Dendritic spine formation and pruning: common cellular mechanisms?

The recent advent of novel high-resolution imaging methods has created a flurry of exciting observations that address a century-old question: what are biological signals that regulate formation and elimination of dendritic spines? Contrary to the traditional belief that the spine is a stable storage site of long-term neuronal memory, the emerging picture is of a dynamic structure that can undergo fast morphological variations. Recent conflicting reports on the regulation of spine morphology lead to the proposal of a unifying hypothesis for a common mechanism involving changes in postsynaptic intracellular Ca2+ concentration, [Ca2+]i: a moderate rise in [Ca2+]i causes elongation of dendritic spines, while a very large increase in [Ca2+]i causes fast shrinkage and eventual collapse of spines. This hypothesis provides a parsimonious explanation for conflicting reports on activity-dependent changes in dendritic spine morphology, and might link these changes to functional plasticity in central neurons.

Synucleins are developmentally expressed, and alpha-synuclein regulates the size of the presynaptic vesicular pool in primary hippocampal neurons.

alpha-, beta-, and gamma-Synuclein, a novel family of neuronal proteins, has become the focus of research interest because alpha-synuclein has been increasingly implicated in the pathogenesis of Parkinson's and Alzheimer's disease. However, the normal functions of the synucleins are still unknown. For this reason, we characterized alpha-, beta-, and gamma-synuclein expression in primary hippocampal neuronal cultures and showed that the onset of alpha- and beta-synuclein expression was delayed after synaptic development, suggesting that these synucleins may not be essential for synapse formation. In mature cultured primary neurons, alpha- and beta-synuclein colocalized almost exclusively with synaptophysin in the presynaptic terminal, whereas little gamma-synuclein was expressed at all. To assess the function of alpha-synuclein, we suppressed expression of this protein with antisense oligonucleotide technology. Morphometric ultrastructural analysis of the alpha-synuclein antisense oligonucleotide-treated cultures revealed a significant reduction in the distal pool of synaptic vesicles. These data suggest that one function of alpha-synuclein may be to regulate the size of distinct pools of synaptic vesicles in mature neurons.

A direct assessment of the role of genetic drift in determining allele frequency variation in populations of Euphydryas editha.

Estimates of allele frequencies at six polymorphic loci were collected over eight generations in two populations of Euphydryas editha. We have estimated, in addition, the effective population size for each generation for both populations with results from mark-recapture and other field data. The variation in allele frequencies generated by random genetic drift was then studied using computer simulations and our direct estimates of effective population size. Substantial differences between observed values and computer-generated expected values assuming drift alone were found for three loci (Got, Hk, Pgi) in one population. These observations are consistent with natural selection in a variable environment.

Transfected synphilin-1 forms cytoplasmic inclusions in HEK293 cells.

The discovery of mutations in the gene for alpha-synuclein in familial Parkinson's disease (PD) has led to an increased interest in this pre-synaptic protein. Synphilin-1, a potential synuclein-binding protein, was cloned using yeast two-hybrid assays. The function of synphilin-1 is currently unknown, although it has been reported to be present along with alpha-synuclein in Lewy bodies in PD. In the present study, we monitored synphilin-1 aggregation directly using fusion proteins of synphilin-1 and green fluorescent protein (EGFP). Transfection of synphilin-EGFP fusion proteins formed cytoplasmic inclusions in HEK293 cells. Although these inclusions overlapped with the distribution of alpha-synuclein, they were unlike Lewy bodies in that they were not eosinophilic, and instead were membrane-bound, lipid-rich cytoplasmic inclusions.

Population structure of a hilltopping butterfly.

The hilltopping behavior of individuals in a closed demographic unit ofEuphydryas editha is examined. Both males and females hilltop but the behavior differs between the sexes. Most males appear to hilltop throughout their reproductive lives. Females hilltop only as virgins. The occurrence of hilltopping in a population is influenced by the density and age structure of the population. In addition, some males appear not to hilltop, but instead "patrol" below the hilltop, a strategy which may be favored in periods of high density. We suggest that in order to understand the ecological and evolutionary significance of hilltopping, its populational aspects need to be investigated as well as individual behaviors, such as territoriality, which hilltopping butterflies may also exhibit.

Extinction, reduction, stability and increase: The responses of checkerspot butterfly (Euphydryas) populations to the California drought.

The California drought of 1975-77 has been correlated with unusual size changes in populations of two species of Euphydryas butterflies. Several populations became extinct, some were dramatically reduced, others remained stable and at least one increased. These differences in population dynamic response are not concordant with predictions made earlier that populations with heavy density-dependent mortality should be more stable in the face of drought than unregulated populations. The different responses are related to the fine details of the relationships between the insects and their host plants, relationships which are variable between populations. Revised predictions are given in the light of better knowledge of the variability and complexity of these insect-host relationships. The diversity of responses underlines the dangers of generalizing about "the ecology" of a taxonomic species. The extinctions support the view that such events are frequent and significant in the biology of populations.

Feasibility and acceptability of the COOP/WONCA charts for identification of functional limitations in rural patients of the People's Republic of China.

The aim of this study was to test whether the COOP/WONCA Charts could help doctors in three specialty outpatient clinics of Hebei Provincial Hospital, China to identify functional limitations in rural patients and promote more patient-centred care. In this descriptive, cross-sectional study, a sample of 113 rural patients from Hebei Province attending outpatient Neurology, Orthopaedic and Cancer clinics completed the COOP/WONCA Charts. The 80 rural patients who indicated significant functional difficulty and 11 doctors then responded to questionnaires to determine perceived usefulness and satisfaction with use of the COOP/WONCA Charts. Respondents remained blind to the other's responses. A focus group interview was conducted to expand overall views on perceived advantages and disadvantages of the charts. Of the 113 patients completing the COOP/WONCA Charts, 71% (n = 80) were considered to report significant difficulty on at least one chart. Fifty-six per cent of patients rated themselves as having significant functional problems in physical fitness and 65% for daily activities. In contrast, a response indicating 'no or little limitation or effect' was given regarding patients' feelings and/or participation in social activities by 75% of patients in all three clinics. There was no statistically significant difference in the proportion of patients with functional impairment between the three clinics when analysed using a Chi-square test. Over 90% of doctors and patients perceived the COOP/WONCA Charts as helpful with 'increased communication' as an outstanding benefit. This study has shown the COOP/WONCA Charts to be feasible and useful tools: to help identify functional limitations in Chinese outpatients; to trigger a more functionally focused patient-centred model of practice; and to encourage appropriate referrals to existing rehabilitation efforts in large Chinese hospitals.

Culture models for the study of estradiol-induced synaptic plasticity.

Estrogen, which classically affects areas of the brain related to reproduction, has also been found to affect brain regions important in learning and memory. Additionally, it has been suggested that estrogen exerts protective effects against neurodegenerative diseases such as Alzheimer's disease. Important mechanisms by which estrogen may confer protection are through the maintenance or modulation of existing synapses, or by the production of new ones. It has now been demonstrated that estrogen can increase synaptogenesis and spine production in the hippocampus, both in vivo as well as in primary hippocampal neurons in culture. The latter model system is the primary focus of this review. Synaptogenesis and spine production have been well characterized in developing and adult animals, and parallels between the synaptic morphology reflecting these processes can be readily observed in high-density primary hippocampal cultures. Moreover, in culture, estrogen induces a variety of ultrastructural modifications, many of which occur in vivo, that have been linked to various in vivo models of learning and memory. For these reasons, high-density hippocampal culture systems should be regarded as valuable tools with which to predict in vivo physiology, and may well be particularly useful for studies of the neuroprotective effects of estrogen.

Comparison of the ID3 algorithm versus discriminant analysis for performing feature selection.

Having obtained disappointing results in a small medical data set despite the fact that our data seemed to be well suited for induction via ID3, we decided to compare the performance of ID3 to discriminant analysis. Performance was gauged by the percentage of correct classification in a second, independent data set. Examples were obtained from a cardiology project on the accuracy of auscultation. There were 107 examples in the first data set and 67 cases in the second. We found that ID3 and discriminant analysis performed equally poorly, with ID3 classifying only 60% of the second set correctly and discriminant analysis classifying 66% of the second set correctly. Also, the ID3 probability statistic for estimating the accuracy of ID3 for classifying further cases was markedly optimistic compared to our actual second data set results. Moreover, with an increase in sample size, ID3 seemed to break down, producing a large, complex decision tree of dubious generality, whereas discriminant analysis, with a larger sample size, used more independent variables but maintained its first set accuracy. These data suggest that there is a need for more sophisticated algorithms than ID3, even at the risk of giving up some computational efficiency.

Morphological plasticity of dendritic spines in central neurons is mediated by activation of cAMP response element binding protein.

While evidence has accumulated in favor of cAMP-associated genomic involvement in long-term synaptic plasticity, the mechanisms downstream of the activated nucleus that underlie these changes in neuronal function remain mostly unknown. Dendritic spines, the locus of excitatory interaction among central neurons, are prime candidates for long-term synaptic modifications. We now present evidence that links phosphorylation of the cAMP response element binding protein (CREB) to formation of new spines; exposure to estradiol doubles the density of dendritic spines in cultured hippocampal neurons, and concomitantly causes a large increase in phosphorylated CREB and in CREB binding protein. Blockade of cAMP-regulated protein kinase A eliminates estradiol-evoked spine formation, as well as the CREB and CREB binding protein responses. A specific antisense oligonucleotide eliminates the phosphorylated CREB response to estradiol as well as the formation of new dendritic spines. These results indicate that CREB phosphorylation is a necessary step in the process leading to generation of new dendritic spines.

Progesterone prevents estradiol-induced dendritic spine formation in cultured hippocampal neurons.

Estradiol has been shown to cause an increase in dendritic spine density in cultured hippocampal neurons, an effect mediated by downregulation of brain-derived neurotrophic factor (BDNF) and glutamic acid decarboxylase (GAD), and the subsequent phosphorylation of cAMP response element binding protein (CREB) in response to enhanced activity levels. Interestingly, progesterone was shown to counteract the effects of estradiol on dendritic spine density in vivo and in vitro. The present study examined how progesterone may act to block the effects of estradiol in the molecular cascade of cellular events leading to formation of dendritic spines. Progesterone did not affect the estradiol-induced downregulation of BDNF or GAD, but it did block the effect of estradiol on CREB phosphorylation. The latter effects of progesterone on the pCREB response and spine formation were reversed by indomethacin, which prevents the conversion of progesterone to the neurosteroid tetrahydroprogesterone (THP). We therefore examined if the progesterone effects were caused by its active metabolite THP. Progesterone treatment caused a 60-fold increase in THP in the culture medium. THP itself enhanced spontaneous GABAergic activity in patch-clamped cultured neurons. Finally, THP blocked the estradiol-induced increase in spine density. These results suggest that progesterone, through conversion to THP, blocks the effects of estradiol on dendritic spines not via a direct nuclear receptor interaction but by counteracting the enhanced excitability produced by estradiol in the cultured network.

CREB activation mediates plasticity in cultured hippocampal neurons.

Activation of cyclic AMP dependent kinase is believed to mediate slow onset, long-term potentiation (LTP) in central neurons. Cyclic-AMP activates a cascade of molecular events leading to phosphorylation of the nuclear cAMP responsive element binding protein (pCREB). Whereas a variety of stimuli lead to activation of CREB, the molecular processes downstream of CREB, which may be relevant to neuronal plasticity, are yet largely unknown. We have recently found that following exposure to estradiol, pCREB mediates the large increase in dendritic spine density in cultured rat hippocampal neurons. We now extend these observations to include other stimuli, such as bicuculline, that cause the formation of new dendritic spines. Such stimuli share with estradiol the same mechanism of action in that both require activity-dependent CREB phosphorylation. Our observations suggest that CREB phosphorylation is a necessary, but perhaps not sufficient, step in the process leading to the generation of new dendritic spines and perhaps to functional plasticity as well.

Regulation of dendritic spine density in cultured rat hippocampal neurons by steroid hormones.

The effects of gonadal steroid hormones on dendritic spines were studied in hippocampal neurons that were dissociated and grown in culture for 2-3 weeks. Exposure to estradiol caused up to a twofold increase in dendritic spine density in these neurons. The effect of estradiol was stereospecific and blocked by the steroid antagonist tamoxifen. The estradiol-induced rise in spine density was blocked by the NMDA antagonist APV, but not by the AMPA/KA antagonist DNQX. The estradiol-induced rise in spine density was blocked by the serine/threonine kinase inhibitor H7, but not by the tyrosine kinase inhibitor genestein, and was partially mimicked by PMA, an activator of protein kinase C. Estradiol also caused an increase in the fluorescence intensity of synaptophysin-immunoreactive terminals, corresponding to presynaptic boutons. Finally, estradiol caused a rise in [Ca]i reactivity of the cultured neurons to topical application of glutamate. These studies are the first to examine receptor and second messenger regulation of dendritic spines, and they illustrate the viability of cultured neurons as a powerful test system to address issues related to the regulation of dendritic spine maturation.

Differential contractile response of cultured microvascular pericytes to vasoactive agents.

OBJECTIVE: Microvascular pericytes may contract in two different ways: In the first, a circumferential or radial mechanical force applied at right angles to the long axis of the vessel may constrict the underlying vessel affecting blood flow and transmural pressure. Retraction and elongation of pericyte processes may also occur tangentially and at right angles to the vessel axis and alter microvessel permeability by changing the amount of ablumenal surface covered or the openness of interendothelial junctions. In this study, cultured pericytes were utilized as a model experimental system to determine if vasoactive stimulation changes their shape in a manner consistent with this hypothesis. METHODS: Pericytes cultured from isolated rate capillaries were subjected to angiotensin II and histamine. Their response was monitored by measuring the area of non-yielding substrate covered by the pericytes and the manner in which their shape changed. Shape changes were quantified by calculating the surface area: perimeter perimeter ratios. RESULTS: Histamine significantly reduced surface area covered and the surface area:perimeter ratio. The pericyte processes retracted, resulting in elongated, spindle-shaped cells. These effects were nullified by the H1 blocker diphenhydramine suggesting a receptor-specific response. Angiotensin II also elicited contraction and reduced surface area, but the cells contracted laterally and longitudinally. The surface area: perimeter ratios also decreased. CONCLUSIONS: These results indicate that pericytes are capable of two types of contractile responses in culture, depending on the specific vasoactive stimulus.

Estradiol increases spine density and NMDA-dependent Ca2+ transients in spines of CA1 pyramidal neurons from hippocampal slices.

To investigate the physiological consequences of the increase in spine density induced by estradiol in pyramidal neurons of the hippocampus, we performed simultaneous whole cell recordings and Ca2+ imaging in CA1 neuron spines and dendrites in hippocampal slices. Four- to eight-days in vitro slice cultures were exposed to 17beta-estradiol (EST) for an additional 4- to 8-day period, and spine density was assessed by confocal microscopy of DiI-labeled CA1 pyramidal neurons. Spine density was doubled in both apical and basal dendrites of the CA1 region in EST-treated slices; consistently, a reduction in cell input resistance was observed in EST-treated CA1 neurons. Double immunofluorescence staining of presynaptic (synaptophysin) and postsynaptic (alpha-subunit of CaMKII) proteins showed an increase in synaptic density after EST treatment. The slopes of the input/output curves of both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) postsynaptic currents were steeper in EST-treated CA1 neurons, consistent with the observed increase in synapse density. To characterize NMDA-dependent synaptic currents and dendritic Ca2+ transients during Schaffer collaterals stimulation, neurons were maintained at +40 mV in the presence of nimodipine, picrotoxin, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). No differences in resting spine or dendritic Ca2+ levels were observed between control and EST-treated CA1 neurons. Intracellular Ca2+ transients during afferent stimulation exhibited a faster slope and reached higher levels in spines than in adjacent dendrites. Peak Ca2+ levels were larger in both spines and dendrites of EST-treated CA1 neurons. Ca2+ gradients between spine heads and dendrites during afferent stimulation were also larger in EST-treated neurons. Both spine and dendritic Ca2+ transients during afferent stimulation were reversibly blocked by D, L-2-amino-5-phosphonovaleric acid (D,L-APV). The increase in spine density and the enhanced NMDA-dependent Ca2+ signals in spines and dendrites induced by EST may underlie a threshold reduction for induction of NMDA-dependent synaptic plasticity in the hippocampus.

Brain-derived neurotrophic factor mediates estradiol-induced dendritic spine formation in hippocampal neurons.

Dendritic spines are of major importance in information processing and memory formation in central neurons. Estradiol has been shown to induce an increase of dendritic spine density on hippocampal neurons in vivo and in vitro. The neurotrophin brain-derived neurotrophic factor (BDNF) recently has been implicated in neuronal maturation, plasticity, and regulation of GABAergic interneurons. We now demonstrate that estradiol down-regulates BDNF in cultured hippocampal neurons to 40% of control values within 24 hr of exposure. This, in turn, decreases inhibition and increases excitatory tone in pyramidal neurons, leading to a 2-fold increase in dendritic spine density. Exogenous BDNF blocks the effects of estradiol on spine formation, and BDNF depletion with a selective antisense oligonucleotide mimics the effects of estradiol. Addition of BDNF antibodies also increases spine density, and diazepam, which facilitates GABAergic neurotransmission, blocks estradiol-induced spine formation. These observations demonstrate a functional link between estradiol, BDNF as a potent regulator of GABAergic interneurons, and activity-dependent formation of dendritic spines in hippocampal neurons.

Estradiol increases dendritic spine density by reducing GABA neurotransmission in hippocampal neurons.

We have previously shown that estradiol causes a twofold rise in dendritic spine density in cultured rat hippocampal neurons, as it does in vivo. More recently, estrogen receptors have been localized to aspiny inhibitory hippocampal interneurons, indicating that their effect on spiny pyramidal neurons may be indirect. We therefore examined the possibility that estradiol affects spine density by regulating inhibition in cultured hippocampal interneurons. Immunocytochemically, estrogen receptors were found to be co-localized with glutamate decarboxylase (GAD)-positive neurons (approximately 21% of total neurons in the culture). Exposure of cultures to estradiol for 1 d caused a marked decrease (up to 80%) in the GAD content of the interneurons, measured both by immunohistochemistry and Western blotting. Also, the number of GAD-positive neurons in the cultures decreased to 12% of the total cell population. Moreover, GABAergic miniature IPSCs were reduced in both size and frequency by estradiol, whereas miniature EPSCs increased in frequency. We then mimicked the proposed effects of estradiol by blocking GABA synthesis with mercaptopropionic acid (MA). Cultures treated with MA expressed a dose-dependent decrease in GABA immunostaining that mimicked that seen with estradiol. MA-treated cultures displayed a significant 50% increase in dendritic spine density over controls, similar to that produced by estradiol. These results indicate that estradiol decreases GABAergic inhibition in the hippocampus, which appears to effectively increase the excitatory drive on pyramidal cells, and thus may provide a mechanism for formation of new dendritic spines.

Neuroprotective agents for clinical trials in Parkinson's disease: a systematic assessment.

BACKGROUND: Current therapies for PD ameliorate symptoms in the early phases of disease but become less effective over time, as the underlying disease progresses. Therapies that slow the progression of PD are needed. However, there have been relatively few clinical trials aimed at demonstrating neuroprotection. The authors sought to identify potential neuroprotective agents for testing in clinical trials. METHODS: First a broad array of compounds were identified by working with clinicians and researchers in academics and industry. Specific criteria were drafted for drug evaluation, including scientific rationale, blood-brain barrier penetration, safety and tolerability, and evidence of efficacy in animal models or humans. Agents were prioritized based on these criteria. RESULTS: The authors identified 59 potential neuroprotective compounds, proposed by 42 clinicians and scientists from 13 countries. After systematic reviews using the specified criteria they found 12 compounds to be attractive candidates for further clinical trials in PD. CONCLUSIONS: Several potential neuroprotective compounds, representing a wide range of mechanisms, are available and merit further investigation in PD.