Axonal growth towards Xenopus skin in vitro is mediated by matrix metalloproteinase activity.

We have previously demonstrated that the growth of peripheral nervous system axons is strongly attracted towards limb buds and skin explants in vitro. Here, we show that directed axonal growth towards skin explants of Xenopus laevis in matrigel is associated with expression of matrix metalloproteinase (MMP)-18 and also other MMPs, and that this long-range neurotropic activity is inhibited by the broad-spectrum MMP inhibitors BB-94 and GM6001. We also show that forced expression of MMP-18 in COS-7 cell aggregates enhances axonal growth from Xenopus dorsal root ganglia explants. Nidogen is the target of MMPs released by cultured skin in matrigel, whereas other components remain intact. Our results suggest a novel link between MMP activity and extracellular matrix breakdown in the control of axonal growth.

Impaired axonal regeneration by isolectin B4-binding dorsal root ganglion neurons in vitro.

The subpopulation of dorsal root ganglion (DRG) neurons recognized by Griffonia simplicifolia isolectin B4 (IB4) differ from other neurons by expressing receptors for glial cell line-derived neurotrophic factor (GDNF) rather than neurotrophins. Additionally, IB4-labeled neurons do not express the laminin receptor, alpha7-integrin (Gardiner et al., 2005), necessary for optimal axonal regeneration in the peripheral nervous system. In cultures of dissociated DRG neurons of adult mice on laminin, robust spontaneous neurite outgrowth from IB4-negative neurons occurs and is strongly enhanced by previous axotomy. In contrast, IB4-labeled neurons show little neurite outgrowth and do not express GAP 43, even after axotomy or culture with GDNF. Moreover, growth of their axons through collagen gels is impaired compared with other DRG neurons. To determine whether the sparse neurite outgrowth of IB4-labeled neurons is attributable to lack of integrin expression, DRG cultures were infected with a herpes simplex 1 vector encoding alpha7-integrin, but its forced expression failed to promote neurite outgrowth in either IB4-labeled or other DRG neurons or in cultured adult retinal ganglion cells. Forced coexpression of both alpha7-integrin and GAP 43 also failed to promote neurite outgrowth in IB4-labeled neurons. In addition, cultured sciatic nerve segments were found to release much lower levels of GDNF, demonstrated by ELISA, than nerve growth factor. These findings together with their impaired intrinsic axonal regeneration capacity may contribute to the known vulnerability of the IB4-labeled population of DRG neurons to peripheral nerve injury.

Effective gene delivery to adult neurons by a modified form of electroporation.

Non-viral methods of transfection of cDNAs into adult neurons and other post-mitotic cells are generally very inefficient. However, the recent development of Nucleofector technology developed by Amaxa Biosystems allows direct delivery of cDNAs into the nucleus, enabling transfection of non-dividing cells. In this study, we describe a reliable method for culturing large numbers of retinal cells from adult rats and using Nucleofection, we were able to transfect cDNA-encoding GFP (jellyfish green fluorescent protein) into retinal ganglion cells (RGCs) with relatively high efficiency (up to 28%). Neuronal GFP expression was observed within 18 h and continued for up to 14 days. This compares with values up to 60% of RGCs expressing GFP following infection with an HSV-1 vector. Adult rat dorsal root ganglion (DRG) neurons were also successfully transfected. Thus, in summary, Nucleofection provides the possibility for a fast and efficient method for cDNA delivery and study of gene function in adult mammalian neurons.

Neurotrophin-independent attraction of growing sensory and motor axons towards developing Xenopus limb buds in vitro.

The mechanisms for directing axons to their targets in developing limbs remain largely unknown though recent studies in mice have demonstrated the importance of neurotrophins in this process. We now report that in co-cultures of larval Xenopus laevis limb buds with spinal cords and dorsal root ganglia of Xenopus and axolotl (Ambystoma mexicanum) axons grow directly to the limb buds over distances of up to 800 microm and in particular to sheets of epidermal cells which migrate away from the limb buds and also tail segments in culture. This directed axonal growth persists in the presence of trk-IgG chimeras, which sequester neurotrophins, and k252a, which blocks their actions mediated via trk receptors. These findings indicate that developing limb buds in Xenopus release diffusible factors other than neurotrophins, able to attract growth of sensory and motor axons over long distances.

Pharmacological characterization of pre- and postsynaptic prostanoid receptors in pig gastric fundus.

This study characterized the subtype of prostanoid receptors on the cholinergic neurones and smooth muscle cells in circularly oriented muscle strips of the pig gastric fundus. Tissues were electrically stimulated (40 V, 4 Hz, 0.25 ms, 2 min) to induce tritium outflow after incubation with [3H]-choline. Indomethacin increased the electrically induced tritium outflow, suggesting an inhibitory effect of endogenous prostanoids. In the presence of indomethacin, PGE2 > PGF2alpha >PGI2 inhibited tritium release while the TP-receptor agonist U-46619 and PGD2 had no effect. The EP2-receptor agonist butaprost had no effect while the EP1- and EP3-receptor agonist sulprostone mimicked the effect of PGE2. The effect of sulprostone was not affected by AH 6809, that antagonizes EP1- and EP2-receptors, suggesting the presence of presynaptic EP3-receptors on the cholinergic nerve endings. All prostanoid receptor agonists, except butaprost, contracted the tissues concentration-dependently; the rank order of potency (U-46619 > sulprostone > PGE2 > PGF2alpha > PGD2 = PGI2) suggests the presence of TP- and EP1- and EP3-receptors on the circular smooth muscle cells.

Characterization of pre- and postsynaptic muscarinic receptors in circular muscle of pig gastric fundus.

1. This study investigated the subtype of muscarinic receptors on the cholinergic neurones and smooth muscle in the circular muscle of the pig gastric fundus. 2. Muscarinic antagonists, except MT-3, concentration-dependently inhibited the contractions induced by a given concentration of acetylcholine. Concentration-response curves by acetylcholine were shifted rightwards in a parallel manner without depression of the maximum by the muscarinic antagonists, except by MT-3 that induced a leftward shift. Correlation of the pIC(50) and pA(2) values with published pK(i) values for the five muscarinic receptor subtypes suggests that the muscarinic receptors on pig gastric fundus circular muscle belong to the M(3) subtype. 3. Electrically-evoked contractions (40 V, 4 Hz, 0.25 ms, 2 min) were concentration-dependently inhibited by the muscarinic antagonists except for methoctramine and AF-DX 116, that increased the amplitude of the electrically-induced contractions in lower concentrations. MT-3 tended to increase the electrically-induced contractions. 4. The antagonists, except MT-3, concentration-dependently increased the electrically-induced tritium outflow (40 V, 4 Hz, 0.25 ms, 2 min) after incubation of the tissues with [(3)H]-choline. MT-3 (3 x 10(-8) and 10(-7) M) decreased the electrically-induced tritium release. Correlation of the pIC(50) values with published pK(i) values for the different muscarinic receptor subtypes yielded a significant and comparable correlation for M(1), M(3), M(4) and M(5) receptors. 5. These results suggest that the postsynaptic receptors in circular muscle of the pig gastric fundus belong to the M(3) subtype. However, the presynaptic receptor could not be clearly defined, although it does certainly not belong to the M(2) subtype.

Presynaptic modulation of cholinergic neurotransmission in the human proximal stomach.

1. This study investigates whether the cholinergic neurones, innervating the human proximal stomach, can be modulated by nitric oxide (NO) or vasoactive intestinal polypeptide (VIP), or via presynaptic muscarinic, alpha(2)- or 5-hydroxytryptamine(4) (5-HT(4)-) receptors. 2. Circular muscle strips, without mucosa, were incubated with [(3)H]-choline to incorporate [(3)H]-acetylcholine into the cholinergic transmitter stores. The basal and electrically-induced release of tritium and [(3)H]-acetylcholine were analysed in a medium containing guanethidine (4 x 10(-6) M), hemicholinium-3 (10(-5) M), physostigmine (10(-5) M) and atropine (10(-6) M). Tissues were stimulated twice for 2 min (S(1) and S(2): 40 V, 1 ms, 4 Hz) and drugs were added before S(2). 3. The NO synthase inhibitor L-N(G)-nitroarginine methyl ester (3 x 10(-4) M) and the NO donor sodium nitroprusside (10(-5) M), as well as VIP (10(-7) M) did not influence the basal release nor the electrically-evoked release. 4. The alpha(2)-adrenoceptor agonist UK-14,304 (10(-5) M) significantly inhibited the electrically-evoked release of [(3)H]-acetylcholine, and this was prevented by the alpha(2)-adrenoceptor antagonist rauwolscine (2 x 10(-6) M). 5. The 5-HT(4)-receptor agonist prucalopride (3 x 10(-7) M) significantly enhanced the electrically-evoked release of [(3)H]-acetylcholine, and the 5-HT(4)-receptor antagonist SB204070 (10(-9) M) prevented this. 6. When atropine (10(-6) M) was omitted from the medium and added before the second stimulation, it significantly increased the release of [(3)H]-acetylcholine. 7. These results suggest that the release of acetylcholine from the cholinergic neurones, innervating the circular muscle in the human proximal stomach, can be inhibited via presynaptic muscarinic auto-receptors and alpha(2)-adrenoceptors, and stimulated via presynaptic 5-HT(4)-receptors. No evidence for modulation by NO or VIP was obtained.