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Calcium-Induced Calcium Release Contributes to Somatic Secretion of Serotonin in Leech Retzius Neurons Citlali Trueta,1,* Sergio Sańchez-Armass,2 Miguel A. Morales,3 Francisco F. De-Miguel1 1

Departamento de Biofı´sica, Instituto de Fisiologıá Celular, Universidad Nacional Autońoma de Me´xico, Apartado postal 70-253, Me´xico, 04510 D.F., Me´xico 2

Facultad de Medicina, Universidad Autońoma de San Luis Potosı´, S.L.P., Me´xico

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Departamento de Biologıá Celular y Fisiologıá, Instituto de Investigaciones Biome´dicas, Universidad Nacional Autońoma de Me´xico

Received 25 September 2003; accepted 13 March 2004

We analyzed the contribution of calcium (Ca2ⴙ)-induced Ca2ⴙ release to somatic secretion in serotonergic Retzius neurons of the leech. Somatic secretion was studied by the incorporation of fluorescent dye FM1-43 upon electrical stimulation with trains of 10 impulses and by electron microscopy. Quantification of secretion with FM1-43 was made in cultured neurons to improve optical resolution. Stimulation in the presence of FM1-43 produced a frequency-dependent number of fluorescent spots. While a 1-Hz train produced 19.5 ⴞ 5.0 spots/soma, a 10-Hz train produced 146.7 ⴞ 20.2 spots/soma. Incubation with caffeine (10 mM) to induce Ca2ⴙ release from intracellular stores without electrical stimulation and external Ca2ⴙ, produced 168 ⴞ 21.7 spots/soma. This staining was reduced by 49% if neurons were preincubated with the Ca2ⴙ- ATPase inhibitor thapsigargin (200 nM). Moreover, in neurons stim-

ulated at 10 Hz in the presence of ryanodine (100 ␮M) to block Ca2ⴙ-induced Ca2ⴙ release, FM1-43 staining was reduced by 42%. In electron micrographs of neurons at rest or stimulated at 1 Hz in the ganglion, endoplasmic reticulum lay between clusters of dense core vesicles and the plasma membrane. In contrast, in neurons stimulated at 20 Hz, the vesicle clusters were apposed to the plasma membrane and flanked by the endoplasmic reticulum. These results suggest that Ca2ⴙ-induced Ca2ⴙ release produces vesicle mobilization and fusion in the soma of Retzius neurons, and supports the idea that neuronal somatic secretion shares common mechanisms with secretion by excitable endocrine cells. © 2004 Wiley

*Present address: Departamento de Neurofisiologıá, Instituto Nacional de Psiquiatrıá “Ramoń de la Fuente Mun˜iz,” Calzada Me´xico-Xochimilco 101, D.F. 14370, Me´xico. Correspondence to: F.F. De-Miguel ([email protected]). Contract grant sponsors: CONACYT and DGEP fellowships (to C.T.). Contact grant sponsor: Human Frontiers Science Program; contract grant number: RG-162/98 (to F.F.M.).

Contract grant sponsor: CONACYT; contract grant number: 40626 (to F.F.M.). © 2004 Wiley Periodicals, Inc. Published online 2 August 2004 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/neu.20055

ABSTRACT:

Periodicals, Inc. J Neurobiol 61: 309 –316, 2004

Keywords: calcium; intracellular calcium release; exocytosis; secretion; serotonin; leech

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INTRODUCTION Increasing evidence is showing that somatic exocytosis of transmitters and peptides occurs from neurons of vertebrates and invertebrates (Chen et al., 1995; Huang and Neher, 1996; Zaidi and Matthews, 1997, 1999; Jaffe et al., 1998; Puopolo et al., 2001; Nguyen and Sargent, 2002; Trueta et al., 2003; Soldo et al., 2003). The characteristics of somatic secretion in most of the neuron types studied are different from those of synapses but similar to those of secretion by excitable endocrine cells. For example, it is produced from dense-core vesicles in the absence of active zones (Zaidi and Matthews, 1997, 1999; Bruns et al., 2000; Puopolo et al., 2001; Trueta et al., 2003); it depends on long depolarizations (Huang and Neher, 1996) or trains of impulses (Soldo et al., 2003; Trueta et al., 2003); its dependence on the intracellular Ca2⫹ concentration is lower than that of synaptic secretion (Huang and Neher, 1996), and it is coupled to excitation through L-type Ca2⫹ channels (Puopolo et al., 2001; Trueta et al., 2003). An exception is chick ciliary ganglion neurons that release acetylcholine from clear vesicles at somatic spines (Nguyen and Sargent, 2002). In the neuronal soma and in excitable endocrine cells, the internal Ca2⫹ concentration required to produce secretion may only be reached by large depolarizations or trains of impulses due to the distance between Ca2⫹ channels and dense-core vesicles, and to the intracellular Ca2⫹ buffering (reviewed by Mansvelder and Kits, 2000). However, in some types of excitable endocrine cells an additional way to increase the intracellular Ca2⫹ concentration to the necessary levels for secretion is by the mechanism of Ca2⫹-induced Ca2⫹ release from intracellular stores (Cheek et al., 1990; Guo et al., 1996; Lemmens et al., 2001; Kang and Holz, 2003). Here we explored the contribution of Ca2⫹-induced Ca2⫹ release to the coupling between excitation and somatic secretion of serotonin in neurons. Our experiments were made using Retzius neurons of the leech, which in culture secrete quanta of serotonin from synapses containing small clear and dense-core vesicles (Henderson et al., 1983; Kuffler et al., 1987) and from large dense-core vesicles in the soma (Bruns et al., 2000; Trueta et al., 2003). Serotonin in the leech modulates neuronal circuits that control behaviors such as swimming (Willard, 1981; Kristan and Nusbaum, 1983; Nusbaum, 1986), feeding (Zhang et al., 2000), and learning (Burrell et al., 2001). Although activation of mechanosensory pathways leads to the activation of Retzius neurons (Szczupak and Kristan, 1995; Velazquez-Ulloa,

et al., 2003), serotonin release modulates integration by mechanosensory neurons (Mar and Drapeau, 1996). Because most of the serotonin in the leech is produced and released by Retzius neurons (McAdoo and Coggeshall, 1976), somatic secretion could be an important source of serotonin for paracrine regulation of behavior. A previous study of somatic secretion using the incorporation of the fluorescent dye FM1-43 showed that stimulation with trains of impulses induces a fluorescent spotted pattern produced by hot spots of exocytosis and subsequent endocytosis, in which clusters of dense-core vesicles act as functional secretion units. The number of fluorescent spots is proportional to the frequency of stimulation (Trueta et al., 2003). In the present study we used a pharmacological approach during electrical stimulation with trains of 10 impulses in the presence of FM1-43 and electron microscopy to analyze the contribution of Ca2⫹-induced Ca2⫹ release to somatic secretion of serotonin.

MATERIALS AND METHODS Isolation and Culture of Neurons Experiments were made using Retzius neurons of the medicinal leech Hirudo medicinalis. For experiments with FM1-43, neurons were individually isolated by suction through a glass pipette (Dietzel et al., 1986) and plated in glass-bottom culture dishes coated with concanavalin-A (Sigma, St. Louis, MO). The culture medium was L-15 (Sigma) supplemented with 6 mg mL⫺1 glucose, 0.1 mg mL⫺1 gentamycin and 2% fetal bovine serum (Gibco, Gaithersburg, MD). Experiments were made at room temperature (20 –25°C).

Stimulation of Secretion Secretion was stimulated and quantified as described by Trueta et al. (2003). In brief, stimulation consisted of a train of 10 action potentials produced by intracellular injection of 10-ms current pulses delivered at 1, 10, or 20 Hz in the presence of FM1-43 (2 ␮M; Molecular Probes; Betz et al., 1992). Although at a frequency of 1 Hz somatic secretion in Retzius neurons is at its basal level, at frequencies of 10 –20 Hz we have reached the maximum amount of somatic secretion (Trueta et al., 2003). Electrical recordings were acquired by an analog-to-digital board Digidata 1200 using Pclamp8 software (Axon Instruments, Union City, CA) and stored in a PC. Before withdrawing the microelectrode, the dye was washed out for 2 min by perfusing in leech Ringer (in mM: NaCl 120; KCl 4; CaCl2 2; Tris-maleate 10, pH 7.4) in which Ca2⫹ was replaced by Mg2⫹ to avoid secretion (Mg2⫹ solution), and containing N-methyl D-glucamine (66 mM). Neurons were then washed for 8 min with normal Ringer solution.

Intracellular Ca2⫹ and Somatic Secretion

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Figure 1 Subcellular distribution of somatic clusters of dense-core vesicles and endoplasmic reticulum. (A) Clusters of dense-core vesicles in the soma of a neuron stimulated at 1 Hz were distant from the plasma membrane (arrows). Endoplasmic reticulum (asterisks) was near the plasma membrane and between the membrane and vesicle clusters. Arrowheads mark rough endoplasmic reticulum. (B) In neurons stimulated at 20 Hz, vesicle clusters were adjacent to the plasma membrane (arrows) and flanked by endoplasmic reticulum (asterisks). Arrowheads as in (A). Scale bar ⫽ 500 nm.

Ca2⫹-induced Ca2⫹ release was evoked using caffeine (10 mM; Thayer et al., 1988; Usachev et al., 1993; Shmigol et al., 1996) in the absence of electrical stimulation, or was blocked by use of 100 ␮M ryanodine (Calbiochem, La Jolla, CA), which at this concentration blocks ryanodine receptors (McPherson et al., 1991; Kirischuk et al., 1996). In some experiments we used 200 nM thapsigargin (Molecular Probes, Eugene, OR) to deplete the intracellular Ca2⫹ stores by inhibiting the ATPase that loads them with Ca2⫹ (Pozzan et al., 1994; Shmigol et al., 1995).

Fluorescence Microscopy Individual neurons were viewed with a Nikon inverted microscope as described by Trueta et al. (2003). Fluorescence images were acquired with a CCD camera coupled to a photomultiplier and to an Argus 10 integrator (all from Hamamatsu Photonics, Japan). Digital images were processed using Metamorph software (Universal Imaging Corp., Downingtown, PA). The number of spots/soma was counted from z series of images applying stereological criteria (Coggeshall and Lekan, 1996). Some of the neurons were scanned with 1-␮m steps with a Bio-Rad MRC 1024 confocal laser-scanning microscope. Seven

images were averaged for each step using the Kalman algorithm. Three-dimensional reconstructions were achieved using Confocal Assistant software (Todd Clerke Brelje).

Electron Microscopy Stimulation of neurons was made in isolated ganglia immersed in leech Ringer. Electrical stimulation was made with 10 trains of 10 impulses at 1 or 20 Hz with 1-min intervals. Ganglia were then perfused with 0.08 M cacodylate buffer (pH 7.4; Sigma, St. Louis, MO), and fixed for 60 min with 0.6% glutaraldehyde and 0.4% paraformaldehyde, followed by postfixation for 60 min in 1% osmium tetroxide, all in cacodylate buffer. Ganglia were serially dehydrated and embedded in Epon. Ultrathin sections were observed in a JEOL 1010 electron microscope (JEOL USA Inc., Peabody, MA). Measurements were made manually from scanned digital images using Metamorph software. Clusters were considered adjacent to the plasma membrane when the distance between the plasma membrane and the nearest vesicle in a cluster was less than 250 nm.

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Statistical Analysis We used the R software (Ihaka and Gentleman, 1996) at the 95% confidence level. Because the data were counts, the square root transformation was applied to obtain variance homogeneity. One-way ANOVA was applied to the data in Figures 2 and 3. The residuals were tested for outliers, symetry, and kurtosis. Variance homogeneity was tested using the Fligner-Killeen test. Normality was assessed with the Shapiro-Wilk test. Multiple comparisons were performed with Tukey’s HSD test. When appropriate, paired or unpaired t-tests were applied to the transformed data. Figures show the mean ⫾ S.E. of the nontransformed data.

RESULTS Ultrastructure of the Soma of Retzius Neurons The ultrastructure of the soma of Retzius neurons in the ganglion was similar to that already described in culture (Bruns et al., 2000; Trueta et al., 2003), with large (100 nm) dense-core vesicles as the only secretory organelles. Dense-core vesicles in the soma of neurons stimulated with 1 Hz trains (n ⫽ 4) were arranged in clusters within the cytoplasm, at a distance from the plasma membrane, as previously seen (Bruns et al., 2000, Trueta et al., 2003). Clusters of vesicles coexisted with mitochondria (Fig. 1) and with membranous electrolucid organelles of irregular shapes (asterisks in Fig. 1), indistinguishable from the endoplasmic reticulum described in other neuron types (Peters et al., 1991). Therefore, we will refer to these membranous organelles as endoplasmic reticulum. Smooth endoplasmic reticulum, which in other neurons operates as a calcium store (Henkart et al., 1978), was predominant (asterisks in Fig. 1), over a small proportion of rough endoplasmic reticulum (arrowheads in Fig. 1). The smooth endoplasmic reticulum was aligned parallel to the plasma membrane and also in the area between the plasma membrane and the vesicle clusters [Fig. 1(A)]. Interestingly, in four neurons stimulated with 20 Hz trains, the clusters of dense-core vesicles were adjacent to the membrane [Fig. 1(B)] and were flanked by endoplasmic reticulum [Fig. 1(B), asterisks]. Similar observations were made in cultured neurons (not shown). The distribution of endoplasmic reticulum in both stimulation conditions supported the possibility that calcium release from these stores could participate in somatic secretion.

Somatic Secretion Somatic secretion was studied quantitatively by the incorporation of fluorescent dye FM1-43 upon stim-

Figure 2 Somatic secretion detected with FM1-43. (A) Phase contrast image of a Retzius neuron in culture with neurites growing from the stump of the primary process. Scale bar ⫽ 60 ␮m. (B,C) Confocal three-dimensional reconstructions of neurons stimulated at 1 and 10 Hz, respectively, in the presence of FM1-43. (D) FM1-43 staining pattern produced by incubation with caffeine (10 mM) in the absence of electrical stimulation and external Ca2⫹. Arrow points to the stump. (E) Number of spots/soma in different experimental conditions. Scale bar ⫽ 30 ␮m.

ulation. Because the FM1-43 staining patterns of Retzius neurons stimulated in culture and in the ganglion are similar (Trueta et al., 2003), these experiments were made using cultured neurons to improve optical resolution. Electrical stimulation with one train of impulses in the presence of FM1-43 produced well-delimited fluorescent spots distributed at the surface of the soma (Fig. 2; Trueta et al., 2003). As shown by the confocal three-dimensional reconstructions in Figure 2, while a train at 1 Hz produced 19.5 ⫾ 5.0 spots/soma (n ⫽ 6 neurons), a train at 10 Hz produced 146.7 ⫾ 20.2 spots/soma (n ⫽ 10 neurons). If Ca2⫹ was replaced with Mg2⫹ in the external solution, the number of spots in neurons stimulated at 10 Hz was only 34 ⫾ 8.0 [Fig. 2(E)], statistically similar to the number of spots produced by a 1-Hz


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Figure 3 Depletion of intracellular Ca2⫹ pools reduces somatic secretion. (A) Fluorescence FM1-43 staining pattern produced by caffeine. (B) Pretreatment of neurons with thapsigargin for 30 min reduced the number of spots produced by caffeine. (C) Fluorescent pattern of neurons incubated with thapsigargin for 30 min in the presence of FM1-43. (D) Number of spots/soma in the conditions above. The dotted line indicates the basal staining level. Fluorescence images are nonconfocal, and were taken at comparable focal planes at the site of contact with the plate. Scale bar ⫽ 10 ␮m.

train. Therefore, the amount of staining at 1 Hz was considered as the basal staining value.

Release of Ca2ⴙ from Intracellular Stores Induced Somatic Secretion As a first approach to test if intracellular Ca2⫹ release may induce somatic secretion, we used caffeine (10 mM) to evoke the release of Ca2⫹ through ryanodine receptors (Thayer et al., 1988; Kirischuk et al., 1996; Shmigol et al., 1996) in the presence of FM1-43. These experiments were made in the absence of electrical stimulation and with 2 mM extracellular Mg2⫹ substituting for Ca2⫹. Incubation of neurons with caffeine produced 168 ⫾ 21.7 spots/soma [Figs. 2(D), 3(A)], a number similar to that produced by stimulation with a 10-Hz train. However, in a different group of nine neurons that had been preincubated with thapsigargin (200 nM) for 30 min to decrease the Ca2⫹ content of intracellular stores (Pozzan et al., 1994; Shmigol et al., 1995), caffeine produced only 86.3 ⫾ 10.3 FM1-43 spots/soma [Fig. 3(B,D)]. This decrease of 49% was significant, and it could have been due, at least in part, to a reduction of the vesicle pool during the leak of Ca2⫹ produced by thapsigargin,

because in eight other neurons incubated for 30 min with thapsigargin in the presence of FM1-43, the number of spots was 58.8 ⫾ 10.8, smaller than that produced by caffeine but significantly higher than the basal staining level [Fig. 3(C,D)].

Ryanodine Reduced Activity-Dependent Somatic Secretion To explore if electrical activity and somatic secretion are functionally coupled by Ca2⫹-induced Ca2⫹ release, neurons were stimulated with a 10-Hz train in the presence of 100 ␮M ryanodine, which at this concentration blocks ryanodine receptors (McPherson et al., 1991; Kirischuk et al., 1996). In this experiment, the number of fluorescent spots was 85.5 ⫾ 16.7 (n ⫽ 11), significantly smaller (42%) than that of control neurons stimulated in the absence of ryanodine (Fig. 4). Interestingly, after restimulation of the same neurons with a 10-Hz train in the presence of FM1-43 and increasing the external Ca2⫹ concentration to 10 mM to increase its entry during depolarizations, the number of fluorescent spots/soma reached 132.2 ⫾ 18.8 [Fig. 3(B)]. This result suggests that somatic secretion can be achieved provided that a

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Figure 4 Blockade of Ca2⫹-induced Ca2⫹ release reduced somatic secretion. (A) Fluorescent FM1-43 spots produced by a 10 Hz train in the presence of ryanodine (100 ␮m). (B) Stimulation of the same neurons in the presence of high (10 mM) external Ca2⫹ increased the number of fluorescent spots. The focal plane is the same as in (A). The asterisk marks nonspecific staining for comparison of (A) and (B). Arrows point to spots produced by the second stimulation. (C) Incubation of neurons with ryanodine in the presence of FM1-43 and Mg2⫹ did not evoke somatic secretion. Images are nonconfocal, and were taken at the area of contact with the plate. Scale bar ⫽ 10 ␮m. (D) Number of spots/soma at each stimulation condition. The discontinuous bar indicates the basal (mean ⫾ S.E.) number of spots.

sufficient intracellular Ca2⫹ concentration is reached, regardless of its origin. Because certain ryanodine concentrations lock its receptors in a subconductance state, producing a leak of Ca2⫹ (Fill and Coronado, 1988), we tested if the reduction of FM1-43 staining produced by ryanodine reflected a reduction of the releasable vesicle pool because of such a Ca2⫹ leak. The number of spots of six neurons incubated with ryanodine (100 ␮M) for 10 min in Mg2⫹ solution with FM1-43 was only 43.7 ⫾ 4.3, similar to the basal staining levels [Fig. 4(C,D)], supporting that the ryanodine concentration used in our experiments had blocked ryanodine receptors.

DISCUSSION Our pharmacological and ultrastructural evidence shows that somatic secretion of serotonin is coupled to electrical activity by Ca2⫹-induced Ca2⫹ release. The main support for this comes from the reduction of

the number of fluorescent FM1-43 spots when secretion was stimulated in the presence of ryanodine or after treatment with thapsigargin. The correlation between the frequency-dependent location of clusters of dense-core vesicles and the number of FM1-43 fluorescent spots in the soma of Retzius neurons suggests that electrical stimulation induces the mobilization of entire vesicle clusters towards the plasma membrane, similar to the movement of vesicles displayed by melanotrophs (Thomas et al., 1990) and chromaffin cells (Steyer and Almers, 1999). Beck et al. (2001) have shown that in Retzius neurons single-action potentials produce Ca2⫹ transients only in submembrane regions of the soma, but trains of impulses increase the Ca2⫹ concentration in central somatic regions including the nucleus, as in some excitable endocrine cells (Cheek et al., 1990; Guo et al., 1996; Lemmens et al., 2001; Kang and Holz, 2003). However, diffusion of Ca2⫹ from the cytoplasm to the vesicle clusters seems unlikely in the absence of a feedback mechanism, because of the


limitations that intracellular buffers impose on intracellular Ca2⫹ diffusion (Neher, 1998). Although the soma of Retzius neurons has a low density of calcium channels (Fernandez de Miguel et al., 1992), they are distributed in hot spots (F.F. De-Miguel, unpublished). Ca2⫹ elevations at these hot spots upon highfrequency trains of impulses may trigger a centripetal saltatory propagation of Ca2⫹ waves in which the smooth endoplasmic reticulum placed between the plasma membrane and the vesicle clusters may act as a relay through the mechanism of Ca2⫹-induced Ca2⫹ release. In chromaffin cells there is a good correlation between the presence of Ca2⫹ microdomains and the sites of fusion of individual vesicles (Becherer et al., 2003). This suggests that in Retzius neurons the increase of the intracellular Ca2⫹ concentration produced by a train of impulses has a double role, triggering vesicle mobilization and promoting their fusion with the plasma membrane. Ca2⫹-induced Ca2⫹ release may help to produce Ca2⫹ waves with the temporal and spatial relationships required for each event. In a more general context the present findings expand our knowledge about the functional role of intracellular Ca2⫹ stores in neurons. It is interesting that frog sympathetic neurons produce Ca2⫹ waves that reach the nucleus (Hernandez-Cruz et al., 1990), and their equivalent rat neurons display somatic Ca2⫹-induced Ca2⫹ release (Hernandez-Cruz et al., 1997) and secretion (Zaidi and Matthews, 1997, 1999). Equivalent information is not yet available in other neuron types. Therefore, the contribution of Ca2⫹-induced Ca2⫹ release to somatic secretion in Retzius neurons may exemplify a mechanism common to neurons and excitable endocrine cells. We thank Rodolfo Delgado and Jorge Sepu´lveda for their assistance in electron microscopy, Bruno Mendez for his assistance in electronics, and Peter A. Mandeville for his advice on statistics.

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