Teresa Audesirk

Estradiol and raloxifene protect cultured SN4741 neurons against oxidative stress

A large body of research has documented neuroprotective effects of estrogen against oxidative stress. Some neurodegenerative diseases such as Parkinsons disease, in which oxidative stress has been implicated as a contributing factor, affect more males than females, suggesting a possible protective effect of estrogen. We used the clonal substantia nigra cell line SN4741 to compare the neuroprotective properties of estrogen and raloxifene against oxidative stress, and to determine whether raloxifene acted as an estrogen agonist or antagonist in this system. We pretreated SN4741 cultures with alpha-estradiol, beta-estradiol, and raloxifene, and exposed them to hydrogen peroxide. Low nanomolar levels of raloxifene, beta-estradiol, and alpha-estradiol all significantly reduced cell death caused by oxidative stress. The estrogen receptor (ER) antagonist ICI 182,780 failed to reverse the neuroprotection by beta-estradiol, suggesting that the effect is not mediated by a classical ER. Western blotting using an antibody to the C-terminus region of ER-alpha revealed two bands, one at approximately 67 kDa (corresponding to ER-alpha) and a more prominent band at approximately 55-56 kDa. These results suggest that, in this cell line, both raloxifene and estrogen may be acting via a non-classical estrogen receptor.

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.

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.

Characterization of pre- and postsynaptic dopamine receptors in Lymnaea

1. The effects of dopamine and several synthetic agonists and antagonists were studied using two identified neurons of the snail Lymnaea stagnalis. 2. In both the buccal-2 (B-2) neurons and the pedal giant (RPeD1) neuron dopamine elicited a hyperpolarizing response at least partly due to potassium efflux. RPeD1 is itself dopaminergic, implicating autoreceptors in its response to dopamine. 3. The following agents were tested: agonists--LY171555, pergolide, SKF38393, (-)-3-PPP, R(-)NPA and dopamine; antagonists--SCH23390, sulpiride, and metaclopramide. Dibutyryl cAMP was applied to determine whether the response is cAMP-mediated. 4. Results indicate that the pharmacological profiles of dopamine receptors on these neurons are inconsistent with those of either D-1, D-2 or autoreceptors in mammals.

Low levels of inorganic lead noncompetitively inhibit μ-calpain

Calpain is a ubiquitous calcium-dependent cysteine protease, whose cytoskeletal protein substrates suggest that it may be important in neuronal differentiation. Lead (Pb2+) is known to substitute for Ca2+ in a variety of intracellular processes, and interferes with the development of hippocampal neurons in vitro. We found that free Pb2+ at 1 nM does not activate calpain in the absence of Ca2+. Pb2+ inhibited the activity of calpain; the degree of calpain inhibition was dependent on an interaction between concentrations of both Ca2+ and Pb2+. In the presence of 1 microM free Ca2+, 10 pM free Pb2+ reduced calpain activity, but in the presence of 100 microM free Ca2+, 1 nM free Pb2+ failed to inhibit calpain. This provides evidence that Pb2+ competes for the Ca2+ binding sites on calpain. In the presence of 40 microM free Ca2+, 1 nM free Pb2+ significantly reduces Vmax without altering Km, suggesting that Pb2+ acts as a noncompetitive inhibitor of calpain. Inhibition of calpain is one mechanism by which Pb2+ may interfere with neuronal development.

Identification of central neurons innervating peripheral chemoreceptive structures in Lymnaea stagnalis

Abstract 1. 1. This mapping study utilized cobalt backfilling to identify central neurons in the brain of Lymnaea stagnalis which possess peripheral axons innervating lips and tentacles. 2. 2. A systematic method of naming cells and cell clusters was developed which can be expanded as new cells are identified. 3. 3. A comparison was made between cells identified here and those identified in other studies. 4. 4. Since it is hypothesized that cells identified by this technique are mechanoreceptive, chemoreceptive, or lip and tentacle motor neurons, this work provides a basis for electrophysiological studies of central neurons mediating the response to food.

Evidence for genetic influences on neurotransmitter content of identified neurones of Lymnaea stagnalis

Neurotransmitter content was measured in two identified giant neurones in isogenic and wild-type populations of the freshwater pond snail Lymnaea stagnalis. The paired serotonergic cerebral giant neurones (LC1 and RC1) have higher transmitter levels and less variability in inbred animals than in wild-type animals. The transmitter content of the unpaired dopaminergic right pedal giant neurone (RPeD1) does not differ between inbred and wild-type animals in either level or variability. It is proposed that serotonin content of the cerebral giant neurones is under partial genetic control, and that animals of the wild-type population may possess a number of different alleles for the genes influencing serotonin levels. Inbreeding resulted in fixation of an allele promoting high serotonin levels. This particular wild-type population is probably already isogenic for genes influencing dopamine content in the right pedal giant neurone.

Culturing Rat Hippocampal Neurons

Cultured neurons are widely used to investigate the mechanisms of neurotoxicity. Embryonic rat hippocampal neurons may be grown as described under a wide variety of conditions to suit differing experimental procedures, including electrophysiology, morphological analysis of neurite development, and various biochemical and molecular analyses.

Double-staining technique allows electrophysiological identification of monoamine-containing neurons

Electrophysiological recording provides important evidence for positive identification of many neurons in gastropods. We describe a technique which combines intracellular recording and injection of a persistent, non-fluorescent dye (Fast Green) with subsequent histofluorescence treatment using a modification of the wholemount glyoxylic acid procedure developed by Barber (1983) to establish the presence or absence of monoamine transmitters in positively identified single gastropod neurons.