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.     

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.     

Sex differences in hippocampal estradiol-induced N-methyl-D-aspartic acid binding and ultrastructural localization of estrogen receptor-alpha

Estradiol increases dendritic spine density and synaptogenesis in the CA1 region of the female hippocampus. This effect is specific to females, as estradiol-treated males fail to show increases in hippocampal spine density. Estradiol-induced spinogenesis in the female is dependent upon upregulation of the N-methyl-D-aspartic acid (NMDA) receptor as well as on non-nuclear estrogen receptors (ER), including those found in dendrites. Thus, in the male, the inability of estradiol to induce spinogenesis may be related to a failure of estradiol to increase hippocampal NMDA receptors as well as a paucity of dendritic ER. In the first experiment, we sought to investigate this possibility by assessing NMDA receptor binding, using [(3)H]-glutamate autoradiography, in estradiol-treated males and females. We found that while estradiol increases NMDA binding in gonadectomized females, estradiol fails to modulate NMDA binding in gonadectomized males. To further investigate sex differences in the hippocampus, we conducted a second separate, but related, ultrastructural study in which we quantified ERalpha-immunoreactivity (ERalpha-ir) in neuronal profiles in the CA1 region of the hippocampus in intact males and females in diestrus and proestrus. Consistent with previous reports in the female, we found ERalpha-ir in several extranuclear sites including dendrites, spines, terminals and axons. Statistical analyses revealed that females in proestrus had a 114.3% increase in ERalpha-labeled dendritic spines compared to females in diestrus and intact males. Taken together, these studies suggest that both the ability of estrogen to increase NMDA binding in the hippocampus and the presence of ERalpha in dendritic spines may contribute to the observed sex difference in estradiol-induced hippocampal spinogenesis.     

Down-regulation of dendritic spine and glutamic acid decarboxylase 67 expressions in the reelin haploinsufficient heterozygous reeler mouse

Heterozygous reeler mice (HRM) haploinsufficient for reelin express approximately 50% of the brain reelin content of wild-type mice, but are phenotypically different from both wild-type mice and homozygous reeler mice. They exhibit, (i) a down-regulation of glutamic acid decarboxylase 67 (GAD(67))-positive neurons in some but not every cortical layer of frontoparietal cortex (FPC), (ii) an increase of neuronal packing density and a decrease of cortical thickness because of neuropil hypoplasia, (iii) a decrease of dendritic spine expression density on basal and apical dendritic branches of motor FPC layer III pyramidal neurons, and (iv) a similar decrease in dendritic spines expressed on the basal dendrite branches of CA1 pyramidal neurons of the hippocampus. To establish whether the defect of GAD(67) down-regulation observed in HRM is responsible for neuropil hypoplasia and decreased dendritic spine density, we studied heterozygous GAD(67) knockout mice (HG(67)M). These mice exhibited a down-regulation of GAD(67) mRNA expression in FPC (about 50%), but they expressed normal amounts of reelin and had no neuropil hypoplasia or down-regulation of dendritic spine expression. These findings, coupled with electron-microscopic observations that reelin colocalizes with integrin receptors on dendritic spines, suggest that reelin may be a factor in the dynamic expression of cortical dendritic spines perhaps by promoting integrin receptor clustering. These findings are interesting because the brain neurochemical and neuroanatomical phenotypic traits exhibited by the HRM are in several ways similar to those found in postmortem brains of psychotic patients.     

Dendritic spine plasticity in gonadatropin-releasing hormone (GnRH) neurons activated at the time of the preovulatory surge

GnRH neuron activity is dependent on gonadal steroid hormone feedback. Altered synaptic input may be one mechanism by which steroids modify GnRH neuron activity. In other neuronal populations, steroid hormones have been shown to elicit profound effects on dendritic spine density, a measure of excitatory synaptic input. The present study examined gonadal steroid feedback effects on GnRH neuron spine density in female GnRH-green fluorescent protein (GFP) mice. Immunocytochemical labeling of GFP in this model reveals fine morphological details of GnRH neurons. Spine density and other features were quantified by confocal analysis. Ovariectomy resulted in a significant reduction in somatic spine density (27%, P < 0.05) compared with sham-operated diestrous females. However, dendritic spine density was unaltered. Positive feedback effects of estradiol on spine density were investigated using a protocol to mimic the GnRH/LH surge. Ten GnRH-GFP mice underwent an established protocol, receiving either estradiol benzoate (1 μg per 20 g body weight) or vehicle (n = 5/group) 32 h prior to being killed during the expected surge. Double-label immunofluorescence showed that all estradiol-treated females expressed cFos in a subpopulation of GnRH neurons. Spine density was determined by confocal analysis of activated (cFos-positive, n = 10 neurons/animal) and nonactivated (cFos-negative, n = 10 neurons/animal) GnRH neurons from estradiol-treated animals and for GnRH neurons (n = 20 neurons/animal) from nonsurged controls (all cFos negative). Activated GnRH neurons (cFos positive) showed a dramatic 60% increase in total spine density (0.78 ± 0.06 spines/μm) compared with nonactivated GnRH neurons (0.50 ± 0.01 spines/μm) in estradiol-treated animals (P < 0.001). Both somatic and dendritic spine density was significantly increased. Spine density was not different between nonactivated GnRH neurons from surged animals (0.50 ± 0.01 spines/μm) and GnRH neurons from nonsurged animals (0.51 ± 0.06 spines/μm). These data demonstrate that positive feedback levels of estradiol stimulate a robust increase in spine density specifically in those GnRH neurons that are activated at the time of the GnRH/LH surge.     

Rapid modulation of spine morphology by the 5-HT2A serotonin receptor through kalirin-7 signaling

The 5-HT_2A serotonin receptor is the most abundant serotonin receptor subtype in the cortex and is predominantly expressed in pyramidal neurons. The 5-HT_2A receptor is a target of several hallucinogens, antipsychotics, anxiolytics, and antidepressants, and it has been associated with several psychiatric disorders, conditions that are also associated with aberrations in dendritic spine morphogenesis. However, the role of 5-HT_2A receptors in regulating dendritic spine morphogenesis in cortical neurons is unknown. Here we show that the 5-HT_2A receptor is present in a subset of spines, in addition to dendritic shafts. It colocalizes with PSD-95 and with multiple PDZ protein-1 (MUPP1) in a subset of dendritic spines of rat cortical pyramidal neurons. MUPP1 is enriched in postsynaptic density (PSD) fractions, is targeted to spines in pyramidal neurons, and enhances the localization of 5-HT_2A receptors to the cell periphery. 5-HT_2A receptor activation by the 5-HT₂ receptor agonist DOI induced a transient increase in dendritic spine size, as well as phosphorylation of p21-activated kinase (PAK) in cultured cortical neurons. PAK is a downstream target of the neuronal Rac guanine nucleotide exchange factor (RacGEF) kalirin-7 that is important for spine remodeling. Kalirin-7 regulates dendritic spine morphogenesis in neurons but its role in neuromodulator signaling has not been investigated. We show that peptide interference that prevents the localization of kalirin-7 to the postsynaptic density disrupts DOI-induced PAK phosphorylation and spine morphogenesis. These results suggest a potential role for serotonin signaling in modulating spine morphology and kalirin-7''s function at cortical synapses.     

Effects of chronic choline and lecithin on mouse hippocampal dendritic spine density

Dendritic spines, which project from the dendrites of central neurons, are thought to contribute to the amount of contact area available for synaptic connections. The density of these spines has been found to correlate with learning and memory function, and there is a progressive decrease in dendritic spine density with aging. In addition, experimental animals given a choline-enriched diet have an increase in neocortical spine density compared to controls. In this study, the dendritic spine density of hippocampal pyramidal cells was examined in aged mice which had received life-long choline enriched, choline deficient or lecithin enriched diets. These treatments had no effect on hippocampal dendritic spine density compared to control. The results indicate that dietary supplementation may have different effects in different brain areas and that the relative increase in learning and memory function in aged animals given a choline or lecithin enriched diet is not due to an increase in hippocampal dendritic spine density.     

Lipocalin-2 controls neuronal excitability and anxiety by regulating dendritic spine formation and maturation

Psychological stress causes adaptive changes in the nervous system directed toward maintaining homoeostasis. These biochemical and structural mechanisms regulate animal behavior, and their malfunction may result in various forms of affective disorders. Here we found that the lipocalin-2 (Lcn2) gene, encoding a secreted protein of unknown neuronal function, was up-regulated in mouse hippocampus following psychological stress. Addition of lipocalin-2 to cultured hippocampal neurons reduced dendritic spine actin's mobility, caused retraction of mushroom spines, and inhibited spine maturation. These effects were further enhanced by inactivating iron-binding residues of Lcn-2, suggesting that they were facilitated by the iron-free form of Lcn-2. Concurrently, disruption of the Lcn2 gene in mice promoted stress-induced increase in spine density and caused an increase in the proportion of mushroom spines. The above changes correlated with higher excitability of CA1 principal neurons and with elevated stress-induced anxiety in Lcn-2(-/-) mice. Our study demonstrates that lipocalin-2 promotes stress-induced changes in spine morphology and function to regulate neuronal excitability and anxiety.     

Progesterone treatment that either blocks or augments the estradiol-induced gonadotropin-releasing hormone surge is associated with different patterns of hypothalamic neural activation.

Progesterone can either augment or inhibit the surge of gonadotropin-releasing hormone (GnRH) that drives the preovulatory luteinizing hormone (LH) surge. This study investigated the central mechanisms through which progesterone might achieve these divergent effects by examining the effects of exogenous steroids on the activation of GnRH neurons and non-GnRH-immunopositive cells in the preoptic area/anterior hypothalamus of steroid-treated ovariectomized ewes. Fos expression (an index of cellular activation) was examined during the estradiol-induced GnRH surge in ewes treated with progesterone using regimes that have been reported to either augment (progesterone pretreatment) or inhibit (progesterone treatment at the time of the surge-inducing estradiol increment) the GnRH surge. Control groups received either no progesterone pretreatment or no surge-inducing estradiol increment. Induction of an LH surge was associated with a significant (p < 0.0001) increase in the proportion of activated GnRH neurons, irrespective of whether ewes received progesterone pretreatment. However, the number of non-GnRH-immunopositive cells activated during the surge was significantly (p < 0.0001) increased in ewes that received the progesterone pretreatment. By contrast, the proportion of GnRH neurons and non-GnRH-immunopositive cells that expressed Fos was significantly (p < 0.0001) reduced in ewes in which the surge was inhibited by progesterone compared to ewes in which a surge was stimulated. These data indicate that (1) progesterone pretreatment increases the activation of non-GnRH cells during the estradiol-induced surge, but does not affect the proportion of GnRH neurons activated and (2) when administered concurrently with a surge-inducing estradiol increment, progesterone prevents the activation of GnRH neurons and non-GnRH cells that is normally associated with the estradiol-induced surge. Therefore, progesterone does not appear to augment the GnRH surge by increasing the proportion of GnRH neurons that are activated by estradiol, whereas inhibition of the GnRH surge involves prevention of the activation of GnRH neurons. Thus, the augmentation and inhibition of the GnRH surge by progesterone appear to be regulated via different effects on the GnRH neurosecretory system.     

Glutamic acid decarboxylase messenger ribonucleic acid is regulated by estradiol and progesterone in the hippocampus.

Ovarian steroids modulate learning, memory, and epileptic seizure activity, functions that are mediated in part by the hippocampus. Normal function depends on precise interactions between the inhibitory gamma-aminobutyric acid (GABA)ergic and excitatory glutamatergic neurons of the hippocampus. To determine whether estradiol and progesterone interact with GABAergic neurons, the levels of mRNA for glutamic acid decarboxylase (GAD), the rate-limiting enzyme for GABA synthesis, were measured by in situ hybridization histochemistry with 35S-labeled riboprobes complimentary to the feline GAD cDNA. The levels of mRNA for GAD were analyzed in selected region of the dorsal hippocampus and medial basal hypothalamus in ovariectomized, ovariectomized estradiol-treated, and ovariectomized estradiol- and progesterone-treated rats. In estradiol-treated rats, GAD mRNA levels increased in GABAergic neurons associated with the CA1 pyramidal cell layer, but not in the stratum oriens of CA1 or any other region of the hippocampus. Estradiol plus progesterone treatment reversed the estradiol-induced increase in GAD mRNA in CA1 and induced a small decrease in the hilus. No effect of estradiol or progesterone was observed in the dorsomedial, ventromedial, or arcuate nuclei of the hypothalamus. Estradiol or progesterone may alter cognitive performance and seizure activity by increasing or decreasing, respectively, the activity of GABAergic neurons in the hippocampus.     

Interactive effects of age and estrogen on cognition and pyramidal neurons in monkey prefrontal cortex

We previously reported that long-term cyclic estrogen (E) treatment reverses age-related impairment of cognitive function mediated by the dorsolateral prefrontal cortex (dlPFC) in ovariectomized (OVX) female rhesus monkeys, and that E induces a corresponding increase in spine density in layer III dlPFC pyramidal neurons. We have now investigated the effects of the same E treatment in young adult females. In contrast to the results for aged monkeys, E treatment failed to enhance dlPFC-dependent task performance relative to vehicle control values (group young OVX+Veh) but nonetheless led to a robust increase in spine density. This response was accompanied by a decline in dendritic length, however, such that the total number of spines per neuron was equivalent in young OVX+Veh and OVX+E groups. Robust effects of chronological age, independent of ovarian hormone status, were also observed, comprising significant age-related declines in dendritic length and spine density, with a preferential decrease in small spines in the aged groups. Notably, the spine effects were partially reversed by cyclic E administration, although young OVX+Veh monkeys still had a higher complement of small spines than did aged E treated monkeys. In summary, layer III pyramidal neurons in the dlPFC are sensitive to ovarian hormone status in both young and aged monkeys, but these effects are not entirely equivalent across age groups. The results also suggest that the cognitive benefit of E treatment in aged monkeys is mediated by enabling synaptic plasticity through a cyclical increase in small, highly plastic dendritic spines in the primate dlPFC.     

beta-Catenin regulates excitatory postsynaptic strength at hippocampal synapses

The precise contribution of the cadherin-beta-catenin synapse adhesion complex in the functional and structural changes associated with the pre- and postsynaptic terminals remains unclear. Here we report a requirement for endogenous beta-catenin in regulating synaptic strength and dendritic spine morphology in cultured hippocampal pyramidal neurons. Ablating beta-catenin after the initiation of synaptogenesis in the postsynaptic neuron reduces the amplitude of spontaneous excitatory synaptic responses without a concurrent change in their frequency and synapse density. The normal glutamatergic synaptic response is maintained by postsynaptic beta-catenin in a cadherin-dependent manner and requires the C-terminal PDZ-binding motif of beta-catenin but not the link to the actin cytoskeleton. In addition, ablating beta-catenin in postsynaptic neurons accompanies a block of bidirectional quantal scaling of glutamatergic responses induced by chronic activity manipulation. In older cultures at a time when neurons have abundant dendritic spines, neurons ablated for beta-catenin show thin, elongated spines and reduced proportion of mushroom spines without a change in spine density. Collectively, these findings suggest that the cadherin-beta-catenin complex is an integral component of synaptic strength regulation and plays a basic role in coupling synapse function and spine morphology.     

Stress duration modulates the spatiotemporal patterns of spine formation in the basolateral amygdala

It has long been hypothesized that morphological and numerical alterations in dendritic spines underlie long-term structural encoding of experiences. Here we investigate the efficacy of aversive experience in the form of acute immobilization stress (AIS) and chronic immobilization stress (CIS) in modulating spine density in the basolateral amygdala (BLA) of male rats. We find that CIS elicits a robust increase in spine density across primary and secondary branches of BLA spiny neurons. We observed this CIS-induced spinogenesis in the BLA 1 d after the termination of CIS. In contrast, AIS fails to affect spine density or dendritic arborization when measured 1 d later. Strikingly, the same AIS causes a gradual increase in spine density 10 d later but without any effect on dendritic arbors. Thus, by modulating the duration of immobilization stress, it is possible to induce the formation of new spines without remodeling dendrites. However, unlike CIS-induced spine formation, the gradual increase in spine density 10 d after a single exposure to AIS is localized on primary dendrites. Finally, this delayed induction of BLA spinogenesis is paralleled by a gradual development of anxiety-like behavior on the elevated plus-maze 10 d after AIS. These findings demonstrate that stressful experiences can lead to the formation of new dendritic spines in the BLA, which is believed to be a locus of storage for fear memories. Our results also suggest that stress may facilitate symptoms of chronic anxiety disorders like post-traumatic stress disorder by enhancing synaptic connectivity in the BLA.     

Reversal of long-term dendritic spine alterations in Alzheimer disease models

Synapse loss is strongly correlated with cognitive impairment in Alzheimer''s disease (AD). We have previously reported the loss of dendritic spines and the presence of dystrophic neurites in both the hippocampi of transgenic mice overexpressing amyloid precursor protein (APP) and in the human brain affected with AD. In the studies reported here we have asked whether the acute alterations in dendritic spines induced by Aβ, as well as the chronic loss of spine density seen in hAPP transgenic mice, are reversible by treatments that restore the cAMP/PKA/CREB signaling pathway or proteasome function to control levels. The results show that both rolipram and TAT-HA-Uch-L1 restore spine density to near control conditions, even in elderly mice. The results suggest that changes in dendritic structure and function that occur after Aβ elevation are reversible even after long periods of time, and that one could envision therapeutic approaches to AD based on this restoration that could work independently of therapies aimed at lowering Aβ levels in the brain.     

Dendritic growth and spine formation in response to estrogen in the developing Purkinje cell

Neurosteroids are synthesized de novo in the brain, and the cerebellar Purkinje cell is a major site for neurosteroid formation. We have demonstrated that the Purkinje cell possesses intranuclear receptor for progesterone and actively produces progesterone de novo from cholesterol only during rat neonatal life, when cerebellar cortical formation occurs dramatically. We have further demonstrated that progesterone promotes dendritic growth, spinogenesis, and synaptogenesis via its receptor in this neuron in the neonate. On the other hand, estrogen may also play an important role in the process of cerebellar cortical formation, because the neonatal rat Purkinje cell possesses estrogen receptor (ER)beta. However, estrogen formation in the neonatal cerebellum is still unclear. In this study, we therefore analyzed the biosynthesis and action of estrogen in Purkinje cells during neonatal life. RT-PCR-Southern and in situ hybridization analyses showed that Purkinje cells expressed the key enzyme of estrogen formation, cytochrome P450 aromatase, in neonatal rats. A specific enzyme immunoassay for estradiol further indicated that cerebellar estradiol concentrations in the neonate were significantly higher than those in the prepuberty and adult. Both in vitro and in vivo studies with newborn rats showed that estradiol promoted dose-dependent dendritic growth of Purkinje cells. Estradiol also increased the density of Purkinje dendritic spines. These effects were inhibited by the ER antagonist tamoxifen. These results suggest that estradiol in the developing Purkinje cell promotes dendritic growth and spinogenesis via ERbeta in this neuron. Estradiol as well as progesterone may contribute to the growth of Purkinje cells during the cerebellar cortical formation.     

Postsynaptic TrkB signaling has distinct roles in spine maintenance in adult visual cortex and hippocampus

In adult primary visual cortex (V1), dendritic spines are more persistent than during development. Brain-derived neurotrophic factor (BDNF) increases synaptic strength, and its levels rise during cortical development. We therefore asked whether postsynaptic BDNF signaling through its receptor TrkB regulates spine persistence in adult V1. This question has been difficult to address because most methods used to alter TrkB signaling in vivo affect cortical development or cannot distinguish between pre- and postsynaptic mechanisms. We circumvented these problems by employing transgenic mice expressing a dominant negative TrkB-EGFP fusion protein in sparse pyramidal neurons of the adult neocortex and hippocampus, producing a Golgi-staining-like pattern. In adult V1, expression of dominant negative TrkB-EGFP resulted in reduced mushroom spine maintenance and synaptic efficacy, accompanied by an increase in long and thin spines and filopodia. In contrast, mushroom spine maintenance was unaffected in CA1, indicating that TrkB plays fundamentally different roles in structural plasticity in these brain areas.     

Acid-sensing ion channel 1a is a postsynaptic proton receptor that affects the density of dendritic spines

Extracellular proton concentrations in the brain may be an important signal for neuron function. Proton concentrations change both acutely when synaptic vesicles release their acidic contents into the synaptic cleft and chronically during ischemia and seizures. However, the brain receptors that detect protons and their physiologic importance remain uncertain. Using organotypic hippocampal slices and biolistic transfection, we found the acid-sensing ion channel 1a (ASIC1a), localized in dendritic spines where it functioned as a proton receptor. ASIC1a also affected the density of spines, the postsynaptic site of most excitatory synapses. Decreasing ASIC1a reduced the number of spines, whereas overexpressing ASIC1a had the opposite effect. Ca(2+)-mediated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) signaling was probably responsible, because acid evoked an ASIC1a-dependent elevation of spine intracellular Ca(2+) concentration, and reducing or increasing ASIC1a levels caused parallel changes in CaMKII phosphorylation in vivo. Moreover, inhibiting CaMKII prevented ASIC1a from increasing spine density. These data indicate that ASIC1a functions as a postsynaptic proton receptor that influences intracellular Ca(2+) concentration and CaMKII phosphorylation and thereby the density of dendritic spines. The results provide insight into how protons influence brain function and how they may contribute to pathophysiology.     

Spinophilin regulates the formation and function of dendritic spines

Spinophilin, a protein that interacts with actin and protein phosphatase-1, is highly enriched in dendritic spines. Here, through the use of spinophilin knockout mice, we provide evidence that spinophilin modulates both glutamatergic synaptic transmission and dendritic morphology. The ability of protein phosphatase-1 to regulate the activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors was reduced in spinophilin knockout mice. Consistent with altered glutamatergic transmission, spinophilin-deficient mice showed reduced long-term depression and exhibited resistance to kainate-induced seizures and neuronal apoptosis. In addition, deletion of the spinophilin gene caused a marked increase in spine density during development in vivo as well as altered filopodial formation in cultured neurons. In conclusion, spinophilin appears to be required for the regulation of the properties of dendritic spines.