Trazodone rescues dysregulated synaptic and mitochondrial nascent proteomes in prion neurodegeneration.

The unfolded protein response (UPR) is rapidly gaining momentum as a therapeutic target for protein misfolding neurodegenerative diseases, in which its overactivation results in sustained translational repression leading to synapse loss and neurodegeneration. In mouse models of these disorders, from Alzheimer's to prion disease, modulation of the pathway-including by the licensed drug, trazodone-restores global protein synthesis rates with profound neuroprotective effects. However, the precise nature of the translational impairment, in particular the specific proteins affected in disease, and their response to therapeutic UPR modulation are poorly understood. We used non-canonical amino acid tagging (NCAT) to measure de novo protein synthesis in the brains of prion-diseased mice with and without trazodone treatment, in both whole hippocampus and cell-specifically. During disease the predominant nascent proteome changes occur in synaptic, cytoskeletal and mitochondrial proteins in both hippocampal neurons and astrocytes. Remarkably, trazodone treatment for just 2 weeks largely restored the whole disease nascent proteome in the hippocampus to that of healthy, uninfected mice, predominantly with recovery of proteins involved in synaptic and mitochondrial function. In parallel, trazodone treatment restored the disease-associated decline in synapses and mitochondria and their function to wild-type levels. In conclusion, this study increases our understanding of how translational repression contributes to neurodegeneration through synaptic and mitochondrial toxicity via depletion of key proteins essential for their function. Further, it provides new insights into the neuroprotective mechanisms of trazodone through reversal of this toxicity, relevant for the treatment of neurodegenerative diseases via translational modulation.

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.

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.

Small Molecules to Improve ER Proteostasis in Disease.

Abnormally high levels of misfolded proteins in the endoplasmic reticulum (ER) lumen result in a stress state that contributes to the progression of several pathological conditions including diabetes, cancer, neurodegeneration, and immune dysregulation. ER stress triggers a dynamic signaling pathway known as the unfolded protein response (UPR). The UPR enforces adaptive or cell death programs by integrating information about the intensity and duration of the stress stimuli. Thus, depending on the disease context, ER stress signaling can be beneficial or detrimental. We discuss current efforts to develop small molecules to target distinct components of the UPR, and their possible applications in treating human disease, focusing on neurodegenerative diseases, metabolic disorders, and cancer.

Central relaxin-3 receptor (RXFP3) activation impairs social recognition and modulates ERK-phosphorylation in specific GABAergic amygdala neurons.

In mammals, the extended amygdala is a neural hub for social and emotional information processing. In the rat, the extended amygdala receives inhibitory GABAergic projections from the nucleus incertus (NI) in the pontine tegmentum. NI neurons produce the neuropeptide relaxin-3, which acts via the Gi/o-protein-coupled receptor, RXFP3. A putative role for RXFP3 signalling in regulating social interaction was investigated by assessing the effect of intracerebroventricular infusion of the RXFP3 agonist, RXFP3-A2, on performance in the 3-chamber social interaction paradigm. Central RXFP3-A2, but not vehicle, infusion, disrupted the capacity to discriminate between a familiar and novel conspecific subject, but did not alter differentiation between a conspecific and an inanimate object. Subsequent studies revealed that agonist-infused rats displayed increased phosphoERK(pERK)-immunoreactivity in specific amygdaloid nuclei at 20 min post-infusion, with levels similar to control again after 90 min. In parallel, we used immunoblotting to profile ERK phosphorylation dynamics in whole amygdala after RXFP3-A2 treatment; and multiplex histochemical labelling techniques to reveal that after RXFP3-A2 infusion and social interaction, pERK-immunopositive neurons in amygdala expressed vesicular GABA-transporter mRNA and displayed differential profiles of RXFP3 and oxytocin receptor mRNA. Overall, these findings demonstrate that central relaxin-3/RXFP3 signalling can modulate social recognition in rats via effects within the amygdala and likely interactions with GABA and oxytocin signalling.

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.

Central relaxin-3 receptor (RXFP3) activation increases ERK phosphorylation in septal cholinergic neurons and impairs spatial working memory.

The medial septum/diagonal band (MS/DB) is a relay region connecting the hypothalamus and brainstem with the hippocampus, and both the MS/DB and dorsal/ventral hippocampus receive strong topographic GABA/peptidergic projections from the nucleus incertus of the pontine tegmentum. The neuropeptide relaxin-3, released by these neurons, is the cognate ligand for a Gi/o-protein-coupled receptor, RXFP3, which is highly expressed within the MS/DB, and both cholinergic and GABAergic neurons in this region of rat brain receive relaxin-3 positive terminals/boutons. Comprehensive in vitro studies have demonstrated that the cell signaling pathways altered by RXFP3 stimulation, include inhibition of forskolin-activated cAMP levels and activation of ERK phosphorylation. In this study we investigated whether intracerebroventricular (icv) injection of RXFP3-A2, a selective relaxin-3 receptor agonist, altered ERK phosphorylation levels in the MS/DB of adult male rats. We subsequently assessed the neurochemical phenotype of phosphorylated (p) ERK-positive neurons in MS/DB after icv RXFP3-A2 administration by dual-label immunostaining for pERK and neuronal markers for cholinergic and GABAergic neurons. Central RXFP3-A2 injection significantly increased levels of pERK immunoreactivity (IR) in MS/DB at 20 and 90 min post-injection, compared to vehicle and naive levels. In addition, RXFP3-A2 increased the number of cells expressing pERK-IR in the MS/DB at 90 (but not 20) min post-injection in cholinergic (but not GABAergic) neurons, which also expressed putative RXFP3-IR. Moreover, icv injection of RXFP3-A2 impaired alternation in a delayed spontaneous T-maze test of spatial working memory. The presence of RXFP3-like IR and the RXFP3-related activation of the MAPK/ERK pathway in MS/DB cholinergic neurons identifies them as a key target of ascending relaxin-3 projections with implications for the acute and chronic modulation of cholinergic neuron activity and function by relaxin-3/RXFP3 signaling.

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.

Acute oral administration of low doses of methylphenidate targets calretinin neurons in the rat septal area.

Methylphenidate (MPD) is a commonly administered drug to treat children suffering from attention deficit hyperactivity disorder (ADHD). Alterations in septal driven hippocampal theta rhythm may underlie attention deficits observed in these patients. Amongst others, the septo-hippocampal connections have long been acknowledged to be important in preserving hippocampal function. Thus, we wanted to ascertain if MPD administration, which improves attention in patients, could affect septal areas connecting with hippocampus. We used low and orally administered MPD doses (1.3, 2.7 and 5 mg/Kg) to rats what mimics the dosage range in humans. In our model, we observed no effect when using 1.3 mg/Kg MPD; whereas 2.7 and 5 mg/Kg induced a significant increase in c-fos expression specifically in the medial septum (MS), an area intimately connected to the hippocampus. We analyzed dopaminergic areas such as nucleus accumbens and striatum, and found that only 5 mg/Kg induced c-fos levels increase. In these areas tyrosine hydroxylase correlated well with c-fos staining, whereas in the MS the sparse tyrosine hydroxylase fibers did not overlap with c-fos positive neurons. Double immunofluorescence of c-fos with neuronal markers in the septal area revealed that co-localization with choline acethyl transferase, parvalbumin, and calbindin with c-fos did not change with MPD treatment; whereas, calretinin and c-fos double labeled neurons increased after MPD administration. Altogether, these results suggest that low and acute doses of methylphenidate primary target specific populations of caltretinin medial septal neurons.

[Effects of methylphenidate on anxiety]. / Efectos del metilfenidato sobre la ansiedad.

The attention deficit disorder with hyperactivity (ADDH) is a widely recognized disorder of unknown etiology. Methylphenidate administration is one of the most commonly used treatments to improve symptoms associated with ADDH. Although it is generally a well tolerated drug, several secondary effects may occur. In particular, this paper will focus on the effects on anxiety, in humans and experimental animal models. It has been shown that acute administration of methylphenidate in adults reduces anxiety, in both animal models and humans. On the other hand, chronic treatment during early ages (postnatal and young subjects) results in higher anxiety in adults. In some cases this effect appears together with higher susceptibility of drug consumption. Thus, we find that, in the literature, methylphenidate is capable of inducing different and opposite effects. Thus, further experiments would be required to elucidate the mechanisms by which methylphenidate exert its actions.

Efectos del metilfenidato sobre la ansiedad / Effects of methylphenidate on anxiety

El trastorno por déficit de atención/hiperactividad (TDAH) es un trastorno neurológico ampliamente reconocido de etiología desconocida. La administración de metilfenidato es uno de los tratamientos más utilizados para la mejora sintomática del TDAH. Aunque es un medicamento en general muy bien tolerado por los pacientes, existen algunos efectos secundarios ajenos a los síntomas de la hiperactividad. En particular, esta revisión se centra en revisar los efectos que la administración aguda o crónica del metilfenidato induce en síntomas de ansiedad en humanos y en modelos animales experimentales. Tanto en modelos animales como en humanos, la administración aguda en adultos tiene un efecto ansiolítico. Por otro lado, en modelos animales, la administración crónica en el período posnatal y adolescentes genera estados de ansiedad en el adulto, aumentando, además, en algunos casos, aunque no en todos, la propensión a la drogodependencia de otras sustancias. Existe disparidad de resultados y serían necesarios más estudios para elucidar los mecanismos por los cuales el metilfenidato ejerce su acción (AU)

The attention deficit disorder with hyperactivity (ADDH) is a widely recognized disorder of unknown etiology. Methylphenidate administration is one of the most commonly used treatments to improve symptoms associated with ADDH. Although it is generally a well tolerated drug, several secondary effects may occur. In particular, this paper will focus on the effects on anxiety, in humans and experimental animal models. It has been shown that acute administration of methylphenidate in adults reduces anxiety, in both animal models and humans. On the other hand, chronic treatment during early ages (postnatal and young subjects) results in higher anxiety in adults. In some cases this effect appears together with higher susceptibility of drug consumption. Thus, we find that, in the literature, methylphenidate is capable of inducing different and opposite effects. Thus, further experiments would be required to elucidate the mechanisms by which methylphenidate exert its actions (AU)