Melatonin regulates neuronal plasticity in the hippocampus

The influence of melatonin on hippocampal evoked potentials initiated by low- and high-frequency electrical stimulations and by two pulses applied in rapid succession was investigated. In confirmation of our previous studies, melatonin attenuated the population spike triggered by low-frequency stimulation (0.03 Hz). High-frequency stimulation (HFS; 100 Hz for 1 sec, three times every 10 sec), which in control slices permanently facilitated neuronal excitability (347% +/- 32%), was also able to amplify the melatonin-depressed potential (467.8% +/- 59.6%). Because melatonin is a hydrophobic molecule, it was dissolved and applied in ethanol. Ethanol (0.4%) by itself reduced the magnitude of HFS-induced potentiation (233.5% +/- 16.8%). The slices stimulated with two pulses separated with a delay longer than 15 msec demonstrated a facilitation of the response to the second stimuli (paired-pulse facilitation; PPF). The influence of melatonin (100 microM) on PPF was biphasic: Shortly after addition of melatonin, PPF was briefly (5-10 min) reversed to paired-pulse inhibition (PPI), which gradually returned to a stable PPF. Ethanol (0.4%) applied without melatonin exerted only a marginal, facilitatory effect on PPF. The delay between two successively applied pulses, shorter than 13 msec, resulted in attenuation of the response to the second stimuli (PPI). Melatonin (100 microM) reversed the attenuation of the second potential within 15-20 min following its application. Ethanol applied by itself at the concentration of 0.4% temporarily (5-10 min), but significantly, depressed the second potential. These results demonstrate the ability of melatonin to modulate specific forms of plasticity in hippocampal pyramidal neurons.     

Genetic and pharmacological inhibition of two-pore domain potassium channel TREK-1 alters depression-related behaviors and neuronal plasticity in the hippocampus in mice

IntroductionThe two‐pore domain potassium channel TREK‐1 is a member of background K⁺ channels that are thought to provide baseline regulation of membrane excitability. Recent studies have highlighted the putative role of TREK‐1 in the action of antidepressants, and its antagonists might be potentially effective antidepressants. However, the mechanisms underlying the actions of TREK‐1 are not yet fully understood.MethodsThe expression of TREK‐1 was examined in a mouse model of chronic unpredictable mild stress (CUMS) using immunoblotting. Neuron‐specific genetic manipulation of TREK‐1 was performed through adeno‐associated virus. Behavioral tests were performed to evaluate depression‐related behaviors. Electrophysiological recordings were used to evaluate synaptic plasticity. Golgi staining was used to examine neuroplasticity.ResultsTREK‐1 expression was increased in the mouse hippocampus after CUMS. Knockdown of TREK‐1 in hippocampal neurons significantly attenuated depressive‐like behaviors and prevented the decrease of CUMS‐induced synaptic proteins in mice. Further examination indicated that neuron‐specific knockdown of TREK‐1 in the hippocampus prevented stress‐induced impairment of glutamatergic synaptic transmission in the CA1 region. Moreover, chronic TREK‐1 inhibition protected against CUMS‐induced depressive‐like behaviors and impairment of synaptogenesis in the hippocampus.ConclusionOur results indicate a role for TREK‐1 in the modulation of synaptic plasticity in a mouse model of depression. These findings will provide insight into the pathological mechanism of depression and further evidence for a novel target for antidepressant treatment.     

Postnatal development of synaptopodin expression in the rodent hippocampus

Synaptopodin is an actin-binding protein of renal podocytes and dendritic spines. We have recently shown that synaptopodin is localized to the spine apparatus, a characteristic organelle of dendritic spines on forebrain neurons. Synaptopodin-deficient mice do not form spine apparatuses, indicating a role of synaptopodin in the formation of this organelle. Here we studied the development of synaptopodin expression in the postnatal rat hippocampus. At birth, synaptopodin mRNA is mainly expressed in CA3 pyramidal neurons. At postnatal day (P) 6, synaptopodin mRNA expression is still strongest in CA3 but is now also found in CA1 pyramidal neurons and granule cells of the suprapyramidal blade of the dentate gyrus. At P9, an almost adult pattern is seen with synaptopodin mRNA expressed by virtually all principal neurons. While synaptopodin mRNA was restricted to cell somata, immunostaining for synaptopodin protein labeled dendritic layers. At birth, no immunoreactivity was visible, while at P5 a weak staining mainly in stratum oriens was observed. At P9, immunolabeling was still strongest in stratum oriens followed by the molecular layer of the dentate gyrus. The adult pattern with strong labeling of all dendritic layers was reached by P12. Together these findings show that synaptopodin expression follows the well-known sequence of hippocampal principal neuron development. Unexpectedly, we also observed synaptopodin mRNA expression in a small population of interneurons as revealed by double labeling with interneuron markers. However, no immunolabeling for synaptopodin was observed in identified interneurons, confirming that the protein is mainly present in spine-bearing principal cells.     

Arc interacts with microtubules/microtubule-associated protein 2 and attenuates microtubule-associated protein 2 immunoreactivity in the dendrites

Arc, activity-regulated cytoskeleton-associated gene, is an immediate early gene, and its expression is regulated by a variety of stimuli, such as electric stimulation and methamphetamine. The function of Arc, however, is unknown. To explore this function, we carried out expression experiments by transfecting green fluorescent protein (GFP)-Arc constructs or by using a protein transduction system in hippocampal cultured neurons. We found that the overexpression of Arc as well as Arc induction by seizure in vivo decreased microtubule-associated protein 2 (MAP2) staining in the dendrites by immunocytochemistry, although MAP2 content was not changed on Western blot. Furthermore, Arc interacted with newly polymerized microtubules and MAP2, leading to blocking of the epitope of MAP2. The data suggest that Arc increased by synaptic activities would trigger dendritic remodeling by interacting with cytoskeletal proteins.     

Melatonin alters the metabolism of the β-amyloid precursor protein in the neuroendocrine cell line PC12

The deposition of amyloid plaques in brain parenchyma is one of the major pathological hallmarks of Alzheimer’s disease (AD). The amyloid in senile plaques is composed of the amyloid β-peptide (Aβ) of 39–43 amino acid residues derived from a larger β-amyloid precursor protein (βAPP). Soluble derivatives of βAPP (sAPP) lacking the cytoplasmic tail, transmembrane domain, and a small portion of the extracellular domain are generated proteolytically by “secretases.” Using cell cultures, the authors analyzed the level of sAPP in neuroblastoma and pheochromocytoma (PC12) cells by immunoblotting samples from conditioned media and cell lysates. Normal levels of secretion of sAPP into conditioned media were severely inhibited by treating cells with melatonin (3–4 mM). The inhibitory effect of melatonin on the secretion of sAPP can be reversed. When the cells that were pretreated with melatonin for 10 h were washed, the normal level of secretion of sAPP was restored. Northern blot analyses indicated that the treatment of PC12 cells with melatonin resulted in a significant decrease in the level of mRNA encoding βAPP, β-actin, and glyceraldehyde-3-phosphate dehydrogenase, and that the treatment of a human neuroblastoma cell line with melatonin resulted in no change in levels of these messages. The secretion of sAPP into the conditioned medium was substantially reduced in the differentiated cells similar to reductions observed in melatonin-treated undifferentiated PC12 cells. Melatonin was found to potentiate the nerve growth factor-mediated differentiation in PC12 cells at 24 h. Taken together, these data suggest that melatonin regulates the metabolism of βAPP and other housekeeping genes in a cell-type specific manner, and that melatonin accelerates the early process of neuronal differentiation.     

Postnatal developmental expression of regulator of G protein signaling 14 (RGS14) in the mouse brain

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and mitogen‐activated protein kinase (MAPK) signaling pathways. In the adult mouse brain, RGS14 mRNA and protein are found almost exclusively in hippocampal CA2 neurons. We have shown that RGS14 is a natural suppressor of CA2 synaptic plasticity and hippocampal‐dependent learning and memory. However, the protein distribution and spatiotemporal expression patterns of RGS14 in mouse brain during postnatal development are unknown. Here, using a newly characterized monoclonal anti‐RGS14 antibody, we demonstrate that RGS14 protein immunoreactivity is undetectable at birth (P0), with very low mRNA expression in the brain. However, RGS14 protein and mRNA are upregulated during early postnatal development, with protein first detected at P7, and both increasing over time until reaching highest sustained levels throughout adulthood. Our immunoperoxidase data demonstrate that RGS14 protein is expressed in regions outside of hippocampal CA2 during development including the primary olfactory areas, the anterior olfactory nucleus and piriform cortex, and the olfactory associated orbital and entorhinal cortices. RGS14 is also transiently expressed in neocortical layers II/III and V during postnatal development. Finally, we show that RGS14 protein is first detected in the hippocampus at P7, with strongest immunoreactivity in CA2 and fasciola cinerea and sporadic immunoreactivity in CA1; labeling intensity in hippocampus increases until adulthood. These results show that RGS14 mRNA and protein are upregulated throughout postnatal mouse development, and RGS14 protein exhibits a dynamic localization pattern that is enriched in hippocampus and primary olfactory cortex in the adult mouse brain. J. Comp. Neurol. 522:186–203, 2014. © 2013 Wiley Periodicals, Inc.     

Different phosphatase-dependent mechanisms mediate long-term depression and depotentiation of long-term potentiation in mouse hippocampal CA1 area

Two types of synaptic depression have been described in the hippocampus, long-term depression and depotentiation of long-term potentiation known to recruit the serine/threonine protein phosphatases PP1, PP2A and PP2B (calcineurin). The contribution of each of these protein phosphatases is controversial. To examine the role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in long-term depression and depotentiation, we analysed the effect of genetically inhibiting calcineurin reversibly in the hippocampus, using the doxycycline-dependent rtTA system in transgenic mice. We show that reducing calcineurin activity has no effect on long-term depression but reversibly affects depotentiation. Consistently, the calcineurin inhibitor FK-506 reproduces the depotentiation impairment observed in the mutant mice but does not affect long-term depression in control animals. In contrast, the PP1/PP2A inhibitor okadaic acid fully blocks both long-term depression and depotentiation. These data demonstrate that the nature of signalling cascades induced by synaptic activity depends on the initial synaptic state. While depression of potentiated synaptic responses requires activation of PP1/PP2A and/or calcineurin, depression of basal synaptic responses depends only on PP1/PP2A activation.     

Partial inhibition of PP1 alters bidirectional synaptic plasticity in the hippocampus

Synaptic plasticity is an important cellular mechanism that underlies memory formation. In brain areas involved in memory such as the hippocampus, long-term synaptic plasticity is bidirectional. Major forms of bidirectional plasticity encompass long-term potentiation (LTP), LTP reversal (depotentiation) and long-term depression (LTD). Protein kinases and protein phosphatases are important players in the induction of both LTP and LTD, and the serine/threonine protein phosphatase-1 (PP1), in particular, has emerged as a key phosphatase in these processes. The goal of the present study was to assess the contribution of PP1 to bidirectional plasticity and examine the impact of a partial inhibition of PP1 on LTP, LTD and depotentiation in the mouse hippocampus. For this, we used transgenic mice expressing an active PP1 inhibitor (I-1*) inducibly in forebrain neurons. We show that partial inhibition of PP1 by I-1* expression alters the properties of bidirectional plasticity by inducing a shift of synaptic responses towards potentiation. At low-frequency stimulation, PP1 inhibition decreases LTD in a frequency-dependent fashion. It favours potentiation over depression at intermediate frequencies and increases LTP at high frequency. Consistently, it also impairs depotentiation. These results indicate that the requirement of bidirectional plasticity for PP1 is frequency-dependent and that a broad range of plasticity is negatively constrained by PP1, a feature that may correlate with the property of PP1 to constrain learning efficacy and promote forgetting.     

Intracerebroventricular administration of growth hormone induces morphological changes in pyramidal neurons of the hippocampus and prefrontal cortex in adult rats

A growing body of evidence suggests that growth hormone (GH) affects synaptic plasticity at both the molecular and electrophysiological levels. However, unclear is whether plasticity that is stimulated by GH is associated with changes in neuron structure. This study investigated the effect of intracerebroventricular (ICV) administration of GH on the morphology of pyramidal neurons of the CA1 region of the dorsal hippocampus and layer III of the prefrontal cortex. Male Wistar rats received daily ICV injections of GH (120 ng) for 7 days, and they were euthanized 21 days later. Changes in neuronal morphology were evaluated using Golgi‐Cox staining and subsequent Sholl analysis. GH administration increased total dendritic length in the CA1 region of the dorsal hippocampus and prefrontal cortex. The Sholl analysis revealed an increase in dendritic length of the third to eighth branch orders in the hippocampus and from the third to sixth branch orders in the prefrontal cortex. Interestingly, GH treatment increased the density of dendritic spines in both brain regions, favoring the presence of mushroom‐like spines only in the CA1 hippocampal region. Our results indicated that GH induces changes in the length of dendritic trees and the density of dendritic spines in two high‐plasticity brain regions, suggesting that GH‐induced synaptic plasticity at the molecular and electrophysiological levels may be associated with these structural changes in neurons.     

SCYL pseudokinases in neuronal function and survival

The generation of mice lacking SCYL1 or SCYL2 and the identiifcation ofScyl1 as the causative gene in the motor neuron disease mouse model muscle deifcient (Scyl1mdf/mdf) demonstrated the importance of the SCY1-like family of protein pseudokinases in neuronal function and survival. Several essential cellular processes such as intracellular trafifcking and nuclear tRNA export are thought to be regulated by SCYL proteins. How-ever, whether deregulation of these processes contributes to the neurodegenerative processes associated with the loss of SCYL proteins is still unclear. Here, I brielfy review the evidence supporting that SCYL proteins play a role in these processes and discuss their possible involvement in the neuronal functions of SCYL pro-teins. I also propose ways to determine the importance of these pathways for the functions of SCYL proteins in vivo.     

M1 muscarinic receptor signaling in mouse hippocampus and cortex

The five subtypes (M1-M5) of muscarinic acetylcholine receptors signal through G(alpha)(q) or G(alpha)(i)/G(alpha)(o). M1, M3 and M5 receptors couple through G(alpha)(q) and function predominantly as postsynaptic receptors in the central nervous system. M1 and M3 receptors are localized to brain regions involved in cognition, such as hippocampus and cortex, but their relative contribution to function has been difficult to ascertain due to the lack of subtype specific ligands. A functional and genetic approach was used to identify the predominant muscarinic receptor subtype(s) mediating responses in mouse hippocampus and cortex, as well as the relative degree of spare muscarinic receptors in hippocampus. The nonselective muscarinic agonist oxotremorine-M stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding in a concentration dependent manner with a Hill slope near unity in wild type mouse hippocampus and cortex. Muscarinic receptor stimulated G(alpha)(q)/11-specific GTP-gamma-35S binding was virtually abolished in both the hippocampus and cortex of M1 receptor knockout (KO) mice. In contrast, there was no loss of signaling in M3 receptor KO mice in either brain region. Muscarinic receptor reserve in wildtype mouse hippocampus was measured by Furchgott analysis after partial receptor alkylation with propylbenzylcholine mustard. Occupation of just 15% of the M1 receptors in mouse hippocampus was required for maximal efficacy of oxotremorine-M-stimulated GTP-gamma-35S binding indicating a substantial level of spare receptors. These findings support a role for the M1 receptor subtype as the primary G(alpha)(q)/11-coupled muscarinic receptor in mouse hippocampus and cortex.     

Neonatal handling selectively alters the phosphorylation of a 47,000 mol. wt. protein in male rat hippocampus

Beginning on postnatal day 1, rat pups were handled for 7 min daily for 23-30 days. This treatment diminished the in vitro phosphorylation of phosphoprotein F-1 [mol. wt. approximately 47 K daltons, protein B-50] in the hippocampus of male rats. Other major phosphoproteins (D-1-2. mol. wt. 80-86 K daltons: E-2-3. mol. wt. 50-55 K daltons) were not influenced by handling. These findings confirm and extend the results of Holmes et al. who observed a decrease in ECS-induced protein kinase activity subsequent to handling.     

Prolonged ampakine exposure prunes dendritic spines and increases presynaptic release probability for enhanced long‐term potentiation in the hippocampus

CX 546, an allosteric positive modulator of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid‐type ionotropic glutamate receptors (AMPARs), belongs to a drug class called ampakines. These compounds have been shown to enhance long‐term potentiation (LTP), a cellular model of learning and memory, and improve animal learning task performance, and have augmented cognition in neurodegenerative patients. However, the chronic effect of CX546 on synaptic structures has not been examined. The structure and integrity of dendritic spines are thought to play a role in learning and memory, and their abnormalities have been implicated in cognitive disorders. In addition, their structural plasticity has been shown to be important for cognitive function, such that dendritic spine remodeling has been proposed as the morphological correlate for LTP. Here, we tested the effect of CX546 on dendritic spine remodeling following long‐term treatment. We found that, with prolonged CX546 treatment, organotypic hippocampal slice cultures showed a significant reduction in CA3–CA1 excitatory synapse and spine density. Electrophysiological approaches revealed that the CA3–CA1 circuitry compensates for this synapse loss by increasing synaptic efficacy through enhancement of presynaptic release probability. CX546‐treated slices showed prolonged and enhanced potentiation upon LTP induction. Furthermore, structural plasticity, namely spine head enlargement, was also more pronounced after CX546 treatment. Our results suggest a concordance of functional and structural changes that is enhanced with prolonged CX546 exposure. Thus, the improved cognitive ability of patients receiving ampakine treatment may result from the priming of synapses through increases in the structural plasticity and functional reliability of hippocampal synapses.     

Specific interaction between Sam68 and neuronal mRNAs: implication for the activity-dependent biosynthesis of elongation factor eEF1A

In cultured hippocampal neurons and in adult brain, the splicing regulatory protein Sam68 is partially relocated to the somatodendritic domain and associates with dendritic polysomes. Transfer to the dendrites is activity-dependent. We have investigated the repertoire of neuronal mRNAs to which Sam68 binds in vivo. By using coimmunoprecipitation and microarray screening techniques, Sam68 was found to associate with a number of plasticity-related mRNA species, including Eef1a1, an activity-responsive mRNA coding for translation elongation factor eEF1A. In cortical neuronal cultures, translation of the Eef1a1 mRNA was strongly induced by neuronal depolarisation and correlated with enhanced association of Sam68 with polysomal mRNAs. The possible function of Sam68 in Eef1a1 mRNA utilization was studied by expressing a dominant-negative, cytoplasmic Sam68 mutant (GFP-Sam68DeltaC) in cultured hippocampal neurons. The level of eEF1A was lower in neurons expressing GFP-Sam68DeltaC than in control neurons, supporting the proposal that endogenous Sam68 may contribute to the translational efficiency of the Eef1a1 mRNA. These findings are discussed in the light of the complex, potentially crucial regulation of eEF1A biosynthesis during long-term synaptic change.     

Running induces widespread structural alterations in the hippocampus and entorhinal cortex

Physical activity enhances hippocampal function but its effects on neuronal structure remain relatively unexplored outside of the dentate gyrus. Using Golgi impregnation and the lipophilic tracer DiI, we show that long-term voluntary running increases the density of dendritic spines in the entorhinal cortex and hippocampus of adult rats. Exercise was associated with increased dendritic spine density not only in granule neurons of the dentate gyrus, but also in CA1 pyramidal neurons, and in layer III pyramidal neurons of the entorhinal cortex. In the CA1 region, changes in dendritic spine density are accompanied by changes in dendritic arborization and alterations in the morphology of individual spines. These findings suggest that physical activity exerts pervasive effects on neuronal morphology in the hippocampus and one of its afferent populations. These structural changes may contribute to running-induced changes in cognitive function.     

Amygdala kindling-induced seizures selectively impair spatial memory. 1. Behavioral characteristics and effects on hippocampal neuronal protein kinase C isoforms.

Protein kinase C (PKC) comprises a family of kinases consisting of nine subspecies that are differentially distributed in the central nervous system. This implies distinct functions. Its involvement is suggested in cellular and molecular mechanisms by which the hippocampus exerts influence on information processing. In this study, it was questioned whether abnormal activity in the neuronal substrate, particularly the hippocampal formation, induced by amygdala kindling indeed impairs spatial memory performance and correlated alpha, beta I/II, and gamma PKC subspecies expression. Rats were trained in a spatial discrimination task (SDT) and simultaneously kindled in the amygdala to induce abnormal, epileptiform activity. Control rats were only trained in the holeboard, a "free choice" maze, in which working (WM) and reference memory (RM) were simultaneously examined. Halfway through and at the end of the experiments the influence of kindling and SDT training on the immunoreactivity for PKC subspecies alpha, beta I/II, and gamma was evaluated in the hippocampal formation. Kindling resulted in a gradual increase in afterdischarge duration and motor seizure (MS) severity. Repeated SDT training ultimately resulted in an asymptotic level of WM and RM performance. As soon as generalized MSs developed, kindled rats failed to improve RM, whereas WM was not influenced. Compared to untrained rats, in trained controls PKC gamma but not PKC alpha beta I/II immunoreactivity was elevated in CA1 pyramidal and dentate gyrus granular cells. Generalized but not partial MSs abolished these alterations in PKC gamma immunoreactivity. The present data indicate that repeated training in a SDT affects the expression of PKC subspecies gamma but not of alpha or beta in the rat hippocampus. Generalized epileptiform activity impair both acquisition of new spatial RM information and PKC gamma expression. It is argued that PKC gamma plays a role in cellular mechanisms through which pathological brain activity impairs certain aspects of spatial memory.     

Cytomegalovirus infection inhibits the expression of N-methyl-d-aspartate receptors in the developing mouse hippocampus and primary neuronal cultures

Cytomegalovirus (CMV) is the most significant infectious cause of developmental brain disorders in humans. The infection occasionally persists and causes neurological disorders. The N-methyl-d-aspartate (NMDA) subtype of glutamate receptors is essential for the development and plasticity of synapses, but also is involved in neuronal excitotoxicity during viral infection. Here we investigated the effects of murine CMV (MCMV) infection on the expression of NMDA receptors in the hippocampal neurons of neonatal mice and primary neuronal cultures. Viral antigen was mostly found in hippocampal pyramidal neurons from the CA1 to CA3. Image analysis of immunohistochemistry demonstrated that the expression of NMDA receptor subunit 1 (NMDA-R1) protein in CA1 neurons of MCMV-infected brain was reduced to 40% of that in uninfected brain. The signal of in situ hybridization for NMDA-R1 mRNA was also decreased in CA1 neurons of MCMV-infected brain. In primary neuronal cultures, reduction of NMDA-R1 expression in MCMV-infected neurons was also detected by immunocytochemistry and Western blotting. These results suggest that reduction of NMDA receptor expression by MCMV infection may cause a decrease in the susceptibility of the neurons to excitotoxic cell death, and may be related to the establishment of viral persistence and functional disturbances in MCMV-infected neurons.     

Isoproterenol increases the phosphorylation of the synapsins and increases synaptic transmission in dentate gyrus, but not in area CA1, of the hippocampus.

Previous studies have shown that either norepinephrine (NE) or isoproterenol (ISO) enhances the slope of the field excitatory postsynaptic potential (EPSP) in the dentate gyrus of the rat hippocampal formation. In contrast, NE and ISO cause no increase in excitatory transmission in area CA1 of the hippocampus. The molecular mechanism underlying this brain region-specific increase in synaptic transmission is not known. The phosphorylation of synapsin I and synapsin II, two homologous presynaptic vesicle-associated proteins, is thought to promote neurotransmitter release. The authors have observed previously NE- and ISO-enhanced phosphorylation of synapsins I and II in the dentate gyrus. The purpose of this study was to determine whether ISO-stimulated phosphorylation also occurs in the CA1, where ISO has no effect on excitatory neurotransmission. These studies were correlated with electrophysiological studies in in vitro hippocampal slices. Superfusion of slices with ISO resulted in an increase in EPSP slope in the dentate but not in area CA1. The enhanced dentate EPSP returned to baseline levels within 30 minutes of washout of the drug. Isoproterenol produced corresponding increases in the phosphorylation of the synapsins in dentate slices but had no effect on these proteins in CA1 slices. Moreover, in dentate slices exposed to a 30-minute wash following incubation with ISO, phosphorylation of the synapsins returned to control levels. This close temporal and brain regional correlation between ISO stimulation of both synapsin phosphorylation and synaptic transmission suggests that the synapsin proteins may play a role in the synaptic potentiation produced by ISO in the dentate.