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In animal models that mimic human third-trimester fetal development, ethanol causes substantial cellular apoptosis in the brain, but for most brain structures, the extent of permanent neuron loss that persists into adulthood is unknown. We injected ethanol into C57BL/6J mouse pups at postnatal day 7 (P7) to model human late-gestation ethanol toxicity, and then used stereological methods to investigate adult cell numbers in several subcortical neurotransmitter systems that project extensively in the forebrain to regulate arousal states. Ethanol treatment caused especially large reductions (34–42%) in the cholinergic cells of the basal forebrain, including cholinergic cells in the medial septal/vertical diagonal band nuclei (Ch1/Ch2) and in the horizontal diagonal band/substantia innominata/nucleus basalis nuclei (Ch3/Ch4). Cell loss was also present in non-cholinergic basal forebrain cells, as demonstrated by 34% reduction of parvalbumin-immunolabeled GABA cells and 25% reduction of total Nissl-stained neurons in the Ch1/Ch2 region. In contrast, cholinergic cells in the striatum were reduced only 12% by ethanol, and those of the brainstem pedunculopontine/lateral dorsal tegmental nuclei (Ch5/Ch6) were not significantly reduced. Similarly, ethanol did not significantly reduce dopamine cells of the ventral tegmental area/substantia nigra or serotonin cells in the dorsal raphe nucleus. Orexin (hypocretin) cells in the hypothalamus showed a modest reduction (14%). Our findings indicate that the basal forebrain is especially vulnerable to alcohol exposure in the late gestational period. Reduction of cholinergic and GABAergic projection neurons from the basal forebrain that regulate forebrain arousal may contribute to the behavioral and cognitive deficits associated with neonatal ethanol exposure.

Serotonin and dopamine are associated with multiple psychiatric disorders. How they interact during development to affect subsequent behavior remains unknown. Knockout of the serotonin transporter or postnatal blockade with selective serotonin reuptake inhibitors (SSRIs) leads to novelty-induced exploration deficits in adulthood, potentially involving the dopamine system. Here, we show in the mouse that raphe nucleus serotonin neurons activate ventral tegmental area dopamine neurons via glutamate co-transmission and that this co-transmission is reduced in animals exposed postnatally to SSRIs. Blocking serotonin neuron glutamate co-transmission mimics this SSRI-induced hypolocomotion, while optogenetic activation of dopamine neurons reverses this hypolocomotor phenotype. Our data demonstrate that serotonin neurons modulate dopamine neuron activity via glutamate co-transmission and that this pathway is developmentally malleable, with high serotonin levels during early life reducing co-transmission, revealing the basis for the reduced novelty-induced exploration in adulthood due to postnatal SSRI exposure.

Early life is a sensitive period, in which enhanced neural plasticity allows the developing brain to adapt to its environment. This plasticity can also be a risk factor in which maladaptive development can lead to long-lasting behavioral deficits. Here, we test how early-life exposure to the selective-serotonin-reuptake-inhibitor (SSRI), fluoxetine, affects motivation, and dopaminergic signaling in adulthood. We show for the first time that mice exposed to fluoxetine in the early postnatal period exhibit a reduction in effort-related motivation. These mice also show blunted responses to amphetamine and reduced dopaminergic activation in a sucrose reward task. Interestingly, we find that the reduction in motivation can be rescued in the adult by administering bupropion, a dopamine-norepinephrine reuptake inhibitor used as an antidepressant and a smoke cessation aid but not by fluoxetine. Taken together, our studies highlight the effects of early postnatal exposure of fluoxetine on motivation and demonstrate the involvement of the dopaminergic system in this process.SIGNIFICANCE STATEMENTThe developmental period is characterized by enhanced plasticity. During this period, environmental factors have the potential to lead to enduring behavioral changes. Here, we show that exposure to the SSRI fluoxetine during a restricted period in early life leads to a reduction in adult motivation. We further show that this reduction is associated with decreased dopaminergic responsivity. Finally, we show that motivational deficits induced by early-life fluoxetine exposure can be rescued by adult administration of bupropion but not by fluoxetine.

Serotonin is a critical neuromodulator involved in development and behavior. Its role in reward is however still debated. Here, we first review classical studies involving electrical stimulation protocols and pharmacological approaches. Contradictory results on the serotonergic' involvement in reward emerge from these studies. These differences might be ascribable to either the diversity of cellular types within the raphe nuclei or/and the specific projection pathways of serotonergic neurons. We continue to review more recent work, using optogenetic approaches to activate serotonergic cells in the Raphe to VTA pathway. From these studies, it appears that activation of this pathway can lead to reinforcement learning mediated through the excitation of dopaminergic neurons by serotonergic neurons co-transmitting glutamate. Finally, given the importance of serotonin during development on adult emotion, the effect of abnormal early-life levels of serotonin on the dopaminergic system will also be discussed. Understanding the interaction between the serotonergic and dopaminergic systems during development and adulthood is critical to gain insight into the specific facets of neuropsychiatric disorders.

The occurrence of dreaming during rapid eye movement (REM) sleep prompts interest in the role of REM sleep in hippocampal-dependent episodic memory. Within the mammalian hippocampus, the dentate gyrus (DG) has the unique characteristic of exhibiting neurogenesis persisting into adulthood. Despite their small numbers and sparse activity, adult-born neurons (ABNs) in the DG play critical roles in memory; however, their memory function during sleep is unknown. Here, we investigate whether young ABN activity contributes to memory consolidation during sleep using Ca 2+ imaging in freely moving mice. We found that contextual fear learning recruits a population of young ABNs that are reactivated during subsequent REM sleep against a backdrop of overall reduced ABN activity. Optogenetic silencing of this sparse ABN activity during REM sleep alters the structural remodeling of spines on ABN dendrites and impairs memory consolidation. These findings provide a causal link between ABN activity during REM sleep and memory consolidation.

The dentate gyrus (DG) of the hippocampus is important for cognition and behavior. However, the circuits underlying these functions are unclear. DG mossy cells (MCs) are potentially important because of their excitatory synapses on the primary cell type, granule cells (GCs). However, MCs also activate GABAergic neurons, which inhibit GCs. We used viral delivery of designer receptors exclusively activated by designer drugs (DREADDs) in mice to implement a gain- and loss-of-function study of MCs in diverse behaviors. Using this approach, manipulations of MCs could bidirectionally regulate behavior. The results suggest that inhibiting MCs can reduce anxiety-like behavior and improve cognitive performance. However, not all cognitive or anxiety-related behaviors were influenced, suggesting specific roles of MCs in some, but not all, types of cognition and anxiety. Notably, several behaviors showed sex-specific effects, with females often showing more pronounced effects than the males. We also used the immediate early gene c-Fos to address whether DREADDs bidirectionally regulated MC or GC activity. We confirmed excitatory DREADDs increased MC c-Fos. However, there was no change in GC c-Fos, consistent with MC activation leading to GABAergic inhibition of GCs. In contrast, inhibitory DREADDs led to a large increase in GC c-Fos, consistent with a reduction in MC excitation of GABAergic neurons, and reduced inhibition of GCs. Together, these results suggest that MCs regulate anxiety and cognition in specific ways. We also raise the possibility that cognitive performance may be improved by reducing anxiety.SIGNIFICANCE STATEMENTThe dentate gyrus (DG) has many important cognitive roles as well as being associated with affective behavior. This study addressed how a glutamatergic DG cell type called mossy cells (MCs) contributes to diverse behaviors, which is timely because it is known that MCs regulate the activity of the primary DG cell type, granule cells (GCs), but how MC activity influences behavior is unclear. We show, surprisingly, that activating MCs can lead to adverse behavioral outcomes, and inhibiting MCs have an opposite effect. Importantly, the results appeared to be task-dependent and showed that testing both sexes was important. Additional experiments indicated what MC and GC circuitry was involved. Together, the results suggest how MCs influence behaviors that involve the DG.

Development passes through sensitive periods, during which plasticity allows for genetic and environmental factors to exert indelible influence on the maturation of the organism. In the context of central nervous system (CNS) development, such sensitive periods shape the formation of neuro-circuits that mediate, regulate, and control behavior. This general mechanism allows for development to be guided by both the genetic blueprint, as well as the environmental context. While allowing for adaptation, such sensitive periods are also windows of vulnerability during which external and internal factors can confer risk to brain disorders by derailing adaptive developmental programs. Our group has been particularly interested in developmental periods that are sensitive to serotonin (5-HT) signaling, and impact behavior and cognition relevant to psychiatry. Specifically, we review a 5-HT-sensitive period that impacts fronto-limbic system development, resulting in cognitive, anxiety, and depression-related behaviors. We discuss preclinical data to establish biological plausibility and mechanistic insights. We also summarize epidemiological findings that underscore the potential public health implications resulting from the current practice of prescribing 5-HT reuptake inhibiting antidepressants during pregnancy. These medications enter the fetal circulation, likely perturb 5-HT signaling in the brain, and may be affecting circuit maturation in ways that parallel our findings in the developing rodent brain. More research is needed to better disambiguate the dual effects of maternal symptoms on fetal and child development from the effects of 5-HT reuptake inhibitors on clinical outcomes in the offspring. Birth Defects Research 109:924-932, 2017. © 2017 Wiley Periodicals, Inc.

Activity in sensory cortical networks reflects both peripheral sensory input and intra‐ and inter-cortical network input. How sensory cortices balance these diverse inputs to provide relatively stable, accurate representations of the external world is not well understood. Furthermore, neuromodulation could alter the balance of these inputs in a state‐ and behavior-dependent manner. Here, we used optogenetic stimulation to directly assay the relative strength of bottom-up (olfactory bulb) and top-down (lateral entorhinal cortex) synaptic inputs to piriform cortex in freely moving rats. Optotrodes in the piriform cortex were used to test the relative strength of these two inputs, in separate animals, with extracellular, monosynaptic evoked potentials. The results suggest a rapid state-dependent shift in the balance of bottom-up and top-down inputs to PCX, with enhancement in the strength of lateral entorhinal cortex synaptic input and stability or depression of olfactory bulb synaptic input during slow-wave sleep compared to waking. The shift is in part due to a state-dependent change in cholinergic tone as assessed with fiber photometry of GCaMP6 fluorescence in basal forebrain ChAT+ neurons, and blockade of the state-dependent synaptic shift with cholinergic muscarinic receptor activation.

The efficacy and duration of memory storage is regulated by neuromodulatory transmitter actions. While the modulatory transmitter serotonin (5-HT) plays an important role in implicit forms of memory in the invertebrate Aplysia, its function in explicit memory mediated by the mammalian hippocampus is less clear. Specifically, the consequences elicited by the spatio-temporal gradient of endogenous 5-HT release are not known. Here we applied optogenetic techniques in mice to gain insight into this fundamental biological process. We find that activation of serotonergic terminals in the hippocampal CA1 region both potentiates excitatory transmission at CA3-to-CA1 synapses and enhances spatial memory. Conversely, optogenetic silencing of CA1 5-HT terminals inhibits spatial memory. We furthermore find that synaptic potentiation is mediated by 5-HT4 receptors, and that systemic modulation of 5-HT4 receptor function can bidirectionally impact memory formation. Collectively, these data reveal powerful modulatory influence of serotonergic synaptic input on hippocampal function and memory formation.

Neurogenesis occurs in a limited number of brain regions during adulthood. Of these, the hippocampus has attracted great interest due to its involvement in memory processing. Moreover, both the hippocampus and the main area that innervates this structure, namely the entorhinal cortex, show remarkable atrophy in patients with Alzheimer's disease (AD). Adult hippocampal neurogenesis is a process that continuously gives rise to newborn granule neurons in the dentate gyrus. These cells coexist with developmentally generated granule neurons in this structure, and both cooperative and competition phenomena regulate the communication between these two types of cells. Importantly, it has been revealed that GSK-3β and tau proteins, which are two of the main players driving AD pathology, are cornerstones of adult hippocampal neurogenesis regulation. We have shown that alterations either promoting or impeding the actions of these two proteins have detrimental effects on the structural plasticity of granule neurons. Of note, these impairments occur both under basal conditions and in response to detrimental and neuroprotective stimuli. Thus, in order to achieve the full effectiveness of future therapies for AD, we propose that attention be turned toward identifying the pathological and physiological actions of the proteins involved in the pathogenesis of this condition., Spanish Ministry of Economy and Competitiveness (SAF-2014-53040-P (Jesu´s A´ vila) and RYC-2015-17189 (María Llorens-Mart´ın)); the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, Spain) (Jesu´s Avila); the Alzheimer’s Association (2015-NIRG-340709 (Mar´ıa Llorens-Mart´ın)); and the Association for Frontotemporal Degeneration (2016 Basic Science Pilot Grant Award (María Llorens-Martín)).

Development passes through sensitive periods, during which plasticity allows for genetic and environmental factors to exert indelible influence on the maturation of the organism. In the context of central nervous system (CNS) development, such sensitive periods shape the formation of neuro-circuits that mediate, regulate, and control behavior. This general mechanism allows for development to be guided by both the genetic blueprint, as well as the environmental context. While allowing for adaptation, such sensitive periods are also windows of vulnerability during which external and internal factors can confer risk to brain disorders by derailing adaptive developmental programs. Our group has been particularly interested in developmental periods that are sensitive to serotonin (5-HT) signaling, and impact behavior and cognition relevant to psychiatry. Specifically, we review a 5-HT-sensitive period that impacts fronto-limbic system development, resulting in cognitive, anxiety, and depression-related behaviors. We discuss preclinical data to establish biological plausibility and mechanistic insights. We also summarize epidemiological findings that underscore the potential public health implications resulting from the current practice of prescribing 5-HT reuptake inhibiting antidepressants during pregnancy. These medications enter the fetal circulation, likely perturb 5-HT signaling in the brain, and may be affecting circuit maturation in ways that parallel our findings in the developing rodent brain. More research is needed to better disambiguate the dual effects of maternal symptoms on fetal and child development from the effects of 5-HT reuptake inhibitors on clinical outcomes in the offspring. Birth Defects Research 109:924-932, 2017. © 2017 Wiley Periodicals, Inc.

Development passes through sensitive periods, during which plasticity allows for genetic and environmental factors to exert indelible influence on the maturation of the organism. In the context of central nervous system development, such sensitive periods shape the formation of neurocircuits that mediate, regulate, and control behavior. This general mechanism allows for development to be guided by both the genetic blueprint as well as the environmental context. While allowing for adaptation, such sensitive periods are also vulnerability windows during which external and internal factors can confer risk to disorders by derailing otherwise resilient developmental programs. Here we review developmental periods that are sensitive to monoamine signaling and impact adult behaviors of relevance to psychiatry. Specifically, we review (1) a serotonin-sensitive period that impacts sensory system development, (2) a serotonin-sensitive period that impacts cognition, anxiety- and depression-related behaviors, and (3) a dopamine- and serotonin-sensitive period affecting aggression, impulsivity and behavioral response to psychostimulants. We discuss preclinical data to provide mechanistic insight, as well as epidemiological and clinical data to point out translational relevance. The field of translational developmental neuroscience has progressed exponentially providing solid conceptual advances and unprecedented mechanistic insight. With such knowledge at hand and important methodological innovation ongoing, the field is poised for breakthroughs elucidating the developmental origins of neuropsychiatric disorders, and thus understanding pathophysiology. Such knowledge of sensitive periods that determine the developmental trajectory of complex behaviors is a necessary step towards improving prevention and treatment approaches for neuropsychiatric disorders.

Serotonin (5-HT) selective reuptake inhibitors (SSRIs) are widely used in the treatment of depression and anxiety disorders, but responsiveness is uncertain and side effects often lead to discontinuation. Side effect profiles suggest that SSRIs reduce dopaminergic (DAergic) activity, but specific mechanistic insight is missing. Here we show in mice that SSRIs impair motor function by acting on 5-HT2C receptors in the substantia nigra pars reticulata (SNr), which in turn inhibits nigra pars compacta (SNc) DAergic neurons. SSRI-induced motor deficits can be reversed by systemic or SNr-localized 5-HT2C receptor antagonism. SSRIs induce SNr hyperactivity and SNc hypoactivity that can also be reversed by systemic 5-HT2C receptor antagonism. Optogenetic inhibition of SNc DAergic neurons mimics the motor deficits due to chronic SSRI treatment, whereas local SNr 5-HT2C receptor antagonism or optogenetic activation of SNc DAergic neurons reverse SSRI-induced motor deficits. Lastly, we find that 5-HT2C receptor antagonism potentiates the antidepressant and anxiolytic effects of SSRIs. Together our findings demonstrate opposing roles for 5-HT2C receptors in the effects of SSRIs on motor function and affective behavior, highlighting the potential benefits of 5-HT2C receptor antagonists for both reduction of motor side effects of SSRIs and augmentation of therapeutic antidepressant and anxiolytic effects.

Adult hippocampal neurogenesis (AHN) is a key process for certain types of hippocampal-dependent learning. Alzheimer’s disease (AD) is accompanied by memory deficits related to alterations in AHN. Given that the increased activity of GSK-3β has been related to alterations in the population of hippocampal granule neurons in AD patients, we designed a novel methodology by which to induce selective GSK-3β overexpression exclusively in newborn granule neurons. To this end, we injected an rtTA-IRES-EGFP-expressing retrovirus into the hippocampus of tTO-GSK-3β mice. Using this novel retroviral strategy, we found that GSK-3β caused a cell-autonomous impairment of the morphological and synaptic maturation of newborn neurons. In addition, we examined whether GSK-3β overexpression in newborn neurons limits the effects of physical activity. While physical exercise increased the number of dendritic spines, the percentage of mushroom spines, and the head diameter of the same in tet-OFF cells, these effects were not triggered in tet-ON cells. This observation suggests that GSK-3β blocks the stimulatory actions of exercise. Given that the activity of GSK-3β is increased in the brains of individuals with AD, these data may be relevant for non-pharmacological therapies for AD., Spanish Ministry of Health (SAF-2014-5040-P), and the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII) (J. A ´ vila); the Japan Society for the promotion of Science (post-doctoral fellowship to M. Llorens-Martín); and the Alzheimer’s Association (2015-NIRG-340709

Visual Abstract, A developing brain shows intense reorganization and heightened neuronal plasticity allowing for environmental modulation of its development. During early life, maternal care is a key factor of this environment and defects in this care can derail adaptive brain development and may result in susceptibility to neuropsychiatric disorders. Nevertheless, the mechanisms by which those maternal interactions immediately impact the offspring’s brain activity to initiate the pathway to pathology are not well understood. We do know that multiple neurotransmitter systems are involved, including the serotonergic system, a key neuromodulator involved in brain development and emotional regulation. We tested the importance of the serotonergic system and pups’ immediate neural response to maternal presence using wireless electrophysiological recordings, a novel approach allowing us to record neural activity during pups’ interactions with their mother. We found that maternal contact modulates the P10–P12 rat pups’ anterior cingulate cortex (ACC) activity by notably increasing local-field potential (LFP) power in low-frequency bands. We demonstrated, by blocking serotonergic receptors, that this increase is mediated through 5-HT2 receptors (5-HT2Rs). Finally, we showed in isolated pups that enhancing serotonergic transmission, using a selective-serotonin-reuptake-inhibitor, is sufficient to enhance LFP power in low-frequency bands in a pattern similar to that observed when the mother is in the nest. Our results highlight a significant contribution of the serotonergic system in mediating changes of cortical activity in pups related to maternal presence.

In humans, damage limited to the hippocampus produces temporally graded retrograde amnesia, with relative sparing of remote compared to recent memory. This observation forms the cornerstone of the idea that as memories mature they are reorganized in a time-dependent manner. In this paper, we evaluate evidence for similar time- dependent reorganization in experimental animals. In the majority of behavioral paradigms examined, these studies provide evidence that memories are gradually reorganized over time, with the hippocampus and cortex playing preferential (although not necessarily mutually exclusive) roles in the expression of recent and remote memory, respectively. This pattern is not observed in all tasks, however. For example, in the water maze hippocampal activity is always necessary for memory expression. Identi- fying situations when the hippocampus is, and is not, required for remote memory expression will help us to understand hippocampal contributions to memory, and, more generally, whether changes in memory organization lead to qualitative changes in the nature of memory.

Although the hippocampus plays a crucial role in the formation of spatial memories, as these memories mature they may become additionally (or even exclusively) dependent on extrahippocampal structures. However, the identity of these extrahippocampal structures that support remote spatial memory is currently not known. Using a Morris water-maze task, we show that the anterior cingulate cortex (ACC) plays a key role in the expression of remote spatial memories in mice. To first evaluate whether the ACC is activated after the recall of spatial memory, we examined the expression of the immediate early gene,c-fos, in the ACC. Fos expression was elevated after expression of a remote (1 month old), but not recent (1 d old), water-maze memory, suggesting that ACC plays an increasingly important role as a function of time. Consistent with the gene expression data, targeted pharmacological inactivation of the ACC with the sodium channel blocker lidocaine blocked expression of remote, but spared recent, spatial memory. In contrast, inactivation of the dorsal hippocampus disrupted expression of spatial memory, regardless of its age. We further showed that inactivation of the ACC blocked expression of remote spatial memory in two different mouse strains, after training with either a hidden or visible platform in a constant location, and using the AMPA receptor antagonist CNQX. Together, our data provide evidence that circuits supporting spatial memory are reorganized in a time-dependent manner, and establish that activity in neurons intrinsic to the ACC is critical for processing remote spatial memories.

Reelin is an extracellular matrix protein that is crucial for neural development and adult brain plasticity. While the Reelin signalling cascade has been reported to be associated with Alzheimer's disease (AD), the role of Reelin in this pathology is not understood. Here we use an in vitro approach to show that Reelin interacts with amyloid-β (Aβ42) soluble species, delays Aβ42 fibril formation and is recruited into amyloid fibrils. Furthermore, Reelin protects against both the neuronal death and dendritic spine loss induced by Aβ42 oligomers. In mice carrying the APP(Swe/Ind) mutation (J20 mice), Reelin overexpression delays amyloid plaque formation and rescues the recognition memory deficits. Our results indicate that by interacting with Aβ42 soluble species, delaying Aβ plaque formation, protecting against neuronal death and dendritic spine loss and preventing AD cognitive deficits, the Reelin pathway deserves consideration as a therapeutic target for the treatment of AD pathogenesis.

Psychiatric disorders have been hypothesized to originate during development, with genetic and environmental factors interacting in the etiology of disease. Therefore, developmentally regulated genes have received attention as risk modulators in psychiatric diseases. Reelin is an extracellular protein essential for neuronal migration and maturation during development, and its expression levels are reduced in psychiatric disorders. Interestingly, several perinatal insults that increase the risk of behavioral deficits alter Reelin signaling. However, it is not known whether a dysfunction in Reelin signaling during perinatal stages increases the risk of psychiatric disorders. Here we used a floxed dab1 allele to study whether a transient decrease in Dab1, a key component of the Reelin pathway, is sufficient to induce behavioral deficits related to psychiatric disorders. We found that transient Dab1 downregulation during perinatal stages leads to permanent abnormalities of structural layering in the neocortex and hippocampus. In contrast, conditional inactivation of the dab1 gene in the adult brain does not result in additional layering abnormalities. Furthermore, perinatal Dab1 downregulation causes behavior impairments in adult mice, such as deficits in memory, maternal care, pre-pulse inhibition, and response to cocaine. Some of these deficits were also found to be present in adolescence. We also show that D-cycloserine rescues the cognitive deficits observed in floxed dab1 mice with layering alterations in the hippocampus and neocortex. Our results indicate a causal relation between the downregulation of Dab1 protein levels during development and the structural and behavioral deficits associated with psychiatric diseases in the adult.

Adult hippocampal neurogenesis (AHN) is crucial for the maintenance of hippocampal function. Several neurodegenerative diseases such as Alzheimer's disease (AD) are accompanied by memory deficits that could be related to alterations in AHN. Here, we took advantage of a conditional mouse model to study the involvement of glycogen synthase kinase-3β (GSK-3β) overexpression (OE) in AHN. By injecting GFP- and PSD95-GFP-expressing retroviruses, we have determined that hippocampal GSK-3β-OE causes dramatic alterations in both dendritic tree morphology and post-synaptic densities in newborn neurons. Alterations in previously damaged neurons were reverted by switching off the transgenic system and also by using a physiological approach (environmental enrichment) to increase hippocampal plasticity. Furthermore, comparative morphometric analysis of granule neurons from patients with AD and from GSK-3β overexpressing mice revealed shared morphological alterations. Taken together, these data indicate that GSK-3β is crucial for hippocampal function, thereby supporting this kinase as a relevant target for the treatment of AD. © 2013 Macmillan Publishers Limited., Spanish Ministry of Health (SAF 2006-02424, BFU-2008-03980 and BFU-2010-21507); the Comunidad de Madrid (SAL/0202/2006); Fundación M. Botín; Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, ISCIII); Fundación R Areces.

In the dentate gyrus of wild-type (wt) mice (a), neuronal precursors maturate and differentiate into mature granule neurons under the influence of pro-neurogenic molecules, such as neurotrophic factors and anti-inflammatory cytokines. In glycogen synthase kinase 3 (GSK-3b)-overexpressing mice (GSK-3b-OE mice) (b), increased neuronal death activates microglia and increases pro-inflammatory cytokine (affecting early neuronal precursor development, as an indirect consequence of GSK-3b overexpression). Throughout the maturational process, morphological alterations are observed in granule neurons of GSK-3b-OE mice. In addition, as a direct consequence of GSK-3b-OE, these cells experience a dramatic reduction of postsynaptic cluster number and volume. Doxycycline treatment did not produce any significant effect on wt mice (c), whereas it successfully reverted the aberrant morphology of granule neurons and normalized pro-inflammatory cytokine levels in GSK-3b-OE mice (d). In addition, the restoration of normal levels of GSK-3b activity produced a drastic increase in the number of postsynaptic clusters (d). The number of postsynaptic clusters is drastically increased in wt mice after environmental enrichment (EE) (e). It is important to note that, in wt mice, EE particularly increased the number of small, newly developed, synaptic contacts, whereas postsynaptic cluster size was increased in GSK-3b-OE animals after EE (f), potentially contributing to enhanced synaptic strength. Interestingly, both doxycycline treatment and EE restored the increased number of ‘pathological’ microglial cells and also normalized pro-inflammatory cytokines to basal levels in GSK-3b-OE mice, thus allowing newborn neurons to develop appropriate morphology and connectivity.

Pharmacologic blockade of monoamine oxidase A (MAOA) or serotonin transporter (5-HTT) has antidepressant and anxiolytic efficacy in adulthood. Yet, genetically conferred MAOA or 5-HTT hypo-activity is associated with altered aggression and increased anxiety/depression. Here we test the hypothesis that increased monoamine signaling during development causes these paradoxical aggressive and affective phenotypes. We find that pharmacologic MAOA blockade during early postnatal development (P2-P21) but not during peri-adolescence (P22-41) increases anxiety- and depression-like behavior in adult (> P90) mice, mimicking the effect of P2-21 5-HTT inhibition. Moreover, MAOA blockade during peri-adolescence, but not P2-21 or P182-201, increases adult aggressive behavior, and 5-HTT blockade from P22-P41 reduced adult aggression. Blockade of the dopamine transporter, but not the norepinephrine transporter, during P22-41 also increases adult aggressive behavior. Thus, P2-21 is a sensitive period during which 5-HT modulates adult anxiety/depression-like behavior, and P22-41 is a sensitive period during which DA and 5-HT bi-directionally modulate adult aggression. Permanently altered DAergic function as a consequence of increased P22-P41 monoamine signaling might underlie altered aggression. In support of this hypothesis, we find altered aggression correlating positively with locomotor response to amphetamine challenge in adulthood. Proving that altered DA function and aggression are causally linked, we demonstrate that optogenetic activation of VTA DAergic neurons increases aggression. It therefore appears that genetic and pharmacologic factors impacting dopamine and serotonin signaling during sensitive developmental periods can modulate adult monoaminergic function and thereby alter risk for aggressive and emotional dysfunction.

Neuronal distal axons have limited access to glucose, their myelin sheaths preventing direct interface with blood, and their axoplasms being at great distances from their cell bodies. Glycogen, the mammalian glucose store, is characterized by extreme branching, which allows packing 55,000 glucoses/glycogen, and maintaining solubility. In neurons, glycogen phosphorylase, the glycogen-digesting enzyme, is located in distal axons where it can generate large amounts of glucose from glycogen.1

In the adult hippocampal dentate gyrus, newborn granule cells grow dendrites into the molecular layer and send axons into the CA3 region. Several molecular markers have been used to analyze production of these new neurons; however, no good markers for new axons have been described. Here we demonstrate that tau protein isoform with three microtubule binding domains (3R-Tau) is a marker of those axons following an antigen retrieval protocol. By using retrovirus-mediated GFP transduction, GFP can be detected in a period of 7-14 days after viral infection. We also provide a >proof of principle> demonstration of the power of that labeling showing modulation of 3R-Tau positive axons under physiological conditions (exercise and aging) as well as in a FTDP-17 neurodegenerative model and Alzheimer's disease models (mice overexpressing AβPPsw, ind or GSK3β). We conclude that 3R-Tau would be an efficient marker and a valuable tool to study new axons in adult neurogenesis as well as in neurodegenerative processes. © 2012-IOS Press and the authors. All rights reserved., Spanish Ministry of Health (SAF 2006-02424; SAF2010-15525); Comunidad de Madrid (SAL/0202/2006); Fundación CIEN (PI 008-09); Fundación M. Botín; Fundación Ramón Areces

Adult hippocampal neurogenesis is thought to be essential for learning and memory, and has been implicated in the pathogenesis of several disorders. Although recent studies have identified key factors regulating neuroprogenitor proliferation in the adult hippocampus, the mechanisms that control the migration and integration of adult-born neurons into circuits are largely unknown. Reelin is an extracellular matrix protein that is vital for neuronal development. Activation of the Reelin cascade leads to phosphorylation of Disabled-1, an adaptor protein required for Reelin signaling. Here we used transgenic mouse and retroviral reporters along with Reelin signaling gain-of-function and loss-of-function studies to show that the Reelin pathway regulates migration and dendritic development of adultgenerated hippocampal neurons. Whereas overexpression of Reelin accelerated dendritic maturation, inactivation of the Reelin signaling pathway specifically in adult neuroprogenitor cells resulted in aberrant migration, decreased dendrite development, formation of ectopic dendrites in the hilus, and the establishment of aberrant circuits. Our findings support a cell-autonomous and critical role for the Reelin pathway in regulating dendritic development and the integration of adult-generated granule cells and point to this pathway as a key regulator of adult neurogenesis. Moreover, our data reveal a novel role of the Reelin cascade in adult brain function with potential implications for the pathogenesis of several neurological and psychiatric disorders. © 2012 the authors., This project was supported by Grant BFU2008-3980 from the Ministerio de Ciencia e Innovación (MICINN), Spain; by grants from the Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) and Caixa Catalunya-Obra Social Foundations to E.S.; by grants from the Spanish Ministry of Science and Innovation (SAF2009-07367 and CONSOLIDER CSD2007-00023) to V.B.; by the Fred Annegers Fellowship from the Epilepsy Foundation (M.M.K.); and by NIH Grant NS058585 to J.M.P. I.R. was recipient of a Formación de Personal Universitario predoctoral fellowship from MINECO (Spain).

Deep brain stimulation (DBS) is an established therapeutic modality for the treatment of movement disorders and an emerging therapeutic approach for the treatment of disorders of mood and thought. For example, recently we have shown that DBS of the fornix may ameliorate cognitive decline associated with dementia. However, like other applications of DBS, the mechanisms mediating these clinical effects are unknown. As DBS modulates neurophysiological activity in targeted brain regions, DBS might influence cognitive function via activity-dependent regulation of hippocampal neurogenesis. Using stimulation parameters analogous to clinical high-frequency DBS, here we addressed this question in mice. We found that acute stimulation of the entorhinal cortex (EC) transiently promoted proliferation in the dentate gyrus (DG). Cells generated as a consequence of stimulation differentiated into neurons, survived for at least several weeks, and acquired normal dentate granule cell (DGC) morphology. Importantly, stimulation-induced promotion of neurogenesis was limited to the DG and not associated with changes in apoptotic cell death. Using immunohistochemical approaches, we found that, once sufficiently mature, these stimulation-induced neurons integrated into hippocampal circuits supporting water-maze memory. Finally, formation of water-maze memory was facilitated 6 weeks (but not 1 week) after bilateral stimulation of the EC. The delay-dependent nature of these effects matches the maturation-dependent integration of adult-generated DGCs into dentate circuits supporting water-maze memory. Furthermore, because the beneficial effects of EC stimulation were prevented by blocking neurogenesis, this suggests a causal relationship between stimulation-induced promotion of adult neurogenesis and enhanced spatial memory.

Despite the impact of schizophrenia and mood disorders, which in extreme cases can lead to death, recent decades have brought little progress in the development of new treatments. Recent studies have shown that Reelin, an extracellular protein that is critical for neuronal development, is reduced in schizophrenia and bipolar disorder patients. However, data on a causal or protective role of Reelin in psychiatric diseases is scarce. In order to study the direct influence of Reelin's levels on behavior, we subjected two mouse lines, in which Reelin levels are either reduced (Reelin heterozygous mice) or increased (Reelin overexpressing mice), to a battery of behavioral tests: open-field, black-white box, novelty-suppressed-feeding, forced-swim-test, chronic corticosterone treatment followed by forced-swim-test, cocaine sensitization and pre-pulse inhibition (PPI) deficits induced by N-methyl-D-aspartate (NMDA) antagonists. These tests were designed to model some aspects of psychiatric disorders such as schizophrenia, mood, and anxiety disorders. We found no differences between Reeler heterozygous mice and their wild-type littermates. However, Reelin overexpression in the mouse forebrain reduced the time spent floating in the forced-swim-test in mice subjected to chronic corticosterone treatment, reduced behavioral sensitization to cocaine, and reduced PPI deficits induced by a NMDA antagonist. In addition, we demonstrate that while stress increased NMDA NR2B-mediated synaptic transmission, known to be implicated in depression, Reelin overexpression significantly reduced it. Together, these results point to the Reelin signaling pathway as a relevant drug target for the treatment of a range of psychiatric disorders. This work was supported by grants from CIBERNED, Plan Nacional de Drogas, Caixa Catalunya Foundation (Obra Social), MICINN and INCRECyT (European Social Fund and JCCM)

In the hippocampus, the production of dentate granule cells (DGCs) persists into adulthood. As adult-generated neurons are thought to contribute to hippocampal memory processing, promoting adult neurogenesis therefore offers the potential for restoring mnemonic function in the aged or diseased brain. Within this regenerative context, one key issue is whether developmentally generated and adult-generated DGCs represent functionally equivalent or distinct neuronal populations. To address this, we labeled separate cohorts of developmentally generated and adult-generated DGCs and used immunohistochemical approaches to compare their integration into circuits supporting hippocampus-dependent memory in intact mice. First, in the water maze task, rates of integration of adult-generated DGCs were regulated by maturation, with maximal integration not occurring until DGCs were five or more weeks in age. Second, these rates of integration were equivalent for embryonically, postnatally, and adult-generated DGCs. Third, these findings generalized to another hippocampus-dependent task, contextual fear conditioning. Together, these experiments indicate that developmentally generated and adult-generated DGCs are integrated into hippocampal memory networks at similar rates, and suggest a functional equivalence between DGCs generated at different developmental stages.

Reelin, an extracellular protein essential for neural migration and lamination, is also expressed in the adult brain. To unravel the function of this protein in the adult forebrain, we generated transgenic mice that overexpress Reelin under the control of the CaMKIIα promoter. Overexpression of Reelin increased adult neurogenesis and impaired the migration and positioning of adult-generated neurons. In the hippocampus, the overexpression of Reelin resulted in an increase in synaptic contacts and hypertrophy of dendritic spines. Induction of long-term potentiation (LTP) in alert-behaving mice showed that Reelin overexpression evokes a dramatic increase in LTP responses. Hippocampal field EPSP during a classical conditioning paradigm was also increased in these mice. Our results indicate that Reelin levels in the adult brain regulate neurogenesis and migration, as well as the structural and functional properties of synapses. These observations suggest that Reelin controls developmental processes that remain active in the adult brain.

Although the clarity of many memories fades with time, some memories may maintain their original precision. Here we used a context discrimination procedure to evaluate whether the hippocampus is important in maintaining precision as memories mature. Spared discrimination in hippocampal-lesioned mice indicated that precise, remote context memories may be supported by extra-hippocampal brain regions.

Previous studies have shown that medial prefrontal cortical regions, such as the anterior cingulate cortex (ACC), play a key role in the expression of remote spatial and contextual memory. To evaluate whether this role is conserved in hippocampal-independent tasks we trained mice in the conditioned taste aversion (CTA) paradigm. Lidocaine-induced inactivation of the ACC blocked the expression of CTA tested one month (remote), but not one day (recent), after conditioning with either a weak or strong unconditioned stimulus (US). These data suggest that the ACC may play a conserved role in remote memory, regardless of memory strength or content.

New neurons are continuously generated in the subgranular zone of the hippocampus throughout adulthood, and there is increasing interest as to whether these new neurons become functionally integrated into memory circuits. This protocol describes the immunohistochemical procedures to visualize the recruitment of new neurons into circuits supporting spatial memory in intact mice. To label adult-generated granule cells, mice are injected with the proliferation marker 5-bromo-2′-deoxyuridine (BrdU). At different delays after BrdU treatment, mice are trained to locate a hidden platform in the Morris water maze, and spatial memory can then be tested in a probe test with the platform removed from the pool. Ninety minutes after this probe test, mice are perfused and tissue is sectioned. Immunohistochemical procedures are used to quantify BrdU-labeled cells and expression of the immediate early gene, Fos. Because Fos expression is regulated by neuronal activity, the degree of overlap between BrdU-labeled and Fos-labeled neurons provides an indication of whether adult-generated granule neurons have been incorporated into spatial memory circuits.

Throughout adulthood, new neurons are continuously added to the dentate gyrus, a hippocampal subregion that is important in spatial learning. Whether these adult-generated granule cells become functionally integrated into memory networks is not known. We used immunohistochemical approaches to visualize the recruitment of new neurons into circuits supporting water maze memory in intact mice. We show that as new granule cells mature, they are increasingly likely to be incorporated into circuits supporting spatial memory. By the time the cells are 4 or more weeks of age, they are more likely than existing granule cells to be recruited into circuits supporting spatial memory. This preferential recruitment supports the idea that new neurons make a unique contribution to memory processing in the dentate gyrus.

An emerging theme in systems neurobiology is that even simple forms of memory depend on activity in a broad network of cortical and subcortical brain regions. One key challenge is to understand how different components of these complex networks contribute to memory. In a new study in Molecular Pain, Tang and colleagues use a novel set of approaches to characterize the role of the anterior cingulate cortex (ACC) in the formation of Pavlovian fear memories.