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Abstract: The rat medial prefrontal cortex, an area considered homologous to the human prefrontal cortex, is a region in which neuronal structural plasticity has been described during adulthood. Some plastic processes such as neurite outgrowth and synaptogenesis are known to be regulated by the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). Since PSA-NCAM is present in regions of the adult CNS which are undergoing structural remodeling, such as the hypothalamus or the hippocampus, we have analyzed the expression of this molecule in the medial prefrontal cortex of adult rats using immunohistochemistry. PSA-NCAM immunoreactivity was found both in cell bodies and in the neuropil of the three divisions of the medial prefrontal cortex. All cell somata expressing PSA-NCAM corresponded to neurons and 5′ bromodeoxyuridine labeling after long survival times demonstrated that these neurons were not recently generated. Many of these PSA-NCAM immunoreactive neurons in the medial prefrontal cortex could be classified as interneurons on the basis of their morphology and glutamate decarboxylase, isoform 67 expression. Some of the PSA-NCAM immunoreactive neurons also expressed somatostatin, neuropeptide Y and calbindin-D28K. By contrast, pyramidal neurons in this cortical region did not appear to express PSA-NCAM. However, some of these principal neurons appeared surrounded by PSA-NCAM immunoreactive puncta. Some of these puncta co-expressed synaptophysin, suggesting the presence of synapses. Since the etiology of some psychiatric disorders has been related to alterations in medial prefrontal cortex structural plasticity, the study of PSA-NCAM expression in this region may open a new approach to the pathophysiology of these mental disorders. [Copyright &y& Elsevier]

Early postnatal development is a critical period for the configuration of neural networks that support social and affective-like behaviors. In this sense, children raised in stressful environments are at high risk to develop maladaptive behaviors immediately or later in life, including anti-social and aggressive behaviors. However, the neurobiological bases of such phenomena remain poorly understood. Here we showed that, at long-term, maternal separation with early weaning (MSEW) decreased the density of somatostatin-expressing (SST+) neurons in the basolateral amygdala (BLA) of females and males, while their activity was only reduced in the medial amygdala (MeA) of males. Interestingly, only MSEW males exhibited long-term behavioral effects, including reduced sociability and social novelty preference in the 3-chamber test (3CH), decreased social interest in the resident-intruder test (RI), and increased aggressivity in both the RI and the tube dominance test (TT). To test whether the manipulation of MeA SST+ neurons was sufficient to reverse these negative behavioral outcomes, we expressed the chemogenetic excitatory receptor hM3Dq in MSEW adult males. We found that the activation of MeA SST+ neurons ameliorated social interest in the RI test and reduced aggression traits in the TT and RI assays. Altogether, our results highlight a role for MeA SST+ neurons in the regulation of aggressivity and social interest and point to the loss of activity of these neurons as a plausible etiological mechanism linking early life stress to these maladaptive behaviors in later life., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The exposure to aversive experiences during early-life affects brain maturation and induces changes in behavior. Additionally, when these experiences coincide with subtle neurodevelopmental alterations, they may contribute to the emergence of psychiatric disorders, such as schizophrenia. Studies in patients and animal models have identified changes in parvalbumin (PV) expressing inhibitory neurons, highlighting their significance in the etiology of this disorder. Most studies have been focused on the cortex, but PV+ neurons also provide inhibitory input to diencephalic regions, particularly to the thalamus (through cells in the thalamic reticular nucleus, TRN) and the habenula. Remarkably, alterations in both nuclei have been described in schizophrenia. Some of these changes in PV+ cells may be mediated by perineuronal nets (PNN), specialized regions of the extracellular matrix that often surround them and regulate their synaptic input and activity. Interestingly, the physiological maturation and integration of PV+ neurons, which involves the assembly of PNN, occurs during early postnatal life. Plasticity molecules associated to inhibitory neurons, such as PSA-NCAM, or NMDA receptors (NMDAR) can also influence the structure and function of these cells. Growing evidence also indicates that glial cells regulate the physiology of PV+ neurons by influencing their maturation and modulating their synaptic connectivity. To explore the impact of early-life aversive experiences and concomitant subtle neurodevelopmental alterations on diencephalic PV+ cells, we analyzed adult male mice subjected to a double-hit model (DHM) of schizophrenia, combining a single injection of an NMDAR antagonist at P7 and post-weaning social isolation. We observed that exploratory behavior, PV+ neurons and their associated PNN, as well as PSA-NCAM and NMDAR expression and glial cells, in the TRN and the habenula were affected by the DHM or one of its factors. To our knowledge, this is the first report on such alterations in these diencephalic structures in an animal model combining neurodevelopmental alterations and early-life stress during adolescence. Our findings complement previous work on PV+ neurons in cortical regions and underscore the importance of studying diencephalic inhibitory networks and their intricate interactions with aversive experiences and neurodevelopmental alterations during early life in the context of schizophrenia., (© 2024. The Author(s).)

Adverse experiences during infancy and adolescence have an important and enduring effect on the brain and are predisposing factors for mental disorders, particularly major depression. This impact is particularly notable in regions with protracted development, such as the prefrontal cortex. The inhibitory neurons of this cortical region are altered by peripubertal stress (PPS), particularly in female mice. In this study we have explored whether the inhibitory circuits of the thalamus are impacted by PPS in male and female mice. This diencephalic structure, as the prefrontal cortex, also completes its development during postnatal life and is affected by adverse experiences. The long-term changes induced by PPS were exclusively found in adult female mice. We have found that PPS increases depressive-like behavior and induces changes in parvalbumin-expressing (PV+) cells of the thalamic reticular nucleus (TRN). We observed reductions in the volume of the TRN, together with those of parameters related to structures/molecules that regulate the plasticity and connectivity of PV+ cells: perineuronal nets, matricellular structures surrounding PV+ neurons, and the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). The expression of the GluN1, but not of GluN2C, NMDA receptor subunit was augmented in the TRN after PPS. An increase in the fluorescence intensity of PV+ puncta was also observed in the synaptic output of TRN neurons in the lateral posterior thalamic nucleus. These results demonstrate that the inhibitory circuits of the thalamus, as those of the prefrontal cortex, are vulnerable to the effects of aversive experiences during early life, particularly in females. This vulnerability is probably related to the protracted development of the TRN and might contribute to the development of psychiatric disorders., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Alcohol consumption and withdrawal alter social behaviour in humans in a sex-dependent manner. The three-chamber test is a widely used paradigm to assess rodents' social behaviour, including sociability and social novelty. Automatic tracking systems are commonly used to score time spent with conspecifics, despite failing to score direct interaction time with conspecifics rather than time in the nearby zone. Thereby, the automatically scored results are usually inaccurate and need manual corrections., New Method: New advances in artificial intelligence (AI) have been used recently to analyze complex behaviours. DeepLabCat is a pose-estimation toolkit that allows the tracking of animal body parts. Thus, we used DeepLabCut, to introduce a scoring model of the three-chamber test to investigate alcohol withdrawal effects on social behaviour in mice considering sex and withdrawal periods. We have compared the results of two automatic pose estimation methods: automatic tracking (AnyMaze) and DeepLabCut considering the manual scoring method, the current gold standard., Results: We have found that the automatic tracking method (AnyMaze) has failed to detect the significance of social deficits in female mice during acute withdrawal. However, tracking the animal's nose using DeepLabCut showed a significant social deficit in agreement with manual scoring. Interestingly, this social deficit was shown only in females during acute and recovered by the protracted withdrawal. DLC and manually scored results showed a higher Spearman correlation coefficient and a lower bias in the Bland-Altman analysis., Conclusion: our approach helps improve the accuracy of scoring the three-chamber test while outperforming commercial automatic tracking systems., Competing Interests: The authors declare no competing interests in this paper., (© 2024 The Authors.)

The retrosplenial cortex (RSC) plays a central role in processing contextual fear conditioning. In addition to corticocortical and thalamocortical projections, the RSC receives subcortical inputs, including a substantial projection from the nucleus incertus in the pontine tegmentum. This GABAergic projection contains the neuropeptide, relaxin-3 (RLN3), which inhibits target neurons via its Gi/o-protein-coupled receptor, RXFP3. To assess this peptidergic system role in contextual fear conditioning, we bilaterally injected the RSC of adult rats with an adeno-associated-virus (AAV), expressing the chimeric RXFP3 agonist R3/I5 or a control AAV, and subjected them to contextual fear conditioning. The R3/I5 injected rats did not display any major differences to control-injected and naïve rats but displayed a significantly delayed extinction. Subsequently, we employed acute bilateral injections of the specific RXFP3 agonist peptide, RXFP3-Analogue 2 (A2), into RSC. While the administration of A2 before each extinction trial had no impact on the extinction process, treatment with A2 before each acquisition trial resulted in delayed extinction. In related anatomical studies, we detected an enrichment of RLN3-immunoreactive nerve fibers in deep layers of the RSC, and a higher level of co-localization of RXFP3 mRNA with vesicular GABA transporter (vGAT) mRNA than with vesicular glutamate transporter-1 (vGLUT1) mRNA across the RSC, consistent with an effect of RLN3/RXFP3 signalling on the intrinsic, inhibitory circuits within the RSC. These findings suggest that contextual conditioning processes in the RSC involve, in part, RLN3 afferent modulation of local inhibitory neurons that provides a stronger memory acquisition which, in turn, retards the extinction process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Contextual fear conditioning is a behavioral paradigm used to assess hippocampal-dependent memory in experimental animals. Perception of the context depends on activation of a distinct population of neurons in the hippocampus and in hippocampal-related areas that process discrete aspects of context perception. In the absence of any putatively associated cue, the context becomes the salient element that may warn of an upcoming aversive event; and in particular conditions, animals generalize this warning to any new or similar context. In this study we evaluated the effects of the number of sessions, the number of unconditioned stimuli per acquisition session and the distribution of extinction sessions to assess fear acquisition and extinction and determine under which conditions generalization occurred in adult, male rats. We observed that the organization and spacing of sessions were relevant factors in the acquisition and extinction of contextual fear memories. Extinction occurred with significantly greater robustness when sessions were spread over two days. Furthermore, results indicated that exposure to a single 0.3 mA, 0.5 s footshock in two different sessions could produce context-specific fear, while more acquisition sessions or more footshocks within a single session produced a generalization of the fear response to a new context. Notably, when generalization occurred, successive re-exposure to the generalized context produced extinction in a similar way to the paired exposure. Together, the present findings identify clear procedural and behavioral parameters amenable to neural systems analysis of three clinically relevant outcomes of contextual fear conditioning, i.e., memory acquisition, storage and extinction., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Crown Copyright © 2024. Published by Elsevier B.V. All rights reserved.)

Nucleus incertus (NI) neurons in the pontine tegmentum give rise to ascending forebrain projections and express the neuropeptide relaxin-3 (RLN3) which acts via the relaxin-family peptide 3 receptor (RXFP3). Activity in the hippocampus and entorhinal cortex can be driven from the medial septum (MS), and the NI projects to all these centers, where a prominent pattern of activity is theta rhythm, which is related to spatial memory processing. Therefore, we examined the degree of collateralization of NI projections to the MS and the medial temporal lobe (MTL), comprising medial and lateral entorhinal cortex (MEnt, LEnt) and dentate gyrus (DG), and the ability of the MS to drive entorhinal theta in the adult rat. We injected fluorogold and cholera toxin-B into the MS septum and either MEnt, LEnt or DG, to determine the percentage of retrogradely labeled neurons in the NI projecting to both or single targets, and the relative proportion of these neurons that were RLN3-positive ( +). The projection to the MS was threefold stronger than that to the MTL. Moreover, a majority of NI neurons projected independently to either MS or the MTL. However, RLN3 + neurons collateralize significantly more than RLN3-negative (-) neurons. In in vivo studies, electrical stimulation of the NI induced theta activity in the MS and the entorhinal cortex, which was impaired by intraseptal infusion of an RXFP3 antagonist, R3(BΔ23-27)R/I5, particularly at ~ 20 min post-injection. These findings suggest that the MS plays an important relay function in the NI-induced generation of theta within the entorhinal cortex., (© 2023. The Author(s).)

Introduction: The septal area provides a rich innervation to the hippocampus regulating hippocampal excitability to different behavioral states and modulating theta rhythmogenesis. However, little is known about the neurodevelopmental consequences of its alterations during postnatal development. The activity of the septohippocampal system is driven and/or modulated by ascending inputs, including those arising from the nucleus incertus (NI), many of which contain the neuropeptide, relaxin-3 (RLN3)., Methods: We examined at the molecular and cellular level the ontogeny of RLN3 innervation of the septal area in postnatal rat brains., Results: Up until P13-15 there were only scattered fibers in the septal area, but a dense plexus had appeared by P17 that was extended and consolidated throughout the septal complex by P20. There was a decrease in the level of colocalization of RLN3 and synaptophysin between P15 and P20 that was reversed between P20 and adulthood. Biotinylated 3-kD dextran amine injections into the septum, revealed retrograde labeling present in the brainstem at P10-P13, but a decrease in anterograde fibers in the NI between P10-20. Simultaneously, a differentiation process began during P10-17, resulting in fewer NI neurons double-labeled for serotonin and RLN3., Discussion: The onset of the RLN3 innervation of the septum complex between P17-20 is correlated with the onset of hippocampal theta rhythm and several learning processes associated with hippocampal function. Together, these data highlight the relevance and need for further analysis of this stage for normal and pathological septohippocampal development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ros-Bernal, Gil-Miravet, Lucerón, Navarro-Sánchez, Castillo-Gómez, Gundlach and Olucha-Bordonau.)

Both early life aversive experiences and intrinsic alterations in early postnatal neurodevelopment are considered predisposing factors for psychiatric disorders, such as schizophrenia. The prefrontal cortex and the hippocampus have protracted postnatal development and are affected in schizophrenic patients. Interestingly, similar alterations have been observed in the retrosplenial cortex (RSC). Studies in patients and animal models of schizophrenia have found alterations in cortical parvalbumin (PV) expressing interneurons, making them good candidates to study the etiopathology of this disorder. Some of the alterations observed in PV+ interneurons may be mediated by perineuronal nets (PNNs), specialized regions of the extracellular matrix, which frequently surround these inhibitory neurons. In this study, we have used a double hit model (DHM) combining a single perinatal injection of an NMDAR antagonist (MK801) to disturb early postnatal development and post-weaning social isolation as an early life aversive experience. We have investigated PV expressing interneurons and PNNs in the hippocampus and the RSC of adult male mice, using unbiased stereology. In the CA1, but not in the CA3 region, of the hippocampus, the number of PNNs and PV + PNN+ cells was affected by the drug treatment, and a significant decrease of these parameters was observed in the groups of animals that received MK801. The percentage of PNNs surrounding PV+ cells was significantly decreased after treatment in both hippocampal regions; however, the impact of isolation was observed only in CA1, where isolated animals presented lower percentages. In the RSC , we observed significant effects of isolation, MK801 and the interaction of both interventions on the studied parameters; in the DHM, we observed a significantly lower number of PV+, PNNs, and PV+PNN+cells when compared to control mice. Similar significant decreases were observed for the groups of animals that were just isolated or treated with MK801. To our knowledge, this is the first report on such alterations in the RSC in an animal model combining neurodevelopmental alterations and aversive experiences during infancy/adolescence. These results show the impact of early-life events on different cortical regions, especially on the structure and plasticity of PV+ neurons and their involvement in the emergence of certain psychiatric disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Klimczak, Rizzo, Castillo-Gómez, Perez-Rando, Gramuntell, Beltran and Nacher.)

Spatial learning and memory processes depend on anatomical and functional interactions between the hippocampus and the entorhinal cortex. A key neurophysiological component of these processes is hippocampal theta rhythm, which can be driven from subcortical areas including the pontine nucleus incertus (NI). The NI contains the largest population of neurons that produce and presumably release the neuropeptide, relaxin-3, which acts via the G i/o -protein-coupled receptor, relaxin-family peptide 3 receptor (RXFP3). NI activation induces general arousal including hippocampal theta, and inactivation induces impairment of spatial memory acquisition or retrieval. The primary aim of this study was to map the NI/relaxin-3 innervation of the parahippocampal cortex (PHC), including the medial and lateral entorhinal cortex, endopiriform cortex, perirhinal, postrhinal, and ectorhinal cortex, the amygdalohippocampal transition area and posteromedial cortical amygdala. Retrograde tracer injections were placed in different parts of the medial and lateral entorhinal cortex, which produced prominent retrograde labeling in the ipsilateral NI and some labeling in the contralateral NI. Anterograde tracer injections into the NI and immunostaining for relaxin-3 produced fiber labeling in deep layers of all parahippocampal areas and some dispersed fibers in superficial layers. Double-labeling studies revealed that both hippocampal projecting and calcium-binding protein-positive (presumed GABAergic) neurons received a relaxin-3 NI innervation. Some of these fibers also displayed synaptophysin (Syn) immunoreactivity, consistent with the presence of the peptide at synapses; and relaxin-3-positive fibers containing Syn bouton-like staining were frequently observed in contact with hippocampal-projecting or calcium-binding protein-positive neuronal somata and more distal elements. Finally, in situ hybridization studies revealed that entorhinal neurons in the superficial layers, and to a lesser extent in deep layers, contain RXFP3 mRNA. Together, our data support functional actions of the NI/relaxin-3-parahippocampal innervation on processes related to memory, spatial navigation and contextual analysis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 García-Díaz, Gil-Miravet, Albert-Gasco, Mañas-Ojeda, Ros-Bernal, Castillo-Gómez, Gundlach and Olucha-Bordonau.)

The impact of stressful events is especially important during early life, because certain cortical regions, especially the prefrontal cortex (PFC), are still developing. Consequently, aversive experiences that occur during the peripubertal period can cause long-term alterations in neural connectivity, physiology and related behaviors. Although sex influences the stress response and women are more likely to develop stress-related psychiatric disorders, knowledge about the effects of stress on females is still limited. In order to analyze the long-term effects of peripubertal stress (PPS) on the excitatory and inhibitory circuitry of the adult PFC, and whether these effects are sex-dependent, we applied an unpredictable chronic PPS protocol based on psychogenic stressors. Using two strains of transgenic mice with specific fluorescent cell reporters, we studied male and diestrus females to know how PPS affects the structure and connectivity of parvalbumin expressing (PV+) interneurons and pyramidal neurons. We also studied the expression of molecules related to excitatory and inhibitory neurotransmission, as well as alterations in the expression of plasticity-related molecules. The structure of pyramidal neurons was differentially affected by PPS in male and female mice: while the former had a decreased dendritic spine density, the latter displayed an increase in this parameter. PPS affected the density of puncta expressing excitatory and inhibitory synaptic markers exclusively in the female mPFC. Similarly, only in female mice we observed an increased complexity of the dendritic tree of PV+ neurons. Regarding the perisomatic innervation on pyramidal and PV + neurons by basket cells, we found a significant increase in the density of puncta in stressed animals, with interesting differences between the sexes and the type of basket cell analyzed. Finally, the PPS protocol also altered the total number of somata expressing the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) when we analyzed both sexes together. These results highlight the strong programming effects of aversive experiences during early life for the establishment of cortical circuitry and the special impact of these stressful events on females., (© 2021 The Authors.)

Telencephalic cognitive and emotional circuits/functions are strongly modulated by subcortical inputs. The main focus of past research on the nature of this modulation has been on the widespread monoamine projections to the telencephalon. However, the nucleus incertus (NI) of the pontine tegmentum provides a strong GABAergic and peptidergic innervation of the hippocampus, basal forebrain, amygdala, prefrontal cortex, and related regions; and represents a parallel source of ascending modulation of cognitive and emotional domains. NI GABAergic neurons express multiple peptides, including neuromedin-B, cholecystokinin, and relaxin-3, and receptors for stress and arousal transmitters, including corticotrophin-releasing factor and orexins/hypocretins. A functional relationship exists between NI neurons and their associated peptides, relaxin-3 and neuromedin-B, and hippocampal theta rhythm, which in turn, has a key role in the acquisition and extinction of declarative and emotional memories. Furthermore, RXFP3, the cognate receptor for relaxin-3, is a G i/o protein-coupled receptor, and its activation inhibits the cellular accumulation of cAMP and induces phosphorylation of ERK, processes associated with memory formation in the hippocampus and amygdala. Therefore, this review summarizes the role of NI transmitter systems in relaying stress- and arousal-related signals to the higher neural circuits and processes associated with memory formation and retrieval., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gil-Miravet, Mañas-Ojeda, Ros-Bernal, Castillo-Gómez, Albert-Gascó, Gundlach and Olucha-Bordonau.)

Social and affective relations occur at every stage of our lives. Impairments in the quality of this "social world" can be exceptionally detrimental and lead to psychopathology or pathological behavior, including schizophrenia, autism spectrum disorder, affective disorders, social phobia or violence, among other things. Exposure to highly stressful or traumatic events, depending on the stage of life in which stress exposure occurs, could severely affect limbic structures, including the amygdala, and lead to alterations in social and affective behaviors. This review summarizes recent findings from stress research and provides an overview of its age-dependent effects on the structure and function of the amygdala, which includes molecular and cellular changes, and how they can trigger deviant social and affective behaviors. It is important to highlight that discoveries in this field may represent a breakthrough both for medical science and for society, as they may help in the development of new therapeutic approaches and prevention strategies in neuropsychiatric disorders and pathological behaviors.

The signaling pathway of the microtubule-associated protein kinase or extracellular regulated kinase (MAPK/ERK) is a common mechanism of extracellular information transduction from extracellular stimuli to the intracellular space. The transduction of information leads to changes in the ongoing metabolic pathways and the modification of gene expression patterns. In the central nervous system, ERK is expressed ubiquitously, both temporally and spatially. As for the temporal ubiquity, this signaling system participates in three key moments: (i) Embryonic development; (ii) the early postnatal period; and iii) adulthood. During embryonic development, the system is partly responsible for the patterning of segmentation in the encephalic vesicle through the FGF8-ERK pathway. In addition, during this period, ERK directs neurogenesis migration and the final fate of neural progenitors. During the early postnatal period, ERK participates in the maturation process of dendritic trees and synaptogenesis. During adulthood, ERK participates in social and emotional behavior and memory processes, including long-term potentiation. Alterations in mechanisms related to ERK are associated with different pathological outcomes. Genetic alterations in any component of the ERK pathway result in pathologies associated with neural crest derivatives and mental dysfunctions associated with autism spectrum disorders. The MAP-ERK pathway is a key element of the neuroinflammatory pathway triggered by glial cells during the development of neurodegenerative diseases, such as Parkinson's and Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis, as well as prionic diseases. The process triggered by MAPK/ERK activation depends on the stage of development (mature or senescence), the type of cellular element in which the pathway is activated, and the anatomic neural structure. However, extensive gaps exist with regards to the targets of the phosphorylated ERK in many of these processes.

The medial prefrontal cortex (mPFC) has been classically defined as the brain region responsible for higher cognitive functions, including the decision-making process. Ample information has been gathered during the last 40 years in an attempt to understand how it works. We now know extensively about the connectivity of this region and its relationship with neuromodulatory ascending projection areas, such as the dorsal raphe nucleus (DRN) or the ventral tegmental area (VTA). Both areas are well-known regulators of the reward-based decision-making process and hence likely to be involved in processes like evidence integration, impulsivity or addiction biology, but also in helping us to predict the valence of our future actions: i.e., what is "good" and what is "bad." Here we propose a hypothesis of a critical period, during which the inputs of the mPFC compete for target innervation, establishing specific prefrontal network configurations in the adult brain. We discuss how these different prefrontal configurations are linked to brain diseases such as addiction or neuropsychiatric disorders, and especially how drug abuse and other events during early life stages might lead to the formation of more vulnerable prefrontal network configurations. Finally, we show different promising pharmacological approaches that, when combined with the appropriate stimuli, will be able to re-establish these functional prefrontocortical configurations during adulthood., (Copyright © 2020 Guirado, Perez-Rando, Ferragud, Gutierrez-Castellanos, Umemori, Carceller, Nacher and Castillo-Gómez.)

The quantification of the expression of different molecules is a key question in both basic and applied sciences. While protein quantification through molecular techniques leads to the loss of spatial information and resolution, immunohistochemistry is usually associated with time-consuming image analysis and human bias. In addition, the scarce automatic software analysis is often proprietary and expensive and relies on a fixed threshold binarization. Here we describe and share a set of macros ready for automated fluorescence analysis of large batches of fixed tissue samples using FIJI/ImageJ. The quantification of the molecules of interest are based on an automatic threshold analysis of immunofluorescence images to automatically identify the top brightest structures of each image. These macros measure several parameters commonly quantified in basic neuroscience research, such as neuropil density and fluorescence intensity of synaptic puncta, perisomatic innervation and col-localization of different molecules and analysis of the neurochemical phenotype of neuronal subpopulations. In addition, these same macro functions can be easily modified to improve similar analysis of fluorescent probes in human biopsies for diagnostic purposes based on the expression patterns of several molecules.

N-Methyl-d-Aspartate receptors (NMDARs) are present in both pyramidal neurons and interneurons of the hippocampus. These receptors play a key role in the structural plasticity of excitatory neurons, but to date little is known about their influence on the remodeling of interneurons. Among hippocampal interneurons, the somatostatin expressing cells in the CA1 stratum oriens are of special interest because of their functional importance and structural characteristics: they display dendritic spines, which change their density in response to different stimuli. In order to understand the role of NMDAR activation on the structural dynamics of the spines of somatostatin expressing interneurons in the CA1 stratum oriens, we have studied entorhino-hippocampal organotypic cultures obtained from mice in which this interneuronal subpopulation expresses constitutively EGFP, and have imaged them in real-time. We have acutely infused the cultures with NMDA, a strong NMDAR agonist, and have analyzed the structural dynamics of somatostatin expressing interneurons, prior and after its administration. The appearance and disappearance rates of their dendritic spines increased 24h after the NMDA infusion and returned to baseline levels 48h afterwards. By contrast, their stability rate decreased 24h after the infusion and also returned to control levels 48h later. The relative density of the dendritic spines remained unaltered throughout the assay. Altogether, our results show that the activation of NMDARs can influence the structural dynamics of interneurons. This is especially important because of the involvement of these receptors in neuronal potentiation/depression and their putative role in the etiopathology of certain neuropsychiatric disorders, such as schizophrenia., (Copyright © 2017 Elsevier B.V. All rights reserved.)

N-methyl-D-aspartate receptors (NMDARs) are present in both pyramidal neurons and interneurons of the hippocampus. These receptors play an important role in the adult structural plasticity of excitatory neurons, but their impact on the remodeling of interneurons is unknown. Among hippocampal interneurons, somatostatin-expressing cells located in the stratum oriens are of special interest because of their functional importance and structural characteristics: they display dendritic spines, which change density in response to different stimuli. In order to understand the role of NMDARs on the structural plasticity of these interneurons, we have injected acutely MK-801, an NMDAR antagonist, to adult mice which constitutively express enhanced green fluorescent protein (EGFP) in these cells. We have behaviorally tested the animals, confirming effects of the drug on locomotion and anxiety-related behaviors. NMDARs were expressed in the somata and dendritic spines of somatostatin-expressing interneurons. Twenty-four hours after the injection, the density of spines did not vary, but we found a significant increase in the density of their en passant boutons (EPB) . We have also used entorhino-hippocampal organotypic cultures to study these interneurons in real-time. There was a rapid decrease in the apparition rate of spines after MK-801 administration, which persisted for 24 h and returned to basal levels afterwards. A similar reversible decrease was detected in spine density. Our results show that both spines and axons of interneurons can undergo remodeling and highlight NMDARs as regulators of this plasticity. These results are specially relevant given the importance of all these players on hippocampal physiology and the etiopathology of certain psychiatric disorders.

The exposure to aversive experiences during early life influences brain development and leads to altered behavior. Moreover, the combination of these experiences with subtle alterations in neurodevelopment may contribute to the emergence of psychiatric disorders, such as schizophrenia. Recent hypotheses suggest that imbalances between excitatory and inhibitory (E/I) neurotransmission, especially in the prefrontal cortex and the amygdala, may underlie their etiopathology. In order to understand better the neurobiological bases of these alterations, we studied the impact of altered neurodevelopment and chronic early-life stress on these two brain regions. Transgenic mice displaying fluorescent excitatory and inhibitory neurons, received a single injection of MK801 (NMDAR antagonist) or vehicle solution at postnatal day 7 and/or were socially isolated from the age of weaning until adulthood (3 months old). We found that anxiety-related behavior, brain volume, neuronal structure, and the expression of molecules related to plasticity and E/I neurotransmission in adult mice were importantly affected by early-life stress. Interestingly, many of these effects were potentiated when the stress paradigm was applied to mice perinatally injected with MK801 ("double-hit" model). These results clearly show the impact of early-life stress on the adult brain, especially on the structure and plasticity of inhibitory networks, and highlight the double-hit model as a valuable tool to study the contribution of early-life stress in the emergence of neurodevelopmental psychiatric disorders, such as schizophrenia., Competing Interests: All authors reported no biomedical financial interests or potential conflicts of interest.

The structure and function of the medial prefrontal cortex (mPFC) is affected in several neuropsychiatric disorders, including schizophrenia and major depression. Recent studies suggest that imbalances between excitatory and inhibitory activity (E/I) may be responsible for this cortical dysfunction and therefore, may underlie the core symptoms of these diseases. This E/I imbalance seems to be correlated with alterations in the plasticity of interneurons but there is still scarce information on the mechanisms that may link these phenomena. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is a good candidate, because it modulates the neuronal plasticity of interneurons and its expression is altered in schizophrenia and major depression. To address this question, we have developed an in vitro model using mPFC organotypic cultures of transgenic mice displaying fluorescent spiny interneurons. After enzymatic depletion of PSA, the spine density of interneurons, the number of synaptic puncta surrounding pyramidal neuron somata and the E/I ratio were strongly affected. These results point to the polysialylation of NCAM as an important factor in the maintenance of E/I balance and the structural plasticity of interneurons. This may be particularly relevant for better understanding the etiology of schizophrenia and major depression.

Dopamine D2 receptors (D2R) in the medial prefrontal cortex (mPFC) are key players in the etiology and therapeutics of schizophrenia. The overactivation of these receptors contributes to mPFC dysfunction. Chronic treatment with D2R agonists modifies the expression of molecules implicated in neuronal structural plasticity, synaptic function, and inhibitory neurotransmission, which are also altered in schizophrenia. These changes are dependent on the expression of the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), a plasticity-related molecule, but nothing is known about the effects of D2R and PSA-NCAM on excitatory neurotransmission and the structure of mPFC pyramidal neurons, two additional features affected in schizophrenia. To evaluate these parameters, we have chronically treated adult rats with PPHT (a D2R agonist) after enzymatic removal of PSA with Endo-N. Both treatments decreased spine density in apical dendrites of pyramidal neurons without affecting their inhibitory innervation. Endo-N also reduced the expression of vesicular glutamate transporter-1. These results indicate that D2R and PSA-NCAM are important players in the regulation of the structural plasticity of mPFC excitatory neurons. This is relevant to our understanding of the neurobiological basis of schizophrenia, in which structural alterations of pyramidal neurons and altered expression of D2R and PSA-NCAM have been found.

Diabetes mellitus patients are at increased risk of developing depression, although the neurobiological bases of this comorbidity are not yet fully understood. These patients show CNS alterations, similar to those found in major depression, including changes in the structure and neurotransmission of excitatory neurons. However, although depressive patients and animal models also display alterations in inhibitory networks, little is known about the effects of diabetes on interneurons. Our main objective was to study the impact of diabetes on interneurons of the medial prefrontal cortex (mPFC), one of the regions most affected by major depression. For this purpose we have induced diabetes with high-dose streptozotozin in transgenic mice displaying fluorescent interneurons. These animals showed a depressive-like behavior (increased immobility time in tail suspension test) in parallel with reductions in interneuronal dendritic arborization and in the expression of GAD67, the enzyme that synthetizes the inhibitory neurotransmitter GABA. However, the levels of PSA-NCAM, a plasticity-related molecule exclusively expressed by interneurons in the mPFC, were unaltered in the different regions and layers of this cortical area. Interestingly, diabetic mice also showed increased levels of synaptophysin, a synaptic vesicle protein. These results indicate that the structure and neurotransmission of interneurons is altered in the mPFC of diabetic mice and suggest that these changes may play a key role in the depressive symptoms associated to diabetes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Excitatory neurons undergo dendritic spine remodeling in response to different stimuli. However, there is scarce information about this type of plasticity in interneurons. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is a good candidate to mediate this plasticity as it participates in neuronal remodeling and is expressed by some mature cortical interneurons, which have reduced dendritic arborization, spine density, and synaptic input. To study the connectivity of the dendritic spines of interneurons and the influence of PSA-NCAM on their dynamics, we have analyzed these structures in a subpopulation of fluorescent spiny interneurons in the hippocampus of glutamic acid decarboxylase-enhanced green fluorescent protein transgenic mice. Our results show that these spines receive excitatory synapses. The depletion of PSA in vivo using the enzyme Endo-Neuraminidase-N (Endo-N) increases spine density when analyzed 2 days after, but decreases it 7 days after. The dendritic spine turnover was also analyzed in real time using organotypic hippocampal cultures: 24 h after the addition of EndoN, we observed an increase in the apparition rate of spines. These results indicate that dendritic spines are important structures in the control of the synaptic input of hippocampal interneurons and suggest that PSA-NCAM is relevant in the regulation of their morphology and connectivity., (© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Stress during childhood and adolescence is a risk factor for psychopathology. Alterations in γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain, have been found following stress exposure and fear experiences and are often implicated in anxiety and mood disorders. Abnormal amygdala functioning has also been detected following stress exposure and is also implicated in anxiety and social disorders. However, the amygdala is not a unitary structure; it includes several nuclei with different functions and little is known on the potential differences the impact of early life stress may have on this system within different amygdaloid nuclei. We aimed here to evaluate potential regional differences in the expression of GABAergic-related markers across several amygdaloid nuclei in adult rats subjected to a peripuberty stress protocol that leads to enhanced basal amygdala activity and psychopathological behaviors. More specifically, we investigated the protein expression levels of glutamic acid decarboxylase (GAD; the principal synthesizing enzyme of GABA) and of GABA-A receptor subunits α2 and α3. We found reduced GAD and GABA-A α3, but not α2, subunit protein levels throughout all the amygdala nuclei examined (lateral, basolateral, basomedial, medial and central) and increased anxiety-like behaviors and reduced sociability in peripubertally stressed animals. Our results identify an enduring inhibition of the GABAergic system across the amygdala following exposure to early adversity. They also highlight the suitability of the peripuberty stress model to investigate the link between treatments targeting the dysfunctional GABAergic system in specific amygdala nuclei and recovery of specific stress-induced behavioral dysfunctions.

Neuronal structural plasticity is known to have a major role in cognitive processes and in the response of the CNS to aversive experiences. This type of plasticity involves processes ranging from neurite outgrowth/retraction or dendritic spine remodeling, to the incorporation of new neurons to the established circuitry. However, the study of how these structural changes take place has been focused mainly on excitatory neurons, while little attention has been paid to interneurons. The exploration of these plastic phenomena in interneurons is very important, not only for our knowledge of CNS physiology, but also for understanding better the etiology of different psychiatric and neurological disorders in which alterations in the structure and connectivity of inhibitory networks have been described. Here we review recent work on the structural remodeling of interneurons in the adult brain, both in basal conditions and after chronic stress or sensory deprivation. We also describe studies from our laboratory and others on the putative mediators of this interneuronal structural plasticity, focusing on the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). This molecule is expressed by some interneurons in the adult CNS and, through its anti-adhesive and insulating properties, may participate in the remodeling of their structure. Finally, we review recent findings on the possible implication of PSA-NCAM on the remodeling of inhibitory neurons in certain psychiatric disorders and their treatments.

Principal neurons in the adult cerebral cortex undergo synaptic, dendritic, and spine remodeling in response to different stimuli, and several reports have demonstrated that the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) participates in these plastic processes. However, there is only limited information on the expression of this molecule on interneurons and on its role in the structural plasticity of these cells. We have found that PSA-NCAM is expressed in mature interneurons widely distributed in all the extension of the cerebral cortex and have excluded the expression of this molecule in most principal cells. Although PSA-NCAM expression is generally considered a marker of immature neurons, birth-dating analyses reveal that these interneurons do not have an adult or perinatal origin and that they are generated during embryonic development. PSA-NCAM expressing interneurons show reduced density of perisomatic and peridendritic puncta expressing different synaptic markers and receive less perisomatic synapses, when compared with interneurons lacking this molecule. Moreover, they have reduced dendritic arborization and spine density. These data indicate that PSA-NCAM expression is important for the connectivity of interneurons in the adult cerebral cortex and that its regulation may play an important role in the structural plasticity of inhibitory networks.

Decreased expression of dopamine D2 receptors (D2R), dysfunction of inhibitory neurotransmission and impairments in the structure and connectivity of neurons in the medial prefrontal cortex (mPFC) are involved in the pathogenesis of schizophrenia and major depression, but the relationship between these changes remains unclear. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM), a plasticity-related molecule, may serve as a link. This molecule is expressed in cortical interneurons and dopamine, via D2R, modulates its expression in parallel to that of proteins related to synapses and inhibitory neurotransmission, suggesting that D2R-targeted antipsychotics/antidepressants may act by affecting the plasticity of mPFC inhibitory circuits. To understand the role of PSA-NCAM in this plasticity, rats were chronically treated with a D2R agonist (PPHT) after cortical PSA depletion. PPHT-induced increases in GAD67 and synaptophysin (SYN) neuropil expression were blocked when PSA was previously removed, indicating a role for PSA-NCAM in this plasticity. The number of PSA-NCAM expressing interneuron somata also increased after PPHT treatment, but the percentages of these cells belonging to different interneuronal subpopulations did not change. Cortical pyramidal neurons did not express PSA-NCAM, but puncta co-expressing this molecule and parvalbumin could be found surrounding their somata. PPHT treatment increased the number of PSA-NCAM and parvalbumin expressing perisomatic puncta, but decreased the percentage of parvalbumin puncta that co-expressed SYN. PSA depletion did not block these effects on the perisomatic region, but increased further the number of parvalbumin expressing puncta and increased the percentage of puncta co-expressing SYN and parvalbumin, suggesting that the polysialylation of NCAM may regulate perisomatic inhibition of mPFC principal neurons. Summarizing, the present results indicate that dopamine acting on D2R influences structural plasticity of mPFC interneurons and point to PSA-NCAM as a key player in this remodeling., (© 2011 Castillo-Gómez et al.)

Changes in the ability of neuronal networks to undergo structural remodeling may be involved in the age-associated cognitive decline. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) declines dramatically during postnatal development, but persists in several regions of the young-adult rat telencephalon, where it participates, through its anti-adhesive properties, in neuronal structural plasticity. However, PSA-NCAM expression during aging has only been studied in the dentate gyrus and the piriform cortex layer II, where it is strongly downregulated in adult (middle-aged) individuals. Using immunohistochemistry, we have observed that in most of the telencephalic areas studied the number of PSA-NCAM expressing cells and the intensity of PSA-NCAM expression in the neuropil remains stable during aging. Old rats only show decreases in the number of PSA-NCAM expressing cells in the lateral amygdala and retrosplenial cortex, and in neuropil expression of stratum lucidum. Given the role of PSA-NCAM in neuronal plasticity, the present results indicate that, even during aging, many regions of the CNS may display neurite, spine or synaptic remodeling.

A "neuroplastic" hypothesis proposes that changes in neuronal structural plasticity may underlie the aetiology of depression and the action of antidepressants. The medial prefrontal cortex (mPFC) is affected by this disorder and shows an intense expression of the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), a plasticity-associated molecule, which is expressed mainly in interneurons. The monoamines serotonin, dopamine and noradrenaline are the principal targets of antidepressant action. Pharmacological manipulation of serotonin levels regulates synaptophysin and PSA-NCAM expression in the adult mPFC. However, the involvement of structural plasticity on the antidepressant effects of dopamine has not been well explored yet. Using immunohistochemistry, we have studied the relationship between dopaminergic fibers and PSA-NCAM expressing neurons in the mPFC and the expression of D2 receptors. In order to evaluate the effects of dopamine in neuronal structural plasticity and on inhibitory neurotransmission, we have analyzed the expression of synaptophysin, PSA-NCAM and GAD67 in the mPFC after cortical dopamine depletion with 6-OHDA and after chronic treatments with the D2 receptor antagonist haloperidol or the D2 receptor agonist PPHT. Many dopaminergic fibers were observed in close apposition to PSA-NCAM expressing neurons and 76% of these cells co-expressed D2 receptor. Both haloperidol treatment and 6-OHDA injection reduced significantly PSA-NCAM, synaptophysin and GAD67 expression in the mPFC. Conversely, PPHT treatment increased the expression of these molecules. Our results give support to the "neuroplastic" hypothesis of depression, suggesting that dopamine acting on D2 receptors may modulate neuronal structural plasticity and inhibitory neurotransmission through changes in PSA-NCAM expression.

New neurons in the adult brain transiently express molecules related to neuronal development, such as the polysialylated form of neural cell adhesion molecule, or doublecortin (DCX). These molecules are also expressed by a cell population in the rat paleocortex layer II, whose origin, phenotype, and function are not clearly understood. We have classified most of these cells as a new cell type termed tangled cell. Some cells with the morphology of semilunar-pyramidal transitional neurons were also found among this population, as well as some scarce cells resembling semilunar, pyramidal. and fusiform neurons. We have found that none of these cells in layer II express markers of glial cells, mature, inhibitory, or principal neurons. They appear to be in a prolonged immature state, confirmed by the coexpression of DCX, TOAD/Ulip/CRMP-4, A3 subunit of the cyclic nucleotide-gated channel, or phosphorylated cyclic adenosine monophosphate response element-binding protein. Moreover, most of them lack synaptic contacts, are covered by astroglial lamellae, and fail to express cellular activity markers, such as c-Fos or Arc, and N-methyl-d-aspartate or glucocorticoid receptors. We have found that none of these cells appear to be generated during adulthood or early youth and that most of them have been generated during embryonic development, mainly in E15.5.

The prefrontal cortex (PFC) of adult rodents is capable of undergoing neuronal remodeling and neuroimaging studies in humans have revealed that the structure of this region also appears affected in different psychiatric disorders. However, the cellular mechanisms underlying this plasticity are still unclear. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) may mediate these structural changes through its anti-adhesive properties. PSA-NCAM participates in neurite outgrowth and synaptogenesis and changes in its expression occur parallel to neuronal remodeling in certain regions of the adult brain. PSA-NCAM is expressed in the hippocampus and temporal cortex of adult humans, but it has not been studied in the PFC. Employing immunohistochemistry on sections from the rostromedial superior frontal gyrus we have found that PSA-NCAM is expressed in the human PFC neuropil following a laminated pattern and in a subpopulation of mature neurons, which lack doublecortin expression. Most of these cells have been identified as interneurons expressing calbindin. The expression of PSA-NCAM in the human PFC is similar to that of rodents. Since this molecule has been linked to the neuronal remodeling found in experimental models of depression, it may also participate in the structural plasticity described in the PFC of depressed patients.

Background: Marked hypertransaminasemia (HT) is not an infrequent situation within clinical practice, which is usually interpreted as primary acute liver damage. The objectives of this study were to determine the incidence, mortality rate and aetiology of marked HT among the general population, and the use therefore as an indicator of primary acute liver damage., Methods: A retrospective study was made of all patients with marked HT (ALT >400 IU/L) which were attended over a two-year period at the Healthcare Department n 11 in the Autonomous Community of Valencia. The computerized medical records and the results of the different supplementary examinations made were reviewed, and an analysis was made of different variables: clinical diagnosis and evolution, other liver function-related biochemical parameters and autoimmune and infection serology markers., Results: A total of 414 patients with marked HT were identified (incidence of 88 cases/100,000 inhab./year), 73 of whom died (mortality rate of 16 deaths/100,000 inhab./year). Of the twenty aetiologies found, the most frequent were extrahepatic cholestasis (28.3%), hypoxic hepatopathy (14.6%) and sepsis (11.9%). The positive predictive value of marked HT as an indicator of primary acute liver damage was 27.7%., Conclusions: Marked HT is a disorder having a remarkable incidence rate among the general population, entailing a high mortality rate. Its aetiology is widely varied, being however the extrahepatic origin predominant.

Recent hypotheses suggest that changes in neuronal structure and connectivity may underlie the etiology of depression. The medial prefrontal cortex (mPFC) is affected by depression and shows neuronal remodeling during adulthood. This plasticity may be mediated by the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), which is intensely expressed in the adult mPFC. As the expression of PSA-NCAM is increased by serotonin in other cerebral regions, antidepressants acting on serotonin reuptake may influence PSA-NCAM expression and thus counteract the effects of depression by modulating neuronal structural plasticity. Using immunohistochemistry, we have studied the relationship between serotoninergic fibers and PSA-NCAM expressing neurons in the adult rat mPFC and the expression of serotonin receptors in these cells. The effects of fluoxetine treatment for 14 days on mPFC PSA-NCAM expression have also been analyzed. Although serotoninergic fibers usually do not contact PSA-NCAM immunoreactive neurons, most of these cells express 5-HT3 receptors. In general, chronic fluoxetine treatment induces significant increases in the number of PSA-NCAM immunoreactive neurons and in neuropil immunostaining and coadministration of the 5-HT3 antagonist ondansetron blocks the effects of fluoxetine on PSA-NCAM expression. These results indicate that fluoxetine, acting through 5-HT3 receptors, can modulate PSA-NCAM expression in the mPFC. This modulation may mediate the structural plasticity of this cortical region and opens new perspectives on the study of the molecular bases of depression.