Fibroblast growth factor 13-mediated regulation of medium spiny neuron excitability and cocaine self-administration.

Cocaine use disorder (CUD) is a prevalent neuropsychiatric disorder with few existing treatments. Thus, there is an unmet need for the identification of new pharmacological targets for CUD. Previous studies using environmental enrichment versus isolation paradigms have found that the latter induces increased cocaine self-administration with correlative increases in the excitability of medium spiny neurons (MSN) of the nucleus accumbens shell (NAcSh). Expanding upon these findings, we sought in the present investigation to elucidate molecular determinants of these phenomena. To that end, we first employed a secondary transcriptomic analysis and found that cocaine self-administration differentially regulates mRNA for fibroblast growth factor 13 (FGF13), which codes for a prominent auxiliary protein of the voltage-gated Na+ (Nav) channel, in the NAcSh of environmentally enriched rats (i.e., resilient behavioral phenotype) compared to environmentally isolated rats (susceptible phenotype). Based upon this finding, we used in vivo genetic silencing to study the causal functional and behavioral consequences of knocking down FGF13 in the NAcSh. Functional studies revealed that knockdown of FGF13 in the NAcSh augmented excitability of MSNs by increasing the activity of Nav channels. These electrophysiological changes were concomitant with a decrease in cocaine demand elasticity (i.e., susceptible phenotype). Taken together, these data support FGF13 as being protective against cocaine self-administration, which positions it well as a pharmacological target for CUD.

TNFR1 signaling converging on FGF14 controls neuronal hyperactivity and sickness behavior in experimental cerebral malaria.

BACKGROUND: Excess tumor necrosis factor (TNF) is implicated in the pathogenesis of hyperinflammatory experimental cerebral malaria (eCM), including gliosis, increased levels of fibrin(ogen) in the brain, behavioral changes, and mortality. However, the role of TNF in eCM within the brain parenchyma, particularly directly on neurons, remains underdefined. Here, we investigate electrophysiological consequences of eCM on neuronal excitability and cell signaling mechanisms that contribute to observed phenotypes. METHODS: The split-luciferase complementation assay (LCA) was used to investigate cell signaling mechanisms downstream of tumor necrosis factor receptor 1 (TNFR1) that could contribute to changes in neuronal excitability in eCM. Whole-cell patch-clamp electrophysiology was performed in brain slices from eCM mice to elucidate consequences of infection on CA1 pyramidal neuron excitability and cell signaling mechanisms that contribute to observed phenotypes. Involvement of identified signaling molecules in mediating behavioral changes and sickness behavior observed in eCM were investigated in vivo using genetic silencing. RESULTS: Exploring signaling mechanisms that underlie TNF-induced effects on neuronal excitability, we found that the complex assembly of fibroblast growth factor 14 (FGF14) and the voltage-gated Na+ (Nav) channel 1.6 (Nav1.6) is increased upon tumor necrosis factor receptor 1 (TNFR1) stimulation via Janus Kinase 2 (JAK2). On account of the dependency of hyperinflammatory experimental cerebral malaria (eCM) on TNF, we performed patch-clamp studies in slices from eCM mice and showed that Plasmodium chabaudi infection augments Nav1.6 channel conductance of CA1 pyramidal neurons through the TNFR1-JAK2-FGF14-Nav1.6 signaling network, which leads to hyperexcitability. Hyperexcitability of CA1 pyramidal neurons caused by infection was mitigated via an anti-TNF antibody and genetic silencing of FGF14 in CA1. Furthermore, knockdown of FGF14 in CA1 reduced sickness behavior caused by infection. CONCLUSIONS: FGF14 may represent a therapeutic target for mitigating consequences of TNF-mediated neuroinflammation.

Glycogen Synthase Kinase 3: Ion Channels, Plasticity, and Diseases.

Glycogen synthase kinase 3ß (GSK3) is a multifaceted serine/threonine (S/T) kinase expressed in all eukaryotic cells. GSK3ß is highly enriched in neurons in the central nervous system where it acts as a central hub for intracellular signaling downstream of receptors critical for neuronal function. Unlike other kinases, GSK3ß is constitutively active, and its modulation mainly involves inhibition via upstream regulatory pathways rather than increased activation. Through an intricate converging signaling system, a fine-tuned balance of active and inactive GSK3ß acts as a central point for the phosphorylation of numerous primed and unprimed substrates. Although the full range of molecular targets is still unknown, recent results show that voltage-gated ion channels are among the downstream targets of GSK3ß. Here, we discuss the direct and indirect mechanisms by which GSK3ß phosphorylates voltage-gated Na+ channels (Nav1.2 and Nav1.6) and voltage-gated K+ channels (Kv4 and Kv7) and their physiological effects on intrinsic excitability, neuronal plasticity, and behavior. We also present evidence for how unbalanced GSK3ß activity can lead to maladaptive plasticity that ultimately renders neuronal circuitry more vulnerable, increasing the risk for developing neuropsychiatric disorders. In conclusion, GSK3ß-dependent modulation of voltage-gated ion channels may serve as an important pharmacological target for neurotherapeutic development.

Inhibition of the Akt/PKB Kinase Increases Nav1.6-Mediated Currents and Neuronal Excitability in CA1 Hippocampal Pyramidal Neurons.

In neurons, changes in Akt activity have been detected in response to the stimulation of transmembrane receptors. However, the mechanisms that lead to changes in neuronal function upon Akt inhibition are still poorly understood. In the present study, we interrogate how Akt inhibition could affect the activity of the neuronal Nav channels with while impacting intrinsic excitability. To that end, we employed voltage-clamp electrophysiological recordings in heterologous cells expressing the Nav1.6 channel isoform and in hippocampal CA1 pyramidal neurons in the presence of triciribine, an inhibitor of Akt. We showed that in both systems, Akt inhibition resulted in a potentiation of peak transient Na+ current (INa) density. Akt inhibition correspondingly led to an increase in the action potential firing of the CA1 pyramidal neurons that was accompanied by a decrease in the action potential current threshold. Complementary confocal analysis in the CA1 pyramidal neurons showed that the inhibition of Akt is associated with the lengthening of Nav1.6 fluorescent intensity along the axonal initial segment (AIS), providing a mechanism for augmented neuronal excitability. Taken together, these findings provide evidence that Akt-mediated signal transduction might affect neuronal excitability in a Nav1.6-dependent manner.

Inhibition of AKT Signaling Alters ßIV Spectrin Distribution at the AIS and Increases Neuronal Excitability.

The axon initial segment (AIS) is a highly regulated subcellular domain required for neuronal firing. Changes in the AIS protein composition and distribution are a form of structural plasticity, which powerfully regulates neuronal activity and may underlie several neuropsychiatric and neurodegenerative disorders. Despite its physiological and pathophysiological relevance, the signaling pathways mediating AIS protein distribution are still poorly studied. Here, we used confocal imaging and whole-cell patch clamp electrophysiology in primary hippocampal neurons to study how AIS protein composition and neuronal firing varied in response to selected kinase inhibitors targeting the AKT/GSK3 pathway, which has previously been shown to phosphorylate AIS proteins. Image-based features representing the cellular pattern distribution of the voltage-gated Na+ (Nav) channel, ankyrin G, ßIV spectrin, and the cell-adhesion molecule neurofascin were analyzed, revealing ßIV spectrin as the most sensitive AIS protein to AKT/GSK3 pathway inhibition. Within this pathway, inhibition of AKT by triciribine has the greatest effect on ßIV spectrin localization to the AIS and its subcellular distribution within neurons, a phenotype that Support Vector Machine classification was able to accurately distinguish from control. Treatment with triciribine also resulted in increased excitability in primary hippocampal neurons. Thus, perturbations to signaling mechanisms within the AKT pathway contribute to changes in ßIV spectrin distribution and neuronal firing that may be associated with neuropsychiatric and neurodegenerative disorders.

Barrel cortex plasticity after photothrombotic stroke involves potentiating responses of pre-existing circuits but not functional remapping to new circuits.

Recovery after stroke is thought to be mediated by adaptive circuit plasticity, whereby surviving neurons assume the roles of those that died. However, definitive longitudinal evidence of neurons changing their response selectivity after stroke is lacking. We sought to directly test whether such functional "remapping" occurs within mouse primary somatosensory cortex after a stroke that destroys the C1 barrel. Using in vivo calcium imaging to longitudinally record sensory-evoked activity under light anesthesia, we did not find any increase in the number of C1 whisker-responsive neurons in the adjacent, spared D3 barrel after stroke. To promote plasticity after stroke, we also plucked all whiskers except C1 (forced use therapy). This led to an increase in the reliability of sensory-evoked responses in C1 whisker-responsive neurons but did not increase the number of C1 whisker-responsive neurons in spared surround barrels over baseline levels. Our results argue against remapping of functionality after barrel cortex stroke, but support a circuit-based mechanism for how rehabilitation may improve recovery.

The E3 ubiquitin ligase IDOL regulates synaptic ApoER2 levels and is important for plasticity and learning.

Neuronal ApoE receptors are linked to learning and memory, but the pathways governing their abundance, and the mechanisms by which they affect the function of neural circuits are incompletely understood. Here we demonstrate that the E3 ubiquitin ligase IDOL determines synaptic ApoER2 protein levels in response to neuronal activation and regulates dendritic spine morphogenesis and plasticity. IDOL-dependent changes in ApoER2 abundance modulate dendritic filopodia initiation and synapse maturation. Loss of IDOL in neurons results in constitutive overexpression of ApoER2 and is associated with impaired activity-dependent structural remodeling of spines and defective LTP in primary neuron cultures and hippocampal slices. IDOL-deficient mice show profound impairment in experience-dependent reorganization of synaptic circuits in the barrel cortex, as well as diminished spatial and associative learning. These results identify control of lipoprotein receptor abundance by IDOL as a post-transcriptional mechanism underlying the structural and functional plasticity of synapses and neural circuits.

Dorsoventral differences in Kv7/M-current and its impact on resonance, temporal summation and excitability in rat hippocampal pyramidal cells.

In rodent hippocampi, the connections, gene expression and functions differ along the dorsoventral (D-V) axis. CA1 pyramidal cells show increasing excitability along the D-V axis, although the underlying mechanism is not known. In the present study, we investigated how the M-current (IM ), caused by Kv7/M (KCNQ) potassium channels, and known to often control neuronal excitability, contributes to D-V differences in intrinsic properties of CA1 pyramidal cells. Using whole-cell patch clamp recordings and the selective Kv7/M blocker 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE991) in hippocampal slices from 3- to 4-week-old rats, we found that: (i) IM had a stronger impact on subthreshold electrical properties in dorsal than ventral CA1 pyramidal cells, including input resistance, temporal summation of artificial synaptic potentials, and M-resonance; (ii) IM activated at more negative potentials (left-shifted) and had larger peak amplitude in the dorsal than ventral CA1; and (iii) the initial spike threshold (during ramp depolarizations) was elevated, and the medium after-hyperpolarization and spike frequency adaptation were increased (i.e. excitability was lower) in the dorsal rather than ventral CA1. These differences were abolished or reduced by application of XE991, indicating that they were caused by IM . Thus, it appears that IM has stronger effects in dorsal than in ventral rat CA1 pyramidal cells because of a larger maximal M-conductance and left-shifted activation curve in the dorsal cells. These mechanisms may contribute to D-V differences in the rate and phase coding of position by CA1 place cells, and may also enhance epileptiform activity in ventral CA1.

Kainate postconditioning restores LTP in ischemic hippocampal CA1: onset-dependent second pathophysiological stress.

Postconditioning can be induced by a broad range of stimuli within minutes to days after an ischemic cerebral insult. A special form is elicited by pharmacological intervention called second pathophysiological stress. The present study aimed to evaluate the effects of low-dose (5 mg/kg) kainate postconditioning with onsets 0, 24 and 48 h after the ischemic insult on the hippocampal synaptic plasticity in a 2-vessel occlusion model in rat. The hippocampal function was tested by LTP measurements of Schaffer collateral-CA1 pyramidal cell synapses in acute slices and the changes in density of Golgi-Cox-stained apical dendritic spines. Postconditioning 0 and 24 h after ischemia was not protective, whereas 48-h-onset postconditioning resulted in the reappearance of a normal spine density (>100,000 spines) 3 days after ischemia, in parallel with the long-term restoration of the damaged LTP function. Similar, but somewhat less effects were observed after 10 days. Our data clearly demonstrate the onset dependence of postconditioning elicited by a subconvulsant dose of kainate treatment in global ischemia, with restoration of the structural plasticity and hippocampal function.

Neuroprotection with a new kynurenic acid analog in the four-vessel occlusion model of ischemia.

Global forebrain ischemia results in damage to the pyramids in the CA1 hippocampal subfield, which is particularly vulnerable to excitotoxic processes. Morphological and functional disintegration of this area leads to a cognitive dysfunction and neuropsychiatric disorders. Treatment with N-methyl-d-aspartate receptor antagonists is a widely accepted method with which to stop the advance of excitotoxic processes and concomitant neuronal death. From a clinical aspect, competitive glycine- and polyamine-site antagonists with relatively low affinity and moderate side-effects are taken into account. Endogenous kynurenic acid acts as an antagonist on the obligatory co-agonist glycine site, and has long been at the focus of neuroprotective trials. In the present study, we estimated the neuroprotective capability of a novel kynurenic acid analog in transient global forebrain ischemia, measuring the rate of hippocampal CA1 pyramidal cell loss and the preservation of long-term potentiation at Schaffer collateral-CA1 synapses. The neuroprotective potential was reflected by a significantly diminished hippocampal CA1 cell loss and preserved long-term potentiation expression. The neuroprotective effect was robust in the event of pretreatment, and also when the drug was administered at the time of reperfusion. This result is beneficial since a putative neuroprotectant proven to be effective as post-treatment is of much greater benefit.

Effects of blood glutamate scavenging on cortical evoked potentials.

It is well known that traumatic or ischemic brain injury is followed by acute excitotoxicity caused by the presence of abnormally high glutamate (Glu) in brain fluids. It has recently been demonstrated that excess Glu can be eliminated from brain into blood following the intravenous administration of oxaloacetate (OxAc), which, by scavenging blood Glu, induces an enhanced and neuroprotective brain-to-blood Glu efflux. In this study, we subjected rats to intravenous OxAc administration (i.v., 12.5, 25, and 50 mg/kg, respectively), and studied its effects on somatosensory evoked cortical potentials (EPs). Against our expectation, the amplitudes of EPs did not decrease but increased in a dose- and time-dependent manner after OxAc administration. Similar effects were observed when blood Glu scavenging was enhanced by combining OxAc (12.5 mg/kgbw) with recombinant glutamate-oxaloacetate transaminase (GOT, 0.14 nmol/100 g rat). On the basis of these results, we suggest that the changes of amplitudes of the EPs involve not only a glutamatergic but also the weakening of a GABAergic component. We cannot rule out the possibility that OxAc penetrates into the brain and improves mitochondrial functions.

A novel kynurenic acid analogue: a comparison with kynurenic acid. An in vitro electrophysiological study.

Kynurenic acid is an endogenous product of the tryptophan metabolism, and as a broad-spectrum antagonist of excitatory amino acid receptors may serve as a protective agent in neurological disorders. The use of kynurenic acid as a neuroprotective agent is rather limited, however, because it has only restricted ability to cross the blood-brain barrier. Accordingly, new kynurenic acid analogues which can readily cross the blood-brain barrier and exert their complex anti-excitotoxic activity are greatly needed. Such a novel analogue, 2-(2-N,N-dimethylaminoethylamine-1-carbonyl)-1H-quinolin-4-one hydrochloride, has been developed and tested. In an in vitro electrophysiological study, in which its properties were compared with those of kynurenic acid, the new analogue behaved quite similarly to kynurenic acid: in the micromolar range, its administration led to a decrease in the amplitudes of the field excitatory postsynaptic potentials in the CA1 region of the hippocampus, while in nanomolar concentrations it did not give rise to inhibition, but, in fact, facilitated the field excitatory postsynaptic potentials. Moreover, the new analogue demonstrated similar protective action against PTZ-induced facilitation to that observed after kynurenic acid administration. The findings strongly suggest that the neuroactive effects of the new analogue are comparable with those of kynurenic acid, but, in contrast with kynurenic acid, it readily crosses the blood-brain barrier. The new analogue may therefore be considered a promising candidate for clinical studies.

Oxaloacetate decreases the infarct size and attenuates the reduction in evoked responses after photothrombotic focal ischemia in the rat cortex.

A traumatic brain injury or a focal brain lesion is followed by acute excitotoxicity caused by the presence of abnormally high glutamate (Glu) levels in the cerebrospinal and interstitial fluids. It has recently been demonstrated that this excess Glu in the brain can be eliminated into the blood following the intravenous administration of oxaloacetate (OxAc), which, by scavenging the blood Glu, induces an enhanced and neuroprotective brain-to-blood Glu efflux. In this study, we subjected rats to a photothrombotic lesion and treated them after the illumination with a single 30-min-long administration of OxAc (1.2 mg/100 g, i.v.). Following induction of the lesion, we measured the infarct size and the amplitudes of the somatosensory evoked potentials (SEPs) as recorded from the skull surface. The photothrombotic lesion resulted in appreciably decreased amplitudes of the evoked potentials, but OxAc administration significantly attenuated this reduction, and also the infarct size assessed histologically. We suggest that the neuroprotective effects of OxAc are due to its blood Glu-scavenging activity, which, by increasing the brain-to-blood Glu efflux, reduces the excess Glu responsible for the anatomical and functional correlates of the ischemia, as evaluated by electrophysiological evoked potential (EP) measurements.

Oxaloacetate restores the long-term potentiation impaired in rat hippocampus CA1 region by 2-vessel occlusion.

Various acute brain pathological conditions are characterized by the presence of elevated glutamate concentrations in the brain interstitial fluids. It has been established that a decrease in the blood glutamate level enhances the brain-to-blood efflux of glutamate, removal of which from the brain may prevent glutamate excitotoxicity and its contribution to the long-lasting neurological deficits seen in stroke. A decrease in blood glutamate level can be achieved by exploiting the glutamate-scavenging properties of the blood-resident enzyme glutamate-oxaloacetate transaminase, which transforms glutamate into 2-ketoglutarate in the presence of the glutamate co-substrate oxaloacetate. The present study had the aim of an evaluation of the effects of the blood glutamate scavenger oxaloacetate on the impaired long-term potentiation (LTP) induced in the 2-vessel occlusion ischaemic model in rat. Transient (30-min) incomplete forebrain ischaemia was produced 72 h before LTP induction. Although the short transient brain hypoperfusion did not induce histologically identifiable injuries in the CA1 region (Fluoro-Jade B, S-100 and cresyl violet), it resulted in an impaired LTP function in the hippocampal CA1 region without damaging the basal synaptic transmission between the Schaffer collaterals and the pyramidal neurons. This impairment could be fended off in a dose-dependent manner by the intravenous administration of oxaloacetate in saline (at doses between 1.5 mmol and 0.1 mumol) immediately after the transient hypoperfusion. Our results suggest that oxaloacetate-mediated blood and brain glutamate scavenging contributes to the restoration of the LTP after its impairment by brain ischaemia.

Kynurenine diminishes the ischemia-induced histological and electrophysiological deficits in the rat hippocampus.

The neuroprotective effect of L-kynurenine sulfate (KYN), a precursor of kynurenic acid (KYNA, a selective N-methyl-D-aspartate receptor antagonist), was studied. KYN (300 mg/kg i.p., applied daily for 5 days) appreciably decreased the number of injured pyramidal cells from 1850+/-100/mm(2) to 1000+/-300/mm(2) (p<0.001) in the CA1 region of the hippocampus in the four-vessel occlusion (4VO)-induced ischemic adult rat brain. A parallel increase in the number of intact, surviving neurons was demonstrated. Post-treatment with KYN (applied immediately right after reperfusion) proved to be much less effective. In parallel with the histology, a protective effect of KYN on the functioning of the CA1 region was observed: long-term potentiation was abolished in the 4VO animals, but its level and duration were restored by pretreatment with KYN. It is concluded that the administration of KYN elevates the KYNA concentration in the brain to neuroprotective levels, suggesting its potential clinical usefulness for the prevention of neuronal loss in neurodegenerative diseases.

Hippocampal (CA1) activities in Wistar rats from different vendors. Fundamental differences in acute ischemia.

Two-vessel occlusion, a frequently used model of global cerebral ischemia in rats, results in a dysfunction predominantly within the CA1 field of the hippocampus; it induces many processes with different time-scales. However, the great divergence in the results of the studies reported in the literature suggests valuable differences in response to hypoperfusion-induced ischemia among the laboratory rats used in these studies. In the present work, the acute effects of two-carotid occlusion-induced global ischemia (2VO) on the CA3 stimulation-evoked population spike activity in the CA1 region of Wistar rats from different suppliers (Charles-River and Harlan) were compared. In the acute electrophysiological experiments, the hippocampal CA1 responses revealed that the Charles-River rats immediately compensated the 2VO much better than did the Harlan rats. However, 3 days later, no difference could be observed between the CA1 activities of these rats. The presented data show that the Wistar rats from different vendors represent an important source of variability in the results of acute experiments on the hippocampal ischemia. These observations draw attention to the importance of the careful choice of the laboratory rats (both strains and breeds) used in such experiments.