The amygdala NT3-TrkC pathway underlies inter-individual differences in fear extinction and related synaptic plasticity.

Fear-related pathologies are among the most prevalent psychiatric conditions, having inappropriate learned fear and resistance to extinction as cardinal features. Exposure therapy represents a promising therapeutic approach, the efficiency of which depends on inter-individual variation in fear extinction learning, which neurobiological basis is unknown. We characterized a model of extinction learning, whereby fear-conditioned mice were categorized as extinction (EXT)-success or EXT-failure, according to their inherent ability to extinguish fear. In the lateral amygdala, GluN2A-containing NMDAR are required for LTP and stabilization of fear memories, while GluN2B-containing NMDAR are required for LTD and fear extinction. EXT-success mice showed attenuated LTP, strong LTD and higher levels of synaptic GluN2B, while EXT-failure mice showed strong LTP, no LTD and higher levels of synaptic GluN2A. Neurotrophin 3 (NT3) infusion in the lateral amygdala was sufficient to rescue extinction deficits in EXT-failure mice. Mechanistically, activation of tropomyosin receptor kinase C (TrkC) with NT3 in EXT-failure slices attenuated lateral amygdala LTP, in a GluN2B-dependent manner. Conversely, blocking endogenous NT3-TrkC signaling with TrkC-Fc chimera in EXT-success slices strengthened lateral amygdala LTP. Our data support a key role for the NT3-TrkC system in inter-individual differences in fear extinction in rodents, through modulation of amygdalar NMDAR composition and synaptic plasticity.

Mitochondria as central hubs in synaptic modulation.

Mitochondria are present in the pre- and post-synaptic regions, providing the energy required for the activity of these very specialized neuronal compartments. Biogenesis of synaptic mitochondria takes place in the cell body, and these organelles are then transported to the synapse by motor proteins that carry their cargo along microtubule tracks. The transport of mitochondria along neurites is a highly regulated process, being modulated by the pattern of neuronal activity and by extracellular cues that interact with surface receptors. These signals act by controlling the distribution of mitochondria and by regulating their activity. Therefore, mitochondria activity at the synapse allows the integration of different signals and the organelles are important players in the response to synaptic stimulation. Herein we review the available evidence regarding the regulation of mitochondrial dynamics by neuronal activity and by neuromodulators, and how these changes in the activity of mitochondria affect synaptic communication.

Biomarkers of hypoxic-ischemic encephalopathy: a systematic review.

BACKGROUND: Current diagnostic criteria for hypoxic-ischemic encephalopathy in the early hours lack objective measurement tools. Therefore, this systematic review aims to identify putative molecules that can be used in diagnosis in daily clinical practice (PROSPERO ID: CRD42021272610). DATA SOURCES: Searches were performed in PubMed, Web of Science, and Science Direct databases until November 2020. English original papers analyzing samples from newborns > 36 weeks that met at least two American College of Obstetricians and Gynecologists diagnostic criteria and/or imaging evidence of cerebral damage were included. Bias was assessed by the Newcastle-Ottawa Scale. The search and data extraction were verified by two authors separately. RESULTS: From 373 papers, 30 met the inclusion criteria. Data from samples collected in the first 72 hours were extracted, and increased serum levels of neuron-specific enolase and S100-calcium-binding protein-B were associated with a worse prognosis in newborns that suffered an episode of perinatal asphyxia. In addition, the levels of glial fibrillary acidic protein, ubiquitin carboxyl terminal hydrolase isozyme-L1, glutamic pyruvic transaminase-2, lactate, and glucose were elevated in newborns diagnosed with hypoxic-ischemic encephalopathy. Moreover, pathway analysis revealed insulin-like growth factor signaling and alanine, aspartate and glutamate metabolism to be involved in the early molecular response to insult. CONCLUSIONS: Neuron-specific enolase and S100-calcium-binding protein-B are potential biomarkers, since they are correlated with an unfavorable outcome of hypoxic-ischemic encephalopathy newborns. However, more studies are required to determine the sensitivity and specificity of this approach to be validated for clinical practice.

TrkC Intracellular Signalling in the Brain Fear Network During the Formation of a Contextual Fear Memory.

Learned fear is orchestrated by a brain fear network that comprises the amygdala, hippocampus and the medial prefrontal cortex. Synaptic plasticity within this network is critical for the formation of proper fear memories. Known for their role in the promotion of synaptic plasticity, neurotrophins position as obvious candidates in the regulation of fear processes. Indeed, recent evidence from our laboratory and others associates dysregulated signalling through neurotrophin-3 and its receptor TrkC with the pathophysiology of anxiety and fear-related disorders. Here, we put wild-type C57Bl/6J mice through a contextual fear conditioning paradigm in order to characterize TrkC activation and expression in the main brain regions involved in (learned) fear - amygdala, hippocampus, and prefrontal cortex - during the formation of a fear memory. We report an overall decreased activation of TrkC in the fear network during fear consolidation and reconsolidation. During reconsolidation, hippocampal TrkC downregulation was accompanied by a decrease in the expression and activation of Erk, a critical signalling pathway in fear conditioning. Moreover, we did not find evidence that the observed decrease of TrkC activation was caused by altered expression of dominant negative form of TrkC, neurotrophin-3, or the PTP1B phosphatase. Our results indicate hippocampal TrkC inactivation through Erk signalling as a potential mechanism in the regulation of contextual fear memory formation.

The role of post-translational modifications in synaptic AMPA receptor activity.

AMPA-type receptors for the neurotransmitter glutamate are very dynamic entities, and changes in their synaptic abundance underlie different forms of synaptic plasticity, including long-term synaptic potentiation (LTP), long-term depression (LTD) and homeostatic scaling. The different AMPA receptor subunits (GluA1-GluA4) share a common modular structure and membrane topology, and their intracellular C-terminus tail is responsible for the interaction with intracellular proteins important in receptor trafficking. The latter sequence differs between subunits and contains most sites for post-translational modifications of the receptors, including phosphorylation, O-GlcNAcylation, ubiquitination, acetylation, palmitoylation and nitrosylation, which affect differentially the various subunits. Considering that each single subunit may undergo modifications in multiple sites, and that AMPA receptors may be formed by the assembly of different subunits, this creates multiple layers of regulation of the receptors with impact in synaptic function and plasticity. This review discusses the diversity of mechanisms involved in the post-translational modification of AMPA receptor subunits, and their impact on the subcellular distribution and synaptic activity of the receptors.

Molecular Mechanisms of Epilepsy: The Role of the Chloride Transporter KCC2.

Epilepsy is a neurological disease characterized by abnormal or synchronous brain activity causing seizures, which may produce convulsions, minor physical signs, or a combination of symptoms. These disorders affect approximately 65 million people worldwide, from all ages and genders. Seizures apart, epileptic patients present a high risk to develop neuropsychological comorbidities such as cognitive deficits, emotional disturbance, and psychiatric disorders, which severely impair quality of life. Currently, the treatment for epilepsy includes the administration of drugs or surgery, but about 30% of the patients treated with antiepileptic drugs develop time-dependent pharmacoresistence. Therefore, further investigation about epilepsy and its causes is needed to find new pharmacological targets and innovative therapeutic strategies. Pharmacoresistance is associated to changes in neuronal plasticity and alterations of GABAA receptor-mediated neurotransmission. The downregulation of GABA inhibitory activity may arise from a positive shift in GABAA receptor reversal potential, due to an alteration in chloride homeostasis. In this paper, we review the contribution of K+-Cl--cotransporter (KCC2) to the alterations in the Cl- gradient observed in epileptic condition, and how these alterations are coupled to the increase in the excitability.

Posttranslational modifications of proteins are key features in the identification of CSF biomarkers of multiple sclerosis.

BACKGROUND: Multiple sclerosis is an inflammatory and degenerative disease of the central nervous system (CNS) characterized by demyelination and concomitant axonal loss. The lack of a single specific test, and the similarity to other inflammatory diseases of the central nervous system, makes it difficult to have a clear diagnosis of multiple sclerosis. Therefore, laboratory tests that allows a clear and definite diagnosis, as well as to predict the different clinical courses of the disease are of utmost importance. Herein, we compared the cerebrospinal fluid (CSF) proteome of patients with multiple sclerosis (in the relapse-remitting phase of the disease) and other diseases of the CNS (inflammatory and non-inflammatory) aiming at identifying reliable biomarkers of multiple sclerosis. METHODS: CSF samples from the discovery group were resolved by 2D-gel electrophoresis followed by identification of the protein spots by mass spectrometry. The results were analyzed using univariate (Student's t test) and multivariate (Hierarchical Cluster Analysis, Principal Component Analysis, Linear Discriminant Analysis) statistical and numerical techniques, to identify a set of protein spots that were differentially expressed in CSF samples from patients with multiple sclerosis when compared with other two groups. Validation of the results was performed in samples from a different set of patients using quantitative (e.g., ELISA) and semi-quantitative (e.g., Western Blot) experimental approaches. RESULTS: Analysis of the 2D-gels showed 13 protein spots that were differentially expressed in the three groups of patients: Alpha-1-antichymotrypsin, Prostaglandin-H2-isomerase, Retinol binding protein 4, Transthyretin (TTR), Apolipoprotein E, Gelsolin, Angiotensinogen, Agrin, Serum albumin, Myosin-15, Apolipoprotein B-100 and EF-hand calcium-binding domain-containing protein. ELISA experiments allowed validating part of the results obtained in the proteomics analysis and showed that some of the alterations in the CSF proteome are also mirrored in serum samples from multiple sclerosis patients. CSF of multiple sclerosis patients was characterized by TTR oligomerization, thus highlighting the importance of analyzing posttranslational modifications of the proteome in the identification of novel biomarkers of the disease. CONCLUSIONS: The model built based on the results obtained upon analysis of the 2D-gels and in the validation phase attained an accuracy of about 80% in distinguishing multiple sclerosis patients and the other two groups.

Brain-derived neurotrophic factor-induced regulation of RNA metabolism in neuronal development and synaptic plasticity.

The neurotrophin brain-derived neurotrophic factor (BDNF) plays multiple roles in the nervous system, including in neuronal development, in long-term synaptic potentiation in different brain regions, and in neuronal survival. Alterations in these regulatory mechanisms account for several diseases of the nervous system. The synaptic effects of BDNF mediated by activation of tropomyosin receptor kinase B (TrkB) receptors are partly mediated by stimulation of local protein synthesis which is now considered a ubiquitous feature in both presynaptic and postsynaptic compartments of the neuron. The capacity to locally synthesize proteins is of great relevance at several neuronal developmental stages, including during neurite development, synapse formation, and stabilization. The available evidence shows that the effects of BDNF-TrkB signaling on local protein synthesis regulate the structure and function of the developing and mature synapses. While a large number of studies have illustrated a wide range of effects of BDNF on the postsynaptic proteome, a growing number of studies also point to presynaptic effects of the neurotrophin in the local regulation of the protein composition at the presynaptic level. Here, we will review the latest evidence on the role of BDNF in local protein synthesis, comparing the effects on the presynaptic and postsynaptic compartments. Additionally, we overview the relevance of BDNF-associated local protein synthesis in neuronal development and synaptic plasticity, at the presynaptic and postsynaptic compartments, and their relevance in terms of disease. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Export and Localization > RNA Localization.

GRASP1 ubiquitination regulates AMPA receptor surface expression and synaptic activity in cultured hippocampal neurons.

The synaptic expression of glutamate receptors of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) type is dynamically controlled by interaction with binding partners and auxiliary proteins. These proteins can be regulated by posttranslational modifications, including ubiquitination. In this work, we investigated the regulation of glutamate receptor interacting protein-associated protein 1 (GRASP1) by ubiquitin-dependent mechanisms and its impact on surface expression and activity of synaptic AMPA receptors. Cotransfection of GFP-ubiquitin decreased myc-GRASP1 protein levels in HEK293T cells, and this effect was inhibited upon transfection of an ubiquitin mutant that cannot be ubiquitinated on Lys48. In addition, transfection of cultured hippocampal neurons with GFP-ubiquitin reduced the dendritic levels of endogenous GRASP1 and decreased the surface expression of GluA1 AMPA receptor subunits, an effect that was partly reversed by cotransfection with GRASP1. Similarly, transfection of hippocampal neurons with GFP-ubiquitin decreased the amplitude of miniature excitatory postsynaptic currents (mEPSCs) mediated by Ca2+ -impermeable AMPA receptors, and this effect was abrogated by cotransfection of GRASP1. Together, the results show a role for ubiquitination in the regulation of the postsynaptic protein GRASP1, which has an impact on the surface distribution of AMPA receptors and on their activity at the synapse.

Transient incubation of cultured hippocampal neurons in the absence of magnesium induces rhythmic and synchronized epileptiform-like activity.

Cell culture models are important tools to study epileptogenesis mechanisms. The aim of this work was to characterize the spontaneous and synchronized rhythmic activity developed by cultured hippocampal neurons after transient incubation in zero Mg2+ to model Status Epilepticus. Cultured hippocampal neurons were transiently incubated with a Mg2+-free solution and the activity of neuronal networks was evaluated using single cell calcium imaging and whole-cell current clamp recordings. Here we report the development of synchronized and spontaneous [Ca2+]i transients in cultured hippocampal neurons immediately after transient incubation in a Mg2+-free solution. Spontaneous and synchronous [Ca2+]i oscillations were observed when the cells were then incubated in the presence of Mg2+. Functional studies also showed that transient incubation in Mg2+-free medium induces neuronal rhythmic burst activity that was prevented by antagonists of glutamate receptors. In conclusion, we report the development of epileptiform-like activity, characterized by spontaneous and synchronized discharges, in cultured hippocampal neurons transiently incubated in the absence of Mg2+. This model will allow studying synaptic alterations contributing to the hyperexcitability that underlies the development of seizures and will be useful in pharmacological studies for testing new drugs for the treatment of epilepsy.

Response of the cerebral vasculature to systemic carbon monoxide administration-Regional differences and sexual dimorphism.

Previous studies about the modulation of the vasculature by CO were performed exclusively in male or sexually immature animals. Understanding the sex differences regarding systemic drug processing and pharmacodynamics is an important feature for safety assessment of drug dosing and efficacy. In this work, we used CORM-A1 as source of CO to examine the effects of this gasotransmitter on brain perfusion and the sex-dependent differences. Dynamic contrast-enhanced imaging (DCE)-based analysis was used to characterize the properties of CO in the modulation of cerebral vasculature in vivo, in adult C57BL/6 healthy mice. Perfusion of the temporal muscle, maxillary vein and in hippocampus, cortex and striatum was analysed for 108 min following CORM-A1 administration of 3 or 5 mg/kg. Under control conditions, brain perfusion was lower in females when compared with males. Under CO treatment, females showed a surprisingly overall reduced perfusion compared with controls (F = 3.452, p = .0004), while no major alterations (or even the expected increase) were observed in males. Cortical structures were only modulated in females. A striking female-dominated vasoconstriction effect was observed in the hippocampus and striatum following administration of CO, in this mixed-sex cohort. As these two regions are implicated in episodic and procedural memory formation, CO may have a relevant impact in learning and memory.

BDNF increases synaptic NMDA receptor abundance by enhancing the local translation of Pyk2 in cultured hippocampal neurons.

The effects of brain-derived neurotrophic factor (BDNF) in long-term synaptic potentiation (LTP) are thought to underlie learning and memory formation and are partly mediated by local protein synthesis. Here, we investigated the mechanisms that mediate BDNF-induced alterations in the synaptic proteome that are coupled to synaptic strengthening. BDNF induced the synaptic accumulation of GluN2B-containing NMDA receptors (NMDARs) and increased the amplitude of NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) in cultured rat hippocampal neurons by a mechanism requiring activation of the protein tyrosine kinase Pyk2 and dependent on cellular protein synthesis. Single-particle tracking using quantum dot imaging revealed that the increase in the abundance of synaptic NMDAR currents correlated with their enhanced stability in the synaptic compartment. Furthermore, BDNF increased the local synthesis of Pyk2 at the synapse, and the observed increase in Pyk2 protein abundance along dendrites of cultured hippocampal neurons was mediated by a mechanism dependent on the ribonucleoprotein hnRNP K, which bound to Pyk2 mRNA and dissociated from it upon BDNF application. Knocking down hnRNP K reduced the BDNF-induced synaptic synthesis of Pyk2 protein, whereas its overexpression enhanced it. Together, these findings indicate that hnRNP K mediates the synaptic distribution of Pyk2 synthesis, and hence the synaptic incorporation of GluN2B-containing NMDARs, induced by BDNF, which may affect LTP and synaptic plasticity.

Alterations in GABAA-Receptor Trafficking and Synaptic Dysfunction in Brain Disorders.

GABAA receptors (GABAAR) are the major players in fast inhibitory neurotransmission in the central nervous system (CNS). Regulation of GABAAR trafficking and the control of their surface expression play important roles in the modulation of the strength of synaptic inhibition. Different pieces of evidence show that alterations in the surface distribution of GABAAR and dysregulation of their turnover impair the activity of inhibitory synapses. A diminished efficacy of inhibitory neurotransmission affects the excitatory/inhibitory balance and is a common feature of various disorders of the CNS characterized by an increased excitability of neuronal networks. The synaptic pool of GABAAR is mainly controlled through regulation of internalization, recycling and lateral diffusion of the receptors. Under physiological condition these mechanisms are finely coordinated to define the strength of GABAergic synapses. In this review article, we focus on the alteration in GABAAR trafficking with an impact on the function of inhibitory synapses in various disorders of the CNS. In particular we discuss how similar molecular mechanisms affecting the synaptic distribution of GABAAR and consequently the excitatory/inhibitory balance may be associated with a wide diversity of pathologies of the CNS, from psychiatric disorders to acute alterations leading to neuronal death. A better understanding of the cellular and molecular mechanisms that contribute to the impairment of GABAergic neurotransmission in these disorders, in particular the alterations in GABAAR trafficking and surface distribution, may lead to the identification of new pharmacological targets and to the development of novel therapeutic strategies.

P2X7 Receptors Mediate CO-Induced Alterations in Gene Expression in Cultured Cortical Astrocytes-Transcriptomic Study.

Carbon monoxide (CO) is an endogenous gasotransmitter that limits inflammation and prevents apoptosis in several tissues, including the brain. Low concentrations of CO are cytoprotective in astrocytes, neurons, and microglia, but the underlying molecular mechanisms remain poorly understood. This work aims at identification of alterations in gene expression conferred by CO in primary cultures of cortical astrocytes, for further disclosure of the molecular mechanism of action of the gasotransmitter. Astrocytes were treated with the CO-releasing molecule CORM-A1 for 40 min, and transcriptional changes were analyzed using RNASeq. A total of 162 genes were differentially expressed in response to CO treatment, and 7 of these genes were selected for further analysis: FosB, Scand1, Rgs10, Actg1, Panx1, Pcbdh21, and Rn18s. The alterations in their expression were further validated using qRT-PCR. An increase in FosB protein expression was also observed after 40 min of CORM-A1 treatment, as determined by a western blot. CO-induced FosB expression and cytoprotection were both abrogated in the presence of the P2X7 receptor antagonist A-438079. Furthermore, CORM-A1 increased phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII), which is a downstream event of P2X7R activation. The functional importance of FosB in CO-induced survival was assessed by knocking down its expression with FosB siRNA. Astrocytes were challenged to death with oxidative stress and cell viability was assessed 24 h later. Downregulation of FosB did not prevent the effects of CO in the inhibition of astrocytic cell death. Nevertheless, the transcriptomic changes observed upon treatment of astrocytes with CO open new opportunities for further studies on CO cytoprotective pathways.

BDNF-Live-Exon-Visualization (BLEV) Allows Differential Detection of BDNF Transcripts in vitro and in vivo.

Bdnf exon-IV and exon-VI transcripts are driven by neuronal activity and are involved in pathologies related to sleep, fear or memory disorders. However, how their differential transcription translates activity changes into long-lasting network changes is elusive. Aiming to trace specifically the network controlled by exon-IV and -VI derived BDNF during activity-dependent plasticity changes, we generated a transgenic reporter mouse for B DNF- l ive- e xon- v isualization (BLEV), in which expression of Bdnf exon-IV and -VI can be visualized by co-expression of CFP and YFP. CFP and YFP expression was differentially activated and targeted in cell lines, primary cultures and BLEV reporter mice without interfering with BDNF protein synthesis. CFP and YFP expression, moreover, overlapped with BDNF protein expression in defined hippocampal neuronal, glial and vascular locations in vivo. So far, activity-dependent BDNF cannot be explicitly monitored independent of basal BDNF levels. The BLEV reporter mouse therefore provides a new model, which can be used to test whether stimulus-induced activity-dependent changes in BDNF expression are instrumental for long-lasting plasticity modifications.

The RNA-Binding Protein hnRNP K Mediates the Effect of BDNF on Dendritic mRNA Metabolism and Regulates Synaptic NMDA Receptors in Hippocampal Neurons.

Brain-derived neurotrophic factor (BDNF) is an important mediator of long-term synaptic potentiation (LTP) in the hippocampus. The local effects of BDNF depend on the activation of translation activity, which requires the delivery of transcripts to the synapse. In this work, we found that neuronal activity regulates the dendritic localization of the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K) in cultured rat hippocampal neurons by stimulating BDNF-Trk signaling. Microarray experiments identified a large number of transcripts that are coimmunoprecipitated with hnRNP K, and about 60% of these transcripts are dissociated from the protein upon stimulation of rat hippocampal neurons with BDNF. In vivo studies also showed a role for TrkB signaling in the dissociation of transcripts from hnRNP K upon high-frequency stimulation (HFS) of medial perforant path-granule cell synapses of male rat dentate gyrus (DG). Furthermore, treatment of rat hippocampal synaptoneurosomes with BDNF decreased the coimmunoprecipitation of hnRNP K with mRNAs coding for glutamate receptor subunits, Ca2+- and calmodulin-dependent protein kinase IIß (CaMKIIß) and BDNF. Downregulation of hnRNP K impaired the BDNF-induced enhancement of NMDA receptor (NMDAR)-mediated mEPSC, and similar results were obtained upon inhibition of protein synthesis with cycloheximide. The results demonstrate that BDNF regulates specific populations of hnRNP-associated mRNAs in neuronal dendrites and suggests an important role of hnRNP K in BDNF-dependent forms of synaptic plasticity.

Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons.

This protocol aims at standardizing the procedure to obtain primary cultures of hippocampal and cerebrocortical neurons for in vitro experiments. Cultures should be prepared from cells isolated during embryonic development when neuronal precursor cells are not yet fully differentiated. This helps increasing the quality and quantity of cells, while offering minimal cell death that often occurs during dissociation of differentiated neurons. Cells plated under the appropriate conditions, either in Petri-dishes or in multi-well plates, will develop and establish synaptic contacts over time since the neuronal culture medium provides the nutrients and trophic factors required for differentiation. In this protocol we describe the methodology for the preparation of both cortical and hippocampal neuronal cultures.

Downregulation of GABAA Receptor Recycling Mediated by HAP1 Contributes to Neuronal Death in In Vitro Brain Ischemia.

Downregulation of GABAergic synaptic transmission contributes to the increase in overall excitatory activity in the ischemic brain. A reduction of GABAA receptor (GABAAR) surface expression partly accounts for this decrease in inhibitory activity, but the mechanisms involved are not fully elucidated. In this work, we investigated the alterations in GABAAR trafficking in cultured rat hippocampal neurons subjected to oxygen/glucose deprivation (OGD), an in vitro model of global brain ischemia, and their impact in neuronal death. The traffic of GABAAR was evaluated after transfection of hippocampal neurons with myc-tagged GABAAR ß3 subunits. OGD decreased the rate of GABAAR ß3 subunit recycling and reduced the interaction of the receptors with HAP1, a protein involved in the recycling of the receptors. Furthermore, OGD induced a calpain-mediated cleavage of HAP1. Transfection of hippocampal neurons with HAP1A or HAP1B isoforms reduced the OGD-induced decrease in surface expression of GABAAR ß3 subunits, and HAP1A maintained the rate of receptor recycling. Furthermore, transfection of hippocampal neurons with HAP1 significantly decreased OGD-induced cell death. These results show a key role for HAP1 protein in the downmodulation of GABAergic neurotransmission during cerebral ischemia, which contributes to neuronal demise.

7th ISN special neurochemistry conference 'Synaptic function and dysfunction in brain diseases'.

This article is part of a mini review series: "Synaptic Function and Dysfunction in Brain Diseases".