Gerald Audesirk

Oxidative stress-mediated down-regulation of bcl-2 promoter in hippocampal neurons.

Generation of oxidative stress/reactive oxygen species (ROS) is one of the causes of neuronal apoptosis. We have examined the effects of ROS at the transcriptional level in an immortalized hippocampal neuronal cell line (H19‐7) and in rat primary hippocampal neurons. Treatment of H19‐7 cells with hydrogen peroxide (150 µm) resulted in a 40% decrease in Bcl‐2 protein and a parallel decrease in bcl‐2 mRNA levels. H19‐7 cells overexpressing bcl‐2 were found to be resistant to ROS‐induced apoptosis. We had previously shown that bcl‐2 promoter activity is positively regulated by the transcription factor cyclic AMP response element binding protein (CREB) in neurons. In the present study, we demonstrate that ROS decreases the activity of luciferase reporter gene driven by a cyclic AMP response element site containing bcl‐2 promoter. Exposure of neurons to ROS for 6 h resulted in basal and fibroblast growth factor‐2‐stimulated phosphorylation/activation of CREB. Chronic 24 h treatment with ROS led to a significant (p < 0.01) decrease in CREB protein and CREB mRNA levels. Adenoviral overexpression of wild type CREB in H19‐7 cells resulted in significant (p < 0.01) protection against ROS‐induced apoptosis through up‐regulation of Bcl‐2 expression whereas dominant negative CREB exaggerated the injury. These findings demonstrate that loss of CREB function contributes to oxidative stress‐induced neuronal dysfunction.

Effects of selective inhibition of protein kinase C, cyclic AMP-dependent protein kinase, and Ca2+-calmodulin-dependent protein kinase on neurite development in cultured rat hippocampal neurons

A variety of experimental evidence suggests that calmodulin and protein kinases, especially protein kinase C, may participate in regulating neurite development in cultured neurons, particularly neurite initiation. However, the results are somewhat contradictory. Further, the roles of calmodulin and protein kinases on many aspects of neurite development, such as branching or elongation of axons vs dendrites, have not been extensively studied. Cultured embryonic rat hippocampal pyramidal neurons develop readily identifiable axons and dendrites. We used this culture system and the new generation of highly specific protein kinase inhibitors to investigate the roles of protein kinases and calmodulin in neurite development. Neurons were cultured for 2 days in the continuous presence of calphostin C (a specific inhibitor of protein kinase C), KT5720 (inhibitor of cyclic AMP‐dependent protein kinase), KN62 (inhibitor of Ca2+‐calmodulin‐dependent protein kinase II), or calmidazolium (inhibitor of calmodulin), each at concentrations from approximately 1 to 10 times the concentration reported in the literature to inhibit each kinase by 50%. The effects of phorbol 12‐myristate 13‐acetate (an activator of protein kinase C) and 4α‐phorbol 12,13‐didecanoate (an inactive phorbol ester) were also tested.

Modulation of neurite branching by protein phosphorylation in cultured rat hippocampal neurons

The control of branching of axons and dendrites is poorly understood. It has been hypothesized that branching may be produced by changes in the cytoskeleton [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419; P. Friedrich, A. Aszodi, MAP2: a sensitive cross-linker and adjustable spacer in dendritic architecture, FEBS Lett. 295 (1991) 5-9]. The assembly and stability of microtubules, which are prominent cytoskeletal elements in both axons and dendrites, are regulated by microtubule-associated proteins, including tau (predominantly found in axons) and MAP2 (predominantly found in dendrites). The phosphorylation state of tau and MAP2 modulates their interactions with microtubules. In their low-phosphorylation states, tau and MAP2 bind to microtubules and increase microtubule assembly and/or stability. Increased phosphorylation decreases these effects. Diez-Guerra and Avila [F.J. Diez-Guerra, J. Avila, MAP2 phosphorylation parallels dendrite arborization in hippocampal neurones in culture, NeuroReport 4 (1993) 412-419] found that protein phosphorylation correlates with neurite branching in cultured rat hippocampal neurons, and hypothesized that increased protein phosphorylation stimulates neurite branching. To test this hypothesis, we cultured rat hippocampal neurons in the presence of specific modulators of serine-threonine protein kinases and phosphatases. Inhibitors of several protein kinases, which would be expected to decrease protein phosphorylation, reduced branching. KT5720, an inhibitor of cyclic AMP-dependent protein kinase, and KN62, an inhibitor of Ca(2+)-calmodulin-dependent protein kinases, inhibited branching of both axons and dendrites. Calphostin C and chelerythrine, inhibitors of protein kinase C, inhibited branching of axons but not dendrites. Treatments that would be expected to increase protein phosphorylation, including inhibitors of protein phosphatases (okadaic acid, cyclosporin A and FK506) and stimulators of PKA (SP-cAMPS) or PKC (phorbol 12-myristate 13-acetate), increased dendrite branching. Only FK506 and phorbol 12-myristate 13-acetate stimulated axon branching. A subset of these agents was tested to confirm their effects on protein phosphorylation in this preparation. Okadaic acid, FK506 and SP-cAMPS all increased protein phosphorylation; KT5720 and KN62 decreased protein phosphorylation. On Western blots, the position of MAP2c extracted from cultures exposed to okadaic acid was slightly shifted toward higher molecular weight, suggesting greater phosphorylation, while the position of MAP2c from cultures exposed to KT5720 and KN62 was slightly shifted toward lower molecular weight, suggesting less phosphorylation. We conclude that protein phosphorylation modulates both dendrite branching and axon branching, but with differences in sensitivity to phosphorylation and/or dephosphorylation by specific kinases and phosphatases.

Amine-containing neurons in the brain of Lymnaea stagnalis: distribution and effects of precursors

Glyoxylic acid-induced fluorescence in whole-brain preparations of the central nervous system of the freshwater pond snail, Lymnaea stagnalis, was used to map the distribution of serotonin-and dopamine-containing neurons. Serotonin and dopamine were easily distinguishable by differences in color of fluorescence. Serotonin-containing neurons were consistently found in the cerebral, pedal, right parietal and visceral ganglia. Dopamine-containing neurons were found in the pedal, and buccal ganglia. Prior incubation of brains in 5-hydroxytryptophan (5-HTP), the immediate precursor to serotonin, produced serotonin-like fluoresence in neurons which do not normally fluoresce. These neurons thus probably possess specific uptake mechanisms for 5-HTP. Since 5-HTP itself fluoresces yellow, the glyoxylic acid technique cannot determine if these neurons contain the enzyme aromatic amino acid decarboxylase, which converts 5-HTP to serotonin, or merely fluoresce because of the 5-HTP taken into the cells.

Research reportRapid, nonaversive conditioning in a freshwater gastropod: I. Effects of age and motivation

The pond snail Lymnaea stagnalis is capable of rapid acquisition of a nonaversive association between food and a novel chemostimulus. Ten to twenty pairings of the unconditioned stimulus, a phagostimulant consisting of a mixture of sucrose and casein digest, and the conditioned stimulus, amyl acetate at 0.004% resulted in feeding movements in response to the conditioned stimulus alone. Four different control groups were used, none of which acquired the response. Age, and age-influenced motivation were found to have a profound effect on learning and on its expression. Old snails which were well fed prior to training failed to acquire the association, while starved old animals learned readily. Starved young snails learned readily, while young snails which were well fed prior to training also acquired the association, but only expressed the learned response after food deprivation.

Behavior of Gastropod Molluscs

Publisher Summary Molluscan behavior is extremely diverse, encompassing the relatively limited behavioral repertoire of clams and limpets and the intelligent, highly flexible behaviors of the cephalopods. Gastropods and cephalopods dominate as subjects for molluscan neurobiology for contrasting reasons: (1) cephalopods for the mammal-like intricacy of their brains and behavior and (2) gastropods for their relative simplicity and their large and individually identifiable neurons. This chapter discusses gastropod behaviors, especially those of species commonly used in neurobiology. Light plays an important role in the life of molluscs. For terrestrial forms, bright light can signal heat and dryness, while shade may represent desirable damp shelter. Feeding is stimulated by the detection of food stimuli by the rhinophores, oral veil, anterior foot, or mouth area. Gastropod feeding, like that of more complex organisms, is influenced by the interaction of several variables, including hunger, satiation, quality of food, intensity of the feeding chemostimulus, sensory adaptation, and habituation.

β-Estradiol influences differentiation of hippocampal neurons in vitro through an estrogen receptor-mediated process

We utilized morphometric analysis of 3 day cultures of hippocampal neurons to determine the effects of both estradiol and the synthetic estrogen receptor modulator raloxifene on several parameters of neuronal growth and differentiation. These measurements included survival, neurite production, dendrite number, and axon and dendrite length and branching. 17 beta-Estradiol (10 nM) selectively stimulated dendrite branching; this effect was neither mimicked by alpha-estradiol, nor blocked by the estrogen receptor antagonist ICI 182780. The selective estrogen receptor modulator raloxifene (100 nM) neither mimicked nor reversed the effects of estradiol on dendritic branching. Western immunoblotting for the alpha and beta subtypes of estrogen receptor revealed the presence of alpha, but not beta, estrogen receptors in our hippocampal cultures. There is growing recognition of the effects of 17 beta-estradiol on neuronal development and physiology, with implications for brain sexual dimorphism, plasticity, cognition, and the maintenance of cognitive function during aging. The role of estradiol in hippocampal neuronal differentiation and function has particular implications for learning and memory. These data support the hypothesis that 17 beta-estradiol is acting via alpha estrogen receptors in influencing hippocampal development in vitro. Raloxifene, prescribed to combat osteoporosis in post-menopausal women, is a selective estrogen receptor modulator with tissue-specific agonist/antagonist properties. Because raloxifene had no effect on dendritic branching, we hypothesize that it does not interact with the alpha estrogen receptor in this experimental paradigm.

Stimulatory and inhibitory effects of inorganic lead on calcineurin.

Calcineurin is a phosphatase with activity dependent on both Ca(2+)/calmodulin binding to the catalytic A subunit and Ca(2+) binding to the regulatory B subunit. We have previously shown that Pb(2+) activates calmodulin with a threshold of about 100 pM free Pb(2+), and that Pb(2+) and Ca(2+) are roughly additive in calmodulin activation (Kern et al., NeuroToxicology 21, 353-364 (2000)). In the present study, we evaluated the effects of Pb(2+), with and without Ca(2+) and calmodulin, on calcineurin activity. In calmodulin-containing, Ca(2+)-free solutions, Pb(2+) activated calcineurin with a threshold of about 100 pM free Pb(2+). Maximum calcineurin activity (comparable to that induced by 10 microM Ca(2+)) was reached at about 200 pM free Pb(2+). Higher Pb(2+) concentrations reduced activity, although some activity remained even at 2000 pM free Pb(2+). Combined with subsaturating Ca(2+) concentrations, as little as 20 pM free Pb(2+) enhanced calcineurin activity, but free Pb(2+) concentrations greater than 200 pM still reduced activity below maximum. Extremely high Ca(2+) concentrations (10 microM) completely reversed the inhibition of activity by 2000 pM free Pb(2+). In the absence of calmodulin, Ca(2+) slightly stimulated calcineurin activity. Pb(2+) did not substitute for Ca(2+) in calmodulin-free activation; in fact, high concentrations of Pb(2+) inhibited Ca(2+)-mediated activation. We tentatively conclude that low concentrations of free Pb(2+) activate calcineurin by activating calmodulin. Higher concentrations reduce calcineurin activity, perhaps by binding to the B subunit.

Oral mechanoreceptors inTritonia diomedea: II. Role in feeding

Summary1.Behavioral analysis of normal feeding inTritonia diomedea revealed a complex series of movements designed to deal with a structurally challenging prey organism, the sea whipVirgularia (Fig. 1). The prey is often swallowed in its entirety during a single bout of feeding which includes movements to bite and swallow the sea whip, and to break its calcareous style (Fig. 2).2.Separate and combined presentation of mechanical and chemical cues similar to those associated with normal feeding demonstrated that each type of sensory stimulus influences the nature of the ingestion movements performed. Mechanical stimuli, in the presence of appropriate chemical cues, inhibit biting and elicit swallowing. Repeated biting is elicited by chemical cues alone. With chemical cues absent, mechanical stimuli within the buccal mass elicit an ambiguous combination of ingestion and ejection movements. A flow diagram of the decision-making process (Fig. 6) is proposed.3.Intracellular stimulation of single central mechanoreceptor neurons described in the previous paper (Audesirk, 1979) often results in movements of the buccal mass, sometimes resembling swallowing and sometimes ejection. Cycles of activity in presumed motor neurons of the buccal ganglia can be elicited either by brief intracellular stimulation of mechanosensory neurons or by pressure on areas of the oral tube known to be densely innervated by these receptors.

Enhancement of dendritic branching in cultured hippocampal neurons by 17β-estradiol is mediated by nitric oxide

Both 17β‐estradiol (E2) and nitric oxide (NO) are important in neuronal development, learning and memory, and age‐related memory changes. There is growing evidence that a number of estrogen receptor‐mediated effects of estradiol utilize nitric oxide as an intermediary. The role of estradiol in hippocampal neuronal differentiation and function has particular implications for learning and memory.