Neurodegeneration and early lethality in superoxide dismutase 2-deficient mice: a comprehensive analysis of the central and peripheral nervous systems.

The contribution of oxidative stress to diabetic complications including neuropathy is widely known. Mitochondrial and cellular damage are associated with the overproduction of reactive oxygen species and decreased levels or function of the cellular antioxidant mitochondrial manganese superoxide dismutase (SOD2). We hypothesized that targeted SOD2 deletion in the peripheral nervous system using cre-lox technology under control of the nestin promoter would accelerate neuropathy in a type 2 model of diabetes, the BKS.db/db mouse. SOD2-deficient mice, however, demonstrated severe gait deformities and seizures and died by 20 days of age. Examination of SOD2 expression levels revealed that SOD2 was lost in brain and reduced in the spinal cord, but appeared normal in dorsal root ganglia and peripheral nerves in SOD2-deficient mice. These findings indicate incomplete targeted knockout of SOD2. Morphological examination revealed cortical lesions similar to spongiform encephalopathy in the brain of SOD2-deficient mice. No lesions were evident in the spinal cord, but changes in myelin within the sciatic and sural nerves including a lack of cohesion between layers of compact myelin were observed. Together, these results indicate that targeted neuronal SOD2 knockout using the nestin promoter results in severe central nervous system degeneration and perinatal lethality in mice. A specific peripheral nervous system-targeting construct is required to examine the consequences of SOD2 knockout in diabetic neuropathy.

Diabetes regulates mitochondrial biogenesis and fission in mouse neurons.

AIMS/HYPOTHESIS: Normal mitochondrial activity is a critical component of neuronal metabolism and function. Disruption of mitochondrial activity by altered mitochondrial fission and fusion is the root cause of both neurodegenerative disorders and Charcot-Marie-Tooth type 2A inherited neuropathy. This study addressed the role of mitochondrial fission in the pathogenesis of diabetic neuropathy. METHODS: Mitochondrial biogenesis and fission were assayed in both in vivo and in vitro models of diabetic neuropathy. Gene, protein, mitochondrial DNA and ultrastructural analyses were used to assess mitochondrial biogenesis and fission. RESULTS: There was greater mitochondrial biogenesis in dorsal root ganglion neurons from diabetic compared with non-diabetic mice. An essential step in mitochondrial biogenesis is mitochondrial fission, regulated by the mitochondrial fission protein dynamin-related protein 1 (DRP1). Evaluation of diabetic neurons in vivo indicated small, fragmented mitochondria, suggesting increased fission. In vitro studies revealed that short-term hyperglycaemic exposure increased levels of DRP1 protein. The influence of hyperglycaemia-mediated mitochondrial fission on cell viability was evaluated by knockdown of Drp1 (also known as Dnm1l). Knockdown of Drp1 resulted in decreased susceptibility to hyperglycaemic damage. CONCLUSIONS/INTERPRETATION: We propose that: (1) mitochondria undergo biogenesis in response to hyperglycaemia, but the increased biogenesis is insufficient to accommodate the metabolic load; (2) hyperglycaemia causes an excess of mitochondrial fission, creating small, damaged mitochondria; and (3) reduction of aberrant mitochondrial fission increases neuronal survival and indicates an important role for the fission-fusion equilibrium in the pathogenesis of diabetic neuropathy.

The anamnestic serologic response to vaccination with a canarypox virus-vectored recombinant West Nile virus (WNV) vaccine in horses previously vaccinated with an inactivated WNV vaccine.

A new recombinant West Nile virus (WNV) vaccine has been licensed for use in horses. Prior to the availability of the recombinant vaccine in 2004, the only equine WNV vaccine available on the market had been an inactivated vaccine. Since the recombinant vaccine only expresses selected viral genes, the question could be posed as to whether a single dose of the recombinant vaccine would be effective in producing an anamnestic serologic response in horses previously vaccinated with an inactivated WNV vaccine. In this study we demonstrate that vaccination of horses with a canarypox-vectored recombinant vaccine, under field conditions, results in a marked anamnestic response in horses previously vaccinated with an inactivated WNV vaccine.

Optimal teaching environment for laparoscopic ventral herniorrhaphy.

The introduction of laparoscopic techniques after residency training has created a new paradigm dependent on laparoscopic workshops. This study tested the benefit of an animate course and evaluated the role of proctoring in learning to perform laparoscopic ventral hernia repair (LVHR). Surgeons who had taken a 1-day LVHR course (n = 59) were polled to determine previous experience with laparoscopic procedures and experience with LVHR after the course. Forty-eight (81%) surgeons completing the course responded. Thirty-two (67%) surgeons had performed 179 LVHRS (mean 5.6) since the course. There were no statistically significant differences between the groups performing and not performing LVHR regarding academic/private practice (P=0.8) or opportunities to perform a ventral herniorrhaphy (P = 0.6). Fifteen (31%) surgeons were precepted in their hospital operating room by the lead author. Thirteen (87%) of precepted surgeons had performed a LVHR compared with 19 (58%) of the 33 surgeons taking the course without a precepted intervention (P = 0.05). Surgeons with experience performing laparoscopic inguinal hernia repair, Nissen fundoplication, and common bile duct exploration were more likely to perform LVHR (P=0.0001). Surgeons performing only laparoscopic cholecystectomy tended to be less likely to perform LVHR, nearing statistical significance (P=0.08). Surgeons with prior advanced laparoscopic surgery experience are thus more likely to perform LVHR after participating in a 1-day course. Surgeons precepted in their hospital operating room were also more likely to perform LVHR. Participation in an animate laboratory and a precepted experience can impact the future performance of advanced laparoscopic surgery.

Prospective randomized evaluation of surgical resident proficiency with laparoscopic suturing after course instruction.

BACKGROUND: Laparoscopic suturing is required to develop competency in advanced laparoscopy. METHODS: Manuals detailing laparoscopic suturing were give to 17 Surgery residents. One week later they performed a suture on a training model. Time (s), accuracy (mm), and knot strength (lb) were recorded. The residents were blindly randomized to intervention (n = 9) and control (n = 8) groups. The intervention residents attended a 60-min course with lecture, video, and individual proctoring. Two weeks later they performed a stitch with standard laparoscopic instruments and a stitch with a suturing assist device. Statistical analysis included a Wilcoxon rank-sum test. RESULTS: The intervention residents decreased their suturing time from the first to the second stitich (732.4-257.6s), the control and residents decreased their time from 500.2 s to 421.8 s. The time required to perform the second stitch showed no significant difference between the two groups (p = 0.46), but the difference in reduced time between the first and second stitch was significant (p = 0.001). Using the suturing assist device for the third suture, the intervention and control groups both decreased their times significantly. The control residents performed almost as quickly as the intervention residents with the suturing; device (p = 0.11). Accuracy and knot strength were not different in any test. CONCLUSIONS: Residents can improve suturing skill with a short didactic course and individual proctoring. A suturing assist device decreases time required by inexperienced surgeons to device perform an intracorporeal tie.

Effectiveness of the ultrasonic coagulating shears, LigaSure vessel sealer, and surgical clip application in biliary surgery: a comparative analysis.

Advancements in laparoscopic surgery are often dictated by the limitations of technical instrumentation. Energy sources other than electrosurgery have become popular with the promise of quick and effective vascular control. With their success surgeons have begun using these on structures other than blood vessels with little or no data establishing their efficacy or safety. This study evaluates alternative energy sources in sealing ductal structures for possible use in liver or gallbladder surgery. After elective cholecystectomy cystic ducts (n = 45) were resealed ex vivo with surgical clips (n = 14), ultrasonic coagulating shears (n = 16), or electrothermal bipolar vessel sealer (n = 15), and bursting pressures were measured. Nineteen additional human cystic ducts were randomized to seal by ultrasonic coagulating shears (n = 9) or electrothermal bipolar vessel sealer (n = 10) and fixed in 10 per cent buffered formalin for histologic evaluation of thermal spread (mm). After this nine adult pigs were randomized to laparoscopic ligation and transection of the common bile duct using surgical clips (n = 3), ultrasonic coagulating shears (n = 3), or electrothermal bipolar vessel sealer (n = 3). The animals underwent necropsy for assessment of seal integrity on the sixth postoperative day. In the ex vivo study the mean cystic duct bursting pressure was 621 mm Hg with surgical clips and 482 mm Hg with the electrothermal bipolar vessel sealer (P = 0.39). The mean cystic duct bursting pressure after ultrasonic coagulating shears was 278 mm Hg, which was statistically less than surgical clips (P = 0.007) and electrothermal bipolar vessel sealer (P = 0.02). The mean thermal spread was 3.5 mm for ultrasonic coagulating shears and 13.4 mm for electrothermal bipolar vessel sealer (P = 0.0002). All animals undergoing ligation and transection of the common bile duct with ultrasonic coagulating shears and electrothermal bipolar vessel sealer developed bile peritonitis by postoperative day 6 as a result of seal leak. All animals undergoing surgical clip ligation and transection of the common bile duct maintained seal integrity. The mean common bile duct pressure above the surgical clip was 12 mm Hg (range 10-14). In conclusion the acute ex vivo study demonstrated a significant difference in the cystic duct bursting pressure between surgical clips and ultrasonic coagulating shears and between electrothermal bipolar vessel sealer and ultrasonic coagulating shears. The ultrasonic coagulating shears and electrothermal bipolar vessel sealer failed to maintain seal integrity in the in vivo animal study. Given the failure of the ultrasonic coagulating shears and electrothermal bipolar vessel sealer in the animal model these energy sources should not be used for transection of the cystic duct or major hepatic ducts during hepatobiliary surgery.

Spatial shaping of cochlear innervation by temporally regulated neurotrophin expression.

Previous work suggested qualitatively different effects of neurotrophin 3 (NT-3) in cochlear innervation patterning in different null mutants. We now show that all NT-3 null mutants have a similar phenotype and lose all neurons in the basal turn of the cochlea. To understand these longitudinal deficits in neurotrophin mutants, we have compared the development of the deficit in the NT-3 mutant to the spatial-temporal expression patterns of brain-derived neurotrophic factor (BDNF) and NT-3, using lacZ reporters in each gene and with expression of the specific neurotrophin receptors, trkB and trkC. In the NT-3 mutant, almost normal numbers of spiral ganglion neurons form, but fiber outgrowth to the basal turn is eliminated by embryonic day (E) 13.5. Most neurons are lost between E13.5 and E15.5. During the period preceding apoptosis, NT-3 is expressed in supporting cells, whereas BDNF is expressed mainly in hair cells, which become postmitotic in an apical to basal temporal gradient. During the period of neuronal loss, BDNF is absent from the basal cochlea, accounting for the complete loss of basal turn neurons in the NT-3 mutant. The spatial gradients of neuronal loss in these two mutants appear attributable to spatial-temporal gradients of neurotrophin expression. Our immunocytochemical data show equal expression of their receptors, TrkB and TrkC, in spiral sensory neurons and thus do not relate to the basal turn loss. Mice in which NT-3 was replaced by BDNF show a qualitative normal pattern of innervation at E13.5. This suggests that the pattern of expression of neurotrophins rather than their receptors is essential for the spatial loss of spiral sensory neurons in NT-3 null mutants.

Identification and characterization of a novel extracellular matrix protein nephronectin that is associated with integrin alpha8beta1 in the embryonic kidney.

The epithelial-mesenchymal interactions required for kidney organogenesis are disrupted in mice lacking the integrin alpha8beta1. None of this integrin's known ligands, however, appears to account for this phenotype. To identify a more relevant ligand, a soluble integrin alpha8beta1 heterodimer fused to alkaline phosphatase (AP) has been used to probe blots and cDNA libraries. In newborn mouse kidney extracts, alpha8beta1-AP detects a novel ligand of 70-90 kD. This protein, named nephronectin, is an extracellular matrix protein with five EGF-like repeats, a mucin region containing a RGD sequence, and a COOH-terminal MAM domain. Integrin alpha8beta1 and several additional RGD-binding integrins bind nephronectin. Nephronectin mRNA is expressed in the ureteric bud epithelium, whereas alpha8beta1 is expressed in the metanephric mesenchyme. Nephronectin is localized in the extracellular matrix in the same distribution as the ligand detected by alpha8beta1-AP and forms a complex with alpha8beta1 in vivo. Thus, these results strongly suggest that nephronectin is a relevant ligand mediating alpha8beta1 function in the kidney. Nephronectin is expressed at numerous sites outside the kidney, so it may also have wider roles in development. The approaches used here should be generally useful for characterizing the interactions of novel extracellular matrix proteins identified through genomic sequencing projects.

Initial results with an electrothermal bipolar vessel sealer.

BACKGROUND: An electrothermal bipolar vessel sealer (EBVS; Ligasure, Valleylab, Boulder, CO, USA) was developed as an alternative to suture ligatures, hemoclips, staplers, and ultrasonic coagulators for ligating vessels and tissue bundles. The EBVS seals vessels up to 7 mm in diameter by denaturing collagen and elastin within the vessel wall and surrounding connective tissue. This study is the first to determine the clinical efficacy and safety of this instrument and delineate its potential timesavings in both experimental (animal) and clinical scenarios. METHODS: A prospective review of the author's clinical experience with the EBVS in laparoscopic and open operations from October 1998 to March 2000 was performed. In addition, five Yorkshire domestic pigs underwent 150-cm small intestine resections (n = 10) using the EBVS (n = 5) and suture ligatures (n = 5). Measurements included time to complete intestinal resection, the number of applications per minute for each method, and the presence of postapplication bleeding. Statistical analysis was performed using Student's t-test. RESULTS: The EBVS was used in 98 cases (46 laparoscopic and 52 open) with a mean of 43 applications (range, 10-150 applications) per case. The operations included 53 colon and/or small bowel resections (54.1%), 24 fundoplications (24.5%), 12 gastric resections (12.2%), 3 splenectomies, 2 pancreatectomies, 1 adrenalectomy, 1 bilateral salpingo-oopherectomy, 1 pancreatic cyst-jejunostomy, and 1 vagotomy with gastrojejunostomy. In all these cases, the EBVS was intended to be the only means of vessel ligation. An alternative ligation technique was required for bleeding in only 13 (0.3%) of more than 4,200 applications of the EBVS. No postoperative hemorrhagic complications occurred. There was an estimated mean reduction in operative time of 39 min per open procedure, and a mean prolongation in operative time of 8 min per laparoscopic procedure when the EBVS was used in lieu of suture ligatures, hemoclips, staplers, or ultrasonic coagulators. In the animal model, the mean time for completion of the intestinal resection was 251.9 s for the EBVS and 702.0 s for ligatures (p < 0.001). The mean number of applications per minute was 7.6 for the EBVS and 1.8 for ligatures (p < 0.001). No postapplication bleeding was seen. CONCLUSIONS: Initial clinical results from the use of EBVS in laparoscopic and open procedures demonstrate it to be safe and effective, reducing operative time in open procedures. Suture ligatures, ties, hemoclips, and other ligating techniques were used rarely (0.3%) after an application of the EBVS. In an experimental animal model, the EBVS was significantly faster and more efficient (more applications per minute) than ligatures for intestinal resection.

Optimal teaching environment for laparoscopic splenectomy.

BACKGROUND: Traditional surgical teaching depends on graduated acquisition of skill learned in residency. The introduction of minimal access techniques after residency training has created a new paradigm dependent on animate course experiences and limited preceptor training. The outcome of performance of a new skill "learned" in these settings has not been assessed. The purpose of this study was to test the benefit of an animate course compared with a precepted operating room experience in learning to perform a laparoscopic splenectomy. METHODS: All attending surgeons who had taken a 1-day course to learn laparoscopic splenectomy (n = 37) and those who had undergone an intraoperative preceptorship (in their hospital) by the lead author (n = 15) were polled to ascertain their previous experience with laparoscopy and with laparoscopic splenectomy since the intervention. The course included lectures, operative videos, and an animal lab. Statistical differences were measured using a t test. RESULTS: Thirty-two of the 37 (86.5%) taking the course and all 15 of the precepted surgeons responded. There was no difference between the groups regarding prior laparoscopic experience (P = 0.73), laparoscopic training during residency (P = 0.74), academic or private practice (P = 0.48), or follow-up since the intervention (P = 0.36). The participants graded the courses (1 to 5, 5 = excellent) at an average of 4.72. Fourteen of 15 precepted surgeons have performed laparoscopic splenectomy as compared with 2 of 32 taking courses (nonprecepted surgeons; P <0.0001). The number of laparoscopic splenectomies performed totaled 112 for precepted surgeons and 4 for nonprecepted surgeons (P = 0.0003). The nonprecepted surgeons performed significantly more open splenectomies than laparoscopic (95 versus 13 respectively, P = 0.02). Reasons quoted not to proceed with laparoscopic splenectomy included waiting for the perfect patient, concern of hilar management, and splenic size. CONCLUSION: Surgeons precepted in their own operating room performed a laparoscopic splenectomy more readily than those gaining experience from a course only (93% versus 6%, respectively) despite no difference in their preintervention experience and having the opportunity to do so. The expectation of the eventual performance of advanced laparoscopic techniques depends on a precepted experience.

Endogenous brain-derived neurotrophic factor and neurotrophin-3 antagonistically regulate survival of axotomized corticospinal neurons in vivo.

Neuronal growth factors regulate the survival of neurons by their survival and death-promoting activity on distinct populations of neurons. The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) promote neuronal survival via tyrosine kinase (Trk) receptors, whereas NGF and BDNF can also induce apoptosis in developing neurons through p75(NTR) receptors in the absence of their respective Trk receptors. Using mutant mice and inactivation of neurotrophins and their receptors with antibodies in rats, we show that endogenous NT-3 induces death of adult BDNF-dependent, axotomized corticospinal neurons (CSNs). When NT-3 is neutralized, the neurons survive even without BDNF, suggesting complete antagonism. Whereas virtually all unlesioned and axotomized CSNs express both trkB and trkC mRNA, p75 is barely detectable in unlesioned CSNs but strongly upregulated in axotomized CSNs by day 3 after lesion, the time point when cell death occurs. Blocking either cortical TrkC or p75(NTR) receptors alone prevents death, indicating that the opposing actions of NT-3 and BDNF require their respective Trk receptors, but induction of death depends on p75(NTR) cosignaling. The results show that neuronal survival can be regulated antagonistically by neurotrophins and that neurotrophins can induce neuronal death in the adult mammalian CNS. We further present evidence that signaling of tyrosine kinase receptors of the trk family can be crucially involved in the promotion of neuronal death in vivo.

Mini-laparoscopic splenectomy.

BACKGROUND AND PURPOSE: Since the introduction of mini-laparoscopic instruments (2- to 3-mm diameter), their utility and safety have been questioned. Their application in cholecystectomy has recently been documented. This study determined the adequacy and safety of these minimally invasive instruments in laparoscopic splenectomy. METHODS: Retrospective review of all 16 mini-laparoscopic splenectomies performed by the authors was carried out. Diagnoses included immune thrombocytopenia (5), spherocytosis (6), and beta-thalassemia, sickle-cell disease, splenic mass, cyst, and splenomegaly in 1 case each. The average age of the patients was 20.1 years (range 4-70 years); seven patients were adults. Ten of the patients were female. The patients' body mass index ranged from 17 to 25 kg/m2. Splenomegaly (at least two times normal size: 100-200 g for children, 400-600 g for adults) was present in each case. A three-trocar technique was used in 15 patients, and a fourth trocar was required in only one case. RESULTS: The average operative time and blood loss were 114 minutes (range 60-195 minutes) and 44 mL (range 10-150 mL), respectively. There were no intraoperative complications, and no patient required transfusion. Conversion to standard laparoscopy or laparotomy did not occur. The mean hospital stay was 1.4 days (range 1-2 days). With an average 20-month follow-up, no wound, septic, or other complications have been identified. All patients or their families (in the case of children) graded the cosmetic outcome as excellent. CONCLUSION: The use of mini-laparoscopic instruments for splenectomy is safe and effective in children and adults with a normal body mass index, even in the case of splenomegaly. Operative times are reasonable, and hospital stays are brief. The postoperative cosmetic appearance is excellent.

Laparoscopic repair of the leaking percutaneous endoscopic gastrostomy.

Peritonitis is an infrequent yet major complication of a percutaneous endoscopic gastrostomy (PEG). Traditionally, patients with peritonitis from leaking PEG tubes underwent open abdominal exploration with repair of the gastrostomy site. We report successful laparoscopic treatment of this significant complication. Surgical techniques and technical aspects of the procedure are discussed.

Hand-assisted laparoscopic splenectomy.

The success of laparoscopic cholecystectomy has resulted in the broad application of minimally invasive techniques in many surgery specialties. The theoretical advantages of laparoscopy over conventional open operations, including less postoperative pain, faster overall recovery, and better cosmetic results have been achieved leading to its acceptance by surgeons and the public alike. Numerous abdominal procedures have been adapted to minimally invasive approaches including bowel resection, inguinal and ventral hernia repair, anti-reflux techniques, and solid organ removal such as splenectomy.

NT-3, like NGF, is required for survival of sympathetic neurons, but not their precursors.

Superior cervical ganglia of postnatal mice with a targeted disruption of the gene for neurotrophin-3 have 50% fewer neurons than those of wild-type mice. In culture, neurotrophin-3 increases the survival of proliferating sympathetic precursors. Both precursor death (W. ElShamy et al., 1996, Development 122, 491-500) and, more recently, neuronal death (S. Wyatt et al., 1997, EMBO J. 16, 3115-3123) have been described in mice lacking NT-3. Consistent with the second report, we found that, in vivo, neurogenesis and precursor survival were unaffected by the absence of neurotrophin-3 but neuronal survival was compromised so that only 50% of the normal number of neurons survived to birth. At the time of neuron loss, neurotrophin-3 expression, assayed with a lacZ reporter, was detected in sympathetic target tissues and blood vessels, including those along which sympathetic axons grow, suggesting it may act as a retrograde neurotrophic factor, similar to nerve growth factor. To explore this possibility, we compared neuron loss in neurotrophin-3-deficient mice with that in nerve growth factor-deficient mice and found that neuronal losses occurred at approximately the same time in both mutants, but were less severe in mice lacking neurotrophin-3. Eliminating one or both neurotrophin-3 alleles in mice that lack nerve growth factor does not further reduce sympathetic neuron number in the superior cervical ganglion at E17.5 but does alter axon outgrowth and decrease salivary gland innervation. Taken together these results suggest that neurotrophin-3 is required for survival of some sympathetic neurons that also require nerve growth factor.

Expression of Trk receptors in the developing mouse trigeminal ganglion: in vivo evidence for NT-3 activation of TrkA and TrkB in addition to TrkC.

Animals lacking neurotrophin-3 (NT-3) are born with deficits in almost all sensory ganglia. Among these, the trigeminal ganglion is missing 70% of the normal number of neurons, a deficit which develops during the major period of neurogenesis between embryonic stages (E) 10.5 and E13.5. In order to identify the mechanisms for this deficit, we used antisera specific for TrkA, TrkB, and TrkC to characterize and compare the expression patterns of each Trk receptor in trigeminal ganglia of wild type and NT-3 mutants between E10.5 and E15.5. Strikingly, TrkA, TrkB, and TrkC proteins appear to be exclusively associated with neurons, not precursors. While some neurons show limited co-expression of Trk receptors at E11.5, by E13. 5 each neuron expresses only one Trk receptor. Neuronal birth dating and cell counts show that in wild-type animals all TrkB- and TrkC-expressing neurons are generated before E11.5, while the majority of TrkA-expressing neurons are generated between E11.5 and E13.5. In mice lacking NT-3, the initial formation of the ganglion, as assessed at E10.5, is similar to that in wild-type animals. At E11.5, however, the number of TrkC-expressing neurons is dramatically reduced and the number of TrkC-immunopositive apoptotic profiles is markedly elevated. By E13.5, TrkC-expressing neurons are virtually eliminated. At E11.5, compared to wild type, the number of TrkB-expressing neurons is also reduced and the number of TrkB immunoreactive apoptotic profiles is increased. TrkA neurons are also reduced in the NT-3 mutants, but the major deficit develops between E12.5 and E13.5 when elevated numbers of TrkA-immunoreactive apoptotic profiles are detected. Normal numbers of TrkA- and TrkB-expressing neurons are seen in a TrkC-deficient mutant. Therefore, our data provide evidence that NT-3 supports the survival of TrkA-, TrkB- and TrkC-expressing neurons in the trigeminal ganglion by activating directly each of these receptors in vivo.

Regulation of neurotrophin-3 expression by epithelial-mesenchymal interactions: the role of Wnt factors.

Neurotrophins regulate survival, axonal growth, and target innervation of sensory and other neurons. Neurotrophin-3 (NT-3) is expressed specifically in cells adjacent to extending axons of dorsal root ganglia neurons, and its absence results in loss of most of these neurons before their axons reach their targets. However, axons are not required for NT-3 expression in limbs; instead, local signals from ectoderm induce NT-3 expression in adjacent mesenchyme. Wnt factors expressed in limb ectoderm induce NT-3 in the underlying mesenchyme. Thus, epithelial-mesenchymal interactions mediated by Wnt factors control NT-3 expression and may regulate axonal growth and guidance.

Characterization of neurotrophin and Trk receptor functions in developing sensory ganglia: direct NT-3 activation of TrkB neurons in vivo.

Spinal sensory ganglia have been shown to contain neuronal subpopulations with different functions and neurotrophin dependencies. Neurotrophins act, in large part, through Trk receptor tyrosine kinases: nerve growth factor (NGF) via TrkA, brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) via TrkB, and neurotrophin-3 (NT-3) via TrkC. In the present paper, we use antibodies to TrkA, TrkB, and TrkC to characterize their expression patterns and to determine which subpopulations of cells are lost in mice lacking individual neurotrophins or Trk receptors. Despite previous reports of Trk receptor mRNAs in neural crest cells, we detect Trk receptor proteins only in neurons and not in neural crest cells or neuronal precursors. Comparisons of neonatal mice deficient in NT-3 or its cognate receptor TrkC have shown that there is a much greater deficiency in spinal sensory neurons in the former, suggesting that NT-3 may activate receptors in addition to TrkC. Using the same antibodies, we show that, during the major period of neurogenesis, NT-3 is required to maintain neurons that express TrkB in addition to those that express TrkC but is not essential for neurons expressing TrkA. Results also indicate that survival of cells expressing both receptors can be maintained by activation of either one alone. NT-3 can thus activate more than one Trk receptor in vivo, which when coexpressed are functionally redundant.

Lack of neurotrophin-3 results in death of spinal sensory neurons and premature differentiation of their precursors.

To understand mechanisms resulting in the absence of two-thirds of spinal sensory neurons in mice lacking NT-3, we have compared dorsal root ganglia development in normal and mutant embryos. The reduction in neurons, achieved by E13, results from several deficits: first, elevated neuronal apoptosis significantly reduces neuronal numbers; second, elevated neurogenesis between E11 and E12, without changes in rates of precursor proliferation or apoptosis, depletes the precursor pool; consequently, the reduced precursor pool prevents increases in neuronal numbers between E12 and E13, when most neurons are born in normal animals. Although deficits occur before final target innervation, we show that NT-3 is expressed at all stages in regions accessible to these neurons or their axons and is only restricted to final targets after innervation.