Effect of intra-BLA overexpression of IGF-1 on the expression of a contextual fear memory trace.

Growth factors, such as insulin-like growth factor 1 (IGF-1), among others are known for their critical involvement in learning and memory processes. IGF-1 regulates cognitive functions, synapse density, neurotransmission, and adult neurogenesis and induces structural and synaptic plasticity-specific changes. Although IGF-1 has been suggested to participate in different memory processes, its role in memories associated with negative emotional experiences still remains to be elucidated. The principal aim of the present study was to test whether IGF-1 overexpression using adenoviral vectors in basolateral amygdala (BLA) influences both the expression and formation of contextual fear memory, as well as the hippocampal structural plasticity associated with such memory trace. We found that IGF-1 overexpression promotes the formation and expression of a specific contextual fear memory trace, and such effect persisted at least 7 days after recall. Moreover, the overexpression of this growth factor in BLA upregulates the activation of the ERK/MAPK pathway in this brain structure. In addition, intra-BLA IGF-1 overexpression causes dorsal hippocampus (DH) structural plasticity modifications promoting changes in the proportion of mature dendritic spines in the CA1 region, after a weak conditioning protocol. The present findings contribute to the knowledge underlying BLA-DH trace memory of fear and reveal important new insights into the neurobiology and neurochemistry of fear acquisition modulated by IGF-1 overexpression. The understanding of how IGF-1 modulates the formation of a fear contextual trace may pave the way for the development of novel therapeutic strategies focused on fear, anxiety, and trauma-related disorders.

Minocycline prevents chronic restraint stress-induced vulnerability to developing cocaine self-administration and associated glutamatergic mechanisms: a potential role of microglia.

Stressful experience-induced cocaine-related behaviors are associated with a significant impairment of glutamatergic mechanisms in the Nucleus Accumbens core (NAcore). The hallmarks of disrupted glutamate homeostasis following restraint stress are the enduring imbalance of glutamate efflux after a cocaine stimulus and increased basal concentrations of extracellular glutamate attributed to GLT-1 downregulation in the NAcore. Glutamate transmission is tightly linked to microglia functioning. However, the role of microglia in the biological basis of stress-induced addictive behaviors is still unknown. By using minocycline, a potent inhibitor of microglia activation with anti-inflammatory properties, we determined whether microglia could aid chronic restraint stress (CRS)-induced glutamate homeostasis disruption in the NAcore, underpinning stress-induced cocaine self-administration. In this study, adult male rats were restrained for 2 h/day for seven days (day 1-7). From day 16 until completing the experimental protocol, animals received a vehicle or minocycline treatment (30 mg/Kg/12h i.p.). On day 21, animals were assigned to microscopic, biochemical, neurochemical or behavioral studies. We confirm that the CRS-induced facilitation of cocaine self-administration is associated with enduring GLT-1 downregulation, an increase of basal extracellular glutamate and postsynaptic structural plasticity in the NAcore. These alterations were strongly related to the CRS-induced reactive microglia and increased TNF-α mRNA and protein expression, since by administering minocycline, the impaired glutamate homeostasis and the facilitation of cocaine self-administration were prevented. Our findings are the first to demonstrate that minocycline suppresses the CRS-induced facilitation of cocaine self-administration and glutamate homeostasis disruption in the NAcore. A role of microglia is proposed for the development of glutamatergic mechanisms underpinning stress-induced vulnerability to cocaine addiction.

Temporal dynamic of the hippocampal structural plasticity associated with the fear memory destabilization/reconsolidation process.

Reconsolidation of a contextual fear memory is a protein synthesis-dependent process in which a previously destabilized memory returns to a stable state. This process has become the subject of many studies due to its importance in memory processing, maintenance and updating, and its potential role as a therapeutical target in fear memory disorders such as phobias and post-traumatic stress disorder. In this sense, understanding the underlying mechanisms of memory reconsolidation is paramount in developing potential treatments for such memory dysfunctions. In the present work, we studied the interaction between two key neural structures involved in the reconsolidation process: the basolateral amygdala complex of the amygdala (BLA) and the dorsal hippocampus (DH). Our results show changes in the structural plasticity of the CA1 region of the DH in the form of dendritic spines density changes associated with the destabilization/reconsolidation process. Furthermore, we demonstrate a modulatory role of BLA over such structural plasticity by infusing different drugs such as ifenprodil, a destabilization blocker, and propranolol, a reconsolidation disruptor, in this brain structure. Altogether our work shows a particular temporal dynamic in the CA1 region of DH that accompanies the destabilization/reconsolidation process and aims to provide new information on the underlying mechanisms of this process that potentially contributes for a better understanding of memory storage, maintenance, expression and updating, and its potential medical applications.

Stress influences the dynamics of hippocampal structural remodeling associated with fear memory extinction.

Fear extinction is defined as a decline in fear-conditioned responses following non-reinforced exposure to a fear conditioned stimulus, therefore the conditioned stimulus gains new predictive properties. Patients with anxiety related disorders (e.g.: PTSD) subjected to extinction-like exposure treatments often experience a relapse of symptoms. Stress is a risk factor for those psychiatric disorders and a critical modulator of fear learning that turns the memory resistant to the extinction process. Dendritic spines are the anatomical sites where neuronal activity reshapes brain networks during learning and memory processes. Thus, we planned to characterize the dynamics of synaptic remodeling before and after contextual fear extinction in the dorsal hippocampus (DH), and how this process is affected by a previous stress experience. Animals with or without previous stress were contextually fear conditioned and one day later trained in an extinction paradigm. Rats were sacrificed one day after conditioning (pre-extinction) or one day after extinction for spine density analysis in the DH. We confirmed that stress exposure induced a deficit in extinction learning. Further, a higher density of dendritic spines, particularly mature ones, was observed in the DH of non-stressed conditioned animals at pre-extinction. Interestingly, after extinction, the spine levels returned to the control values. Conversely, stressed animals did not show such spines boost (pre-extinction) or any other change (post-extinction). In contrast, such standard dynamics of dendritic changes as well as the behavioral extinction was recovered when stressed animals received an intra-basolateral amygdala infusion of midazolam prior to stress. Altogether, these findings suggest that stress hinders the normal dynamic of dendritic remodeling after fear extinction and this could be part of the neurobiological substrate that makes those memories resistant to be extinguished.

Excitation/inhibition imbalance and impaired synaptic inhibition in hippocampal area CA3 of Mecp2 knockout mice.

Rett syndrome (RTT) is a neurodevelopment disorder associated with intellectual disabilities and caused by loss-of-function mutations in the gene encoding the transcriptional regulator Methyl-CpG-binding Protein-2 (MeCP2). Neuronal dysfunction and changes in cortical excitability occur in RTT individuals and Mecp2-deficient mice, including hippocampal network hyperactivity and higher frequency of spontaneous multiunit spikes in the CA3 cell body layer. Here, we describe impaired synaptic inhibition and an excitation/inhibition (E/I) imbalance in area CA3 of acute slices from symptomatic Mecp2 knockout male mice (referred to as Mecp2(-/y) ). The amplitude of TTX-resistant miniature inhibitory postsynaptic currents (mIPSC) was smaller in CA3 pyramidal neurons of Mecp2(-/y) slices than in wildtype controls, while the amplitude of miniature excitatory postsynaptic currents (mEPSC) was significantly larger in Mecp2(-/y) neurons. Consistently, quantitative confocal immunohistochemistry revealed significantly lower intensity of the alpha-1 subunit of GABAA Rs in the CA3 cell body layer of Mecp2(-/y) mice, while GluA1 puncta intensities were significantly higher in the CA3 dendritic layers of Mecp2(-/y) mice. In addition, the input/output (I/O) relationship of evoked IPSCs had a shallower slope in CA3 pyramidal neurons Mecp2(-/y) neurons. Consistent with the absence of neuronal degeneration in RTT and MeCP2-based mouse models, the density of parvalbumin- and somatostatin-expressing interneurons in area CA3 was not affected in Mecp2(-/y) mice. Furthermore, the intrinsic membrane properties of several interneuron subtypes in area CA3 were not affected by Mecp2 loss. However, mEPSCs are smaller and less frequent in CA3 fast-spiking basket cells of Mecp2(-/y) mice, suggesting an impaired glutamatergic drive in this interneuron population. These results demonstrate that a loss-of-function mutation in Mecp2 causes impaired E/I balance onto CA3 pyramidal neurons, leading to a hyperactive hippocampal network, likely contributing to limbic seizures in Mecp2(-/y) mice and RTT individuals.

Hippocampal dendritic spines remodeling and fear memory are modulated by GABAergic signaling within the basolateral amygdala complex.

GABAergic signaling in the basolateral amygdala complex (BLA) plays a crucial role on the modulation of the stress influence on fear memory. Moreover, accumulating evidence suggests that the dorsal hippocampus (DH) is a downstream target of BLA neurons in contextual fear. Given that hippocampal structural plasticity is proposed to provide a substrate for the storage of long-term memories, the main aim of this study is to evaluate the modulation of GABA neurotransmission in the BLA on spine density in the DH following stress on contextual fear learning. The present findings show that prior stressful experience promoted contextual fear memory and enhanced spine density in the DH. Intra-BLA infusion of midazolam, a positive modulator of GABAa sites, prevented the facilitating influence of stress on both fear retention and hippocampal dendritic spine remodeling. Similarly to the stress-induced effects, the blockade of GABAa sites within the BLA ameliorated fear memory emergence and induced structural remodeling in the DH. These findings suggest that GABAergic transmission in BLA modulates the structural changes in DH associated to the influence of stress on fear memory.

Activity-dependent BDNF release and TRPC signaling is impaired in hippocampal neurons of Mecp2 mutant mice.

Dysfunction of the neurotrophin brain-derived neurotrophic factor (BDNF) is implicated in Rett syndrome (RTT), but the state of its releasable pool and downstream signaling in mice lacking methyl-CpG-binding protein-2 (Mecp2) is unknown. Here, we show that membrane currents and dendritic Ca(2+) signals evoked by recombinant BDNF or an activator of diacylglycerol (DAG)-sensitive transient receptor potential canonical (TRPC) channels are impaired in CA3 pyramidal neurons of symptomatic Mecp2 mutant mice. TRPC3 and TRPC6 mRNA and protein levels are lower in Mecp2 mutant hippocampus, and chromatin immunoprecipitation (ChIP) identified Trpc3 as a target of MeCP2 transcriptional regulation. BDNF mRNA and protein levels are also lower in Mecp2 mutant hippocampus and dentate gyrus granule cells, which is reflected in impaired activity-dependent release of endogenous BDNF estimated from TRPC currents and dendritic Ca(2+) signals in CA3 pyramidal neurons. These results identify the gene encoding TRPC3 channels as a MeCP2 target and suggest a potential therapeutic strategy to boost impaired BDNF signaling in RTT.

Hippocampal structural plasticity accompanies the resulting contextual fear memory following stress and fear conditioning.

The present research investigated the resulting contextual fear memory and structural plasticity changes in the dorsal hippocampus (DH) following stress and fear conditioning. This combination enhanced fear retention and increased the number of total and mature dendritic spines in DH. Intra-basolateral amygdala (BLA) infusion of midazolam prior to stress prevented both the enhancement of fear retention and an increase in the density of total and mature dendritic spines in DH. These findings emphasize the role of the stress-induced attenuation of GABAergic neurotransmission in BLA in the promoting influence of stress on fear memory and on synaptic remodeling in DH. In conclusion, the structural remodeling in DH accompanied the facilitated fear memory following a combination of fear conditioning and stressful stimulation.

A BDNF sensitive mechanism is involved in the fear memory resulting from the interaction between stress and the retrieval of an established trace.

The present study investigates the fear memory resulting from the interaction of a stressful experience and the retrieval of an established fear memory trace. Such a combination enhanced both fear expression and fear retention in adult Wistar rats. Likewise, midazolam intra-basolateral amygdala (BLA) infusion prior to stress attenuated the enhancement of fear memory thus suggesting the involvement of a stress-induced reduction of the GABAergic transmission in BLA in the stress-induced enhancing effect. It has been suggested that, unlike the immediate-early gene Zif268 which is related to the reconsolidation process, the expression of hippocampal brain-derived neurotrophic factor (BDNF) is highly correlated with consolidation. We therefore evaluate the relative contribution of these two neurobiological processes to the fear memory resulting from the above-mentioned interaction. Intra-dorsal hippocampus (DH) infusions of either the antisense Zif268 or the inhibitor of the protein degradation (Clasto-Lactacystin ß-Lactone), suggested to be involved in the retrieval-dependent destabilization process, did not affect the resulting contextual memory. In contrast, the knockdown of hippocampal BDNF mitigated the stress-induced facilitating influence on fear retention. In addition, the retrieval experience elevated BDNF level in DH at 60 min after recall exclusively in stressed animals. These findings suggest the involvement of a hippocampal BDNF sensitive mechanism in the stress-promoting influence on the fear memory following retrieval.

Role of amygdala astrocytes in different phases of contextual fear memory.

Growing evidence indicates a critical role of astrocytes in learning and memory. However, little is known about the role of basolateral amygdala complex (BLA-C) astrocytes in contextual fear conditioning (CFC), a paradigm relevant to understand and generate treatments for fear- and anxiety-related disorders. To get insights on the involvement of BLA-C astrocytes in fear memory, fluorocitrate (FLC), a reversible astroglial metabolic inhibitor, was applied at critical moments of the memory processing in order to target the acquisition, consolidation, retrieval and reconsolidation process of the fear memory. Adult Wistar male rats were bilaterally cannulated in BLA-C. Ten days later they were infused with different doses of FLC (0.5 or 1 nmol/0.5 µl) or saline before or after CFC and before or after retrieval. FLC impaired fear memory expression when administered before and shortly after CFC, but not one hour later. Infusion of FLC prior and after retrieval did not affect the memory. Our findings suggest that BLA-C astrocytes are critically involved in the acquisition/early consolidation of fear memory but not in the retrieval and reconsolidation. Furthermore, the extinction process was presumably not affected (considering that peri-retrieval administration could also affect this process).

Hyperforin modulates dendritic spine morphology in hippocampal pyramidal neurons by activating Ca(2+) -permeable TRPC6 channels.

The standardized extract of the St. John's wort plant (Hypericum perforatum) is commonly used to treat mild to moderate depression. Its active constituent is hyperforin, a phloroglucinol derivative that reduces the reuptake of serotonin and norepinephrine by increasing intracellular Na(+) concentration through the activation of nonselective cationic TRPC6 channels. TRPC6 channels are also Ca(2+) -permeable, resulting in intracellular Ca(2+) elevations. Indeed, hyperforin activates TRPC6-mediated currents and Ca(2+) transients in rat PC12 cells, which induce their differentiation, mimicking the neurotrophic effect of nerve growth factor. Here, we show that hyperforin modulates dendritic spine morphology in CA1 and CA3 pyramidal neurons of hippocampal slice cultures through the activation of TRPC6 channels. Hyperforin also evoked intracellular Ca(2+) transients and depolarizing inward currents sensitive to the TRPC channel blocker La(3+) , thus resembling the actions of the neurotrophin brain-derived neurotrophic factor (BDNF) in hippocampal pyramidal neurons. These results suggest that the antidepressant actions of St. John's wort are mediated by a mechanism similar to that engaged by BDNF.

HDAC activity is required for BDNF to increase quantal neurotransmitter release and dendritic spine density in CA1 pyramidal neurons.

Molecular mechanisms involved in the strengthening and formation of synapses include the activation and repression of specific genes or subsets of genes by epigenetic modifications that do not alter the genetic code itself. Chromatin modifications mediated by histone acetylation have been shown to be critical for synaptic plasticity at hippocampal excitatory synapses and hippocampal-dependent memory formation. Considering that brain-derived neurotrophic factor (BDNF) plays an important role in synaptic plasticity and behavioral adaptations, it is not surprising that regulation of this gene is subject to histone acetylation changes during synaptic plasticity and hippocampal-dependent memory formation. Whether the effects of BDNF on dendritic spines and quantal transmitter release require histone modifications remains less known. By using two different inhibitors of histone deacetylases (HDACs), we describe here that their activity is required for BDNF to increase dendritic spine density and excitatory quantal transmitter release onto CA1 pyramidal neurons in hippocampal slice cultures. These results suggest that histone acetylation/deacetylation is a critical step in the modulation of hippocampal synapses by BDNF. Thus, mechanisms of epigenetic modulation of synapse formation and function are novel targets to consider for the amelioration of symptoms of intellectual disabilities and neurodegenerative disorders associated with cognitive and memory deficits.

Hippocampal CA1 pyramidal neurons of Mecp2 mutant mice show a dendritic spine phenotype only in the presymptomatic stage.

Alterations in dendritic spines have been documented in numerous neurodevelopmental disorders, including Rett Syndrome (RTT). RTT, an X chromosome-linked disorder associated with mutations in MECP2, is the leading cause of intellectual disabilities in women. Neurons in Mecp2-deficient mice show lower dendritic spine density in several brain regions. To better understand the role of MeCP2 on excitatory spine synapses, we analyzed dendritic spines of CA1 pyramidal neurons in the hippocampus of Mecp2(tm1.1Jae) male mutant mice by either confocal microscopy or electron microscopy (EM). At postnatal-day 7 (P7), well before the onset of RTT-like symptoms, CA1 pyramidal neurons from mutant mice showed lower dendritic spine density than those from wildtype littermates. On the other hand, at P15 or later showing characteristic RTT-like symptoms, dendritic spine density did not differ between mutant and wildtype neurons. Consistently, stereological analyses at the EM level revealed similar densities of asymmetric spine synapses in CA1 stratum radiatum of symptomatic mutant and wildtype littermates. These results raise caution regarding the use of dendritic spine density in hippocampal neurons as a phenotypic endpoint for the evaluation of therapeutic interventions in symptomatic Mecp2-deficient mice. However, they underscore the potential role of MeCP2 in the maintenance of excitatory spine synapses.

Impairment of glutamate homeostasis in the nucleus accumbens core underpins cross-sensitization to cocaine following chronic restraint stress.

Though the facilitating influence of stress on drug abuse is well documented, the mechanisms underlying this interaction have yet to be fully elucidated. The present study explores the neurobiological mechanisms underpinning the sensitized response to the psychomotor-stimulating effects of cocaine following chronic restraint stress (CRS), emphasizing the differential contribution of both subcompartments of the nucleus accumbens (NA), the core (NAcore) and shell (NAshell), to this phenomenon. Adult male Wistar rats were restrained for 2 h/day for 7 days and, 2 weeks after the last stress exposure (day 21), all animals were randomly assigned to behavioral, biochemical or neurochemical tests. Our results demonstrated that the enduring CRS-induced increase in psychostimulant response to cocaine was paralleled by an increase of extracellular dopamine levels in the NAcore, but not the NAshell, greater than that observed in the non-stress group. Furthermore, we found that CRS induced an impairment of glutamate homeostasis in the NAcore, but not the NAshell. Its hallmarks were increased basal extracellular glutamate concentrations driven by a CRS-induced downregulation of GLT-1, blunted glutamate levels in response to cocaine and postsynaptic structural remodeling in pre-stressed animals. In addition, ceftriaxone, a known GLT-1 enhancer, prevented the CRS-induced GLT-1 downregulation, increased basal extracellular glutamate concentrations and changes in structural plasticity in the NAcore as well as behavioral cross-sensitization to cocaine, emphasizing the biological importance of GLT-1 in the comorbidity between chronic stress exposure and drug abuse. A future perspective concerning the paramount relevance of the stress-induced disruption of glutamate homeostasis as a vulnerability factor to the development of stress and substance use disorders during early life or adulthood of descendants is provided.

Network hyperexcitability in hippocampal slices from Mecp2 mutant mice revealed by voltage-sensitive dye imaging.

Dysfunctions of neuronal and network excitability have emerged as common features in disorders associated with intellectual disabilities, autism, and seizure activity, all common clinical manifestations of Rett syndrome (RTT), a neurodevelopmental disorder caused by loss-of-function mutations in the transcriptional regulator methyl-CpG-binding protein 2 (MeCP2). Here, we evaluated the consequences of Mecp2 mutation on hippocampal network excitability, as well as synapse structure and function using a combination of imaging and electrophysiological approaches in acute slices. Imaging the amplitude and spatiotemporal spread of neuronal depolarizations with voltage-sensitive dyes (VSD) revealed that the CA1 and CA3 regions of hippocampal slices from symptomatic male Mecp2 mutant mice are highly hyperexcitable. However, only the density of docked synaptic vesicles and the rate of release from the readily releasable pool are impaired in Mecp2 mutant mice, while synapse density and morphology are unaffected. The differences in network excitability were not observed in surgically isolated CA1 minislices, and blockade of GABAergic inhibition enhanced VSD signals to the same extent in Mecp2 mutant and wild-type mice, suggesting that network excitability originates in area CA3. Indeed, extracellular multiunit recordings revealed a higher level of spontaneous firing of CA3 pyramidal neurons in slices from symptomatic Mecp2 mutant mice. The neuromodulator adenosine reduced the amplitude and spatiotemporal spread of VSD signals evoked in CA1 of Mecp2 mutant slices to wild-type levels, suggesting its potential use as an anticonvulsant in RTT individuals. The present results suggest that hyperactive CA3 pyramidal neurons contribute to hippocampal dysfunction and possibly to limbic seizures observed in Mecp2 mutant mice and RTT individuals.

Stress-induced vulnerability to develop cocaine addiction depends on cofilin modulation.

Actin dynamics in dendritic spines can be associated with the neurobiological mechanisms supporting the comorbidity between stress exposure and cocaine increase rewards. The actin cytoskeleton remodeling in the nucleus accumbens (NA) has been implicated in the expression of stress-induced cross-sensitization with cocaine. The present study evaluates the involvement of cofilin, a direct regulator of actin dynamics, in the impact of stress on vulnerability to cocaine addiction. We assess whether the neurobiological mechanisms that modulate repeated-cocaine administration also occur in a chronic restraint stress-induced cocaine self-administration model. We also determine if chronic stress induces alterations in dendritic spines through dysregulation of cofilin activity in the NA core. Here, we show that the inhibition of cofilin expression in the NA core using viral short-hairpin RNA is sufficient to prevent the cocaine sensitization induced by chronic stress. The reduced cofilin levels also impede a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor surface expression enhancement and promote the reduction of head diameter in animals pre-exposed to stress after a cocaine challenge in the NA core. Moreover, downregulation of cofilin expression prevents facilitation of the acquisition of cocaine self-administration (SA) in male rats pre-exposed to chronic stress without modifying performance in sucrose SA. These findings reveal a novel, crucial role for cofilin in the neurobiological mechanisms underpinning the comorbidity between stress exposure and addiction-related disorders.

Activity-dependent release of endogenous BDNF from mossy fibers evokes a TRPC3 current and Ca2+ elevations in CA3 pyramidal neurons.

Multiple studies have demonstrated that brain-derived neurotrophic factor (BDNF) is a potent modulator of neuronal structure and function in the hippocampus. However, the majority of studies to date have relied on the application of recombinant BDNF. We herein report that endogenous BDNF, released via theta burst stimulation of mossy fibers (MF), elicits a slowly developing cationic current and intracellular Ca(2+) elevations in CA3 pyramidal neurons with the same pharmacological profile of the transient receptor potential canonical 3 (TRPC3)-mediated I(BDNF) activated in CA1 neurons by brief localized applications of recombinant BDNF. Indeed, sensitivity to both the extracellular BDNF scavenger tropomyosin-related kinase B (TrkB)-IgG and small hairpin interference RNA-mediated TRPC3 channel knockdown confirms the identity of this conductance as such, henceforth-denoted MF-I(BDNF). Consistent with such activity-dependent release of BDNF, these MF-I(BDNF) responses were insensitive to manipulations of extracellular Zn(2+) concentration. Brief theta burst stimulation of MFs induced a long-lasting depression in the amplitude of excitatory postsynaptic currents (EPSCs) mediated by both AMPA and N-methyl-d-aspartate (NMDA) receptors without changes in the NMDA receptor/AMPA receptor ratio, suggesting a reduction in neurotransmitter release. This depression of NMDAR-mediated EPSCs required activity-dependent release of endogenous BDNF from MFs and activation of Trk receptors, as it was sensitive to the extracellular BDNF scavenger TrkB-IgG and the tyrosine kinase inhibitor k-252b. These results uncovered the most immediate response to endogenously released--native--BDNF in hippocampal neurons and lend further credence to the relevance of BDNF signaling for synaptic function in the hippocampus.

Fasting induces remodeling of the orexigenic projections from the arcuate nucleus to the hypothalamic paraventricular nucleus, in a growth hormone secretagogue receptor-dependent manner.

OBJECTIVE: Arcuate nucleus (ARC) neurons producing Agouti-related peptide (AgRP) and neuropeptide Y (NPY; ARCAgRP/NPY neurons) are activated under energy-deficit states. ARCAgRP/NPY neurons innervate the hypothalamic paraventricular nucleus (PVH), and ARC→PVH projections are recognized as key regulators of food intake. Plasma ghrelin levels increase under energy-deficit states and activate ARCAgRP/NPY neurons by acting on the growth hormone secretagogue receptor (GHSR). Here, we hypothesized that activation of ARCAgRP/NPY neurons in fasted mice would promote morphological remodeling of the ARCAgRP/NPY→PVH projections in a GHSR-dependent manner. METHODS: We performed 1) fluorescent immunohistochemistry, 2) imaging of green fluorescent protein (GFP) signal in NPY-GFP mice, and 3) DiI axonal labeling in brains of ad libitum fed or fasted mice with pharmacological or genetic blockage of the GHSR signaling and then estimated the density and strength of ARCAgRP/NPY→PVH fibers by assessing the mean fluorescence intensity, the absolute area with fluorescent signal, and the intensity of the fluorescent signal in the fluorescent area of the PVH. RESULTS: We found that 1) the density and strength of ARCAgRP/NPY fibers increase in the PVH of fasted mice, 2) the morphological remodeling of the ARCAgRP/NPY→PVH projections correlates with the activation of PVH neurons, and 3) PVH neurons are not activated in ARC-ablated mice. We also found that fasting-induced remodeling of ARCAgRP/NPY→PVH fibers and PVH activation are impaired in mice with pharmacological or genetic blockage of GHSR signaling. CONCLUSION: This evidence shows that the connectivity between hypothalamic circuits controlling food intake can be remodeled in the adult brain, depending on the energy balance conditions, and that GHSR activity is a key regulator of this phenomenon.

Dendritic spine pathologies in hippocampal pyramidal neurons from Rett syndrome brain and after expression of Rett-associated MECP2 mutations.

Rett syndrome (RTT) is an X chromosome-linked neurodevelopmental disorder associated with the characteristic neuropathology of dendritic spines common in diseases presenting with mental retardation (MR). Here, we present the first quantitative analyses of dendritic spine density in postmortem brain tissue from female RTT individuals, which revealed that hippocampal CA1 pyramidal neurons have lower spine density than age-matched non-MR female control individuals. The majority of RTT individuals carry mutations in MECP2, the gene coding for a methylated DNA-binding transcriptional regulator. While altered synaptic transmission and plasticity has been demonstrated in Mecp2-deficient mouse models of RTT, observations regarding dendritic spine density and morphology have produced varied results. We investigated the consequences of MeCP2 dysfunction on dendritic spine structure by overexpressing ( approximately twofold) MeCP2-GFP constructs encoding either the wildtype (WT) protein, or missense mutations commonly found in RTT individuals. Pyramidal neurons within hippocampal slice cultures transfected with either WT or mutant MECP2 (either R106W or T158M) showed a significant reduction in total spine density after 48 h of expression. Interestingly, spine density in neurons expressing WT MECP2 for 96 h was comparable to that in control neurons, while neurons expressing mutant MECP2 continued to have lower spine density than controls after 96 h of expression. Knockdown of endogenous Mecp2 with a specific small hairpin interference RNA (shRNA) also reduced dendritic spine density, but only after 96 h of expression. On the other hand, the consequences of manipulating MeCP2 levels for dendritic complexity in CA3 pyramidal neurons were only minor. Together, these results demonstrate reduced dendritic spine density in hippocampal pyramidal neurons from RTT patients, a distinct dendritic phenotype also found in neurons expressing RTT-associated MECP2 mutations or after shRNA-mediated endogenous Mecp2 knockdown, suggesting that this phenotype represent a cell-autonomous consequence of MeCP2 dysfunction.

Memory consolidation impairment induced by Interleukin-1ß is associated with changes in hippocampal structural plasticity.

Pro-inflammatory cytokines, particularly Interleukin-1ß (IL-1ß), can affect cognitive processes such as learning and memory. The aim of this study was to establish whether the effect of IL-1ß on contextual fear memory is associated with changes in hippocampal structural plasticity. We also studied the effect of α-melanocyte-stimulating hormone (α-MSH), a potent anti-inflammatory and neuro-protective peptide. Different groups of animals were implanted bilaterally in dorsal hippocampus (DH). After recovery they were conditioned for contextual fear memory and received the different treatments (vehicle, IL-1ß, α-MSH or IL-1ß + α-MSH). Memory was assessed 24 hs after conditioning and immediately after rats were perfused for dendritic spine analysis. Our results show that local hippocampal administration of IL-1ß just after memory encoding induced impairment in contextual memory and a reduction in the total density of CA1 hippocampal dendritic spines, particularly the mature ones. α-MSH administration reversed the IL-1ß induced changes. The results suggest that neuro-inflammation induced by IL-1ß interferes with experience-dependent structural plasticity in DH whereas α-MSH has a beneficial modulatory role in preventing this effect.