Ion conductances related to development of repetitive firing in mouse retinal ganglion neurons in situ

ABSTRACT: In the retina, the ability to encode graded depolarizations into spike trains of variable frequency appears to be a specific property of retinal ganglion neurons (RGNs). To deduce the developmental changes in ion conductances underlying the transition from single to repetitive firing, patch-clamp recordings were performed in the isolated mouse retina between embryonic day 15 (E15) and postnatal day 5 (P5). Immature neurons of the E15 retina were selected according to their capacity to generate voltage-activated Na+ currents (I(Na)(v)). Identification of P5 RGNs was based on retrograde labeling, visualization of the axon, or the amplitude of I(Na)(v). At E15, half of the cells were excitable but none of them generated more than one spike. At P5, all cells were excitable and a majority discharged in tonic fashion. Ion conductances subserving maintenance of repetitive discharge were identified at P5 by exposure to low extracellular Ca2+, Cd2+, and charybdotoxin, all of which suppressed repetitive discharge. omega-Conotoxin GVIA and nifedipine had no effect. We compared passive membrane properties and a variety of voltage-activated ion channels at E15 and P5. It was found that the density of high voltage-activated (HVA) Ca2+ currents increased in parallel with the development of repetitive firing, while the density of Ni2+-sensitive low voltage-activated (LVA) Ca2+ currents decreased. Changes in density and activation kinetics of tetrodotoxin-sensitive Na+ currents paralleled changes in firing thresholds and size of action potentials, but seemed to be unrelated to maintenance of repetitive firing. Densities of A-type K+ currents and delayed rectifier currents did not change. The results suggest that HVA Ca2+ channels, and among them a toxin-resistant subtype, are specifically engaged in activation of Ca2+-sensitive K+ conductance and thereby account for frequency coding in postnatal RGNs.     

Reduced light response of neuronal firing activity in the suprachiasmatic nucleus and optic nerve of cryptochrome-deficient mice

To examine roles of the Cryptochromes (Cry1 and Cry2) in mammalian circadian photoreception, we recorded single-unit neuronal firing activity in the suprachiasmatic nucleus (SCN), a primary circadian oscillator, and optic nerve fibers in vivo after retinal illumination in anesthetized Cry1 and Cry2 double-knockout (Cry-deficient) mice. In wild-type mice, most SCN neurons increased their firing frequency in response to retinal illumination at night, whereas only 17% of SCN neurons responded during the daytime. However, 40% of SCN neurons responded to light during the daytime, and 31% of SCN neurons responded at night in Cry-deficient mice. The magnitude of the photic response in SCN neurons at night was significantly lower (1.3-fold of spontaneous firing) in Cry-deficient mice than in wild-type mice (4.0-fold of spontaneous firing). In the optic nerve near the SCN, no difference in the proportion of light-responsive fibers was observed between daytime and nighttime in both genotypes. However, the response magnitude in the light-activated fibers (ON fibers) was high during the nighttime and low during the daytime in wild-type mice, whereas this day-night difference was not observed in Cry-deficient mice. In addition, we observed day-night differences in the spontaneous firing rates in the SCN in both genotypes and in the fibers of wild-type, but not Cry-deficient mice. We conclude that the low photo response in the SCN of Cry-deficient mice is caused by a circadian gating defect in the retina, suggesting that Cryptochromes are required for appropriate temporal photoreception in mammals.     

Cranial sensory neuron development in the absence of brain-derived neurotrophic factor in BDNF/Bax double null mice

To investigate the role of brain-derived neurotrophic factor (BDNF) in differentiation of cranial sensory neurons in vivo, we analyzed development of nodose (NG), petrosal (PG), and vestibular (VG) ganglion cells in genetically engineered mice carrying null mutations in the genes encoding BDNF and the proapoptotic Bcl-2 homolog Bax. In bax(-/-) mutants, ganglion cell numbers were increased significantly compared to wild-type animals, indicating that naturally occurring cell death in these ganglia is regulated by Bax signaling. Analysis of bdnf(-/-)bax(-/-) mutants revealed that, although the Bax null mutation completely rescued cell loss in the absence of BDNF, it did not rescue the lethality of the BDNF null phenotype. Moreover, despite rescue of BDNF-dependent neurons by the bax null mutation, sensory target innervation was abnormal in double null mutants. Vagal sensory innervation to baroreceptor regions of the cardiac outflow tract was completely absent, and the density of vestibular sensory innervation to the cristae organs was markedly decreased, compared to wild-type controls. Moreover, vestibular afferents failed to selectively innervate their hair cell targets within the cristae organs in the double mutants. These innervation failures occurred despite successful navigation of sensory fibers to the peripheral field, demonstrating that BDNF is required locally for afferent ingrowth into target tissues. In addition, the bax null mutation failed to rescue expression of the dopaminergic phenotype in a subset of NG and PG neurons. These data demonstrate that BDNF signaling is required not only to support survival of cranial sensory neurons, but also to regulate local growth of afferent fibers into target tissues and, in some cells, transmitter phenotypic expression is required.     

Development of anterior gustatory epithelia in the palate and tongue requires epidermal growth factor receptor.

We characterized the gustatory phenotypes of neonatal mice having null mutations for epidermal growth factor receptor (egfr(-/-)), brain-derived neurotrophic factor (bdnf(-/-)), or both. We counted the number and diameter of fungiform taste buds, the prevalence of poorly differentiated or missing taste cells, and the incidence of ectopic filiform-like spines, each as a function of postnatal age and anterior/posterior location. Egfr(-/-) mice and bdnf(-/-) mice had similar reductions in the total number of taste buds on the anterior portions of the tongue and palate. Nonetheless, there were significant differences in their gustatory phenotypes. EGFR deficiency selectively impaired the development of anterior gustatory epithelia in the mouth. Only bdnf(-/-) mice had numerous taste buds missing from the foliate, vallate, and posterior fungiform papillae. Only egfr(-/-) fungiform taste papillae had robust gustatory innervation, markedly reduced cytokeratin 8 expression in taste cells, and a high incidence of a filiform-like spine. Egfr/bdnf double-null mutant mice had a higher frequency of failed fungiform taste bud differentiation. In bdnf(-/-) mice taste cell development failed because of sparse gustatory innervation. In contrast, in young egfr(-/-) mice the abundance of axons innervating fungiform papillae and the normal numbers of geniculate ganglion neurons implicate gustatory epithelial defects rather than neural defects.     

Serotonin transporter function, but not expression, is dependent on brain-derived neurotrophic factor (BDNF): in vivo studies in BDNF-deficient mice

In the present study, we used high-speed chronoamperometry to examine serotonin (5-HT) transporter (5-HTT) function in vivo in 2-, 5-, and 10-month-old brain-derived neurotrophic factor (BDNF)+/- mice. The rate of clearance of exogenously applied 5-HT was measured in CA3 region of hippocampus. In 2-month-old mice, the rate of 5-HT clearance did not differ between BDNF+/+ and BDNF+/- mice. In BDNF+/+ mice, 5-HT clearance rate (Tc) increased markedly with age. In contrast, Tc remained relatively static in BDNF+/- mice across 2-, 5-, and 10-month age groups. At 5 months of age, female BDNF+/+ mice had a lower maximal velocity (Vmax) for 5-HT clearance than male BDNF+/+ mice. There was a similar trend in 5-month-old BDNF+/- mice, but this did not reach statistical significance. There was an age-dependent increase in KT value for 5-HT clearance (i.e., decreased in vivo affinity of 5-HTT), but no significant effect of genotype or gender. 5-HTT density, as measured by [3H]cyanoimipramine binding, was not different between BDNF+/+ and BDNF+/- mice, although there was a significant increase in 5-HTT binding with age. The selective 5-HT reuptake inhibitor fluvoxamine (50 and 100 pmol) significantly decreased 5-HT clearance in BDNF+/+ mice, but not in BDNF+/- mice. Our data suggest that the profoundly reduced ability of 5- and 10-month-old BDNF+/- mice to clear 5-HT is not because of a decrease in the total number of 5-HTTs, but may be due to functional deficits in the 5-HTT, e.g., in the machinery/signaling required for insertion of 5-HTTs into the plasma membrane and/or activation of the 5-HTT once it is positioned to take up 5-HT from extracellular fluid.     

Evidence that brain-derived neurotrophic factor is required for basal neurogenesis and mediates,in part,the enhancement of neurogenesis by dietary restriction in the hippocampus of adult mice

To determine the role of brain-derived neurotrophic factor (BDNF) in the enhancement of hippocampal neurogenesis resulting from dietary restriction (DR), heterozygous BDNF knockout (BDNF +/-) mice and wild-type mice were maintained for 3 months on DR or ad libitum (AL) diets. Mice were then injected with bromodeoxyuridine (BrdU) and killed either 1 day or 4 weeks later. Levels of BDNF protein in neurons throughout the hippocampus were decreased in BDNF +/- mice, but were increased by DR in wild-type mice and to a lesser amount in BDNF +/- mice. One day after BrdU injection the number of BrdU-labeled cells in the dentate gyrus of the hippocampus was significantly decreased in BDNF +/- mice maintained on the AL diet, suggesting that BDNF signaling is important for proliferation of neural stem cells. DR had no effect on the proliferation of neural stem cells in wild-type or BDNF +/- mice. Four weeks after BrdU injection, numbers of surviving labeled cells were decreased in BDNF +/- mice maintained on either AL or DR diets. DR significantly improved survival of newly generated cells in wild-type mice, and also improved their survival in BDNF +/- mice, albeit to a lesser extent. The majority of BrdU-labeled cells in the dentate gyrus exhibited a neuronal phenotype at the 4-week time point. The reduced neurogenesis in BDNF +/- mice was associated with a significant reduction in the volume of the dentate gyrus. These findings suggest that BDNF plays an important role in the regulation of the basal level of neurogenesis in dentate gyrus of adult mice, and that by promoting the survival of newly generated neurons BDNF contributes to the enhancement of neurogenesis induced by DR.     

蝎毒耐热蛋白对大鼠急性分离海马神经元兴奋性的影响

应用全细胞膜片钳记录技术在电流钳模式下观察经持续高温等特殊处理后分离纯化的30～50 kDa蝎毒耐热蛋白(scorpion venom heat resistant protein,SVHRP)(国家发明专利,专利号ZL01 106166.92)对急性分离大鼠海马神经元兴奋性的影响.结果发现SVHRP可致海马神经元兴奋性降低.神经元经1×10-2 μg/mL SVHRP处理后动作电位发放模式改变,发放频率减少.在52个受检细胞中,有45个细胞产生位相放电(占86.54%);7个细胞产生重复放电(占13.46%).在产生位相放电的45个细胞中,有8个细胞在SVHRP处理后仍可以诱发出位相放电(占17.78%);37个细胞在SVHRP处理后无法诱导出位相放电(占82.22%),SVHRP处理后动作电位的产生与处理前相比,有显著差异(P＜0.01,n=45);在产生重复放电的7个细胞中,在1×10-2μg/mL SVHRP作用后均不能再次诱发出重复放电,而是产生一个动作电位或不再产生动作电位,药物处理前产生的动作电位个数为14.57±1.00,SVHRP处理后产生动作电位的个数为0.57±0.20,二者之间有显著性差异(P＜0.01,n=7).1×10-4 μg/mLSVHRP处理后,诱发动作电位产生的基强度由(75.10±8.99)pA增加到(119.85±12.73)pA(P＜0.01,n=8);阈电位由(-41.17±2.15)mV升至(-32.40±1.48)mV(P＜0.01,n=8);动作电位峰值由(68.49±2.33)mV下降至(54.71±0.81)mV(P＜0.01,n=8).由于神经元超兴奋性被认为是癫痫发作的基本机制之一,因此上述结果表明SVHRP有可能通过降低海马神经元兴奋性发挥其抗癫痫作用,这为蝎毒药物的进一步开发提供理论依据.     

Retinotopic map refinement requires spontaneous retinal waves during a brief critical period of development

During retinocollicular map development, spontaneous waves of action potentials spread across the retina, correlating activity among neighboring retinal ganglion cells (RGCs). To address the role of retinal waves in topographic map development, we examined wave dynamics and retinocollicular projections in mice lacking the beta2 subunit of the nicotinic acetylcholine receptor. beta2(-/-) mice lack waves during the first postnatal week, but RGCs have high levels of uncorrelated firing. By P8, the wild-type retinocollicular projection remodels into a refined map characterized by axons of neighboring RGCs forming focal termination zones (TZs) of overlapping arbors. In contrast, in P8 beta2(-/-) mice, neighboring RGC axons form large TZs characterized by broadly distributed arbors. At P8, glutamatergic retinal waves appear in beta2(-/-) mice, and later, visually patterned activity appears, but the diffuse TZs fail to remodel. Thus, spontaneous retinal waves that correlate RGC activity are required for retinotopic map remodeling during a brief early critical period.     

GABA(B) receptors in 5-HT transporter- and 5-HT1A receptor-knock-out mice: further evidence of a transduction pathway shared with 5-HT1A receptors

The functional properties of GABA(B) receptors were examined in the dorsal raphe nucleus (DRN) and the hippocampus of knock-out mice devoid of the 5-HT transporter (5-HTT-/-) or the 5-HT(1A) receptor (5-HT(1A)-/-). Electrophysiological recordings in brain slices showed that the GABA(B) receptor agonist baclofen caused a lower hyperpolarization and neuronal firing inhibition of DRN 5-HT cells in 5-HTT-/- versus 5-HTT+/+ mice. In addition, [(35)S]GTP-gamma-S binding induced by GABA(B) receptor stimulation in the DRN was approximately 40% less in these mutants compared with wild-type mice. In contrast, GABA(B) receptors appeared functionally intact in the hippocampus of 5-HTT-/-, and in both this area and the DRN of 5-HT(1A)-knock-out mice. The unique functional changes of DRN GABA(B) receptors closely resembled those of 5-HT(1A) autoreceptors in 5-HTT-/- mice, further supporting the idea that both receptor types are coupled to a common pool of G-proteins in serotoninergic neurons.     

Chronic hypertension in ANP knockout mice: contribution of peripheral resistance

Atrial Natriuretic Peptide (ANP) exerts a chronic hypotensive effect which is mediated by a reduction in total peripheral resistance (TPR). Mice with a homozygous disruption of the pro-ANP gene (-/-) fail to synthesize ANP and develop chronic hypertension in comparison to their normotensive wild-type (+/+) siblings. In order to determine whether alterations in basal hemodynamics underlie the hypertension associated with lack of endogenous ANP activity, we used anesthetized mice to measure arterial blood pressure (ABP) and heart rate (HR), as well as cardiac output (CO) by thermodilution technique. -/- (n = 7) and +/+ (n = 10) mice of comparable weight and age were used. Stroke volume (SV) and TPR were derived from CO, HR, and ABP by a standard formula. ABP (mm Hg) was significantly higher in -/- (132+/-4) (P < 0.0001) than in +/+ mice (95+/-2). CO (ml min(-1)), HR(beats min(-1))and SV (microl beat(-1)) did not differ significantly between -/- and +/+ mice (CO -/- = 7.3+/-0.5, +/+ = 8.3+/-0.6; HR -/- = 407+/-22, +/+ = 462+/-21; SV -/- = 17.6+/-1.1, +/+ = 17.6+/-1.7). However, TPR (mm Hg ml(-1) min(-1)) was significantly elevated in -/- mice (18.4+/-0.7) compared to +/+ mice (12.3+/-1) (P = 0.0003). Autonomic ganglion blockade with a mixture of hexamethonium and pentolinium was followed by comparable percent reductions in CO (-/- = 28+/-4, +/+ = 29+/-3), HR (-/- = 9+/-4, +/+ = 16+/-4) and SV(-/- = 21+/-4, +/+ = 15+/-6) in both genotypes. However, the concomitant decrease in ABP (%) in -/- (41+/-2) was significantly greater than in +/+ (23+/-4) mice (P = 0.0009) and was accompanied by a significant reduction in TPR. We conclude that the hypertension associated with lack of endogenous ANP is due to elevated TPR, which is determined by an increase in cardiovascular autonomic tone.     

Connective tissue growth factor is necessary for retinal capillary basal lamina thickening in diabetic mice.

Experimental prevention of basal lamina (BL) thickening of retinal capillaries ameliorates early vascular changes caused by diabetes. Connective tissue growth factor (CTGF) is upregulated early in diabetes in the human retina and is a potent inducer of expression of BL components. We hypothesize that CTGF is causally involved in diabetes-induced BL thickening of retinal capillaries. To test this hypothesis, we compared the effects of streptozotocin (STZ)-induced diabetes on retinal capillary BL thickness between wild-type mice (CTGF+/+) and mice lacking one functional CTGF allele (CTGF+/-). Differences in BL thickness were calculated by quantitative analysis of electron microscopic images of transversally sectioned capillaries in and around the inner nuclear layer of the retina. We show that BL thickening was significant in diabetic CTGF+/+ mice compared with control CTGF+/+ mice, whereas diabetes did not significantly induce BL thickening in CTGF+/- mice. We conclude that CTGF expression is necessary for diabetes-induced BL thickening and suggest that reduction of CTGF levels may be protective against the development of diabetic retinopathy.     

Aryl hydrocarbon receptor-dependent induction of apoptosis by 2,3,7,8-tetrachlorodibenzo-p-dioxin in cerebellar granule cells from mouse

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a prototypical environmental contaminant with neurotoxic properties that alters neurodevelopment and behavior. TCDD is a ligand of the aryl hydrocarbon receptor (AhR), which is a key signaling molecule to fully understand the toxic and carcinogenic properties of dioxin. Much effort is underway to unravel the molecular mechanisms and the signaling pathways involved in TCDD-induced neurotoxicity, and to define its molecular targets in neurons. We have used cerebellar granule cells (CGC) from wild-type (AhR+/+) and AhR-null (AhR-/-) mice to characterize the cell death that takes place in neurons after TCDD toxicity. TCDD induced cell death in CGC cultures from wild-type mice with an EC(50) of 127±21 nM. On the contrary, when CGC neurons from AhR-null mice were treated with TCDD no significant cell death was observed. The role of AhR in TCDD-induced death was further assessed by using the antagonists resveratrol and α-naphtoflavone, which readily protected against TCDD toxicity in AhR+/+ CGC cultures. AhR+/+ CGC cultures treated with TCDD showed nuclear fragmentation, DNA laddering, and increased caspase 3 activity, similarly to what was found by the use of staurosporine, a well-established inducer of apoptosis. Finally, the AhR pathway was active in CGC because TCDD could induce the expression of the target gene cytochrome P450 1A2 in AhR+/+ CGC cultures. All together these results support the hypothesis that TCDD toxicity in CGC neurons involves the AhR and that it takes place mainly through an apoptotic process. AhR could be then considered a novel target in neurotoxicity and neurodegeneration whose down-modulation could block certain xenobiotic-related adverse effects in CNS.     

Amphetamine-induced locomotion and gene expression are altered in BDNF heterozygous mice

Administration of amphetamine overstimulates medium spiny neurons (MSNs) by releasing dopamine and glutamate from afferents in the striatum. However, these afferents also release brain-derived neurotrophic factor (BDNF) that protects striatal MSNs from overstimulation. Intriguingly, all three neurochemicals increase opioid gene expression in MSNs. In contrast, striatal opioid expression is less in naive BDNF heterozygous (BDNF(+/-)) vs. wild-type (WT) mice. This study was designed to determine whether partial genetic depletion of BDNF influences the behavioral and molecular response to an acute amphetamine injection. An acute injection of amphetamine [5 mg/kg, intraperitoneal (i.p.)] or saline was administered to WT and BDNF(+/-) mice. WT and BDNF(+/-) mice exhibited similar locomotor activity during habituation, whereas BDNF(+/-) mice exhibited more prolonged locomotor activation during the third hour after injection of amphetamine. Three hours after amphetamine injection, there was an increase of preprodynorphin mRNA in the caudate putamen and nucleus accumbens (Acb) and dopamine D(3) receptor mRNA levels were increased in the Acb of BDNF(+/-) and WT mice. Striatal/cortical trkB and BDNF, and mesencephalic tyrosine hydroxylase mRNA levels were only increased in WT mice. These results indicate that BDNF modifies the locomotor responses of mice to acute amphetamine and differentially regulates amphetamine-induced gene expression.     

Attenuation of allergen-induced responses in CCR6-/- mice is dependent upon altered pulmonary T lymphocyte activation

We have established a defect in CCR6-/- mice in response to a cockroach allergen airway challenge characterized by decreased IL-5 production, reduced CD4+ T and B cells as well as decreased eosinophil accumulation. To determine the nature of the defect in CCR6-/- mice T lymphocyte populations from allergen-sensitized wild-type mice were transferred into sensitized CCR6-/- mice. The reconstituted response was characterized by an increase in IL-5 levels, eosinophil accumulation, and serum IgE levels in recipient CCR6-/- mice. Analysis of lymphocytes from draining lymph nodes of CCR6+/+ and CCR6-/- sensitized or challenged mice demonstrated a significant decrease in IL-5 and IL-13 production in CCR6-/- mice. In contrast, the systemic response in allergen-rechallenged spleen cells demonstrated no significant alteration in allergen-induced cytokine production. Transfer of isolated splenic T lymphocytes from sensitized CCR6+/+ mice induced airway hyperresponsiveness in wild-type but not CCR6-/- naive mice, suggesting that T cells alone were not sufficient to induce airway hyperresponsiveness in CCR6-/- mice. Additional analysis demonstrated decreased CD11c+, CD11b+ and CD11c, and B220 subsets of dendritic cells in the lungs of CCR6-/- mice after allergen challenge. Using in vitro cell mixing studies with isolated pulmonary CD4+ T cells and CD11c+ cells from CCR6+/+ or CCR6-/- mice, we demonstrate alterations in both CCR6-/- T cells and CCR6-/- pulmonary APCs to elicit IL-5 responses. Altogether, the defect in CCR6-/- mice appears to be primarily due to an alteration in T cell activation, but also appears to include local pulmonary APC defects.     

Hormone secretion in transgenic rats and electrophysiological activity in their gonadotropin releasing-hormone neurons

Expression of GFP in GnRH neurons has allowed for studies of individual GnRH neurons. We have demonstrated previously the preservation of physiological function in male GnRH-GFP mice. In the present study, we confirm using biocytin-filled GFP-positive neurons in the hypothalamic slice preparation that GFP-expressing somata, axons, and dendrites in hypothalamic slices from GnRH-GFP rats are GnRH1 peptide positive. Second, we used repetitive sampling to study hormone secretion from GnRH-GFP transgenic rats in the homozygous, heterozygous, and wild-type state and between transgenic and Wistar males after ~4 yr of backcrossing. Parameters of hormone secretion were not different between the three genetic groups or between transgenic males and Wistar controls. Finally, we performed long-term recording in as many GFP-identified GnRH neurons as possible in hypothalamic slices to determine their patterns of discharge. In some cases, we obtained GnRH neuronal recordings from individual males in which blood samples had been collected the previous day. Activity in individual GnRH neurons was expressed as total quiescence, a continuous pattern of firing of either low or relatively high frequencies or an intermittent pattern of firing. In males with both intensive blood sampling (at 6-min intervals) and recordings from their GnRH neurons, we analyzed the activity of GnRH neurons with intermittent activity above 2 Hz using cluster analysis on both data sets. The average number of pulses was 3.9 ± 0.6/h. The average number of episodes of firing was 4.0 ± 0.6/h. Therefore, the GnRH pulse generator may be maintained in the sagittal hypothalamic slice preparation.     

GABAergic inhibition shapes frequency tuning and modifies response properties in the superior olivary nucleus of the leopard frog

The role of gamma-aminobutyric acid (GABA)ergic inhibition in shaping the excitatory frequency tuning of 74 neurons in the superior olivary nucleus of the leopard frog, Rana pipiens, was studied using iontophoretic application of the GABA(A) receptor antagonist, bicuculline methiodide. For 37 neurons, bicuculline application broadened and/or changed the configuration of the excitatory frequency-tuning curve. Results indicate that GABA-mediated inhibition not only sharpens the tuning curves of neurons but also plays a critical role in creating new frequency tuning properties in the superior olivary nucleus. Bicuculline application affected other neuronal response properties as well. Spontaneous firing rate increased 11-338% for 18 of 59 neurons. For 32 of 58 neurons there was an increase in stimulus-evoked discharge rate and a change in rate-level function. There was no qualitative effect on the discharge pattern of 60 neurons, though 2 tonically responding neurons did show an increase (> 30%) in response duration. Additional roles for GABAergic inhibition in monaural signal analysis are discussed.     

Exercise Does Not Protect against MPTP-Induced Neurotoxicity in BDNF Happloinsufficent Mice

Exercise has been demonstrated to potently protect substantia nigra pars compacta (SN) dopaminergic neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. One mechanism proposed to account for this neuroprotection is the upregulation of neurotrophic factors. Several neurotrophic factors, including Brain Derived Neurotrophic Factor (BDNF), have been shown to upregulate in response to exercise. In order to determine if exercise-induced neuroprotection is dependent upon BDNF, we compared the neuroprotective effects of voluntary exercise in mice heterozygous for the BDNF gene (BDNF+/-) with strain-matched wild-type (WT) mice. Stereological estimates of SNpc DA neurons from WT mice allowed 90 days exercise via unrestricted running demonstrated complete protection against the MPTP-induced neurotoxicity. However, BDNF+/- mice allowed 90 days of unrestricted exercise were not protected from MPTP-induced SNpc DA neuron loss. Proteomic analysis comparing SN and striatum from 90 day exercised WT and BDNF+/- mice showed differential expression of proteins related to energy regulation, intracellular signaling and trafficking. These results suggest that a full genetic complement of BDNF is critical for the exercise-induced neuroprotection of SNpc DA neurons.     

Unimpaired osmotic water permeability and fluid secretion in bile duct epithelia of AQP1 null mice

The mechanisms by which fluid moves across the luminal membrane of cholangiocyte epithelia are uncertain. Previous studies suggested that aquaporin-1 (AQP1) is an important determinant of water movement in rat cholangiocytes and that cyclic AMP mediates the movement of these water channels from cytoplasm to apical membrane, thereby increasing the osmotic water permeability. To test this possibility we measured agonist-stimulated fluid secretion and osmotically driven water transport in isolated bile duct units (IBDUs) from AQP1 wild-type (+/+) and null (-/-) mice. AQP1 expression was confirmed in a mouse cholangiocyte cell line and +/+ liver. Forskolin-induced fluid secretion, measured from the kinetics of IBDU luminal expansion, was 0.05 fl/min and was not impaired in -/- mice. Osmotic water permeability (P(f)), measured from the initial rate of IBDU swelling in response to a 70-mosM osmotic gradient, was 11.1 x 10(-4) cm/s in +/+ mice and 11.5 x 10(-4) cm/s in -/- mice. P(f) values increased by approximately 50% in both +/+ and -/- mice following preincubation with forskolin. These findings provide direct evidence that AQP1 is not rate limiting for water movement in mouse cholangiocytes and does not appear to be regulated by cyclic AMP in this species.     

Superoxide dismutase 1 protects retinal cells from oxidative damage

Bolstering the endogenous oxidative damage defense system is a good strategy for development of treatments to combat neurodegenerative diseases in which oxidative damage plays a role. A first step in such treatment development is to determine the role of various components of the defense system in cells that degenerate. In this study, we sought to determine the role of superoxide dismutase 1 (SOD1) in two models of oxidative damage-induced retinal degeneration. In one model, paraquat is injected into the vitreous cavity and then enters retinal cells and generates reactive oxygen species (ROS) that cause progressive retinal damage. Assessment of retinal function with serial electroretinograms (ERGs) showed that sod1 -/- mice were much more sensitive than sod1 +/+ mice to the damaging effects of paraquat, while sod1 +/- mice showed intermediate sensitivity. Compared to sod1 +/+ mice, sod1 -/- mice showed greater paraquat-induced oxidative damage and apoptosis. In the second model, mice were exposed to hyperoxia for several weeks, and sod1 -/- mice showed significantly greater reductions in ERG amplitudes than sod1 +/+ mice. In both of these models, transgenic mice carrying a sod1 transgene driven by a beta-actin promoter showed less oxidative stress-induced reduction in ERG amplitudes. These data demonstrate that SOD1 protects retinal cells against paraquat- and hyperoxia-induced oxidative damage and suggest that overexpression of SOD1 should be considered as one component of ocular gene therapy to prevent oxidative damage-induced retinal degeneration.     

Serum TRPM1 Autoantibodies from Melanoma Associated Retinopathy Patients Enter Retinal ON-Bipolar Cells and Attenuate the Electroretinogram in Mice

Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous malignant melanoma and the presence of autoantibodies that label neurons in the inner retina. The visual symptoms and electroretinogram (ERG) phenotype characteristic of MAR resemble the congenital visual disease caused by mutations in TRPM1, a cation channel expressed by both melanocytes and retinal bipolar cells. Four serum samples from MAR patients were identified as TRPM1 immunoreactive by 1. Labeling of ON-bipolar cells in TRPM1+/+ but not TRPM1−/− mouse retina, 2. Labeling of TRPM1-transfected CHO cells; and 3. Attenuation of the ERG b-wave following intravitreal injection of TRPM1-positive MAR IgG into wild-type mouse eyes, and the appearance of the IgG in the retinal bipolar cells at the conclusion of the experiment. Furthermore, the epitope targeted by the MAR autoantibodies was localized within the amino-terminal cytoplasmic domain of TRPM1. Incubation of live retinal neurons with TRPM1-positive MAR serum resulted in the selective accumulation of IgG in ON-bipolar cells from TRPM1+/+ mice, but not TRPM1−/− mice, suggesting that the visual deficits in MAR are caused by the uptake of TRPM1 autoantibodies into ON-bipolar cells, where they bind to an intracellular epitope of the channel and reduce the ON-bipolar cell response to light.