Cell Tissue Res (2004) 316:295–303 DOI 10.1007/s00441-004-0886-6
REGULAR ARTICLE
Tiziana Meneghel-Rozzo · Aldo Rozzo · Lisa Poppi · Marjan Rupnik
In vivo and in vitro development of mouse pancreatic b-cells in organotypic slices Received: 10 February 2004 / Accepted: 10 March 2004 / Published online: 15 April 2004 Springer-Verlag 2004
Abstract Taking tissue slices of the embryonic and newborn pancreas is a novel approach for the study of the perinatal development of this gland. The aim of this study was to describe the morphology and physiology of in vivo and in vitro developing b-cells. In addition, we wanted to lay a foundation for the functional analysis of other pancreatic cells, either alone or as part of an integrative pancreatic physiology approach. We used cytochemistry and light microscopy to detect specific markers and the whole-cell patch-clamp to assess the function of single bcells. The insulin signal in the embryonic b-cells was condensed to a subcellular compartment and redistributed throughout the cytosol during the first 2 days after birth. The hormone distribution correlated well with the development of membrane excitability and hormone release competence in b-cells. Endocrine cells survived in the organotypic tissue culture and maintained their physiological properties for weeks. We conclude that our preparation fulfills the criteria for a method of choice to characterize the function of developing pancreas in wildtype and genetically modified mice that die at birth. We suggest organotypic culture for in vitro studies of the development and regeneration of b-cells. Keywords Newborn pancreas · Tissue slices · Organotypic culture · In vitro maturation · Development · Mouse (NMRI)
This work was supported by the European Commission (grant QLG1-CT-2001-02233 to TMR, AR and MR), the DFG Research Center for Molecular Physiology of the Brain (CMPB) and the Max-Planck Society (MR) T. Meneghel-Rozzo · A. Rozzo · M. Rupnik ()) European Neuroscience Institute Gttingen, Waldweg 33, 37073 Gttingen, Germany e-mail:
[email protected] L. Poppi Dipartimento di Sanit Pubblica, Patologia Comparata ed Igiene Veterinaria, Agripolis str. Romea 16, 35020 Legnaro (PD), Italy
Introduction A functional adult b-cell is an insulin-positive cell with more than 10,000 insulin vesicles distributed throughout the cytosol (Dean 1973; Bratanova-Tochkova et al. 2002). It releases insulin in response to glucose and tolbutamide stimulation (Ashcroft and Rorsman 1989). A b-cell is also characterized by a large resting KATP conductance (Speier and Rupnik 2003), a hyperpolarized membrane potential at low glucose and an almost complete inactivation of voltage-activated Na+ current at the resting membrane potential (Plant 1988). When plasma glucose increases, the plasma membrane depolarizes and typically exhibits an intermittent electrical activity (Dean and Matthews 1970). The depolarization is associated with an increase in cytosolic Ca2+ activity (Ashcroft and Rorsman 1989). Several laboratories have shown that embryonic rat bcells are significantly less sensitive to glucose stimulation as compared to adults (Hole et al. 1988; Bliss and Sharp 1994; Weinhaus et al. 1995), although the critical components of the insulin secretory machinery and glucose metabolism are present (Bergsten et al. 1998). It has been suggested that metabolism of nutrient secretagogues fails to couple to mechanisms involved in the membrane depolarization (Hole et al. 1988) and in a subsequent increase in cytosolic Ca2+ (Weinhaus et al. 1995). Development of glucose-dependent insulin release in embryonic mice has not been studied to date. Recently, a number of knockout mouse models were reported showing early onset endocrine defects, e.g., Pax4, Pax6 and Arx knockout animals (Sosa-Pineda et al. 1997; St-Onge et al. 1997; Collombat et al. 2003). In addition, mice with mutation in genes involved in the secretory machinery, e.g., SNAP-25 and CAPS1, die at birth (Washbourne et al. 2002). Functional analysis of these mouse models is thus only possible at the perinatal stage (Sorensen et al. 2003; Sedej et al. 2004). This is the first report on the biology of developing mouse pancreatic b-cells in tissue slices. The major advantages of fresh slices are: rapid preparation, optimal handling of limited biological material, reduced me-
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chanical stress, and the absence of enzymatic disturbance. Our primary aim was to characterize the morphology of slices, and describe the necessary conditions for using thick slices in immunocytochemistry, confocal microscopy and whole-cell patch-clamping. We wanted to assess the development of the glucose-dependent secretory phenotype in the neonatal pancreas. This information could serve as a solid base for work on genetically manipulated mice, with either a defect in the secretory machinery or changed developmental programs. In addition, it is an approach of choice for assessment of the functional development of other tissues in the pancreas and of the integrative physiology of the pancreas.
Fig. 1a–g Morphology of a newborn pancreatic tissue slice. a Reflected light micrography of a pancreatic slice from P1 mouse (tail region, hilus upper left). The duct of Wirsung runs from the left upper corner to the middle, where it branches out. A thin layer of agarose (arrowhead) gives mechanical support to the tissue. b Magnification of the islet of Langerhans (arrowhead). c Transmission light micrography of a fresh pancreatic slice. The numerous islets are aligned around the central duct and surrounded by exocrine tissue. d, e Fluorescence light micrography of an islet of Langerhans in a tissue slice using glucagon (red) and insulin (green) antibody in d and f P0 mouse. Note the accumulation of cytosolic insulin in b-cells (arrowheads). e, g The islet from a P2 mouse. Note a uniform distribution of cytosolic insulin (arrowheads). f and g are fourfold close-ups of the parts of d and e. Scale bars 2 mm (a), 100 m (b), 250 m (c), 30 m (d, e)
Materials and methods Animals Pregnant NMRI mice (ZTE, University of Gttingen, Germany) were killed by cervical dislocation and embryos were extracted by cesarean section on embryonic day 19 (E19). Neonatal NMRI mice up to postnatal day 3 (P0–P3; both sexes) were kept with their mothers. Embryos and neonatal mice were killed by decapitation. Preparation of tissue slices The pancreata were injected under the organ capsule with lowmelting point agarose 1.9% (BMA, USA) at 37C. Blown-up tissue was cooled down with the ice-cold solution, extracted from the
297 Table 1 Primary antibodies used for immunocytochemistry
Antigen
Marker
Host
Dilution
Source
Bromodeoxyuridine (BrdU) Cytokeratin-20 (CK20) Glucagon Glut-2 Insulin Insulin Tau
Proliferation Ductal cells a-cells Glucose membrane transporter 2 b-cells b-cells Axons and neuronal cells
Mouse Mouse Rabbit Rabbit Rabbit Mouse Mouse
1:200 1:100 1:500 1:200 1:125 1:1,000 1:100
Chemicon Dianova Dako Chemicon Progen Sigma Zymed Lab.
animal, and then immersed in agarose of the same density. The blocks of agarose containing the injected pancreata were cut into 100-m slices using a vibratome (Fig. 1A, B; Leica VT1000S, Germany; Speier and Rupnik 2003). Slices were then stored in carbogen (5% CO2, 95% O2) bubbled with extracellular solution at room temperature and used within 8 h. Alternatively, they were placed on the air-medium interface of the Millicell semiporous membranes (pore size of 0.4 m, Millipore Co., USA) and cultivated up to several weeks in a humid incubator at 37C with 5% CO2. The agarose and the semiporous membrane support preserved the tissue architecture without cell migration or cell cluster flattening as observed in isolated islets (own data, not shown). The glucose concentration was low (3.33 mM) to avoid excessive b-cell stimulation. The morphology of slices was documented by using wide-field light microscopy equipped with a CCD camera (Axiovert 25, Zeiss, Germany; Coolpix 995, Nikon, Japan). Solutions The extracellular solution contained (mM): NaCl 125, KCl 2.5, MgCl2 1, CaCl2 2, NaH2PO4 1.25, NaHCO3 26, Na pyruvate 2, ascorbic acid 0.5, myo-inositol 3, lactic acid 6 and glucose between 3 and 15, pH 7.3 (with carbogen at 35C). The intracellular solution contained (mM): KCl 150, HEPES 10, MgCl2 2, EGTA 0.05, ATPNa2 2, pH 7.2 (with KOH). Tissue slices were incubated in CMRL medium-1066 (Invitrogen Co., UK) without l-glutamine and supplemented with 10% fetal bovine serum, 26.2 mM NaHCO3, 100 U/ml penicillin and 100 g/ml streptomycin. All chemicals were from Sigma (USA) unless otherwise indicated. Histochemistry and microscopy Slices were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 1 h at room temperature. To block the unspecific binding and to ensure deeper penetration of antibodies we used a blocking solution composed of 3% normal goat serum, 3% BSA and 0.3% Triton in PBS. The slices were incubated with primary antibodies in the blocking solution overnight at 4C. Table 1 shows the primary antibodies used in this study, their dilutions and sources. Secondary antibodies (Alexa 488, Alexa 647; goat anti-mouse and anti-rabbit IgG; Molecular Probes, USA) were diluted 1:500 and applied for 1 h at room temperature. For double staining we coapplied both primary antibodies and later both secondary antibodies. Finally, slices were mounted using a solution of 95% glycerol in PBS. Lectins (FITC labeled glycoproteins extracted from Bandeiraea simplicifolia, GSI-B4) were diluted 1:50 and used to mark the endothelial cells (Laitinen 1987). The fluorescence immunostaining was acquired using a confocal microscope (Leica TCS SP2 AOBS; Leica Microsystems, Germany) using 488 nm (Ar) and 633 nm (He-Ne) lasers for excitation. The excitation light was limited to a narrow range around laser peak with an acousto-optical beam splitter (AOBS; Leica Microsystems). The emission was detected at 500–540 nm (green channel) and 660–750 nm (red channel) using a spectrophotometer (SP2, Leica Microsystems). The excitation crosstalk was minimized by the sequential scanning and the images were processed by Leica confocal software.
Paraffin-embedded slices Pancreata were collected from ten newborn mice and fixed by formalin (40% in 10% PBS) using injection and immersion. Following dehydration in graded alcohols and xylene, the pancreata were embedded in paraffin blocks and cut to 5-m sections. The sections were mounted on glass slides, deparaffinized in xylene, hydrated in descending ethanol series and stained with hematoxylin-eosin (HE; Kaltek-Sigma) and with Gomori’s staining (silver impregnation technique; Gomori 1941). The images were collected by transmitted light microscopy (Olympus AX 70, Japan). Electrophysiology Borosilicate glass capillaries (GC150F-15; WPI, USA) were pulled on a horizontal pipette puller (P-97 Sutter Instruments, USA) to resistances of 3–6 MW in 150 mM KCl. The slices were held at the bottom of the recording chamber (400 l) by a platinum frame with nylon fibers and superfused with the extracellular solution (35C, 1.5 ml/min). The slices were viewed with an upright microscope (60 water, NA 1, Eclipse E600FN, Nikon). The glucose concentration throughout the experiment was 3 mM unless otherwise indicated. Recordings were performed in the standard whole-cell mode via a patch-clamp lock-in amplifier (1.6 kHz; SWAM IIc, Celica, Slovenia). The current-clamp mode was used to record the membrane potential (Vm). We recorded membrane currents after applying different voltage protocols, to study voltage-dependent Na+ and K+ channels as well as to monitor ATP-sensitive K+ (KATP) conductance. A continuous sine voltage (11 mV RMS) was applied to measure the resting membrane capacitance (Cm), a parameter proportional to the cell surface area, reporting a balance between exo- and endocytotic events (Neher and Marty 1982). All the acquired parameters were transferred to a PC via an A/D converter (PCI-6035E, National Instruments, USA) and recorded on the hard disk using WinWCP V3.2.9 software (John Dempster, University of Strathclyde, UK). The same software was used to apply voltage protocols, to analyze data and export them to Sigma Plot 2001 (version 7.0, SPSS Inc., USA). Statistics are given as means € SEM. The statistical significance for the comparison was assessed using Student’s t-test.
Results Morphology of fresh pancreatic slices From each pancreas we obtained about 20–30 tissue slices with well preserved morphology (Fig. 1a, b). The islets were mainly aligned along the ducts while the exocrine tissue was peripheral (Fig. 1c). Even under a low microscope magnification, islets of Langerhans were easily distinguished from the exocrine tissue by color, cell size and typical ellipsoid shape (100, Fig. 1c). Smaller islets and small insulin-positive cell clusters (10–15 cells) were only visible after immunostaining.
298 Fig. 2a–d Reticulin capsule in newborn mouse islets. a Lectin staining of the connective tissue around and within the islet in P0 mouse pancreas (v blood vessels, arrowheads capsule). b Insulin (green) and CK-20 (red) antibody staining showing close association between ductal cells and the islets. c Gomori staining of the histological section of a newborn mouse pancreas. Islets are indicated with asterisks. d Higher magnification of the reticulin (black arrow) (i islet of Langerhans, d duct, e exocrine tissue with zymogen granules, v blood vessels with erythrocytes). Scale bars 50 m (a), 20 m (b), 18 m (c, d)
Using specific antibodies raised against insulin and glucagon, it was possible to study the distribution of insulin- and glucagon-positive cells. At all studied pre- or postnatal days the glucagon- and insulin-positive cells were located both centrally and peripherally in the islet. Glucagon-positive cells often formed small clusters (Fig. 1d, e) and presented about 10% of the islet; the rest were insulin-positive cells. In embryonic and P0 pancreata, insulin staining was limited to perinuclear accumulation (Fig. 1d, f), while from P2 on the insulin signal was, as in adults (Speier and Rupnik 2003), homogeneously distributed throughout the cytoplasm (Fig. 1e, g). Islet capsule, blood vessels and ducts In human (Gomori 1941), rat (Bonner-Weir 1991; Hisaoka et al. 1990) and mouse islets (Bonner-Weir 1991) the existence of a capsule surrounding the islet has already been described. It has not yet been reported that such a layer of collagen fibers would surround the islet already at birth. We initially used lectin to specifically label collagen structures in the pancreas. Lectin strongly labeled the exocrine cells and endothelium of the blood vessels (Laitinen 1987), but only weakly stained the b-cells (Fig. 2a). However, a thin layer that could represent a capsule was detected in addition to a dense net of blood vessels around and within the islets (Fig. 2a). To confirm
the presence and to identify the composition of the capsule a metal impregnation technique was used. Gomori’s method selectively stained the collagen structure called reticulin (Gomori 1941; Lamar-Jones 2002). As shown in Fig. 2b, c, the islets from P0 mice were surrounded by a thin layer of reticular fibers, which physically separated islets from the exocrine tissue, ducts and vessels, occasionally with septa separating the endocrine cell mass (Fig. 2b). The islets of Langerhans in the embryonic and newborn mice were closely associated with the pancreatic ducts (Figs. 1, 2b–d). The ducts were recognized by their characteristic histological architecture in paraffin sections (Fig. 2c, d) and by their intermediate cytokeratin filaments (Fig. 2b; Yoon et al. 1999; Coulombe et al. 2002), which were stained with the antibody against CK-20, a specific ductal cell marker (Fig. 2d; Bouwens et al. 1994; Bertelli et al. 2000). Innervation in pancreatic islets Immunostaining with anti-Tau antibody was used to assess the morphology and anatomical arrangement of the intrapancreatic neurons in relation to the islets. Intrapancreatic neurons were assembled into ganglia, projecting dendrites into the surrounding exocrine tissue and neighboring islets (Fig. 3a, c). Ganglia in the slices were connected to each other as was also reported in cat pancreas (King et al. 1988). At least two types of intrapan-
299 Fig. 3a–d Intrapancreatic innervation. a Composite fluorescent micrography of Tau (green) and Glut-2 (red) antibody staining in a tissue slice from the newborn mouse. Ganglia are clearly associated with the islets (asterisks). b Tau antibody (red)-positive bigger ganglion (arrow) innervating an islet stained by insulin antibody (green). c Higher magnification of the smaller ganglion from a (white box). Numerous processes (arrowheads) enter the islets to form a close association with endocrine cells. d Glut-2 (red) and Tau (green) antibody staining of a neuroinsular complex (NIC) in an acute slice of P2 mouse pancreas. Note the close association between nerve cell bodies and b-cells. Scale bars 100 m (a), 40 m (b), 25 m (c), 10 mm (d)
creatic ganglia were distinguished based on their size, shape and number of neurons (Tiscornia 1977; King et al. 1988; Love and Szebeni 1999). In the newborn mice we found numerous large ellipsoid ganglia, containing from 7 to 30 neurons (Fig. 3b). Smaller, triangular intrapancreatic ganglia contained three to six neurons (Fig. 3c). In addition, we also found a specific neuroendocrine association with an unknown function, so-called “neuroinsular complex type I” (NIC; Fujita 1959; Persson-Sjgren et al. 2001; Fig. 3d). Organotypic culture of pancreatic slices After a week of organotypic culturing in 3.33 mM glucose, most of the cells outside the islets disappeared (Fig. 4); however, cells inside the islets survived for over 12 weeks (not shown). In 5 mM glucose or higher, also neurons, ductal cells and some exocrine cells survived in the culture. During the first week of the organotypic culture the embryonic and P0 islets grew and changed their shape from an elongated ellipsoid to a rounder ellipsoid, similar to that found in the adult pancreas (Fig. 4b). The length, width and volume of the islets were 70€17 m, 135€27 m, and 350,000 m3 in fresh newborn islets and
110€17 m, 255€45 m, and 1,600,000 m3 in cultured islets (n>50 in each group). The increase in islet size could be due to the cell growth (McEvoy 1981) or to the increased cell number (Lazarow et al. 1973). To study the cell growth we measured the resting membrane capacitance. The b-cells in culture grew in size (from 3.6 to 5.6 pF) during the first week, reaching the size comparable to the adult b-cells (5.6 pF; Speier and Rupnik 2003), bigger than in fresh P3 b-cells (4.9 pF; see Table 2). The volume of a typical b-cell in a newborn islet was calculated to be 900 m3 and such an islet would contain about 400 cells. In cultured islets the volume of a single cell increased to 1,800 m3, suggesting an additional increase in the cell number to about 1,000 cells per islet. To assay cell proliferation we performed immunohistochemical labeling with bromodeoxyuridine (BrdU; Scaglia et al. 1997). Both glucagon- and insulin-positive cells kept their capacity to proliferate in culture (Fig. 4c) with at least one division per cell in 7 days of culturing. Functional b-cell discrimination The identification of b-cells inside the islets was a key step for the electrophysiological characterization. The
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Fig. 4a–c Organotypic culture of pancreatic slices. a Composite transmission light micrography of a fresh pancreatic slice from E19 mouse; and b the same slice after 7 days in the culture. Note the increased size of the islets and absence of the exocrine tissue. c Table 2 In vivo and in vitro development of b-cell electrophysiological parameters. Numbers in parentheses are the number of cells tested or portion of cells showing a measured parameter. Significant differences in the means of the parameters are indicated in bold
Cell size (pF) Rest Vm (mV) I(Na+) (pA) +
I(K ) (pA) KATP (nS) DCm (fF)
Glucagon (red) and BrdU (green) antibody staining of the islet performed after 24 h exposure to BrdU. Note BrdU staining in numerous b-cell nuclei and also a-cell nuclei (arrowheads). Scale bars 250 m (a, b), 30 m (c)
Fresh
Fresh
Cultured
In vivo
In vitro
E19P0
P2P3
E19P0
(p)
(p)
3.6€0.3 (10) 54€5 (7) 365€31 (4/9) +1,176€156 (8) 1.8€0.5 (9) 29.9€4.6 (4/6)
4.9€0.4 (12) 77€4 (10) 704€66 (9/9) 1,318€134 (10) 2.2€0.6 (11) 89.5€14.4 (8/10)
5.6€0.2 (11) 75€8 (11) 1,437€74 (6/10) 1,762€152 (11) 4.2€0.8 (11) 56.7€16.2 (9/11)
0.023
0.0002
0.004
0.043
0.0025
10E8
response to glucose is the functional feature of insulinreleasing cells in vivo, but it has not yet been demonstrated in perinatal mice. We designed a fast approach to discriminate b-cells by comparing the responses to a square depolarization to 20 mV after two pre-pulses to 75 and 150 mV (Fig. 5a). The cells were defined as b-cells if the Na+ currents elicited by depolarization from 75 mV were smaller. This approach was used in all cells studied and 37 in 41 cells showed either a typical hyperpolarized half inactivation (25/37) or the absence of Na+ current (12/37; Table 2). The functional diagnosis in b-cells from perinatal mice yielded a comparable proportion of b- and non-b-cells as observed in the morphological analysis. Another parameter that changed during the early postnatal life was the amplitude of voltage-activated Na+ currents (Fig. 5a). The amplitude of the Na+ current increased about twofold between E19 and P2. An even larger increase in the amplitude of Na+ current was measured after 1 week in the culture, where the Na+ currents increased fourfold (Table 2). To further check the validity of Na+ current b-cell diagnostics in embryonic and in postnatal b-cells, it was necessary to test their response to the classical secretagogues. The coapplication of high glucose (10 mM) and tolbutamide (100 M) only slightly depolarized the b-
0.50
0.016
0.59
0.021
0.004
0.14
Fig. 5a, b Functional development of b-cells. a Inactivation of voltage-activated Na+ currents at subphysiological membrane potentials. Depolarization to 20 mV from 75 mV and 150 mV (top) generated inward currents in embryonic (E19), P2 and 7 days cultured newborn b-cells (P0D7). b Typical increase in membrane capacitance triggered by a train of depolarizing pulses from 60 mV to +10 mV (top) in embryonic (E19), P2 and cultured embryonic b-cells
cells in E19 and P0 slices. In contrast, such a treatment elicited a typical adult-like electrical activity in b-cells from P2 and P3 slices, where the induced depolarization was followed by sustained electrical activity (firing) or by
301
intermittent electrical activity (bursting) comparable to that observed in adults (Dean and Matthews 1970; Speier and Rupnik 2003). Electrophysiological properties of b-cells Two macroscopic K+ currents were measured: (1) voltage-activated K+ currents and (2) ATP-sensitive K+ currents. To evoke voltage-activated K+ currents a depolarizing step from 60 mV to +40 mV (150 ms) was applied. All b-cells showed these currents and the amplitude of these currents was stable during the first days after birth and in organotypic culture (Table 2). KATP channels were also present in all tested b-cells and their steady-state conductance after dialysis of cytosol with 2 mM ATP was between 2 nS in fresh and 4 nS in cultured slices (Table 2). Under our experimental conditions with 2.5 mM [K+]o and 150 mM [K+]in, the EK was at 110 mV, but in contrast to adult b-cells the resting Vm after dialysis of embryonic and early postnatal b-cells was more positive (Table 2). A train of depolarizing pulses was applied in order to activate voltage-activated Ca2+ channels, and induce Ca2+ influx and exocytotic release of insulin (Fig. 5b). The Cm changes in the embryonic and P0 b-cells were significantly lower than those in P2 and P3 in mice. During the first postnatal days the number of responsive cells increased from 66% to 80%. The amplitude of Cm change increased threefold (Fig. 5b, Table 2). After 1 week in culture, 82% of b-cells responded to a stimulatory train, but the amplitude of the Cm change did not increase further due to culturing (Fig. 5b, Table 2).
Discussion Pancreas tissue slices We have shown that our new preparation is suitable for the study of the morphology and physiology of mouse pancreas using various approaches: from immunostaining and confocal microscopy to electrophysiology and organotypic culturing. The volume of newborn pancreata is small, and the b-cells are sensitive to mechanical and enzymatic treatment, and subject to active differentiation. All these features are poorly compatible with the classical cell isolation techniques. In characterization of mice lacking important secretory proteins and with homozygous perinatal lethality, the major obstacle is often the quantity of the biological material, due to small size and reduced Mendelian frequencies. The tissue slicing of pancreas has a high yield in retrieving endocrine cells while the entire gland can be isolated and subsequently cut. Important also is the speed of preparation, which is limited to some tens of minutes. Control and test conditions can be tested in parallel on tissue from the same donor, either fresh or cultured, significantly reducing the statistical variability, and the num-
ber of experiments and animals needed. In addition, pancreatic b-cells are not the only tissue that could be studied in this preparation; it provides fast and easy access to the physiology of other endocrine cells, exocrine cells, blood vessels, ductal system, peripheral nervous tissue and adipose tissue. Morphology of acute pancreatic slices The resistance during the penetration of patch pipette into the islets and the visual effect of the pipette’s positive pressure on the outer surface of the islet suggested that a thin collagen capsule enwraps the islet also in newborn pancreas, similarly to that observed in adults (BonnerWeir 1991). The Gomori and lectin fluorescent staining showed the capsule as intertwine reticulin fibers. The capsule may not only have a mechanical role supporting the islet structure but also an important function in limiting the diffusion of hormones between the islets and surrounding exocrine tissue. It might also divide one islet into functional subdomains. The presence of reticulin septa in adult islets offers a morphological support for the suggested functional clustering of b-cells (Meda 1995). The b-cells that are connected via gap junctions (Meda et al. 1991) were supposed to be functionally grouped into territories (Meda 1995). However, in newborn islets, reticular septa were hard to visualize, suggesting that they may further develop during the morphological maturation involving the cell hyperplasia and hypertrophy. The insulin-positive immunostaining of the embryonic b-cells shows that the insulin biosynthetic pathway is already established before birth as was also found in newborn rats (Bergsten et al. 1998). However, apparently the final stages leading to exocytosis are not yet functional (Hole et al. 1988; Bliss and Sharp 1994). Accordingly, we were not able to detect the secretory response from the embryonic and P0 b-cells evoked by a train of depolarizations. Innervation of pancreatic islets The close association between neurons and endocrine cells in pancreas has been previously observed in various animal species (Legg 1967; Unsicker 1984; Ahrn 1999). Similarly, Orci et al. (1973) reported the specialized membrane junctions in the rat pancreas, additionally suggesting the existence of synapses. In mouse, we found a number of dendrites projecting into the islet, which agrees with previous data (Lundquist and Ericson 1978). The nerve fibers contacting the islets were both adrenergic and cholinergic (Lundquist and Ericson 1978; Unsicker 1984). Further studies will be necessary to assess the detailed role of this tight neuroendocrine association in the control of hormone release.
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b-cell discrimination The cell location in the newborn islets cannot be used as a sole criterion to select b-cells due to the small number of cells and the distribution of insulin- and glucagon-positive cells (Fig. 1). Also the cell size cannot be used because newborn b-cells are smaller than adult cells and therefore not different from non-b-cells (Table 2). In P2 and P3 newborns, typical Na+ channel inactivation could be correlated to the electrical activity induced by glucose and tolbutamide. In embryonic b-cells, which do not react to secretagogues, Na+ current inactivation thus appeared to be the only, but in our opinion reliable, parameter for bcell discrimination (Plant 1988; Gpel et al. 1999). In vivo functional development of b-cells Based on in vivo development we were able to group the measured b-cells into two groups (see Table 2). In the first group we put non-mature late embryonic and early newborn stage (E19–P0) with insulin-positive cells, and in the second group mature newborn stage (P2–P3) showing both electrical and secretory activity. We found out that full glucose-dependent insulin release in mice appears only during the 2nd day after birth. This result correlates well with the recently described phenotype of Arx knockout mice, which do not contain a-cells. These mice are born without phenotypic discrepancies, but die of severe hypoglycemia and dehydration 48 h after birth (Collombat et al. 2003). The b-cells from mice older than 2 days are functionally adult. The resting Vm also differed between the two groups, with the b-cells from the first group being more depolarized than the second group (Table 2). During the development the b-cells progressively hyperpolarized to reach more or less adult resting membrane potential at P2. A similar progressive hyperpolarization has also been found in developing colonic cells (Beskid and Pacha 2003). The rather depolarized resting Vm in embryonic bcells might also be a result of insufficient paracrine insulin activity. The low interstitial insulin may result in lower activity or no upregulation of Na+/K+ pump as was reported for the skeletal muscles (Clausen 2003). A dedicated paracrine secretion of insulin in addition to the endocrine has recently been demonstrated by two-photon based secretory assays (Takahashi et al. 2002). In vitro development of b-cells In the organotypic culture in low glucose all the noninsular structures (exocrine pancreas, ducts, vessels and neurons) disappeared. Durant et al. (2003) attributed these changes to apoptotic events and macrophages. We used the clearance of the non-endocrine tissue to improve the accessibility to the islets for patch-clamp pipettes. This can be even more appreciated when the islet morphology has been changed due to genetic modification, e.g. Pax6
mice, where the proper islets do not form. Such clusters of insulin-positive cells are indistinguishable from exocrine tissue in the differential interference contrast microscopy usually used for patching in slices (Rozzo et al., in preparation). In the organotypic culture the islets survived for many weeks, but after a couple of weeks the excitability of the cells rapidly declined (not shown). Therefore we limited our study to the in vitro functional development during the 1st week of culturing. During this week b-cells developed to an apparently more mature stage. Initially, we observed a pronounced increase in the size of islets, number of endocrine cells and redistribution of cytosolic insulin. In addition to hypertrophy and hyperplasia, bcells improved their endocrine function. Their resting membrane potential progressively hyperpolarized, and they gained the ability to respond to secretagogues and to exocytose during a train of depolarizations. These results open up the possibility for future screening of factors guiding developmental as well as regeneration process. Morphological characterization of embryonic and perinatal b-cells shows that these cells contain insulin, though its subcellular distribution is different from that of mature b-cells. However, these immature b-cells already possess electrophysiological features that are so typical that they can be readily used to diagnose them from other endocrine cells. This fact also opens up possibilities for early characterization of surrogate insulin-producing cells differentiated from either embryonic or adult stem cells (Blyszczuk et al. 2003). Acknowledgements We thank Marion Niebeling and Heiko Rhse for excellent technical support.
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