After peripheral nerve injury, neurotrophins play a key role in the regeneration of damaged axons which can be augmented by exercise, although the distinct roles played by neurons and Schwann cells are unclear. In this study, we evaluated the requirement for the neurotrophin, brain derived neurotrophic factor (BDNF), in neurons and Schwann cells, for the regeneration of peripheral axons after injury. Common fibular or tibial nerves in thy-1-YFP-H mice were cut bilaterally and repaired using a graft of the same nerve from transgenic mice lacking BDNF in Schwann cells (BDNF-/-) or wild-type mice (WT). Two weeks post-repair, axonal regeneration into BDNF-/- grafts were markedly less than WT grafts, emphasizing the importance of Schwann cell BDNF. Nerve regeneration was enhanced by treadmill training post-transection, regardless of the BDNF content of the nerve graft. We further tested the hypothesis that training-induced increases in BDNF in neurons allow regenerating axons to overcome a lack of BDNF expression in cells in the pathway through which they regenerate. Nerves in mice lacking BDNF in YFP+ neurons (SLICK) were cut and repaired with BDNF-/- and WT nerves. SLICK axons lacking BDNF did not regenerate into grafts lacking Schwann cell BDNF. Treadmill training could not rescue the regeneration into BDNF-/- grafts if the neurons also lacked BDNF. Both Schwann cell- and neuron-derived BDNF are thus important for axon regeneration in cut peripheral nerves.

We investigated the extent of misdirection of regenerating axons when that regeneration was enhanced using treadmill training. Retrograde fluorescent tracers were applied to the cut proximal stumps of the tibial and common fibular nerves two or four weeks after transection and surgical repair of the mouse sciatic nerve. The spatial locations of retrogradely labeled motoneurons were studied in untreated control mice and in mice receiving two weeks of treadmill training, either according to a continuous protocol (10 m/min, one hour/day, five day/week) or an interval protocol (20 m/min for two minutes, followed by a five minute rest, repeated 4 times, five days/week). More retrogradely labeled motoneurons were found in both treadmill trained groups. The magnitude of this increase was as great as or greater than that found after using other enhancement strategies. In both treadmill trained groups, the proportions of motoneurons labeled from tracer applied to the common fibular nerve that were found in spinal cord locations reserved for tibial motoneurons in intact mice was no greater than in untreated control mice and significantly less than found after electrical stimulation or chondroitinase treatment. Treadmill training in the first two weeks following peripheral nerve injury produces a marked enhancement of motor axon regeneration without increasing the propensity of those axons to choose pathways leading to functionally inappropriate targets.

The sodium-bicarbonate cotransporter NBCn1 (SLC4A7) is an acid-base transporter that normally moves Na(+) and HCO(3)(-) into the cell. This membrane protein is sensitive to cellular and systemic pH changes. We examined NBCn1 expression and localization in the brain and its response to chronic metabolic acidosis. Two new NBCn1 antibodies were generated by immunizing a rabbit and a guinea pig. The antibodies stained neurons in a variety of rat brain regions, including hippocampal pyramidal neurons, dentate gyrus granular neurons, posterior cortical neurons, and cerebellar Purkinje neurons. Choroid plexus epithelia were also stained. Double immunofluorescence labeling showed that NBCn1 and the postsynaptic density protein PSD-95 were found in the same hippocampal CA3 neurons and partially colocalized in dendrites. PSD-95 was pulled down from rat brain lysates with the GST/NBCn1 fusion protein and was also coimmunoprecipitated with NBCn1. Chronic metabolic acidosis was induced by feeding rats with normal chow or 0.4 M HCl-containing chow for 7 days. Real-time PCR and immunoblot showed upregulation of NBCn1 mRNA and protein in the hippocampus of acidotic rats. NBCn1 immunostaining was enhanced in CA3 neurons, posterior cortical neurons, and cerebellar granular cells. Intraperitoneal administration of N-methyl-d-aspartate caused neuronal death determined by caspase-3 activity, and this effect was more severe in acidotic rats. Administering N-methyl-d-aspartate also inhibited NBCn1 upregulation in acidotic rats. We conclude that NBCn1 in neurons is upregulated by chronic acid loads, and this upregulation is associated with glutamate excitotoxicity.

The role of neurotrophin-4/5 in the enhancement of axon regeneration in peripheral nerves produced by treadmill training was studied in mice. Common fibular nerves of animals of the H strain of thy-1-YFP mice, in which a subset of axons in peripheral nerves is marked by the presence of yellow fluorescent protein, were cut and surgically repaired using nerve grafts from non-fluorescent mice. Lengths of profiles of fluorescent regenerating axons were measured using optical sections made through whole mounts of harvested nerves. Measurements from mice that had undergone one hour of daily treadmill training at modest speed (10 m/min) were compared to those of untrained (control) mice. Modest treadmill training resulted in fluorescent axon profiles that were nearly twice as long as controls at one, two and four week survival times. Similar enhanced regeneration was found when cut nerves of wild type mice were repaired with grafts from neurotrophin-4/5 knockout mice or grafts made acellular by repeated freezing/thawing. No enhancement was produced by treadmill training in neurotrophin-4/5 knockout mice, irrespective of the nature of the graft used to repair the cut nerve. Much as had been observed previously for the effects of brief electrical stimulation, the effects of treadmill training on axon regeneration in cut peripheral nerves are independent of changes produced in the distal segment of the cut nerve and depend on the promotion of axon regeneration by changes in NT-4/5 expression by cells in the proximal nerve segment.

The sodium/bicarbonate cotransporter NBCn1 regulates intracellular pH (pHi) and transepithelial HCO–3 movement. We examined NBCn1 expression and activity under acidic conditions and its contribution to ammonium movement in the mouse medullary thick ascending limb (MTAL) cell line ST‐1. Incubating cells at pH 6.8 for 24 h lowered the mean steady‐state pHi from 7.10 to 6.56. Immunoblot showed NBCn1 upregulation after acidic incubation. The Cl/HCO3 exchanger AE2 was present but its expression remained unaffected by acidic incubation. Acidic incubation decreased 14C‐methylammonium/ammonia (MA) accumulation by 22%. The 14C‐MA accumulation after acidic incubation was unaffected by 0.5 mM DIDS, whereas the accumulation in controls was reduced by DIDS. In CO2/HCO–3‐free solution, cells at normal pH accumulated 14C‐MA over 7 min and reached a plateau. In CO2/HCO–3 solution, however, cells markedly accumulated 14C‐MA over 30 min. This CO2/HCO–3‐dependent accumulation was reduced by DIDS. Cl– removal reduced the accumulation and this reduction was more substantial with DIDS. Expressed in Xenopus oocytes, NBCn1 increased CO2/HCO–3‐dependent 14C‐MA accumulation, which was abolished by replacing Na+. Two‐electrode voltage clamp exhibited negligible current upon NH4Cl application. We conclude that NBCn1 in the MTAL is upregulated under acidic conditions and the upregulation enhances ammonium uptake.