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.