [see: (Vizi and Kiss 1998)], also by serotonin (5-HT) via presynaptic 5-HT1B heteroreceptors (Maura and Raiteri 1986; Cassel et al. 1995; Vizi and Kiss 1998).

Both M2 and 5-HT1B receptors belong to the large family of G-protein coupled receptors (GPCRs). During the last two decades much evidence has been accumulated showing that the availability and function of GPCRs at the neuronal cell surface is regulated by the neuronal environment, which – following receptor stimulation – induces intraneuronal trafficking of GPCRs. The trafficking process involves several steps: internalization of the receptors by endocytosis, followed by their intraneuronal sequestration, their eventual degradation (down-regulation) and/or de-novo synthesis and finally, recycling of the GPCRs to the plasma membrane surface; [for reviews see: (Koenig and Edwardson 1997; Bloch et al. 1999, 2003; Bernard et al. 2006)]. Thus, changes in the neuronal environment of GPCRs may lead to changes in the responsiveness of these receptors to the corresponding neurotransmitter and thereby affect the transmission of information from the pre- to the postsynaptic cell.

We have long been interested in the consequences of changes in the neuronal environment on the presynaptic modulation of transmitter release and their possible implication in cognitive processes. In view of the significant importance of cholinergic/serotonergic interactions in the hippocampus and other brain structures on both the presynaptic (see above) and the cognitive level [e.g. (Cassel and Jeltsch 1995)], we lesioned hippocampal afferent neurons either by non-selective techniques (aspirative or electrolytic lesions) or by neuron-specific lesion techniques (stereotaxic injections of cholinergic or serotonergic neurotoxins: 192IgG saporin, or 5,7-dihydroxytryptamine, respectively); in most of these experiments, the neuronal environment of the presynaptic axon terminals was further modified by transplantation of fetal septal or fetal raphe cells into the denervated hippocampus [e.g. (Cassel et al. 1995; Jackisch et al. 1999; Suhr et al. 1999; Birthelmer et al. 2002a,b)].

To further study such questions on a more cellular level, we have developed primary cell cultures from the fetal [embryonic day 17 (ED 17)] septal (Ehret et al. 2001) or the fetal (ED 15) raphe region (Birthelmer et al. 2007). In these cultures we were able to evoke the release of ACh or 5-HT, respectively, by electrical field stimulation and to show, that this release was modulated by several presynaptic receptors. Neuronal cell cultures are especially well suited to study the influence of the neuronal environment on the expression of functional receptors, especially because it is very easy to modify the culture conditions. It was therefore, the aim of the present investigation to study the influence of exogenous muscarinic agonists and of manipulations of endogenous ACh levels on the expression and/or function of muscarinic autoreceptors in cultured septal cholinergic neurons. Moreover, we were interested in the question of how co-cultivation of both septal and raphe cell cultures, i.e. whether the presence of 5-HT releasing cells, affected the expression and/or function of the M2 autoreceptor or the 5-HT1B heteroreceptor, respectively, in cholinergic neurons.

Materials and methods

Chemicals and drugs were purchased from the following sources: [methyl-³H]choline chloride from Amersham Biosciences (Freiburg, Germany); 5-[1,2-3H(N)] hydroxytryptamine creatinine sulphate ([3H]5-HT; 21,8 Ci/mmol) from NEN Life Sciences (Frankfurt, Germany); 3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenyl]benzamide (GR-55,562); 6-nitro-2-(1-piperazinyl)-quinoline (6-nitroquipazine) from Biotrend (Köln, Germany); atropine sulphate, oxotremorine sesquifumarate, tetrodotoxin (TTX) from Sigma-Aldrich (Munich, Germany). The following drugs were kindly donated: 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrollo[3,2-b]pyrid-5-one (CP-93,129) from Pfizer (Groton, CT, USA), carbachol from Merck (Darmstadt, Germany), donepezil hydrochloride from Eisai Co. Ltd (Koishikawa, Japan) and (+)oxaprotilin (CGP 12104A) from Novartis (Basel, Switzerland). Sources of products used for cell cultivation and immunocytochemistry are indicated hereafter or in preceding papers from our group (see below).

Preparation of cell cultures

Cell suspensions from the fetal septal region of the rat (cholinergic cultures) at ED 17 were prepared exactly as described previously (Ehret et al. 2001), plated at a density of about 650 000 cells/cm² on circular glass cover slips (5 mm diameter) and cultured for up to 14 days [day 14 in vitro (DIV 14)] at 37°C in a humidified air/5% CO2 atmosphere. The growth medium consisted of Dulbecco’s modified Eagle’s medium (Nut. Mix. F-12 with Glutamax-1, InVitrogen GmbH, Karlsruhe, Germany) containing 10% NU-serum TM IV (Schubert & Weiss, München, Germany), a mixture of insulin, transferrin, and sodium selenite (5 μg/mL, 5 μg/mL, and 5 ng/mL; Sigma-Aldrich, München, Germany), a mixture of penicillin and streptomycin (50 U/mL and 50 μg/mL; InVitrogen GmbH) as well as mouse nerve growth factor (10 ng/mL) and human neurotrophin-3 (50 ng/mL; Alomone/ICS, München, Germany). The growth medium was changed every 3–4 days.

Treatment of septal cell cultures during growth with various drugs was performed (starting from DIV 3 until DIV 14, or for 24 h only, i.e. from DIV 13 to DIV 14) by the addition to the culture medium of aliquots of sterile-filtrated concentrated stock solutions of the corresponding drugs. To remove these drugs from the medium at DIV 14, the cell cultures were washed 3–5 times with pre-warmed (37°C) superfusion medium (see below) before starting the pre-incubation with [³H]choline. Moreover, pre-perfusion of the cell cultures (for 49 min at a rate of 0.6 mL/min) during the release experiment (see below) further contributed to the washout of drugs present during growth of the cultures.

Septal/raphe co-cultures (cholinergic/serotonergic co-cultures) were prepared in a similar manner, using at the starting day both rat fetal septum tissue (at ED 17) and rat fetal raphe tissue (at ED 15). To obtain the septal or raphe region, respectively, dissection of the fetal brains was performed according to the literature (Dunnett and Björklund 1992; Svendsen 1995). The tissue pieces obtained were dissociated using the same steps of trituration, trypsinization, and centrifugation as described previously [see: (Ehret et al. 2001) and (Birthelmer et al. 2007)]. Finally, cells of septal and raphe origin were plated on the circular glass cover slips at a density of about 325 000 cells/cm² (for each fetal brain region) and cultured for up to 14 days as above. Treatment of septal/raphe co-cultures with 10 μmol/L GR-55,562 during growth (starting from DIV 3 until DIV 14) was performed as described above for septal mono-cultures.

Immunostaining of cells

Mono-cultures of cholinergic neurons or serotonergic neurons were characterized by immunocytochemical techniques as described previously [see: (Ehret et al. 2001) and (Birthelmer et al. 2007)]. In the present investigation, only the immunocytochemical characterization of co-cultured septal and raphe neurons will be shown.

Vesicular acetylcholine transporter

Cultured cells were washed with 0.1 mol/L sodium phosphate buffer (PB; pH 7.35) and fixed for 30 min with PB containing 4% paraformaldehyde. Following fixation, the cultures were washed for 30 min in PB and then treated with 2% normal horse serum (Gibco, Invitrogen GmbH, Life Technologies, Karlsruhe, Germany) and 0.1% Triton X-100 in PB for 30 min. Cells were then incubated for 12 h at 4°C with a polyclonal vesicular acetylcholine transporter (VAChT) antibody (goat anti-VAChT affinity purified polyclonal antibody; Chemicon International, Temecula, CA, USA) 1 : 500, 0.1% Triton X-100, and 2% normal horse serum in PB. Cultures were washed with PB (60 min) and incubated with Cy3 conjugated donkey anti-goat antibody (1 : 500; Rockland, Gilbertsville, PA, USA) in PB for 1 h at 24°C. The stained cultures were briefly washed with water and mounted in Moviol® (Hoechst, Frankfurt, Germany).

Tryptophan hydroxylase

Immunostaining of the co-cultures for tryptophan hydroxylase (TrpH) was performed as described for VAChT (see above) using the following antibodies: a polyclonal TrpH antibody (sheep anti-TrpH affinity purified polyclonal antibody; Chemicon International; 1 : 500) and a Cy3 conjugated donkey anti-sheep antibody (Rockland; 1:500).

Electrically-evoked release of acetylcholin and serotonin

Circular cover slips (cell culture discs) with either septal cholinergic neurons, or with co-cultured septal/raphe cells were grown for DIV 14 and then transferred to a 35-mm Petri dish filled with pre-warmed, modified Krebs–Henseleit buffer [KH buffer, composition in mmol/L: NaCl, 118; KCl, 4.8; CaCl2, 1.3; MgSO4, 1.2; NaHCO3, 25; KH2PO4, 1.2; glucose, 11; ascorbic acid, 0.57; and Na2EDTA, 0.03; saturated with carbogen (95% O2, 5% CO2), pH adjusted to 7.4]. Cells were washed carefully with this medium 3–5 times (see also above). Subsequently, the ‘cell culture discs’ were incubated for 30 min at 37°C in 2 mL KH buffer containing either 0.1 μmol/L [3H]choline (for ACh-release experiments), or 0.1 μmol/L [3H]5-HT (+1 μmol/L oxaprotilin, for 5-HT release experiments) under an atmosphere of carbogen. Oxaprotilin was routinely present during the pre-incubation phase of 5-HT release experiments to prevent false labeling of noradrenergic neurons. These might be present in the culture as a result of the anatomical proximity of the L coeruleus and the raphe region in the embryonic brain [see (Birthelmer et al. 2007) and Fig. 5 in Dunnett and Björklund (1992)]. Following incubation, the cover slips were carefully washed with pre-warmed, gassed KH buffer, transferred to 12 superfusion chambers (two cell culture discs, back to back, per chamber) and superfused continuously with KH buffer (25°C, gassed with carbogen) at a rate of 0.6 mL/min in the presence of hemicholinium-3 (10 μmol/L, ACh release experiments) or 6-nitroquipazine (1 μmol/L, 5-HT release experiments). Hemicholinium-3 and 6-nitroquipazine are potent inhibitors of the reuptake of [³H]choline (which results from the degradation of released [³H]ACh), and [³H]5-HT, respectively, thereby increasing the amounts of the evoked overflows of [³H] and improving the signal-to-noise ratio. In addition to these two drugs, further drugs were present throughout superfusion in some experiments as indicated. Collection of 4-min fractions was started after 49 min of superfusion. During superfusion electrical field stimulation was performed (360 rectangular pulses at 3 Hz, 0.5 ms, 9 V/chamber, and 90–100 mA) either twice (after 57 [S1] and 89 [S2] min of superfusion), or three times (after 58 [S1], 86 [S2] and 114 [S3] min of superfusion). Drugs to be tested were added to the superfusion medium from 12 min before S2 or S3 onwards. At the end of the experiment, the radioactivity of superfusate samples and ‘cell culture discs’ (cells dissolved in 250 μL Soluene 350; Packard, Frankfurt, Germany) was determined by liquid scintillation counting.

Figure 5.

 Effects of oxotremorine and CP-93,129 on the electrically-evoked overflow of [³H] from septal cell cultures (black columns), or from co-cultures of septal and raphe cells (gray columns), respectively, pre-incubated with [³H]choline. Both cell cultures were prepared and the effects of the agonists measured directly in parallel. At day 14 in vitro (DIV) the cell culture discs were washed carefully with superfusion medium, pre-incubated with [³H]choline, superfused and electrically stimulated three times (S1, S2, and S3) as described in Materials and methods. (a) Oxotremorine (1 and 10 μmol/L), or (b) CP-93,129 (1 and 10 μmol/L) were added to the superfusion medium before S2 or S3, respectively. Their effect on the evoked overflow is expressed as the ratio Sn/S1 in percent of the corresponding controls (no drugs during Sn). For data on the evoked overflow of [³H] at S1, see Table 2. Statistics: *p < 0.05, **p < 0.01, and ***p < 0.001 versus corresponding controls (no drugs during Sn); comparison of co-cultures versus septal cell cultures: ###p < 0.001, NS: not significant; means ± SEM; n, number of cell culture discs; data from 6 to 7 independent septal cultures or co-cultures, respectively (non-parametric anova with Kruskal–Wallis test; Mann–Whitney test).

Calculations and statistics

The fractional rate of tritium outflow (as a percentage of cell tritium per 4 min) was calculated as: (amount of tritium outflow [in dpm per 4 min]) × 100/(amount of tritium [in dpm] in the ‘cell culture discs’ at the start of the respective 4-min period); in the absence of electrical stimulation these fractional rate values represent the basal outflow of tritium. The stimulation-evoked overflow of tritium at Sn was expressed as a percentage of the tritium content of the cell culture discs just before the onset of the respective stimulation period. It was calculated following subtraction of the basal outflow; the latter was assumed to decline linearly from the 4-min fraction immediately before the onset of the stimulation to the 4-min fraction, 12–16 min after the onset of the stimulation. Effects of drugs added before Sn were estimated as the ratio of the overflow evoked by the corresponding stimulation periods (Sn/S1) and then compared with the appropriate control ratios (same age of culture; no drug addition before Sn). Effects of drugs added before Sn on the basal outflow of [3H] were determined as the ratio (bn/b1) of the fractional rates of [3H]outflow of the fractions preceding the corresponding stimulation periods and then compared with the appropriate control ratios (same age of culture; no drug addition before Sn).

Significance of differences was tested using anova followed by Bonferroni’s test (parametric), or non-parametric anova (Kruskal–Wallis test) followed by Dunn’s test, or by the Mann–Whitney test (see also Results). All data are shown as means ± SEM; n, total number of ‘cell culture discs’.

Results

Functional expression of the muscarinic autoreceptor in cell cultures from the fetal septum

Following pre-incubation with [³H]choline, primary cultures of the rat fetal septal region release [³H]ACh during electrical field stimulation and this release is modulated by presynaptic muscarinic autoreceptors (Ehret et al. 2001). To illustrate the experimental setup of the present investigation, Fig. 1 shows the time course of [³H]outflow from such septal cell cultures, the effects of electrical field stimulations and the inhibitory effect of the muscarinic agonist oxotremorine (1 and 10 μmol/L).

Figure 1.

 Time course of spontaneous and electrically-evoked [3H]outflow from septal cell cultures (at day 14 in vitro), following pre-incubation with [³H]choline. Cells from the fetal (ED 17) septal region were grown for 14 days on poly-d-lysine coated glass cover slips, carefully washed, pre-incubated with [³H]choline, superfused continuously with physiological buffer and stimulated three times (S1, S2, and S3) by electrical fields (360 pulses, 3 Hz, 0.5 ms, and 90 mA). (a) control experiments; (b) oxotremorine 1 or 10 μmol/L was added to the superfusion medium before S2 or S3, respectively, as shown by the horizontal bars. For the amount of [3H]overflow evoked at S1 see Table 1, for the extent of the effects of oxotremorine on the evoked overflow (Sn/S1 ratio) see Figure 2; means ± SEM; n, number of cell culture discs.

In the present study, we investigated whether addition of drugs to the growth medium of the septal cell cultures from DIV 3 to DIV 14 affected the functional expression of the muscarinic autoreceptor. At DIV 14, all drugs added to the culture medium during growth were carefully washed away before pre-incubation of the cells with [³H]choline. Autoreceptor function was then determined as the effect of 1 or 10 μmol/L oxotremorine on the electrically-evoked overflow of [³H] from cell culture discs pre-labeled with [³H]choline (see also Fig. 1). Please note that in these experiments septal cultures, in which no drugs were present during growth from DIV 3 to DIV 14 (0 μmol/L drug), were always run directly in parallel.

Figure 2a shows that the presence of the muscarinic agonist oxotremorine (10 or 100 μmol/L) during growth of the cell cultures from DIV 3 to DIV 14 completely abolished the inhibitory effect of 1 μmol/L oxotremorine at DIV 14. In a similar manner, the presence of another muscarinic agonist, carbachol (1 or 100 μmol/L), during the same period significantly reduced or abolished the effects of 1 μmol/L oxotremorine (Fig. 2b). Interestingly, if oxotremorine (100 μmol/L) or carbachol (100 μmol/L) were present in the growth medium for 24 h only, i.e. from DIV 13 to DIV 14, the inhibitory effect of 1 μmol/L oxotremorine on DIV 14 was also abolished [102.3 ± 12.6% of controls [n = 5; not significant (NS)] following 100 μmol/L oxotremorine for 24 h, or 85.5 ± 8.8% of controls (n = 9; NS) following 100 μmol/L carbachol for 24 h]. On the other hand, addition of the muscarinic antagonist atropine (10 μmol/L) during growth of the septal cell cultures from DIV 3 to DIV 14 did not change the inhibitory effect of 1 μmol/L oxotremorine, which amounted to 73.4 ± 6.1% of controls (= 6) in the absence and to 72.2 ± 5.5% of controls (= 4) in the presence of atropine during growth.

Figure 2.

 Effects of oxotremorine (1 μmol/L) on the electrically-evoked overflow of [³H] from septal cell cultures pre-incubated with [³H]choline. During development of the cultures either (a) oxotremorine (0, 10, or 100 μmol/L) or (b) carbachol (0, 1, or 100 μmol/L) was present in the growth medium from day 3 in vitro (DIV 3) to DIV 14. At DIV 14 the cell culture discs were washed carefully with superfusion medium, pre-incubated with [³H]choline superfused and electrically stimulated twice (S1, S2) as described in Materials and methods. Oxotremorine (1 μmol/L) was added to the medium before S2. Its effect on the evoked overflow is expressed as the ratio S2/S1 in percent of the corresponding controls (no oxotremorine during S2). For data on the evoked overflow of [³H] at S1 see Table 1. Statistics: *p < 0.05, **p < 0.01, and ***p < 0.001 versus corresponding controls (no oxotremorine during S2); #p < 0.05, ##p < 0.01, and ###p < 0.001 versus no agonist during DIV 3 to DIV 14; means ± SEM; n, number of cell culture discs; data from 2 to 3 independent septal cultures (unpaired t-test or anova with Bonferroni multiple comparisons test).

Assuming a spontaneous firing of cholinergic cells during growth in the cultures, we also modified the culture conditions with the aim of either increasing (Fig. 3a) or decreasing (Fig. 3b) the levels of endogenous ACh in the growth medium. In the experiments summarized in Fig. 3a, the ACh esterase inhibitor donepezil (1 μmol/L) was present from DIV 3 to DIV 14, whereas in those depicted in Fig. 3b, TTX (0.1 μmol/L) was present during the same cultivation period. Again (as above), cultures were grown directly in parallel, in which donepezil or TTX, respectively, were absent from the growth medium (no pre-treatment); only the corresponding values from cultures grown directly in parallel are shown in Fig. 3(a or b), respectively.

Figure 3.

 Effects of oxotremorine on the electrically-evoked overflow of [³H] from septal cell cultures pre-incubated with [³H]choline. During the development of the cultures either (a) donepezil (1 μmol/L) or (b) tetrodotoxin (TTX, 0.1 μmol/L) was present in the growth medium from day 3 in vitro (DIV 3) to DIV 14. At DIV 14 the cell culture discs were washed carefully with superfusion medium, pre-incubated with [³H]choline superfused and electrically stimulated three times (S1, S2, and S3), as described in Materials and methods. Oxotremorine was added to the medium before S2 or S3, respectively as indicated on the abscissa. Its effect on the evoked overflow is expressed as the ratio Sn/S1 in percent of the corresponding controls (no oxotremorine during Sn). For data on the evoked overflow of [³H] at S1 see Table 1. Statistics: *p < 0.05, ***p < 0.001 versus corresponding controls (no oxotremorine during Sn); #p < 0.05 versus pre-treatment with 1 μmol/L donepezil from DIV 3 to DIV 14; means ± SEM; n, number of cell culture discs; data from 3 to 4 independent septal cell cultures (Mann–Whitney test or non-parametric anova with Kruskal–Wallis test).

As evident from Fig. 3a, oxotremorine (0.1 and 1 μmol/L) significantly inhibited the electrically-evoked overflow of [³H] from septal cells pre-incubated with [³H]choline, but only in the absence of donepezil during growth. However, in the presence of donepezil the effects of the muscarinic agonist were reduced and no longer differed from the corresponding control values (i.e. donepezil during growth but no oxotremorine at S2 or S3).

Figure 3b shows that oxotremorine (0.1 and 1 μmol/L) significantly inhibited the evoked overflow of [³H] from septal cell cultures pre-incubated with [³H]choline both in the absence or presence of TTX (0.1 μmol/L) during growth, and without significant differences in between the growth conditions. It should be noted, however, that the presence of TTX (0.1 μmol/L) during growth from DIV 3 to DIV 14 significantly (< 0.001) reduced the evoked overflow of [³H] at S1 from the cells at DIV 14 by about −56% (see Table 1).

Table 1.   Effects of drugs added to the growth medium (from DIV 3 to DIV 14) on the evoked overflow of [³H] (in percent of cell-[³H]) following pre-incubation of cultures from the rat fetal septal area with [³H]choline
Addition to growth medium (DIV 3–DIV 14)Evoked [³H]overflow S1 in percent of cell-³Hn
  1. aSimilar statistical significances were obtained by comparison of the S1-values to control-S1-values from experiments done directly in parallel. As the latter control-S1-values did not differ significantly, they were taken together under ‘none’. Statistics: ***< 0.001, *< 0.05 versus ‘none’ (non-parametric anova + Kruskal–Wallis test); means ± SEM; n, number of cell culture discs.

None (mean ± SEM of all corresponding controlsa)0.661 ± 0.047101
TTX (0.1 μmol/L)0.292 ± 0.056***21
Carbachol (1 μmol/L)0.431 ± 0.03814
Carbachol (100 μmol/L)0.364 ± 0.025*18
Oxotremorine (10 μmol/L)0.652 ± 0.07817
Oxotremorine (100 μmol/L)0.383 ± 0.0399
Atropin (10 μmol/L)0.469 ± 0.0377
Donepezil (1 μmol/L)0.657 ± 0.05615
CP-93,129 (10 μmol/L)0.273 ± 0.035***12

Also, pre-treatment of the septal cultures for 11 days with both the 5-HT1B receptor agonist CP-93,129 (10 μmol/L) and the highest concentration of carbachol (100 μmol/L) significantly (< 0.05) diminished the evoked overflow at S1, whereas some of the other additions showed inhibitory tendencies only (NS).

Functional expression of M-autoreceptors and the 5-HT1B heteroreceptors on cholinergic neurons in co-cultures of cells from the fetal septal and raphe region

Using the same culture conditions, it was also possible to obtain serotonergic cell cultures from the fetal (ED 15) raphe region (Birthelmer et al. 2007). To check the influence of other neuronal systems such as serotonergic neurons on the functional expression of presynaptic receptors in septal cholinergic neurons, we established co-cultures from fetal septal and fetal raphe neurons. For this purpose the fetal (ED 17) septal region and fetal (ED 15) raphe region were dissected on the same day. After several dissociation and sedimentation steps (see Materials and methods), a mixture of both cell types was plated on the cell cultures discs at an initial cell density similar to that used for septal cholinergic mono-cultures (see Materials and methods). As described for septal mono-cultures (Ehret et al. 2001) and raphe mono-cultures (Birthelmer et al. 2007), the co-cultured cells grew also well on poly-d-lysine coated glass cover slips and consisted of a mixture of non-neuronal and neuronal cells. Fig. 4 shows that both cholinergic neurons (stained with an antibody against the vesicular ACh transporter; VAChT) and serotonergic neurons (stained with an antibody against TrpH) co-existed in these cell cultures.

Figure 4.

 Microscopic aspect of co-cultivated septal and raphe cells. Fetal septal cells prepared at ED 17 and fetal raphe cells prepared at ED 15 from Wistar rat embryos were co-cultivated as described in Materials and methods for 2 weeks. At DIV 14 (a) serotonergic cells were stained using an antibody against tryptophan hydroxylase (TrpH-AB); in (b) cholinergic cells (in the same region of the cell layer) were visualized using an antibody against the vesicular ACh transporter (VAT-AB); in (c) the figures (a) and (b) are merged; horizontal bar: 20 μm.

Moreover, during electrical field stimulation of these co-cultures, an evoked overflow of [³H] was detectable not only following pre-incubation of the cell culture discs with [³H]choline, but also following their pre-incubation with [³H]5-HT (see Table 2). Table 2 also shows that following pre-incubation with [³H]choline, both the accumulation of [³H] and the evoked [³H]overflow at S1 were significantly lower in co-cultured septal neurons than in septal mono-cultures; moreover, the evoked [³H]overflow at S1 was not affected by the presence of the 5-HT1B receptor antagonist GR-55,562 (1 μmol/L) throughout superfusion. Finally, it is evident from Table 2 that the evoked overflow of [³H] from co-cultures pre-incubated with [³H]5-HT was about 14 times higher than that from co-cultures pre-incubated with [³H]choline.

Table 2.    [³H]Accumulation and electrically-evoked overflow of [³H] from septal mono-cultures and from septal/raphe co-cultures pre-incubated with either [³H]choline or [³H]5-HT. Concentrations of drugs present throughout superfusion: hemicholinium-3: 10 μmol/L; GR-55,562 and 6-nitroquipazine: 1 μmol/L
CulturePre-incubation withDrug present during growth (DIV 3 to DIV 14)Drugs throughout [³H]accumulation (pmoles/disc) S1 (percent of cell-[³H]) n
  1. Statistics: *< 0.05, **< 0.01, and ***< 0.001, versus septal mono-culture; ##< 0.01, versus no drug present during growth (anova + Kruskal–Wallis test); means ± SEM; n, number of cell culture discs.

Septal mono-culture[³H]cholineNoneHemicholinium-32.28 ± 0.070.83 ± 0.1135
Septal/raphe co-culture[³H]cholineNoneHemicholinium-31.85 ± 0.06*0.46 ± 0.03*81
Septal/raphe co-culture[³H]cholineGR-55,562 (10 μmol/L)Hemicholinium-31.11 ± 0.10***##0.29 ± 0.04***##16
Septal/raphe co-culture[³H]cholineNoneHemicholinium-3 GR-55,562 1.39 ± 0.23**0.34 ± 0.03**13
Septal/raphe co-culture[³H]5-HTNone6-nitroquipazine3.15 ± 0.10**6.57 ± 0.21***23

Besides the muscarinic autoreceptor [see above and (Ehret et al. 2001)], cultured cholinergic neurons from the fetal septal region are also endowed with 5-HT1B heteroreceptors. Accordingly, the evoked overflow of [³H] from cultured fetal septal cells at DIV 14 was inhibited by the selective 5-HT1B receptor agonist CP-93,129 (1 μmol/L; 68.9 ± 3.5% of controls, < 0.001 vs. controls), an effect which was significantly diminished by the simultaneous presence of the 5-HT1B receptor selective antagonist GR-55,562 (1 μmol/L; 85.2 ± 2.0% of controls; < 0.01 vs. CP-93,129 given alone, Mann–Whitney test). GR-55,562 given alone did not affect the evoked overflow of [³H] from cultured septal cells pre-incubated with [³H]choline (Table 2).

Figure 5a depicts the effects of the muscarinic agonist oxotremorine and Fig. 5b that of the 5-HT1B receptor agonist CP-93,129 on the evoked overflow of [³H] in (1) septal mono-cultures (black columns) pre-incubated with [³H]choline and (2) on septal/raphe co-cultures (gray columns, pre-incubated with [³H]choline as well). Both types of cultures were prepared and investigated on the same experimental days under identical conditions (i.e. directly in parallel). The figure shows that the inhibitory effect of oxotremorine was similar in both cultures (although it tended to be smaller in co-cultured neurons), whereas the effect of CP-93,129 was no longer detectable in the co-cultures. The inhibitory effects of CP-93,129 were also abolished if the septal/raphe co-cultures were grown in the presence of the 5-HT1B receptor selective antagonist GR-55,562 (10 μmol/L) from DIV 3 to DIV 14 [effects in percent of the corresponding controls: 1 μmol/L CP-93,129: 116.2 ± 9.6 (n = 12, NS); 10 μmol/L CP-93,129: 97.6 ± 18.2 (n = 12; NS)].

Interestingly, when septal mono-cultures were grown in the presence of the 5-HT1B receptor agonist CP-93,129 from DIV 3 to DIV 14, the effects of 1 and 10 μmol/L CP-93,129 on the evoked overflow of [³H] from cultured septal cells pre-incubated with [³H]choline were also absent [effects in percent of the corresponding controls: 1 μmol/L CP-93,129: 106.6 ± 9.9 (n = 8, NS); 10 μmol/L CP-93,129: 104.2 ± 15.8 (n = 8; NS)]. Moreover, the presence of the 5-HT1B antagonist GR-55,562 during growth also significantly affected the accumulation of [³H]choline by the cells, as well as the evoked overflow of [³H] at S1 (see Table 2).

Discussion

Presynaptic modulation of ACh release in septal mono-cultures

In a previous study (Ehret et al. 2001), we have shown that primary cell cultures obtained from the fetal rat septum develop a dense network of non-neuronal and neuronal cells, the latter of which can be stained in part by an antibody against a marker enzyme of cholinergic neurons, choline acetyltransferase. We also reported that – following pre-incubation with [³H]choline – electrical field stimulation of such cultures elicits an overflow of [³H] which consisted of about 78% of authentic [³H]ACh and was both Ca2+-dependent and TTX sensitive. Therefore, we concluded that electrical field stimulation of similar cell cultures lead to an action-potential-evoked, exocytotic release of ACh (Ehret et al. 2001). Moreover, it was shown that the M-receptor agonist oxotremorine, as well as opioid and adenosine receptor agonists inhibited this evoked release of ACh, suggesting that presynaptic M-, opioid- and adenosine (A1) receptors are present on these cultured cholinergic neurons (Ehret et al. 2001). Although the type of the M-autoreceptor involved was not further characterized in this study, it should be noted that the cultures were prepared from the fetal septal area of the rat and that it has been shown that the M2-autoreceptor type prevails in the target area of the septo-hippocampal cholinergic projection.

As the present study shows, cholinergic neurons in these cultures are also endowed with 5-HT1B heteroreceptors, given that the inhibitory effect of the 5-HT1B selective agonist CP-93,129 [see: (Hoyer et al. 1994)] on the electrically-evoked release of ACh was significantly diminished by the 5-HT1B selective antagonist GR-55,562 [e.g. (Walsh et al. 1995)].

The first main observation of the present investigation is that the function of the M2 autoreceptor, which was evident starting from post-natal day 4 in hippocampal slices (Goldbach et al. 1998), but was not yet detectable in septal cell cultures at DIV 7 (Ehret et al. 2001), can be inhibited either by the continuous (from DIV 3 to DIV 14) or transient (24 h) presence of muscarinic agonists like oxotremorine or carbachol during growth; the inhibitory effect of the muscarinic agonist oxotremorine at DIV 14 was significantly reduced or even abolished following these growth conditions (Fig. 2 and accompanying text). Agonist exposure has also been shown to regulate M-receptor function in developing oligodendrocytes (Molina-Holgado et al. 2003). In contrast, as evident from the present data, autoreceptor development was not affected by the competitive M-receptor antagonist atropine during growth, suggesting that the mechanism of receptor trafficking (or down-regulation) involves as a first step its stimulation by an agonist, followed by receptor phosphorylation, β-arrestin binding, etc. [see: (Bernard et al. 2006)].

Assuming a spontaneous firing of the cholinergic neurons during development of the cultures, we also tried to increase the levels of the endogenous M receptor agonist, ACh, by the continuous presence (from DIV 3 to DIV 14) of the clinically used ACh esterase inhibitor donepezil (Seltzer 2005). In agreement with the effects of exogenously added agonists (see above), the function of M2 autoreceptors appeared to be reduced when ACh esterase was inhibited during growth of the cultures (Fig. 3a). In support of this conclusion, it has been shown using immunocytochemistry at light and electron microscopic levels, that acute and chronic ACh esterase inhibition regulates the intracellular distribution and localization of M2 and M4 receptors in striatal neurons (Liste et al. 2002). Moreover, in ACh esterase deficient mice striatal M2 receptors were almost absent at the membrane, but accumulated in the endoplasmic reticulum and the Golgi complex (Bernard et al. 2003). Finally, using the same techniques, a redistribution of striatal M2 (Bernard et al. 1998) and M4 (Bernard et al. 1999) receptors was also observed following acute stimulation of muscarinic receptors with oxotremorine; this is in agreement with the present data.

On the other hand, our attempts to decrease the levels of endogenously released ACh by blocking spontaneous action potential generation and propagation with TTX (0.1 μmol/L) from DIV 3 to DIV 14 did not significantly increase the effects of oxotremorine at DIV 14. It should be noted, however, that following growth of cholinergic neurons in the presence of TTX the evoked release of ACh was significantly reduced by more than 50%, suggesting that TTX inhibited the maturation of cholinergic neurons in the culture although we did not check the density of the cells or the protein content in these cultures. In agreement with this suggestion, studies on the development of neurons in organotypic slice cultures have shown that the presence of TTX inhibited cortical axon branch formation (Uesaka et al. 2005), and altered spine maturation (Drakew et al. 1999; Frotscher et al. 2000). Finally, TTX has also been found to abolish survival promotion by tachykinins in mesencephalic dopaminergic cell cultures (Salthun-Lassalle et al. 2005).

Taken together, the consequences of adding direct and indirect muscarinic agonists during growth of the septal cell cultures suggest that M receptor agonists, but not antagonists, are able to suppress the function of muscarinic autoreceptors in cultured cholinergic neurons. However, whether these findings are the consequence of an agonist-induced inhibition of the development of M2 autoreceptors or whether they are due to alterations in signal transduction pathways or to acute agonist-induced down-regulation of the M2 autoreceptor [i.e. a reduced number of receptors subsequent to a decrease in the corresponding mRNA levels (Fukamauchi et al. 1991; Habecker and Nathanson 1992; Steel and Buckley 1993)] cannot be determined by the techniques used in the present study. Alternatively, increased rates of intracellular M2 autoreceptor redistribution/trafficking [for reviews see: (Koenig and Edwardson 1997; Bloch et al. 1999, 2003; Bernard et al. 2006)] are also possible.

Presynaptic modulation of ACh release in septal/raphe co-cultures 

The present investigation shows that co-cultivation of rat embryonic septal cells (obtained at ED 17) and raphe cells (obtained at ED 15) yielded mixed cultures of neuronal and non-neuronal cells. From immunocytochemical data, the presence of both cholinergic and serotonergic neurons in these co-cultures is evident (Fig. 4). Moreover, as in septal mono-cultures, electrical field stimulation of cells pre-incubated with [³H]choline evokes an overflow of [³H] which, in agreement with the observations on septal mono-cultures, most probably represents the release of ACh. In contrast to septal mono-cultures, however (data not shown), an electrically-evoked overflow of [³H] from these co-cultures can also be elicited following pre-incubation of the cell culture discs with [³H]5-HT (Table 2). In agreement with studies on raphe mono-cultures (Birthelmer et al. 2007; submitted), it can be assumed that the evoked [³H]overflow from co-cultures pre-incubated with [³H]5-HT represents action potential-induced, exocytotic release of 5-HT. Moreover, as already discussed in the previous study (Birthelmer et al. 2007), we have to assume that serotonergic neurons in these co-cultures fire spontaneously during growth and thus release 5-HT into the culture medium. Taken together, these observations suggest that cholinergic and serotonergic neurons co-exist in these cultures and that, in contrast to the situation of the septal mono-cultures, the cholinergic neurons grow under the constant influence of endogenous 5-HT released from developing and spontaneously firing serotonergic neurons.

The second main finding of the present study is the observation that the function of the M2 autoreceptor in the co-cultures seems to be rather unaffected by the presence of serotonergic neurons during growth; the effect of oxotremorine, although slightly smaller than in mono-cultures, did not differ significantly from that in co-cultures (Fig. 5a). Although M2 and 5-HT1B receptor activation may involve similar signal transduction mechanisms [i.e. inhibition of presynaptic Ca2+-channels, activation of presynaptic hyperpolarizing K+-channels, inhibition of adenylate cyclase; for review see: (Boehm and Kubista 2002; Krejci et al. 2004; Sari 2004; Kubista and Boehm 2006)], a heterologous regulation of M2 mRNA expression via common signal transduction steps [e.g. (Habecker and Nathanson 1992)] is not supported by the present findings.

On the other hand, as suggested by the significantly reduced electrically-evoked release of [³H]ACh during the first stimulation period (S1, see Table 2), the presence of 5-HT releasing serotonergic cells in these cultures seems to inhibit the development of cholinergic neurons. In agreement with this interpretation is the observation that, following the presence of the selective 5-HT1B receptor agonist CP-93,129 during growth (from DIV 3 until DIV 14), the evoked release of [³H]ACh at S1 in septal mono-cultures was significantly diminished (Table 1). It has also been proposed in the literature that 5-HT plays an important role not only in the development of serotonergic neurons themselves (Whitaker-Azmitia and Azmitia 1986), but also in that of other neurons [e.g. (Whitaker-Azmitia 1991; Beique et al. 2004; Sodhi and Sanders-Bush 2004)], in in vitro differentiation (Menegola et al. 2004), and in adult neurogenesis (Brezun and Daszuta 1999)]. Nevertheless, it might be argued from the present experimental setup (see Materials and methods) that, in contrast to the septal mono-cultures, only half of the number of fetal septal cells was plated on the cell culture discs at the start of the co-cultures, a condition which is also illustrated by the lower [³H]accumulation of the cells (Table 2). However, this fact alone does not explain the reduced release of [³H]ACh in the co-cultures, since the S1-values shown in Table 2 are expressed in percent of the [³H] content of the cells and [³H]choline is assumed to be preferentially (but not exclusively) accumulated by cholinergic neurons via the high affinity choline carrier. Taken together, we therefore suggest that the number of ACh-releasing cholinergic axon terminals is reduced under the influence of 5-HT that is released by serotonergic neurons during growth (or by exogenous 5-HT agonists in the growth medium); on the other hand, their endowment with M2 autoreceptors appears to be unaffected (see above). However, regarding the role of 5-HT in the development of cholinergic neurons in-vitro, it cannot be completely excluded that other factors might also be involved, such as cell to cell contacts or changes in the concentrations of neurotrophic factors released by the cells during co-cultivation.

The latter remark is in contrast to the third main observation of this investigation, namely that the function of the 5-HT1B heteroreceptor on cholinergic neurons in the co-cultures seems to be completely inhibited: the effects of the 5-HT1B agonist CP-93,129 on the evoked release of ACh was no longer detectable in septal/raphe co-cultures (Fig. 5b). Interestingly, the same phenomenon was observed in septal mono-cultures treated from DIV 3 to DIV 14 with an exogenous 5-HT agonist CP-93,129 (10 μmol/L). On the other hand, also in septal/raphe co-cultures grown from DIV 3 to DIV 14 in the presence of the selective 5-HT1B antagonist GR-55,562 (10 μmol/L), the 5-HT1B receptor was no longer detectable at DIV 14.

Several explanations for these somewhat contradictory phenomenon are possible: (i) as mentioned above, it could be speculated that the high levels of 5-HT released during electrical stimulation of the cell culture discs (see Table 2) might compete with the exogenous agonist CP-93,129 for the 5-HT1B heteroreceptor; in this case, however, the evoked release of [³H]ACh in co-cultures should be increased by 5-HT1B antagonists like GR-55,562, an effect which was not observed (Table 2); (ii) desensitization and down-regulation of the 5-HT1B receptor because of high concentrations of 5-HT during growth of the cholinergic neurons could occur, as shown in an opossum kidney cell line exposed to 5-HT (Pleus and Bylund 1992; Unsworth and Molinoff 1992); in this context it should be noted that the 5-HT1B antagonist GR-55,562 has been shown to strongly increase the release of 5-HT from serotonergic neurons in culture (Birthelmer et al. 2007). This would account for the inhibitory influence of this antagonist (if present during growth from DIV 3 to DIV 14) on both the development of cholinergic neurons [see values for [³H]choline accumulation and the evoked ACh release (S1 values) in Table 1] and on the function of the 5-HT1B heteroreceptor; and (iii) finally, 5-HT1B receptor redistribution/trafficking following agonist stimulation, like that observed for the M2 receptor (see above), might also take place in cholinergic neurons originating from the fetal septum. To our knowledge, this is the first time that a similar observation was made for presynaptic 5-HT1B heteroreceptors.

Conclusive remarks

Primary cell cultures of the fetal rat septum are a useful model to study the expression of functional presynaptic receptors in vitro. From our present data, we conclude that the ‘neuronal environment’ strongly affects either the expression and/or function of presynaptic receptors: high concentrations of exogenous or endogenous neurotransmitter agonists appear to significantly reduce the inhibitory potency of both presynaptic auto- and heteroreceptors. Most probably, similar events are also important for the functioning of human brain: changes in the neuronal environment under pathological (e.g. neurodegenerative diseases) and physiological conditions (e.g. age-dependent decline of specific transmitter systems), but also under therapeutic regimens (e.g. treatment of patients with drugs which increase extracellular concentrations of neurotransmitters, such as monoamine reuptake inhibitors, ACh esterase inhibitors, amphetamines, etc.) may affect the expression and/or function of presynaptic auto- and heteroreceptors.

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (Ja 244/4 and Ja 244/5). Moreover, we are much indebted to Dr Jean-Christophe Cassel, Strasbourg (France) and to Dr Sandra Dieni, Melbourne (Australia) for many helpful comments on the manuscript.

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