Faculty Bibliography

Numerous studies have documented the mechanisms that regulate intracellular pH (pH(i)) in hippocampal neurons in response to an acid load. Here, we studied the response of pH(i) to depolarization in cultured hippocampal neurons. Elevation of external K(+) (6-30 mm) elicited an acid transient followed by a large net alkaline shift. Similar responses were observed in acutely dissociated hippocampal neurons. In Ca(2+)-free media, the acid response was curtailed and the alkaline shift enhanced. DIDS blocked the alkaline response and revealed a prolonged underlying acidification that was highly dependent on Ca(2+) entry. Similar alkaline responses could be elicited by AMPA, indicating that this rise in pH(i) was a depolarization-induced alkalinization (DIA). The DIA was found to consist of Cl(-)-dependent and Cl(-)-independent components, each accounting for approximately one-half of the peak amplitude. The Cl(-)-independent component was postulated to arise from operation of the electrogenic Na(+)-HCO(3)(-) cotransporter NBCe1. Quantitative PCR and single-cell multiplex reverse transcription-PCR demonstrated message for NBCe1 in our hippocampal neurons. In neurons cultured from Slc4a4 knock-out (KO) mice, the DIA was reduced by approximately one-half compared with wild type, suggesting that NBCe1 was responsible for the Cl(-)-independent DIA. In Slc4a4 KO neurons, the remaining DIA was virtually abolished in Cl(-)-free media. These data demonstrate that DIA of hippocampal neurons occurs via NBCe1, and a parallel DIDS-sensitive, Cl(-)-dependent mechanism. Our results indicate that, by activating net acid extrusion in response to depolarization, hippocampal neurons can preempt a large, prolonged, Ca(2+)-dependent acidosis

Sleep deprivation (SD) can have a negative impact on cognitive function, but the mechanism(s) by which SD modulates memory remains unclear. We have previously shown that astrocyte-derived adenosine is a candidate molecule involved in the cognitive deficits following a brief period of SD (Halassa et al., 2009). In this study, we examined whether genetic disruption of soluble N-ethylmaleimide-sensitive factor attached protein (SNARE)-dependent exocytosis in astrocytes (dnSNARE mice) or pharmacological blockade of A1 receptor signaling using an adenosine A1 receptor (A1R) antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT), could prevent the negative effects of 6 h of SD on hippocampal late-phase long-term potentiation (L-LTP) and hippocampus-dependent spatial object recognition memory. We found that SD impaired L-LTP in wild-type mice but not in dnSNARE mice. Similarly, this deficit in L-LTP resulting from SD was prevented by a chronic infusion of CPT. Consistent with these results, we found that hippocampus-dependent memory deficits produced by SD were rescued in dnSNARE mice and CPT-treated mice. These data provide the first evidence that astrocytic ATP and adenosine A1R activity contribute to the effects of SD on hippocampal synaptic plasticity and hippocampus-dependent memory, and suggest a new therapeutic target to reverse the hippocampus-related cognitive deficits induced by sleep loss.

Sustained increase in intraocular pressure represents a major risk factor for eye disease, yet the cellular mechanisms of pressure transduction in the posterior eye are essentially unknown. Here we show that the mouse retina expresses mRNA and protein for the polymodal transient receptor potential vanilloid 4 (TRPV4) cation channel known to mediate osmotransduction and mechanotransduction. TRPV4 antibodies labeled perikarya, axons, and dendrites of retinal ganglion cells (RGCs) and intensely immunostained the optic nerve head. Muller glial cells, but not retinal astrocytes or microglia, also expressed TRPV4 immunoreactivity. The selective TRPV4 agonists 4alpha-PDD and GSK1016790A elevated [Ca(2+)](i) in dissociated RGCs in a dose-dependent manner, whereas the TRPV1 agonist capsaicin had no effect on [Ca(2+)](RGC). Exposure to hypotonic stimulation evoked robust increases in [Ca(2+)](RGC). RGC responses to TRPV4-selective agonists and hypotonic stimulation were absent in Ca(2+)-free saline and were antagonized by the nonselective TRP channel antagonists Ruthenium Red and gadolinium, but were unaffected by the TRPV1 antagonist capsazepine. TRPV4-selective agonists increased the spiking frequency recorded from intact retinas recorded with multielectrode arrays. Sustained exposure to TRPV4 agonists evoked dose-dependent apoptosis of RGCs. Our results demonstrate functional TRPV4 expression in RGCs and suggest that its activation mediates response to membrane stretch leading to elevated [Ca(2+)](i) and augmented excitability. Excessive Ca(2+) influx through TRPV4 predisposes RGCs to activation of Ca(2+)-dependent proapoptotic signaling pathways, indicating that TRPV4 is a component of the response mechanism to pathological elevations of intraocular pressure

Production of new neurons in the adult hippocampus decreases with age; this decline may underlie age-related cognitive impairment. Here we show that continuous depletion of the neural stem cell pool, as a consequence of their division, may contribute to the age-related decrease in hippocampal neurogenesis. Our results indicate that adult hippocampal stem cells, upon exiting their quiescent state, rapidly undergo a series of asymmetric divisions to produce dividing progeny destined to become neurons and subsequently convert into mature astrocytes. Thus, the decrease in the number of neural stem cells is a division-coupled process and is directly related to their production of new neurons. We present a scheme of the neurogenesis cascade in the adult hippocampus that includes a proposed 'disposable stem cell' model and accounts for the disappearance of hippocampal neural stem cells, the appearance of new astrocytes, and the age-related decline in the production of new neurons

Conventional stable isotope labeling with amino acids in cell culture (SILAC) requires extensive metabolic labeling of proteins and therefore is difficult to apply to cells that do not divide or are unstable in SILAC culture. Using two different sets of heavy amino acids for labeling allows for straightforward SILAC quantitation using partially labeled cells because the two cell populations are always equally labeled. Here we report the application of this labeling strategy to primary cultured neurons. We demonstrated that protein quantitation was not compromised by incomplete labeling of the neuronal proteins. We used this method to study neurotrophin-3 (NT-3) signaling in primary cultured neurons. Surprisingly our results indicate TrkB signaling is a major component of the signaling network induced by NT-3 in cortical neurons. In addition, involvement of proteins such as VAMP2, Scamp1, and Scamp3 suggests that NT-3 may lead to enhanced exocytosis of synaptic vesicles

In adult skin, stem cells in the hair follicle bulge cyclically regenerate the follicle, whereas a distinct stem cell population maintains the epidermis. The degree to which all bulge cells have equal regenerative potential is not known. We found that Sonic hedgehog (Shh) from neurons signals to a population of cells in the telogen bulge marked by the Hedgehog response gene Gli1. Gli1-expressing bulge cells function as multipotent stem cells in their native environment and repeatedly regenerate the anagen follicle. Shh-responding perineural bulge cells incorporate into healing skin wounds where, notably, they can change their lineage into epidermal stem cells. The perineural niche (including Shh) is dispensable for follicle contributions to acute wound healing and skin homeostasis, but is necessary to maintain bulge cells capable of becoming epidermal stem cells. Thus, nerves cultivate a microenvironment where Shh creates a molecularly and phenotypically distinct population of hair follicle stem cells

Tropomyosin-related kinase B receptor (TrkB) is a neurotrophin receptor important for the synaptic plasticity underlying hippocampal-dependent learning and memory. Because this receptor is widely expressed in hippocampal neurons, the precise location of TrkB activation is likely important for its specific actions. The goal of this study was to identify the precise sites of TrkB activation in the mouse hippocampal formation and to determine any changes in the distribution of activated TrkB under conditions of enhanced brain-derived neurotrophic factor (BDNF) expression and hippocampal excitability. Using electron microscopy, we localized TrkB phosphorylated at tyrosine 816 (pTrkB) in the hippocampal formation of male and female mice under conditions of naturally low circulating estradiol and naturally high circulating estradiol, when BDNF expression, TrkB signaling, and synaptic plasticity are enhanced. To compare relative amounts of pTrkB in each group, we counted profiles containing pTrkB-immunoreactivity (pTrkB-ir) in all hippocampal subregions. pTrkB-ir was in axons, axon terminals, dendrites, and dendritic spines of neurons in the hippocampal formation, but the majority of pTrkB-ir localized to presynaptic profiles. pTrkB-ir also was abundant in glial profiles, which were further identified as microglia using immunofluorescence and confocal microscopy. Axonal and glial pTrkB-ir and pTrkB-ir in the CA1 stratum radiatum were more abundant in high-estradiol states (proestrus females) than low-estradiol states (estrus and diestrus females and males). These findings suggest that presynaptic TrkB is positioned to modulate estradiol-mediated and BDNF-dependent synaptic plasticity. Furthermore, they suggest a novel role for TrkB in microglial function in the neuroimmune system

Lithium, a drug that has long been used to treat bipolar disorder and some other human pathogenesis, has recently been shown to stimulate neural precursor growth. However, the involved mechanism is not clear. Here, we show that lithium induces proliferation but not survival of neural precursor cells. Mechanistic studies suggest that the effect of lithium mainly involved activation of the transcription factor NF-AT and specific induction of a subset of proliferation-related genes. While NF-AT inactivation by specific inhibition of its upstream activator calcineurin antagonized the effect of lithium on the proliferation of neural precursor cells, specific inhibition of the NF-AT inhibitor GSK-3beta, similar to lithium treatment, promoted neural precursor cell proliferation. One important function of lithium appeared to increase inhibitory phosphorylation of GSK-3beta, leading to GSK-3beta suppression and subsequent NF-AT activation. Moreover, lithium-induced proliferation of neural precursor cells was independent of its role in inositol depletion. These findings not only provide mechanistic insights into the clinical effects of lithium, but also suggest an alternative therapeutic strategy for bipolar disorder and other neural diseases by targeting the non-canonical GSK-3beta-NF-AT signaling

Introduction: Pulmonary function evaluation in patients with cystic fibrosis (CF) has demonstrated disparity between spirometric and oscillometric assessments. Most studies have indicated that oscillometry may appear normal despite significant abnormalities on spirometry. However, normal values for impulse oscillometry (IOS) in pediatric populations are limited and vary by study. The present study assessed the role for IOS by assessment of both the acute response to bronchodilator and the chronic response to treatment. Methods: Patients hospitalized with exacerbations of CF were evaluated with both spirometry and oscillometry. Data were obtained pre and post bronchodilator administration and related to published normative data. When feasible, lung volumes were assessed by plethysmography. Serial testing was performed during and following standard therapy which included vigorous chest physical therapy and intravenous antibiotics targeted to the predominate organism isolated from sputum or bronchoscopy specimens. Results: Data were available in 5 patients with CF with age ranging from 5 to 44 years. Abnormal spirometry was evident in 4 subjects. Although FEV₁/FVC was mildly reduced in these subjects (68+5%), the predominant abnormality was reduction in vital capacity (50+12%). HRCT demonstrated severe mucous plugging in multiple airways and bronchoscopy in one patient confirmed total occlusion of the bronchial lumen form respiratory secretions. Despite these spirometric and radiographic abnormalities, oscillometric assessment of resistance assessed was within published normal limits in these subjects. However, a positive response to bronchodilator was observed in 3 patients and serial testing in one subject demonstrated further improvement in airway resistance by IOS. These changes in oscillometric data occurred with minimal change in FVC and FEV₁. Conclusions: Although IOS parameters in an individual patient may be within published normal limits, reduction in resistance may be apparent either acutely post bronchodilator or chronically following treatment. These improvements in IOS parameters may not be apparent on spirometry, providing a potential role for IOS in the evaluation of patients with CF. These data suggest that improvement in post bronchodilator measurements of airway resistance may be a useful adjunct to guide the appropriate length of treatment for CF exacerbations

BACKGROUND: Models of autism spectrum disorders (ASD) as neural disconnection syndromes have been predominantly supported by examinations of abnormalities in corticocortical networks in adults with autism. A broader body of research implicates subcortical structures, particularly the striatum, in the physiopathology of autism. Resting state functional magnetic resonance imaging has revealed detailed maps of striatal circuitry in healthy and psychiatric populations and vividly captured maturational changes in striatal circuitry during typical development. METHODS: Using resting state functional magnetic resonance imaging, we examined striatal functional connectivity (FC) in 20 children with ASD and 20 typically developing children between the ages of 7.6 and 13.5 years. Whole-brain voxelwise statistical maps quantified within-group striatal FC and between-group differences for three caudate and three putamen seeds for each hemisphere. RESULTS: Children with ASD mostly exhibited prominent patterns of ectopic striatal FC (i.e., functional connectivity present in ASD but not in typically developing children), with increased functional connectivity between nearly all striatal subregions and heteromodal associative and limbic cortex previously implicated in the physiopathology of ASD (e.g., insular and right superior temporal gyrus). Additionally, we found striatal functional hyperconnectivity with the pons, thus expanding the scope of functional alterations implicated in ASD. Secondary analyses revealed ASD-related hyperconnectivity between the pons and insula cortex. CONCLUSIONS: Examination of FC of striatal networks in children with ASD revealed abnormalities in circuits involving early developing areas, such as the brainstem and insula, with a pattern of increased FC in ectopic circuits that likely reflects developmental derangement rather than immaturity of functional circuits