Postnatal development of the relaxin-3 innervation of the rat medial septum.

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.

Nucleus incertus projections to rat medial septum and entorhinal cortex: rare collateralization and septal-gating of temporal lobe theta rhythm activity.

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.

Relaxin-3 Innervation From the Nucleus Incertus to the Parahippocampal Cortex of the Rat.

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.

Involvement of the Nucleus Incertus and Relaxin-3/RXFP3 Signaling System in Explicit and Implicit Memory.

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 Gi/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.

Becoming Stressed: Does the Age Matter? Reviewing the Neurobiological and Socio-Affective Effects of Stress throughout the Lifespan.

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.

MAP/ERK Signaling in Developing Cognitive and Emotional Function and Its Effect on Pathological and Neurodegenerative Processes.

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.

Fernando de Castro: Cajal's Man on the Peripheral Nervous System.

Santiago Ramón y Cajal developed his initial scientific career working alone. After the publication of his opus magna ("Textura del sistema nervioso del hombre y los vertebrados") and the general recognition of the scientific environments that crystallized with the concession of the International Moscow Prize (1900), the Spanish Government decided to officially support Cajal with a laboratory and the first salaries to pay collaborators. Is then when the Spanish Neurological School births: in 1902, Francisco Tello is the first one to be incorporated. With new additions, Cajal's work is complimented in new aspects, including Neuropathologies. Fernando de Castro is one of his youngest direct disciples, one of the closest and more beloved. Fernando de Castro worked from 1916 in Cajal's lab, until the death of El Maestro. He was specially committed by Cajal to unravel different aspects of the structure of the peripheral ganglia: sensitive and vegetative. Afterward, Fernando de Castro described by first time the nature of arterial chemoreceptors in the carotid body. While trying to confirm his anatomical description with physiological demonstrations, and accumulating delays because of scientific decision and the sociopolitical circumstances in Spain, Corneille Heymans was awarded with the Nobel Prize in Physiology or Medicine 1938 for his contributions to the knowledge of cardiorespiratory reflexes. The Karolinska Institutet forgot Heinrich Hering and Fernando de Castro in their decision. Undoubtedly, Fernando de Castro was the most important disciple of Cajal working in the different structures of the peripheral nervous system, and this work is now reviewed here. Anat Rec, 303:1206-1214, 2020. © 2019 American Association for Anatomy.

Early intervention with ABA prevents neuroinflammation and memory impairment in a triple transgenic mice model of Alzheimer´s disease.

Neuroinflammation and insulin resistance in the brain are intimately linked to neurodegenerative disorders, including Alzheimer's disease. Even though traditionally Alzheimer´s disease has been associated to Aß deposits and hyperphosphorylated Tau intracellular tangles, several studies show that neuroinflammation may be the initial cause that triggers degeneration. Accordingly, a number of natural supplements that improves brain insulin sensitivity and reduce neuroinflammation have been proposed as good choices in the therapeutic prevention of cognitive decline. Further supporting this evidence, we show that phytohormone Abscisic Acid, can prevent memory impairment and neuroinflammation markers in a triple transgenic mouse model, where no peripheral inflammatory changes have occurred. Moreover, our data strongly suggests that early intervention is critical for good prognosis, and that cognitive improvement requires longer treatment than recovering neuroinflammation markers.

Modulation of forebrain function by nucleus incertus and relaxin-3/RXFP3 signaling.

The nucleus incertus (NI) in the pontine tegmentum sends ascending projections to the midbrain, hypothalamus, amygdala, basal forebrain, hippocampus, and prefrontal cortex, and has a postulated role in modulating several forebrain functions. A substantial population of GABAergic NI neurons expresses the neuropeptide, relaxin-3, which acts via the Gi/o -protein-coupled receptor, RXFP3, present throughout the forebrain target regions. Broad and specific manipulations of these systems by activation or inhibition of the NI or modulating RXFP3 signaling have revealed key insights into the likely influence of the NI/relaxin-3/RXFP3 system on modalities including arousal, feeding, stress responses, anxiety and addiction, and attention and memory. This range of actions corresponds to a likely impact of NI/(relaxin-3) projections on multiple integrated circuits, but makes it difficult to draw conclusions about a generalized function for this network. This review will focus on the key physiological process of oscillatory theta rhythm and the neural circuits that promote it during behavioral activation, highlighting the ability of NI and relaxin-3/RXFP3 signaling systems to modulate these circuits. A better understanding of these mechanisms may provide a way to therapeutically adjust malfunction of forebrain activity present in several pathological conditions.

GABAergic Neurons in the Rat Medial Septal Complex Express Relaxin-3 Receptor (RXFP3) mRNA.

The medial septum (MS) complex modulates hippocampal function and related behaviors. Septohippocampal projections promote and control different forms of hippocampal synchronization. Specifically, GABAergic and cholinergic projections targeting the hippocampal formation from the MS provide bursting discharges to promote theta rhythm, or tonic activity to promote gamma oscillations. In turn, the MS is targeted by ascending projections from the hypothalamus and brainstem. One of these projections arises from the nucleus incertus in the pontine tegmentum, which contains GABA neurons that co-express the neuropeptide relaxin-3 (Rln3). Both stimulation of the nucleus incertus and septal infusion of Rln3 receptor agonist peptides promotes hippocampal theta rhythm. The Gi/o-protein-coupled receptor, relaxin-family peptide receptor 3 (RXFP3), is the cognate receptor for Rln3 and identification of the transmitter phenotype of neurons expressing RXFP3 in the septohippocampal system can provide further insights into the role of Rln3 transmission in the promotion of septohippocampal theta rhythm. Therefore, we used RNAscope multiplex in situ hybridization to characterize the septal neurons expressing Rxfp3 mRNA in the rat. Our results demonstrate that Rxfp3 mRNA is abundantly expressed in vesicular GABA transporter (vGAT) mRNA- and parvalbumin (PV) mRNA-positive GABA neurons in MS, whereas ChAT mRNA-positive acetylcholine neurons lack Rxfp3 mRNA. Approximately 75% of Rxfp3 mRNA-positive neurons expressed vGAT mRNA (and 22% were PV mRNA-positive), while the remaining 25% expressed Rxfp3 mRNA only, consistent with a potential glutamatergic phenotype. Similar proportions were observed in the posterior septum. The occurrence of RXFP3 in PV-positive GABAergic neurons gives support to a role for the Rln3-RXFP3 system in septohippocampal theta rhythm.

Implications of glial nitric oxide in neurodegenerative diseases.

Nitric oxide (NO) is a pleiotropic janus-faced molecule synthesized by nitric oxide synthases (NOS) which plays a critical role in a number of physiological and pathological processes in humans. The physiological roles of NO depend on its local concentrations, as well as its availability and the nature of downstream target molecules. Its double-edged sword action has been linked to neurodegenerative disorders. Excessive NO production, as the evoked by inflammatory signals, has been identified as one of the major causative reasons for the pathogenesis of several neurodegenerative diseases. Moreover, excessive NO synthesis under neuroinflammation leads to the formation of reactive nitrogen species and neuronal cell death. There is an intimate relation between microglial activation, NO and neuroinflammation in the human brain. The role of NO in neuroinflammation has been defined in animal models where this neurotransmitter can modulate the inflammatory process acting on key regulatory pathways, such as those associated with excitotoxicity processes induced by glutamate accumulation and microglial activation. Activated glia express inducible NOS and produce NO that triggers calcium mobilization from the endoplasmic reticulum, activating the release of vesicular glutamate from astroglial cells resulting in neuronal death. This change in microglia potentially contributes to the increased age-associated susceptibility and neurodegeneration. In the current review, information is provided about the role of NO, glial activation and age-related processes in the central nervous system (CNS) that may be helpful in the isolation of new therapeutic targets for aging and neurodegenerative diseases.

Persistent phagocytic characteristics of microglia in the substantia nigra of long-term Parkinsonian macaques.

Patients with Parkinson's disease show persistent microglial activation in the areas of the brain where the degeneration of dopaminergic neurons takes place. The reason for maintaining this activated state is still unknown, but it is thought that this persistent microglial activation may contribute to the degeneration of dopaminergic neurons. In this study, we report the microanatomical details of microglia and the relationship between microglia and neurons in the substantia nigra pars compacta of Parkinsonian monkeys years after insult with MPTP. We observed that microglial cells appear polarized toward dopaminergic neurons in MPTP-treated macaques compared to untreated animals and present clear phagocytic characteristics, such as engulfing gliaptic contacts, an increase in Golgi apparatus protein machinery and ball-and-chain phagocytic buds. These results demonstrate that activated microglia maintain phagocytic characteristics years after neurotoxin insult, and phagocytosis may be a key contributor to the neurodegenerative process.

Effects of pharmacological agents, sleep deprivation, hypoxia and transcranial magnetic stimulation on electroencephalographic rhythms in rodents: towards translational challenge models for drug discovery in Alzheimer's disease.

Different kinds of challenge can alter spontaneous ongoing electroencephalographic (EEG) rhythms in animal models, thus providing paradigms to evaluate treatment effects in drug discovery. The effects of challenges represented by pharmacological agents, hypoxia, sleep deprivation and transcranial magnetic stimulation (TMS) on EEG rhythms are here reviewed to build a knowledge platform for innovative translational models for drug discovery in Alzheimer's disease (AD). It has been reported that antagonists of cholinergic neurotransmission cause synchronisation of spontaneous ongoing EEG rhythms in terms of enhanced power of EEG low frequencies and decreased power of EEG high frequencies. Acetylcholinesterase inhibitors and serotonergic drugs may restore a normal pattern of EEG desynchronisation. Sleep deprivation and hypoxia challenges have also been reported to elicit abnormal synchronisation of spontaneous ongoing EEG rhythms in rodents. The feasibility and reproducibility of TMS have been demonstrated in rodents but information on a consistent modulation of EEG after TMS manipulation is very limited. Transgenic mice over-expressing human amyloid precursor protein complementary DNAs (cDNAs) harbouring the 'Swedish' mutation and PS-1 cDNAs harbouring the A264E mutation, which recapitulate some of the pathological features of AD, exhibit alterations of spontaneous ongoing EEG rhythms at several low and high frequencies. This does not appear, however, to be a consequence of beta-amyloid deposition in the brain. The present review provides a critical evaluation of changes of spontaneous ongoing EEG rhythms due to the experimental manipulations described above, in order to stimulate the promote more adherent models fitting dynamics in humans.

ROCK/Cdc42-mediated microglial motility and gliapse formation lead to phagocytosis of degenerating dopaminergic neurons in vivo.

The role of microglial motility in the context of adult neurodegeneration is poorly understood. In the present work, we investigated the microanatomical details of microglia-neuron interactions in an experimental mouse model of Parkinson's disease following the intraperitoneal injection of MPTP. The specific intoxication of dopaminergic neurons induces the cellular polarization of microglia, leading to the formation of body-to-body neuron-glia contacts, called gliapses, which precede neuron elimination. Inhibiting ROCK/Cdc42-mediated microglial motility in vivo blocks the activating features of microglia, such as increased cell size and number of filopodia and diminishes their phagocyting/secreting domains, as the reduction of the Golgi apparatus and the number of microglia-neuron contacts has shown. High-resolution confocal images and three-dimensional rendering demonstrate that microglia engulf entire neurons at one-to-one ratio, and the microglial cell body participates in the formation of the phagocytic cup, engulfing and eliminating neurons in areas of dopaminergic degeneration in adult mammals.

Dopamine modulation affects the performance of parkinsonian patients in a precision motor task measured by an antropomorphic device.

Several parameters related to the timing, grip force and load force involved in a precision grasping task were studied in patients with Parkinson's disease (PD) at different moments of medication and healthy controls, using a sensorized anthropomorphic device which was totally adapted to the human hand. The aim of this work was to carry out an accurate study of the reach-load-grip-hold-place-release subtasks to identify any physical motor impairment, its relation to medication and Parkinsonian strategies. Twenty seven patients in ON and OFF-like medication moments, and twenty seven age-matched controls took part in the experiment, which consisted of using the index finger and the thumb to perform a precision motor task involving different experimental objects. Visual cues were used as distracting elements. Results showed several motor parameters impaired in OFF-like medication moment but attenuated in ON state, suggesting a medication effect on the performance of the task.

CCL2-expressing astrocytes mediate the extravasation of T lymphocytes in the brain. Evidence from patients with glioma and experimental models in vivo.

CCL2 is a chemokine involved in brain inflammation, but the way in which it contributes to the entrance of lymphocytes in the parenchyma is unclear. Imaging of the cell type responsible for this task and details on how the process takes place in vivo remain elusive. Herein, we analyze the cell type that overexpresses CCL2 in multiple scenarios of T-cell infiltration in the brain and in three different species. We observe that CCL2+ astrocytes play a part in the infiltration of T-cells in the brain and our analysis shows that the contact of T-cells with perivascular astrocytes occurs, suggesting that may be an important event for lymphocyte extravasation.

Microglial glucocorticoid receptors play a pivotal role in regulating dopaminergic neurodegeneration in parkinsonism.

Among the pathogenic processes contributing to dopaminergic neuron (DN) death in Parkinson disease (PD), evidence points to non-cell-autonomous mechanisms, particularly chronic inflammation mounted by activated microglia. Yet little is known about endogenous regulatory processes that determine microglial actions in pathological states. We examined the role of glucocorticoid receptors (GRs), activated by glucocorticoids released in response to stress and known to regulate inflammation, in DN survival. Overall GR level was decreased in substantia nigra of PD patients and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. GR changes, specifically in the microglia after MPTP treatment, revealed a rapid augmentation in the number of microglia displaying nuclear localization of GR. Mice with selective inactivation of the GR gene in macrophages/microglia (GR(LysMCre)) but not in DNs (GR(DATCre)) showed increased loss of DNs after MPTP intoxication. This DN loss in GR(LysMCre) mice was not prevented by corticosterone treatment, in contrast to the protection observed in control littermates. Moreover, absence of microglial GRs augmented microglial reactivity and led to their persistent activation. Analysis of inflammatory genes revealed an up-regulation of Toll-like receptors (TLRs) by MPTP treatment, particularly TLR9, the level of which was high in postmortem parkinsonian brains. The regulatory control of GR was reflected by higher expression of proinflammatory genes (e.g., TNF-α) with a concomitant decrease in anti-inflammatory genes (e.g., IL-1R2) in GR(LysMCre) mice. Indeed, in GR(LysMCre) mice, alterations in phosphorylated NF-κB levels indicated its protracted activation. Together, our data indicate that GR is important in curtailing microglial reactivity, and its deregulation in PD could lead to sustained inflammation-mediated DN injury.

No Lewy pathology in monkeys with over 10 years of severe MPTP Parkinsonism.

The recent knowledge that 10 years after transplantation surviving human fetal neurons adopt the histopathology of Parkinson's disease suggests that Lewy body formation takes a decade to achieve. To determine whether similar histopathology occurs in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-primate models over a similar timeframe, the brains of two adult monkeys made parkinsonian in their youth with intermittent injections of MPTP were studied. Despite substantial nigral degeneration and increased alpha-synuclein immunoreactivity within surviving neurons, there was no evidence of Lewy body formation. This suggests that MPTP-induced oxidative stress and inflammation per se are not sufficient for Lewy body formation, or Lewy bodies are human specific.