Neuropathological and Motor Impairments after Incomplete Cervical Spinal Cord Injury in Pigs.

Humans, primates, and rodents with cervical spinal cord injury (SCI) show permanent sensorimotor dysfunction of the upper/forelimb as consequence of axonal damage and local neuronal death. This work aimed at characterizing a model of cervical SCI in domestic pigs in which hemisection with excision of 1 cm of spinal cord was performed to reproduce the loss of neural tissue observed in human neuropathology. Posture and motor control were assessed over 3 months by scales and kinematics of treadmill locomotion. Histological measurements included lesion length, atrophy of the adjacent spinal cord segments, and neuronal death. In some animals, the retrograde neural tracer aminostilbamidine was injected in segments caudal to the lesion to visualize propriospinal projection neurons. Neuronal loss extended for 4-6 mm from the lesion borders and was more severe in the ipsilateral, caudal spinal cord stump. Axonal Wallerian degeneration was observed caudally and rostrally, associated with marked atrophy of the white matter in the spinal cord segments adjacent to the lesion. The pigs showed chronic monoplegia or severe monoparesis of the foreleg ipsilateral to the lesion, whereas the trunk and the other legs had postural and motor impairments that substantially improved during the first month post-lesion. Adaptations of the walking cycle such as those reported for rats and humans ameliorated the negative impact of focal neurological deficits on locomotor performance. These results provide a baseline of behavior and histology in a porcine model of cervical spinal cord hemisection that can be used for translational research in SCI therapeutics.

Expression of the cold thermoreceptor TRPM8 in rodent brain thermoregulatory circuits.

The cold- and menthol-activated ion channel transient receptor potential channel subfamily M member 8 (TRPM8) is the principal detector of environmental cold in mammalian sensory nerve endings. Although it is mainly expressed in a subpopulation of peripheral sensory neurons, it has also been identified in non-neuronal tissues. Here, we show, by in situ hybridization (ISH) and by the analysis of transgenic reporter expression in two different reporter mouse strains, that TRPM8 is also expressed in the central nervous system. Although it is present at much lower levels than in peripheral sensory neurons, we found cells expressing TRPM8 in restricted areas of the brain, especially in the hypothalamus, septum, thalamic reticular nucleus, certain cortices and other limbic structures, as well as in some specific nuclei in the brainstem. Interestingly, positive fibers were also found traveling through the major limbic tracts, suggesting a role of TRPM8-expressing central neurons in multiple aspects of thermal regulation, including autonomic and behavioral thermoregulation. Additional ISH experiments in rat brain demonstrated a conserved pattern of expression of this ion channel between rodent species. We confirmed the functional activity of this channel in the mouse brain using electrophysiological patch-clamp recordings of septal neurons. These results open a new window in TRPM8 physiology, guiding further efforts to understand potential roles of this molecular sensor within the brain.

Enhanced spinal cord microstimulation using conducting polymer-coated carbon microfibers.

Intraspinal microstimulation (ISMS) may help to restore motor functions after spinal cord injury. ISMS caudal to the lesion activates motoneurons and evokes selective movements with graded force in rats and other mammals. We investigated the safety and effectiveness of conducting polymer (CP)-coated carbon microfibers (CMFs) for ISMS. 7-µm-diameter CMFs coated with poly(3,4-ethylenedioxythiophene) doped with poly[(4-styrenesulfonic acid)-co-(maleic acid)] (PEDOT:PSS-co-MA) were used to apply current-controlled biphasic electric pulses at the cervical spinal cord (C7) of anesthetized rats. Electrode performance and motoneuron activation, as readout by voltage transients, cyclic voltammetry, electrochemical impedance spectroscopy, electromyography (EMG) and foreleg kinematics, were investigated as a function of microfiber length (50 µm vs. 250 µm) and presence of polymer coating. The microfibers were very effective in activating specific spinal motoneurons, with the lowest stimulus thresholds varying between -28 µA and -46 µA in the cathodic phase. EMG and kinematic thresholds decreased when the microfiber tip approached the targeted motor nucleus (triceps brachii, t.b.) from the dorsal spinal cord surface. ISMS with polymer-coated CMFs produced higher electrical activity in the t.b. fascicles compared to bare CMFs. PEDOT:PSS-co-MA coating of 250-µm CMFs avoided the generation of unsafe overvoltages for biphasic pulses up to -80/+40 µA in vivo, although the positive effect of the conducting polymer was lost after the application of a few thousands of electric pulses. Thus, CP-coated CMFs may provide an effective and minimally invasive electrode for ISMS; however, polymer optimization is still required to improve its electrical stability and safety for long-term use. Statement of significance Intraspinal microstimulation may restore motor functions after spinal cord injury. In the present study we demonstrate that carbon microfibers (CMFs) coated with the conducting polymer PEDOT:PSS-co-MA can be advantageously used for this purpose. These microfibers allow for both effective and temporarily safe electrical activation of spinal motor circuits with high spatial resolution. The presence of the polymer enhances the effectiveness of the electrical stimuli to recruit spinal motoneurons. Thus, conducting polymer-coated CMFs have potential for the development of advanced neuroprosthetic devices, although further improvements are needed regarding their electrochemical and mechanical stability.

Biofunctionalized Conducting Polymer/Carbon Microfiber Electrodes for Ultrasensitive Neural Recordings.

Carbon microfibers (MFs) coated with conducting polymers may provide a solution for long-term recording of activity from individual or small groups of neurons. Attaching cell adhesion molecules to the electro-sensitive surface might further improve electrode-neuron contact, thus enhancing signal stability and fidelity. We fabricated biofunctionalized microelectrodes consisting of 7-µm diameter carbon MFs coated with poly(3,4-ethylenedioxythiophene) doped with poly[(4-styrenesulfonic acid)-co-(maleic acid)] ( PEDOT: PSS-co-MA), and linked N-Cadherin to the polymer surface. These electrodes were tested for recording artificially generated electric potentials, as well as multiunit activity (MUA), sharp wave-ripple complexes (SWRs), and field excitatory postsynaptic potentials (fEPSPs) in rat hippocampal slices. The effects of electrode length and functionalization were compared. PEDOT: PSS-co-MA coating improved electric current detection and reduced the electrical noise but had no significant effect on the amplitude of recorded biopotentials. Surface biofunctionalization lowered the electric current flow, and further reduced the electrical noise. Additionally, it increased the amplitude of the recorded MUA, finally doubling the signal-to-noise ratio achieved with bare carbon MFs. Biofunctionalization benefits were apparent only for potentials from cells putatively adjacent to the microelectrode. Analysis of fEPSPs excluded adverse effects of functionalized electrodes in basal synaptic transmission. These results demonstrate the possibility of enhancing the amplitude and signal-to-noise ratio of neural recordings by coating the microelectrodes with conducting polymers modified with neural cell adhesion molecules, and support the use of biofunctionalized MFs in advanced neuroprosthetic devices.

Endocytosis of synaptic ADAM10 in neuronal plasticity and Alzheimer's disease.

A disintegrin and metalloproteinase 10 (ADAM10), a disintegrin and metalloproteinase that resides in the postsynaptic densities (PSDs) of excitatory synapses, has previously been shown to limit ß-amyloid peptide (Aß) formation in Alzheimer's disease (AD). ADAM10 also plays a critical role in regulating functional membrane proteins at the synapse. Using human hippocampal homogenates, we found that ADAM10 removal from the plasma membrane was mediated by clathrin-dependent endocytosis. Additionally, we identified the clathrin adaptor AP2 as an interacting partner of a previously uncharacterized atypical binding motif in the ADAM10 C-terminal domain. This domain was required for ADAM10 endocytosis and modulation of its plasma membrane levels. We found that the ADAM10/AP2 association was increased in the hippocampi of AD patients compared with healthy controls. Long-term potentiation (LTP) in hippocampal neuronal cultures induced ADAM10 endocytosis through AP2 association and decreased surface ADAM10 levels and activity. Conversely, long-term depression (LTD) promoted ADAM10 synaptic membrane insertion and stimulated its activity. ADAM10 interaction with the synapse-associated protein-97 (SAP97) was necessary for LTD-induced ADAM10 trafficking and required for LTD maintenance and LTD-induced changes in spine morphogenesis. These data identify and characterize a mechanism controlling ADAM10 localization and activity at excitatory synapses that is relevant to AD pathogenesis.

The influence of cold temperature on cellular excitability of hippocampal networks.

The hippocampus plays an important role in short term memory, learning and spatial navigation. A characteristic feature of the hippocampal region is its expression of different electrical population rhythms and activities during different brain states. Physiological fluctuations in brain temperature affect the activity patterns in hippocampus, but the underlying cellular mechanisms are poorly understood. In this work, we investigated the thermal modulation of hippocampal activity at the cellular network level. Primary cell cultures of mouse E17 hippocampus displayed robust network activation upon light cooling of the extracellular solution from baseline physiological temperatures. The activity generated was dependent on action potential firing and excitatory glutamatergic synaptic transmission. Involvement of thermosensitive channels from the transient receptor potential (TRP) family in network activation by temperature changes was ruled out, whereas pharmacological and immunochemical experiments strongly pointed towards the involvement of temperature-sensitive two-pore-domain potassium channels (K(2P)), TREK/TRAAK family. In hippocampal slices we could show an increase in evoked and spontaneous synaptic activity produced by mild cooling in the physiological range that was prevented by chloroform, a K(2P) channel opener. We propose that cold-induced closure of background TREK/TRAAK family channels increases the excitability of some hippocampal neurons, acting as a temperature-sensitive gate of network activation. Our findings in the hippocampus open the possibility that small temperature variations in the brain in vivo, associated with metabolism or blood flow oscillations, act as a switch mechanism of neuronal activity and determination of firing patterns through regulation of thermosensitive background potassium channel activity.

Blocking ADAM10 synaptic trafficking generates a model of sporadic Alzheimer's disease.

We describe here an innovative, non-transgenic animal model of Alzheimer's disease. This model mimics early stages of sporadic disease, which represents the vast majority of cases. The model was obtained by interfering with the complex between a disintegrin and metalloproteinase domain containing protein 10 (ADAM10), the main α-secretase candidate, and its partner, synapse-associated protein 97, a protein of the postsynaptic density-membrane associated guanylate kinase family. Association of ADAM10 with synapse-associated protein 97 governs enzyme trafficking and activity at synapses. Interfering with the ADAM10/synapse-associated protein 97 complex for 2 weeks by means of a cell-permeable peptide strategy is sufficient to shift the metabolism of the amyloid precursor protein towards amyloidogenesis and allows the reproduction of initial phases of sporadic Alzheimer's disease. After 2 weeks of treatment, we detected progressive Alzheimer's disease-like neuropathology, with an increase of ß-amyloid aggregate production and of tau hyperphosphorylation, and a selective alteration of N-methyl-d-aspartic acid receptor subunit composition in the postsynaptic compartment of mouse brain. Behavioural and electrophysiological deficits were also induced by peptide treatment.

Learning, AMPA receptor mobility and synaptic plasticity depend on n-cofilin-mediated actin dynamics.

Neuronal plasticity is an important process for learning, memory and complex behaviour. Rapid remodelling of the actin cytoskeleton in the postsynaptic compartment is thought to have an important function for synaptic plasticity. However, the actin-binding proteins involved and the molecular mechanisms that in vivo link actin dynamics to postsynaptic physiology are not well understood. Here, we show that the actin filament depolymerizing protein n-cofilin is controlling dendritic spine morphology and postsynaptic parameters such as late long-term potentiation and long-term depression. Loss of n-cofilin-mediated synaptic actin dynamics in the forebrain specifically leads to impairment of all types of associative learning, whereas exploratory learning is not affected. We provide evidence for a novel function of n-cofilin function in synaptic plasticity and in the control of extrasynaptic excitatory AMPA receptors diffusion. These results suggest a critical function of actin dynamics in associative learning and postsynaptic receptor availability.

A postsynaptic signaling pathway that may account for the cognitive defect due to IL1RAPL1 mutation.

BACKGROUND: Interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) gene mutations are associated with cognitive impairment ranging from nonsyndromic X-linked mental retardation to autism. IL1RAPL1 belongs to a novel family of Toll/IL-1 receptors, whose expression in the brain is upregulated by neuronal activity. Currently, very little is known about the function of this protein. We previously showed that IL1RAPL1 interacts with the neuronal calcium sensor NCS-1 and that it regulates voltage-gated calcium channel activity in PC12 cells. RESULTS: Here we show that IL1RAPL1 is present in dendritic spine where it interacts with PSD-95, a major component of excitatory postsynaptic compartment. Using gain- and loss-of-function experiments in neurons, we demonstrated that IL1RAPL1 regulates the synaptic localization of PSD-95 by controlling c-Jun terminal kinase (JNK) activity and PSD-95 phosphorylation. Mice carrying a null mutation of the mouse Il1rapl1 gene show a reduction of both dendritic spine density and excitatory synapses in the CA1 region of the hippocampus. These structural abnormalities are associated with specific deficits in hippocampal long-term synaptic plasticity. CONCLUSION: The interaction of IL1RAPL1 with PSD-95 discloses a novel pathophysiological mechanism of cognitive impairment associated with alterations of the JNK pathway leading to a mislocalization of PSD-95 and abnormal synaptic organization and function.

ERK activation in axonal varicosities modulates presynaptic plasticity in the CA3 region of the hippocampus through synapsin I.

Activity-dependent changes in the strength of synaptic connections in the hippocampus are central for cognitive processes such as learning and memory storage. In this study, we reveal an activity-dependent presynaptic mechanism that is related to the modulation of synaptic plasticity. In acute mouse hippocampal slices, high-frequency stimulation (HFS) of the mossy fiber (MF)-CA3 pathway induced a strong and transient activation of extracellular-regulated kinase (ERK) in MF giant presynaptic terminals. Remarkably, pharmacological blockade of ERK disclosed a negative role of this kinase in the regulation of a presynaptic form of plasticity at MF-CA3 contacts. This ERK-mediated inhibition of post-tetanic enhancement (PTE) of MF-CA3 synapses was both frequency- and pathway-specific and was observed only with HFS at 50 Hz. Importantly, blockade of ERK was virtually ineffective on PTE of MF-CA3 synapses in mice lacking synapsin I, 1 of the major presynaptic ERK substrates, and triple knockout mice lacking all synapsin isoforms displayed PTE kinetics resembling that of wild-type mice under ERK inhibition. These findings reveal a form of short-term synaptic plasticity that depends on ERK and is finely tuned by the firing frequency of presynaptic neurons. Our results also demonstrate that presynaptic activation of the ERK signaling pathway plays part in the activity-dependent modulation of synaptic vesicle mobilization and transmitter release.

Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1) controls activation of extracellular signal-regulated kinase (ERK) signaling in the striatum and long-term behavioral responses to cocaine.

BACKGROUND: Ras-extracellular signal-regulated kinase (Ras-ERK) signaling is central to the molecular machinery underlying cognitive functions. In the striatum, ERK1/2 kinases are co-activated by glutamate and dopamine D1/5 receptors, but the mechanisms providing such signaling integration are still unknown. The Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1), a neuronal specific activator of Ras-ERK signaling, is a likely candidate for coupling these neurotransmitter signals to ERK kinases in the striatonigral medium spiny neurons (MSN) and for modulating behavioral responses to drug abuse such as cocaine. METHODS: We used genetically modified mouse mutants for Ras-GRF1 as a source of primary MSN cultures and organotypic slices, to perform both immunoblot and immunofluorescence studies in response to glutamate and dopamine receptor agonists. Mice were also subjected to behavioral and immunohistochemical investigations upon treatment with cocaine. RESULTS: Phosphorylation of ERK1/2 in response to glutamate, dopamine D1 agonist, or both stimuli simultaneously is impaired in Ras-GRF1-deficient striatal cells and organotypic slices of the striatonigral MSN compartment. Consistently, behavioral responses to cocaine are also affected in mice deficient for Ras-GRF1 or overexpressing it. Both locomotor sensitization and conditioned place preference are significantly attenuated in Ras-GRF1-deficient mice, whereas a robust facilitation is observed in overexpressing transgenic animals. Finally, we found corresponding changes in ERK1/2 activation and in accumulation of FosB/DeltaFosB, a well-characterized marker for long-term responses to cocaine, in MSN from these animals. CONCLUSIONS: These results strongly implicate Ras-GRF1 in the integration of the two main neurotransmitter inputs to the striatum and in the maladaptive modulation of striatal networks in response to cocaine.

Synaptic vesicle docking: sphingosine regulates syntaxin1 interaction with Munc18.

Consensus exists that lipids must play key functions in synaptic activity but precise mechanistic information is limited. Acid sphingomyelinase knockout mice (ASMko) are a suitable model to address the role of sphingolipids in synaptic regulation as they recapitulate a mental retardation syndrome, Niemann Pick disease type A (NPA), and their neurons have altered levels of sphingomyelin (SM) and its derivatives. Electrophysiological recordings showed that ASMko hippocampi have increased paired-pulse facilitation and post-tetanic potentiation. Consistently, electron microscopy revealed reduced number of docked vesicles. Biochemical analysis of ASMko synaptic membranes unveiled higher amounts of SM and sphingosine (Se) and enhanced interaction of the docking molecules Munc18 and syntaxin1. In vitro reconstitution assays demonstrated that Se changes syntaxin1 conformation enhancing its interaction with Munc18. Moreover, Se reduces vesicle docking in primary neurons and increases paired-pulse facilitation when added to wt hippocampal slices. These data provide with a novel mechanism for synaptic vesicle control by sphingolipids and could explain cognitive deficits of NPA patients.

Profilin2 contributes to synaptic vesicle exocytosis, neuronal excitability, and novelty-seeking behavior.

Profilins are actin binding proteins essential for regulating cytoskeletal dynamics, however, their function in the mammalian nervous system is unknown. Here, we provide evidence that in mouse brain profilin1 and profilin2 have distinct roles in regulating synaptic actin polymerization with profilin2 preferring a WAVE-complex-mediated pathway. Mice lacking profilin2 show a block in synaptic actin polymerization in response to depolarization, which is accompanied by increased synaptic excitability of glutamatergic neurons due to higher vesicle exocytosis. These alterations in neurotransmitter release correlate with a hyperactivation of the striatum and enhanced novelty-seeking behavior in profilin2 mutant mice. Our results highlight a novel, profilin2-dependent pathway, regulating synaptic physiology, neuronal excitability, and complex behavior.