Extracellular matrix glycoproteins inhibit neurite outgrowth of different types of identified leech neurons in culture

Neuroscience 137 (2006) 1165–1176

EXTRACELLULAR MATRIX GLYCOPROTEINS INHIBIT NEURITE OUTGROWTH OF DIFFERENT TYPES OF IDENTIFIED LEECH NEURONS IN CULTURE N. FLORES-ABREU, J. VARGAS AND F. F. DE-MIGUEL*

determine the outgrowth pattern that characterizes each type of adult neurons? The differences in the regenerating capabilities of neurons suggest that each neuron type responds in different way to the combination of growthpromoting and -inhibiting influences in the external milieu. Among these influences, the family of extracellular matrix (ECM) molecules may produce a wide range of effects on the outgrowth patterns of neurons (Sanes, 1989; Giancotti and Ruoslahti, 1999). While one neuron type may grow extensively on certain ECM proteins, other neuron types may not grow at all or avoid them (Bixby and Harris, 1991; Lein and Higgins, 1989; Müller et al., 1990). In this paper we compare quantitatively the outgrowth patterns formed by different types of identified neurons in culture and explore the contribution of inhibitory ECM glycoproteins to the formation of their distinctive outgrowth patterns. Our experiments were made with identified neurons isolated from the CNS of the leech and kept in culture. The CNS of the leech contains 21 similar segmental ganglia that lie ventrally along the animal. Each ganglion contains a similar number of neurons distributed in a stereotyped manner. Many neuron types have been identified by their position, electrical activity pattern, synaptic connections and contribution to behavior (Nicholls and Baylor, 1968; Rela and Szczupak, 2003; Rodriguez et al., 2004; Taylor et al., 2003; Briggman et al., 2005). In addition, adult leech neurons maintain the ability to regenerate neurites, specific connections and behaviors after injury (Baylor and Nicholls, 1971; Jansen and Nicholls, 1972; Wallace et al., 1977; Muller and Scott, 1979; Duan et al., 2005). This capability has also allowed the isolation of individual neurons and their culture, where they regrow and form selective synapses with appropriate targets (Fuchs et al., 1981; for review see Fernandez de Miguel and Drapeau, 1995). Identified leech neurons in culture regenerate characteristic outgrowth patterns that act as fingerprints for their identification (Chiquet and Acklin, 1986; Grumbacher-Reinert, 1989). The internal side of the capsules that enwrap the ganglion contains a useful substrate for analyzing how different individual neurons interact with an ECM they encounter in the animal, in the absence of soluble factors and cell to cell interactions (Fernandez de Miguel, 1997). The combination of each neuron type with the mesh of ECM molecules inside the ganglion capsules produces distinctive outgrowth patterns, which in some cases resemble those produced by the same neuron in the developing or adult nervous system (Fernandez de Miguel, 1997). For example, anterior pagoda (AP) neurons cultured on ganglion capsules regenerate a bipolar outgrowth pattern with

Departamento de Biofísica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apartado postal 70-253, México 04510 D.F., Mexico

Abstract—We explored the contribution of inhibitory peanutbinding extracellular matrix glycoproteins to the regeneration of characteristic outgrowth patterns by different types of identified neurons. Adult leech neurons were isolated one by one and plated in culture on a substrate that consisted of the capsules that encase the CNS ganglia. On the inside surface of this substrate, a combination of growth-promoting and -inhibiting extracellular matrix glycoproteins regulates the regeneration of distinctive outgrowth patterns by different neuron types. The role of inhibitory glycoproteins that bind to peanut lectin was studied by perturbation experiments in which peanut lectin was added to the culture medium. The effects of peanut lectin on the outgrowth patterns depended on the specific cell type that was tested. Anterior pagoda neurons, which on capsules produce a bipolar outgrowth pattern, in the presence of the lectin multiplied the number of primary neurites and the total neurite length and also lost their bipolarity. Annulus erector motoneurons, which on capsules grow poorly, in the presence of peanut lectin sprouted 70% more neurites and duplicated their total neurite length. By contrast, Retzius neurons which grow profusely on ganglion capsules, in the presence of peanut lectin increased the number of primary neurites without increasing their total neurite length or branch points. When neurons were plated on plastic, peanut lectin added to the culture medium did not affect the growth of neurons, thus showing that the effects of peanut lectin were induced by blocking the binding of neurons to inhibitory glycoproteins on the capsules. These results show that regeneration of different neuron types has different regulation by inhibitory extracellular matrix molecules. © 2005 Published by Elsevier Ltd on behalf of IBRO. Key words: neurite outgrowth, regeneration, extracellular matrix, inhibitory proteins, PNA, leech.

Inhibitory proteins have an active role in the development and regeneration of the nervous system (Villegas-Perez et al., 1988; Schwab and Caroni, 1988; Masuda-Nakagawa and Nicholls, 1991; Nicholls and Saunders, 1996; Sandvig et al., 2004). The effects of these proteins include the blockade of cell migration, the abolition of the sprouting, extension and branching of neurites (for reviews see Caroni et al., 1988; Schwab et al., 1993; Roskies and O’Leary, 1994; Dent et al., 2004; Kuan et al., 2004). To what extent does the balance between inhibitory and growth-promoting proteins *Corresponding author. Tel: 52-55-5622-5622; fax: 52-55-5622-5607. E-mail address: [email protected] (F. F. De-Miguel). Abbreviations: AE, annulus erector; AP, anterior pagoda; ECM, extracellular matrix; PNA, peanut. 0306-4522/06$30.00⫹0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2005.10.036

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Fig. 1. Perturbation of the outgrowth pattern of AP neurons by PNA lectin. (A) Four subsequent stages of the development of the bipolar pattern of AP neurons at 12 h intervals. The formation of the pattern is characterized by the early extension of one neurite with branches followed by the production of the second primary neurite after a delay. In this period there is also retraction of some secondary neurites (arrowhead at 36 h). (B) Three examples of the bipolar pattern of AP neurons by 48 h growing. (C) Increase in the number of neurites and loss of the bipolar pattern in the presence of PNA lectin. Four subsequent images of the development of the pattern of AP neurons are shown at 12 h intervals. Arrows point to some of the neurites. Note the persistence of all of the neurites, showing a lack of retraction. (D) Three examples of the outgrowth pattern of AP neurons after 48 h regenerating neurites in the presence of PNA. Scale bar⫽20 ␮m.

two primary neurites and some branches (De-Miguel and Vargas, 2000), similar to the pattern of these neurons by embryonic day 9 (Gao and Macagno, 1987). Retzius neurons, which are the major serotonin releasing neurons in the animal (Trueta et al., 2003; De-Miguel and Trueta, 2005), on capsules form multiple robust neurites with large growth cones (Fernandez de Miguel, 1997). In contrast to the extensive sprouting of Retzius neurons, annulus erector (AE) motoneurons grow poorly on capsules (De-Miguel and Vargas, 1997). However the three neuron types plated on leech laminin on concanavalin A as substrates sprout multiple long neurites and branches, demonstrating their extensive regenerative potential (Chiquet and Acklin, 1986; De-Miguel and Vargas, 1997). Therefore, the celldependent differences in the extent of growth displayed on ganglion capsules do not reflect their intrinsic growth capabilities, but their sensitivities to the concerted regulation by a cocktail of ECM proteins, including growth-promoting and inhibitory molecules.

The use of lectins to identify different ECM glycoproteins in the capsules has allowed the detection of a set of inhibitory glycoproteins by their specific binding to peanut (PNA) lectin. The use of PNA lectin in the culture medium as a way to block the binding of AP neurons with these proteins, and therefore avoid selectively their inhibitory effects, induces the production of supernumerary neurites (De-Miguel et al., 2002). Here we extended this study to analyze to which extent the different growing capabilities of AP, AE and Retzius neurons are due to a cell-dependent intrinsic sensitivity to inhibiting ECM proteins. This was tested by the quantitative analysis of the outgrowth patterns of neurons plated on ganglion capsules in the presence or absence of PNA lectin.

EXPERIMENTAL PROCEDURES Isolation and culture of neurons Identified neurons were isolated from the CNS of adult leeches, Hirudo medicinalis using the procedure described by Dietzel et al.

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in leech Ringer’s solution containing 0.2% triton X-100 (Sigma). This procedure eliminates living elements in the capsules and improves the visualization of the neuronal processes without affecting neurite outgrowth (Fernandez de Miguel, 1997). Individual capsules were sterilized by washing them several times in L-15 containing 1 mg/ml gentamycin and were then transferred to glass-bottomed wells. Neurons were plated on the internal side of the capsule.

Growth on different substrates and perturbation experiments For perturbation experiments, 1.0 ␮g/ml of PNA lectin was added to the culture medium in which Rz, AP and AE neurons were plated. Neurons were also plated in the absence of lectin and to test that PNA lectin did not have growth promoting effects by itself, neurons were plated on 10-␮l micro well dishes coated with 2 mg/ml PNA for 2 h and on plastic with soluble 1.0 ␮g/ml PNA lectin added to the culture medium.

Microscopy and quantization of outgrowth patterns Neurons plated on ganglion capsules were examined with Nomarski optics and neurons plated on plastic or on lectin-coated plastic dishes were viewed under phase contrast optics. Images were digitized with a CCD camera coupled to an inverted microscope. The uneven topology of the capsules made it necessary to take images at different focal planes and to reconstruct the neuritic trees by digital pasting various segments of images by using Adobe Photoshop software (Adobe Systems, Mountain View, CA, USA). The outgrowth patterns were quantified from digital images, using Metamorph Imaging System 3.6 software (Universal Imaging, West Chester, PA, USA). The number of primary branches, the total neurite length and the number of branch points were measured manually at 12 h intervals for 2 days. Our results are expressed as mean values⫾S.E.M. Group differences were analyzed by a two tail Student t-test, adjusting the confidence level to 95%.

Electrical recordings

Fig. 2. Quantitative differences induced by PNA lectin on the neurite length (A), number of primary neurites (B) and number of branches (C) of AP neurons. Asterisks indicate significant differences at each time.

(1986). In brief, nerve cords were dissected, and ganglion capsules were opened to expose the cell somata. The ganglia were kept in L-15 culture medium (Gibco, Mexico City, Mexico) supplemented with 6 mg/ml glucose, 0.1 mg/ml gentamycin (Sigma, St. Louis, MO, USA) and 2% heat-inactivated fetal calf serum (FSC; Gibco) and were incubated for 1 h in 2 mg/ml collagenase/dispase (Boehringer-Mannheim, Darmstadt, Germany). After the enzyme treatment Rz, AP and AE neurons were identified visually by their size and location in the ganglion, sucked out, and rinsed several times in L-15 to sterilize them and remove debris. Only neurons with primary stumps (remaining portions of the primary process) were used in these experiments, because the stump is the preferential region for neurite growth. Neurons were plated with their stump contacting the substrate.

Isolation of ganglion capsules Cell-free ganglion capsules were obtained by opening the ganglion with forceps and removing all neurons by cutting with scissors the axon bundles that form the connective nerve and nerve roots. With this procedure, the neuropil and neuronal packet were removed as a whole. The capsules were then incubated for 30 min

Intracellular recordings were made from every neuron to confirm its identity and physiological conditions after 48 h growing. Intracellular recordings were made with borosilicate glass microelectrodes (FHC Inc, Bowdoinham, ME, USA) with tip resistances ranging from18 –25 M⍀ when filled with 3 M KCl. The microelectrodes were coupled to pre-amplifiers (Almost Perfect Electronics, Basel, Switzerland). Data were acquired by an analog-to-digital board Digidata 1200 (Axon Instruments, Foster City, CA, USA) using Axoscope 8.0 or Pclamp 8.0 and stored in a PC for further analysis.

RESULTS Outgrowth patterns of AP neurons Attachment of AP neurons to the inner side of ganglion capsules triggered outgrowth of neurites over the following hours. Fig. 1A shows the development of the outgrowth pattern of an AP neuron during 48 h at 12 h intervals. As shown before, the extension of primary neurites was accompanied by the retraction secondary neurites (De-Miguel and Vargas, 2002). After 48 h growing, seven AP neurons had formed the characteristic bipolar pattern. Three of these neurons that had grown for 48 h are shown in Fig. 1B. The addition of 1 ␮g/ml of PNA lectin to the culture medium in which AP neurons were cultured induced an

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Fig. 3. Lack of growth-promoting activity of PNA lectin on AP neurons. (A) Three examples of neurons growing on plastic as substrate. (B) Three other examples of AP neurons growing on plastic coated with PNA lectin. (C) Three neurons growing on plastic with lectin added to the culture medium. Although the extent of growth is different from one neuron to another, the characteristics of the outgrowth pattern remained similar in the three experimental conditions. Scale bar⫽30 ␮m.

increase in the number of primary neurites, the total neurite length and the number of branch points (Fig. 1C–D). The differences with respect to AP neurons growing in the absence of the lectin were significant by 12 h of growth and these differences remained for a further 36 h (Fig. 2). In addition, in the presence of PNA neurites were oriented in multiple directions (Fig. 1C–D) and we did not detect retraction of branches in any of the neurons. The quantitative data at 12 h intervals comparing the patterns of AP neurons growing with and without PNA lectin are in Fig. 2. As can be seen, by 48 h, the total neurite length in the presence of PNA lectin was double that observed without the lectin, 612.0⫾120.0 ␮m in the presence of PNA compared with 290.0⫾63.0 ␮m in controls. It is noteworthy that by 48 h, the curve relating neurite extension with time in the absence of PNA lectin almost reached saturation. By contrast while in the presence of PNA lectin the kinetics of growth remained linear. The differences between both curves were significant at every time, as shown by the asterisks. The number of primary neurites was also duplicated in the presence of PNA lectin, and by 48 h, the control AP neurons had produced 3.0⫾0.5 neurites, while

in the presence of PNA neurons had 6.0⫾1.0 neurites. Again, the differences between both groups had been settled by 12 h and remained 36 h later. Interestingly, the number of branch points had a three-fold increase and by 48 h growing there were 3.25⫾1.5 branches in control neurons and 9.6⫾2.6 in the presence of PNA lectin. The coefficient of the number of branch points per neurite at 48 h of growth was 1.08 without PNA and 1.42 with PNA, thus being consistent with a reduction of retraction of secondary neurites in the presence of PNA lectin. To test whether the increase in growth of AP neurons was due to the binding of the lectin to ECM glycoproteins and not to a direct activation of neuronal receptors, we plated neurons on plastic (n⫽11), on plastic coated with PNA lectin (n⫽11) and on plastic, adding the soluble lectin to the culture medium (n⫽10 neurons). As shown in Fig. 3, the outgrowth patterns of AP neurons growing in the tree experimental conditions were similar, all consisting of long lamellar neurites with filopodia. The differences in the extent of growth of neurons in Fig. 3C when compared with those in Fig. 3A or B are because images of neurons in C were from a set of neurons in a different experiment.

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Fig. 4. Perturbation of the outgrowth pattern of AE neurons by PNA lectin. (A) Four subsequent stages of the development of the pattern of AE neurons at 12 h intervals. There were some small neurites sent in different directions. (B) Two other examples of the pattern of AE neurons by 48 h growing. (C) PNA lectin induced an increase in the number of neurites. Four subsequent images of the formation of the pattern are shown at 12 h intervals. There was no detectable retraction of neurites. (D) Two examples of the outgrowth pattern of AE neurons in the presence of PNA after 48 h of growth. Arrows in all cases point to different neurites. Scale bar⫽20 ␮m.

However, the similarities in the general characteristics of the outgrowth patterns confirm that the perturbing effects of PNA lectin on the outgrowth pattern of AP neurons plated on capsules were produced by the blockade of the ECM glycoproteins.

Outgrowth pattern of AE motoneurons Plating AE motoneurons inside the ganglion capsules triggered the production of small number of neurites oriented in different directions and with some bifurcations. As in the

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Fig. 5. Quantitative differences induced by PNA lectin in the neurite length (A), number of primary neurites (B) and number of branches (C) of AE motoneurons. Asterisks indicate significant differences.

case of AP neurons, some of the secondary branches were retracted at early stages of growth. Fig. 4A shows an AE neuron growing at 12 h intervals. Two other neurons that had grown for 48 h are shown in Fig. 4B. In the 16 AE neurons included in this study, the electrical activity pattern recorded with intracellular microelectrodes was similar to that already described in culture (De-Miguel and Vargas, 1997), consisting of small spikes produced upon large depolarizations by current injection. The addition of PNA lectin to the culture medium perturbed the outgrowth pattern of AE neurons by increasing the number and length of their primary neurites and also by increasing the number of branches. Fig. 4C shows an example of the development of this pattern at 12 h intervals

and Fig. 4D shows two other examples of neurons after 48 h growing. As can be seen, the increase of growth of AE neurons induced by PNA lectin was also established before 12 h and was kept until 48 h growing. As shown in Fig. 5, by this time, the total neurite length had reached 524.0⫾46.0 ␮m without PNA (n⫽8) and 1158.0⫾198.0 ␮m in the presence of PNA (n⫽8). Conversely the number of primary neurites increased by 70%, from 5.6⫾0.4 in controls to 9.5⫾2.6 with PNA and the number of branch points was almost tripled, from 5.4⫾2.0 without PNA to 13.8⫾4.0 with PNA. The coefficient of the number of branches per neurite was 0.93 without PNA and 1.45 with PNA, similar to the values of AP neurons in both conditions, thus suggesting that PNA had induced either,

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Fig. 6. Lack of growth-promoting activity of PNA lectin on AE motoneurons. (A) Three examples of neurons growing on plastic as substrate. (B) Three other examples of AE neurons growing on plastic coated with PNA lectin. (C) Three neurons from a different batch growing on plastic with lectin added to the culture medium. Although the extent of growth was smaller, the general characteristics of the pattern remained the same. Scale bar⫽60 ␮m.

an increase in the number of secondary branches or a decrease in their retraction. AE motoneurons plated directly on plastic (n⫽12; Fig. 6A), on plastic coated with PNA lectin (n⫽12; Fig. 6B) or on plastic and adding PNA lectin to the culture medium (n⫽11; Fig. 6C), also developed similar outgrowth patterns, with large lamellae and filopodia at their tips, similar to those formed by AE neurons plated on Con A (De-Miguel and Vargas, 1997). Neurons in Fig. 6 had been growing for only 12 h, yet the extent of growth confirms their enormous potential for outgrowth. In addition, this control shows that the effect of the lectin was through the binding to ECM glycoproteins and not a direct effect on the neurons.

Outgrowth patterns of Retzius neurons In contrast to AP or AE neurons, Retzius neurons grow profusely on ganglion capsules, as they also do on other substrates. This suggests that Retzius neurons may be less sensitive to the inhibitory effects of PNA-binding glycoproteins. Seven Retzius neurons plated inside the ganglion capsules regenerated their processes rapidly, forming multiple robust branched and multidirectional neurites that were guided by large growth cones (Fernandez de Miguel, 1997). Four moments of the development of the pattern of a Retzius neuron as well as two other neurons that had grown for 48 h are presented in

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Fig. 7. Perturbation of the outgrowth pattern of Retzius neurons by PNA lectin. (A) Four subsequent stages of the development of the bipolar pattern at 12 h intervals. The normal pattern on capsules was characterized by the rapid production of multiple neurites. (B) Three examples neurons by 48 h growing. (C) Successive images of one neuron in the presence of PNA at 12 h intervals. Arrows point to some of the neurites. Note the lack of retractions of neurites. (D) Two other examples of the outgrowth pattern of Retzius neurons in the presence of PNA after 48 h of growth. Scale bar⫽80 ␮m.

Fig. 7A and B, respectively. The retraction of neurites was not a detectable component of the formation of the outgrowth pattern. Although at first glance, the addition of 1 ␮g/ml of PNA to the culture medium did not change the regeneration pattern of Retzius neurons (Fig. 7C–D), the quantification made from seven neurons showed significant differences. While the total neurite length with and without PNA lectin remained similar during the first 48 h growing (1523.16⫾192.32 without PNA and 1758.0⫾

453.32 ␮m in the presence of PNA lectin) by 48 h, there was a significant increase in the number of primary neurites which by 48 h was 12.4⫾3.49 without PNA and 19.3⫾2.5 in the presence of the lectin (Fig. 8B). PNA lectin also produced an increase in the number of branch points at 12 and 36 h, although the values were statistically similar at 24 and 48 h. By 48 h the number of branch points was 13.14⫾3.4 in the absence of PNA and 14.83⫾2.5 in the presence of PNA (Fig. 8C). In contrast to the results found with AP and AE neurons,

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Fig. 8. Quantitative differences induced by PNA lectin in the neurite length (A), number of primary neurites (B) and number of branches (C) of Retzius cells. Significant differences are indicated by the asterisks.

the number of branches per neurite at 48 h was decreased from 1.05 without PNA to 0.76 with PNA. Our controls plating Retzius neurons directly on plastic (n⫽13), on plastic pre-coated with PNA lectin (n⫽15) and on plastic plus PNA added to the culture medium (n⫽10) also gave results that could be detected by 12 h of growth (Fig. 9). While Retzius neurons did not grow directly on plastic (Fig. 9A), they extended big lamellae when the plastic was coated with 2 mg/ml of PNA lectin (Fig. 9B), thus showing that, at least at this high concentration, the lectin could promote growth by Retzius neurons. However, since Retzius neurons plated on plastic did not grow in the presence of soluble PNA

(Fig. 9C), it is likely that the effects produced by the lectin on Retzius neurons plated on ganglion capsules were due to its binding to the ECM glycoproteins and not to the activation of neuronal receptors.

DISCUSSION Our results demonstrate that the characteristic branching patterns of different neuron types plated inside the ganglion capsules are influenced by PNA-binding inhibitory ECM glycoproteins anchored to the ganglion capsules.

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Fig. 9. Growth-promoting activity of PNA lectin on Retzius neurons. (A) Three examples of neurons growing on plastic as substrate. (B) Three other examples of Retzius neurons growing on plastic coated with PNA lectin. On PNA lectin neurons generated lamellar extensions with filopodia at their tips. (C) Three examples of neurons growing on plastic with lectin added to the culture medium. Soluble lectin at a concentration that affects the outgrowth pattern of Retzius neurons plated on capsules failed to induce growth of neurons plated on plastic. Scale bar⫽80 ␮m.

In the nervous systems of mammalians, a lesion induces the expression of cocktails inhibitory proteins (For review see He and Koprivica, 2004). Since the ECM on ganglion capsules contains a mixture of growth-promoting and inhibiting molecules (Masuda-Nakagawa and Wiedemann, 1992; De-Miguel et al., 2002), its final influence on the outgrowth pattern seems to depend on the balance exerted by both or several types of influences on the neuron type. The results presented here are consistent with previous observations showing that PNA-binding glycoproteins have inhibitory actions on the outgrowth pattern of AP neurons and allows us to extend this conclusion to other neuron types. The early effects of PNA lectin increasing the formation of neurites resemble the effects of antibodies applied to block other types of proteins that inhibit regeneration (Schwab, 2002), and also resemble the effects of other PNA-binding proteins which inhibit cell migration and neurite extension in the chick (Davies et al., 1990; Krull et al., 1995; Vermeren et al., 2000). This also suggests that in general, PNA-binding proteins have purely inhibitory effects, in contrast to the diverse effects pro-

duced by other ECM molecules such as tenascin, which promotes growth in some neurons while it inhibits growth by other cell types (Masuda-Nakagawa and Wiedemann, 1992; Pesheva and Probstmeier, 2000; Jones and Jones, 2000). Our comparisons made in three different types of neuron suggest that PNA-binding inhibitory proteins have a combination of conserved and specific effects in different types of cells, inhibiting the production of neurites at early stages, reducing the rate at which neurons grow, restricting the production of secondary neurites and inducing their absorption. These last two effects were seen originally in AP cells (De-Miguel and Vargas, 2002) and now became apparent in AE neurons and to a certain extent, in Retzius neurons. The cell-specific regulation of the outgrowth patterns suggests the activation of different types of receptors. For example, the effect of PNA lectin on the number of branches displayed by Retzius neurons is opposite to that produced in AE and AP neurons (although a direct effect of the lectin on Retzius neurons cannot be ruled out). The types of receptors activated by PNA-binding proteins

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remain unknown, however, several types of proteins that inhibit regeneration in vertebrates activate common receptors, for example the Rho family (for review see Sandvig et al., 2004). Therefore, it may be possible that inhibitory PNA binding proteins in vertebrates and invertebrates, may use their glycosylation sites for an early recognition of different types of receptors from the same family. The inhibition of the formation of branches imposed by PNA-binding proteins is similar to the effect of semaphorin 3 (Sema 3) on the development of cortical neurons in culture (Dent et al., 2004). This analogy is another example of similar actions induced by proteins from different molecular families and at different phylogenetic levels. On the other hand, that PNA-binding proteins have much wider effects on the outgrowth patterns may be due to a blocking effect of PNA lectin on different ECM proteins, since four PNA-binding glycoproteins or at least subunits have been detected in ganglion capsule homogenates (De-Miguel et al., 2002). The presence of active inhibitory molecules in the capsules of adult animals is striking and we have no evidence as to their function under normal conditions. This also occurs for members of the Nogo family, which are expressed in the adult CNS of mammalians (Huber et al., 2002). However, the well-developed regenerative capabilities of adult central neurons in the leech may be explained, at least in part, by the rapid regulatory effects induced by the signaling from these ECM proteins, in addition to the rapid expression of multiple genes and proteins by the neurons and their environment (Luthi, 1994; Blackshaw et al., 2004; Wang et al., 2005). In a more general context, the different responses of neurons to the regulatory effects of the inhibitory proteins in the ECM may exemplify a more general phenomenon used by different neuron types to regulate the formation of their branching patterns, not only after a lesion but also during development. This later assumption may be supported by the bipolar pattern developed by regenerating AP neurons on capsules, which resembles their own outgrowth pattern by developmental day 9 (Gao and Macagno, 1987). Later on, AP neurons require pressure sensory neurons, which develop earlier and their branches pioneer the formation of the peripheral branches of AP neurons (Gan and Macagno, 1997). Since each of the three neuronal types studied here displayed different perturbation characteristics, our results are consistent with the original hypothesis that the regulation of different branching patterns that characterize different neuron types has different dependencies on inhibitory ECM glycoproteins. Acknowledgments—This work was supported by a CONACYT 40626 grant to FFDM. N.F. received a fellowship from Probetel UNAM, and J.V. was supported by UNAM and CONACYT fellowships.

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(Accepted 21 October 2005) (Available online 15 December 2005)