Pharmacologic rescue of motor and sensory function by the neuroprotective compound P7C3 following neonatal nerve injury.

Nerve injuries cause pain, paralysis and numbness that can lead to major disability, and newborns often sustain nerve injuries during delivery that result in lifelong impairment. Without a pharmacologic agent to enhance functional recovery from these injuries, clinicians rely solely on surgery and rehabilitation to treat patients. Unfortunately, patient outcomes remain poor despite application of the most advanced microsurgical and rehabilitative techniques. We hypothesized that the detrimental effects of traumatic neonatal nerve injury could be mitigated with pharmacologic neuroprotection, and tested whether the novel neuroprotective agent P7C3 would block peripheral neuron cell death and enhance functional recovery in a rat neonatal nerve injury model. Administration of P7C3 after sciatic nerve crush injury doubled motor and sensory neuron survival, and also promoted axon regeneration in a dose-dependent manner. Treatment with P7C3 also enhanced behavioral and muscle functional recovery, and reversed pathological mobilization of spinal microglia after injury. Our findings suggest that the P7C3 family of neuroprotective compounds may provide a basis for the development of a new neuroprotective drug to enhance recovery following peripheral nerve injury.

The tyrosine phosphatase STEP constrains amygdala-dependent memory formation and neuroplasticity.

STriatal-Enriched protein tyrosine Phosphatase (STEP; PTPN5) is expressed in brain regions displaying adult neuroplasticity. STEP modulates neurotransmission by dephosphorylating regulatory tyrosine residues on its substrates. In this way, STEP inactivates extracellular-signal-regulated kinase 1/2 (ERK1/2), limiting the duration and spatial distribution of ERK signaling. Two additional substrates, the tyrosine kinase Fyn and the NR2B subunit of the N-methyl-d-aspartic acid receptor, link STEP to glutamate receptor internalization in the synapse. Thus, STEP may act through parallel pathways to oppose the development of experience-dependent synaptic plasticity. We examined the hypothesis that the absence of STEP facilitates amygdala-dependent behavioral and synaptic plasticity (i.e., fear conditioning and long-term potentiation) using STEP-deficient mice (STEP KO). These mice show no detectable expression of STEP in the brain along with increases in Tyr phosphorylation of STEP substrates. Here we demonstrate that STEP KO mice also display augmented fear conditioning as measured by an enhancement in conditioned suppression of instrumental response when a fear-associated conditioned stimulus was presented. Deletion of STEP also increases long-term potentiation and ERK phosphorylation in the lateral amygdala. The current experiments demonstrate that deletion of STEP can enhance experience-induced neuroplasticity and memory formation and identifies STEP as a target for pharmacological treatment aimed at improving the formation of long-term memories.

Rett syndrome induced pluripotent stem cell-derived neurons reveal novel neurophysiological alterations.

Rett syndrome (RTT) is a neurodevelopmental autism spectrum disorder caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Here, we describe the first characterization and neuronal differentiation of induced pluripotent stem (iPS) cells derived from Mecp2-deficient mice. Fully reprogrammed wild-type (WT) and heterozygous female iPS cells express endogenous pluripotency markers, reactivate the X-chromosome and differentiate into the three germ layers. We directed iPS cells to produce glutamatergic neurons, which generated action potentials and formed functional excitatory synapses. iPS cell-derived neurons from heterozygous Mecp2(308) mice showed defects in the generation of evoked action potentials and glutamatergic synaptic transmission, as previously reported in brain slices. Further, we examined electrophysiology features not yet studied with the RTT iPS cell system and discovered that MeCP2-deficient neurons fired fewer action potentials, and displayed decreased action potential amplitude, diminished peak inward currents and higher input resistance relative to WT iPS-derived neurons. Deficiencies in action potential firing and inward currents suggest that disturbed Na(+) channel function may contribute to the dysfunctional RTT neuronal network. These phenotypes were additionally confirmed in neurons derived from independent WT and hemizygous mutant iPS cell lines, indicating that these reproducible deficits are attributable to MeCP2 deficiency. Taken together, these results demonstrate that neuronally differentiated MeCP2-deficient iPS cells recapitulate deficits observed previously in primary neurons, and these identified phenotypes further illustrate the requirement of MeCP2 in neuronal development and/or in the maintenance of normal function. By validating the use of iPS cells to delineate mechanisms underlying RTT pathogenesis, we identify deficiencies that can be targeted for in vitro translational screens.

Endogenous ATP involvement in mustard-oil-induced central sensitization in trigeminal subnucleus caudalis (medullary dorsal horn).

Central sensitization represents a sustained hypersensitive state of dorsal horn nociceptive neurons that can be evoked by peripheral inflammation or injury to nerves and tissues. It reflects neuroplastic changes such as increases in neuronal spontaneous activity, receptive field size, and responses to suprathreshold stimuli and a decrease in activation threshold. We recently demonstrated that purinergic receptor mechanisms in trigeminal subnucleus caudalis (Vc; medullary dorsal horn) are also involved in the initiation and maintenance of central sensitization in brain stem nociceptive neurons of trigeminal subnucleus oralis. The aim of the present study was to investigate whether endogenous ATP is involved in the development of central sensitization in Vc itself. The experiments were carried out on urethan/alpha-chloralose anesthetized and immobilized rats. Single neurons were recorded and identified as nociceptive-specific (NS) in the deep laminae of Vc. During continuous saline superfusion (0.6 ml/h it) over the caudal medulla, Vc neuronal central sensitization was readily induced by mustard oil application to the tooth pulp. However, this mustard-oil-induced central sensitization could be completely blocked by continuous intrathecal superfusion of the wide-spectrum P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2, 4-disulphonic acid tetra-sodium (33-100 microM) and by apyrase (an ectonucleotidase enzyme, 30 units/ml). Superfusion of the selective P2X1, P2X3 and P2X(2/3) receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (6-638 microM) partially blocked the Vc central sensitization. The two P2X receptor antagonists did not significantly affect the baseline nociceptive properties of the Vc neurons. These findings implicate endogenous ATP as an important mediator contributing to the development of central sensitization in nociceptive neurons of the deep laminae of the dorsal horn.

LTP gets culture.

In a recent breakthrough, a methodology has been developed for studying persistent enhancement of excitatory synaptic transmission in primary cultures of hippocampus. Results from the use of this method have already pointed to a previously unsuspected differential role for synaptic versus extrasynaptic NMDA receptors in lasting synaptic potentiation.

NMDA receptor regulation by Src kinase signalling in excitatory synaptic transmission and plasticity.

Regulation of postsynaptic glutamate receptors is one of the main mechanisms for altering synaptic efficacy in the central nervous system. Recent studies have given insight into the upregulation of the NMDA receptor by Src family tyrosine kinases, which bind to scaffolding proteins in the NMDA receptor complex. Src acts as a common step in signalling cascades that link G-protein-coupled receptors with protein kinase C via the intermediary cell-adhesion kinase beta. This signalling to NMDA receptors is required for long-term potentiation in the CA1 region of the hippocampus.

CAKbeta/Pyk2 kinase is a signaling link for induction of long-term potentiation in CA1 hippocampus.

Long-term potentiation (LTP) is an activity-dependent enhancement of synaptic efficacy, considered a model of learning and memory. The biochemical cascade producing LTP requires activation of Src, which upregulates the function of NMDA receptors (NMDARs), but how Src becomes activated is unknown. Here, we show that the focal adhesion kinase CAKbeta/Pyk2 upregulated NMDAR function by activating Src in CA1 hippocampal neurons. Induction of LTP was prevented by blocking CAKbeta/Pyk2, and administering CAKbeta/Pyk2 intracellularly mimicked and occluded LTP. Tyrosine phosphorylation of CAKbeta/Pyk2 and its association with Src was increased by stimulation that produced LTP. Finally, CAKbeta/Pyk2-stimulated enhancement of synaptic AMPA responses was prevented by blocking NMDARS, chelating intracellular Ca(2+), or blocking Src. Thus, activating CAKbeta/Pyk2 is required for inducing LTP and may depend upon downstream activation of Src to upregulate NMDA receptors.

Regulation of neuregulin signaling by PSD-95 interacting with ErbB4 at CNS synapses.

Neuregulins (NRGs) and their receptors, the ErbB protein tyrosine kinases, are essential for neuronal development, but their functions in the adult CNS are unknown. We report that ErbB4 is enriched in the postsynaptic density (PSD) and associates with PSD-95. Heterologous expression of PSD-95 enhanced NRG activation of ErbB4 and MAP kinase. Conversely, inhibiting expression of PSD-95 in neurons attenuated NRG-mediated activation of MAP kinase. PSD-95 formed a ternary complex with two molecules of ErbB4, suggesting that PSD-95 facilitates ErbB4 dimerization. Finally, NRG suppressed induction of long-term potentiation in the hippocampal CA1 region without affecting basal synaptic transmission. Thus, NRG signaling may be synaptic and regulated by PSD-95. A role of NRG signaling in the adult CNS may be modulation of synaptic plasticity.

Neuronal plasticity: increasing the gain in pain.

We describe those sensations that are unpleasant, intense, or distressing as painful. Pain is not homogeneous, however, and comprises three categories: physiological, inflammatory, and neuropathic pain. Multiple mechanisms contribute, each of which is subject to or an expression of neural plasticity-the capacity of neurons to change their function, chemical profile, or structure. Here, we develop a conceptual framework for the contribution of plasticity in primary sensory and dorsal horn neurons to the pathogenesis of pain, identifying distinct forms of plasticity, which we term activation, modulation, and modification, that by increasing gain, elicit pain hypersensitivity.

The Lurcher mutation of an alpha-amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid receptor subunit enhances potency of glutamate and converts an antagonist to an agonist.

A point mutation of the GluRdelta2 (A654T) glutamate receptor subunit converts it into a functional channel, and a spontaneous mutation at this site is thought to be responsible for the neurodegeneration of neurons in the Lurcher mouse. This mutation is located in a hydrophobic region of the M3 domain of this subunit, and this alanine is conserved throughout many of the glutamate receptors. We show here that site-directed mutagenesis of the homologous alanine (A636T; GluR1-L(c)) in the GluR1 AMPA receptor subunit alters its channel properties. The apparent potencies of both kainate and glutamate were increased 85- and 2000-fold, respectively. Furthermore, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)was converted from a competitive antagonist into a potent agonist. Our results demonstrate that a single amino acid within or near the putative second transmembrane region of the GluR1 subunit is critical for the binding/gating properties of this AMPA receptor.

P2Y(1) purinoceptor-mediated Ca(2+) signaling and Ca(2+) wave propagation in dorsal spinal cord astrocytes.

ATP is known to act as an extracellular messenger mediating the propagation of Ca(2+) waves in astrocyte networks. ATP mediates Ca(2+) waves by activating P2Y purinoceptors, which mobilize intracellular Ca(2+) in astrocytes. A number of P2Y purinoceptor subtypes have been discovered, but it is not known which P2Y subtypes participate in transmitting astrocyte Ca(2+) waves. Here, we show that ATP analogs that are selective agonists for the P2Y(1) subtype of purinoceptor caused release of intracellular Ca(2+) in astrocytes from the dorsal spinal cord. The Ca(2+) responses were blocked by adenosine-3'-phospho-5'-phosphosulfate, an antagonist known to selectively inhibit P2Y(1) but not other P2Y purinoceptor subtypes. Also, we show that P2Y(1) mRNA is expressed in dorsal spinal cord astrocytes. Furthermore, expression of P2Y(1) in an astrocytoma cell line lacking endogenous purinoceptors was sufficient to permit propagation of intercellular Ca(2+) waves. Finally, Ca(2+) wave propagation in dorsal spinal cord astrocytes was suppressed by pharmacologically blocking P2Y(1) purinoceptors. Together, these results indicate that dorsal spinal astrocytes express functional P2Y(1) purinoceptors, which participate in the transmission of Ca(2+) waves. Ca(2+) waves in astrocytes have been implicated as a major signaling pathway coordinating glial and neuronal activity; therefore, P2Y(1) purinoceptors may represent an important link in cell-cell signaling in the CNS.

Src potentiation of NMDA receptors in hippocampal and spinal neurons is not mediated by reducing zinc inhibition.

The protein-tyrosine kinase Src is known to potentiate the function of NMDA receptors, which is necessary for the induction of long-term potentiation in the hippocampus. With recombinant receptors composed of NR1-1a/NR2A or NR1-1a/2B subunits, Src reduces voltage-independent inhibition by the divalent cation Zn2+. Thereby the function of recombinant NMDA receptors is potentiated by Src only when the Zn2+ level is sufficient to cause tonic inhibition. Here we investigated whether the Src-induced potentiation of NMDA receptor function in neurons is caused by reducing voltage-independent Zn2+ inhibition. Whereas chelating extracellular Zn2+ blocked the Src-induced potentiation of NR1-1a/2A receptors, we found that Zn2+ chelation did not affect the potentiation of NMDA receptor (NMDAR) currents by Src applied into hippocampal CA1 or CA3 neurons. Moreover, Src did not alter the Zn2+ concentration-inhibition relationship for NMDAR currents in CA1 or CA3 neurons. Also, chelating extracellular Zn2+ did not prevent the upregulation of NMDA single-channel activity by endogenous Src in membrane patches from spinal dorsal horn neurons. Taking these results together we conclude that Src-induced potentiation of NMDAR currents is not mediated by reducing Zn2+ inhibition in hippocampal and dorsal horn neurons.

Src, a molecular switch governing gain control of synaptic transmission mediated by N-methyl-D-aspartate receptors.

The N-methyl-D-aspartate (NMDA) receptor is a principal subtype of glutamate receptor mediating fast excitatory transmission at synapses in the dorsal horn of the spinal cord and other regions of the central nervous system. NMDA receptors are crucial for the lasting enhancement of synaptic transmission that occurs both physiologically and in pathological conditions such as chronic pain. Over the past several years, evidence has accumulated indicating that the activity of NMDA receptors is regulated by the protein tyrosine kinase, Src. Recently it has been discovered that, by means of up-regulating NMDA receptor function, activation of Src mediates the induction of the lasting enhancement of excitatory transmission known as long-term potentiation in the CA1 region of the hippocampus. Also, Src has been found to amplify the up-regulation of NMDA receptor function that is produced by raising the intracellular concentration of sodium. Sodium concentration increases in neuronal dendrites during high levels of firing activity, which is precisely when Src becomes activated. Therefore, we propose that the boost in NMDA receptor function produced by the coincidence of activating Src and raising intracellular sodium may be important in physiological and pathophysiological enhancement of excitatory transmission in the dorsal horn of the spinal cord and elsewhere in the central nervous system.

Gain control of NMDA-receptor currents by intracellular sodium.

The influx of Na+ is fundamental to electrical signalling in the nervous system and is essential for such basic signals as action potentials and excitatory postsynaptic potentials. During periods of bursting or high levels of discharge activity, large increases in intracellular Na+ concentration ([Na+]i) are produced in neuronal soma and dendrites. However, the intracellular signalling function of raised postsynaptic [Na+]i is unknown. Here we show that [Na+]i regulates the function of NMDA (N-methyl-D-aspartate) receptors, a principal subtype of glutamate receptor. NMDA-receptor-mediated whole-cell currents and NMDA-receptor single-channel activity were increased by raising [Na+]i and channel activity decreased upon lowering [Na+]i; therefore, the activity of NMDA channels tracks changes in [Na+]i. We found that the sensitivity of the channel to Na+ was set by a Src kinase that is associated with the channel. Raising [Na+]i selectively increased synaptic responses mediated by NMDA receptors, but not by non-NMDA receptors. Thus, the change in postsynaptic [Na+]i that occurs during neuronal activity is a signal for controlling the gain of excitatory synaptic transmission. This mechanism may be important for NMDA-receptor-dependent plasticity and toxicity in the central nervous system.

Src, N-methyl-D-aspartate (NMDA) receptors, and synaptic plasticity.

The protein tyrosine kinase Src is expressed widely in the central nervous system and is abundant in neurons. Over the past several years, evidence has accumulated showing that one function of Src is to regulate the activity of N-methyl-D-aspartate (NMDA) receptors and other ion channels. NMDA receptors are a principal subtype of glutamate receptor that mediates fast excitatory transmission at most central synapses. Recently it has been discovered that, by means of up-regulating the function of NMDA receptors, Src mediates the induction of long-term potentiation (LTP) in the CA1 region of the hippocampus. This finding led to a new model for induction of LTP whereby tetanic stimulation produces a rapid activation of Src, causing enhanced NMDA receptor function. This enhanced NMDA receptor function boosts the entry of Ca2+, which may thereby trigger the downstream signalling cascade, ending in potentiation of non-NMDA receptors. This functional role for Src may be important in physiological and pathophysiological processes in the central nervous system.

P2Y and P2U receptors differentially release intracellular Ca2+ via the phospholipase c/inositol 1,4,5-triphosphate pathway in astrocytes from the dorsal spinal cord.

In astrocytes, raising intracellular Ca2+ concentration is a principal mechanism for transducing extracellular signals following activation of cell-surface receptors. Receptors that may be activated by purine nucleotides, P2 receptors, are known to be expressed by astrocytes from dorsal spinal cord; these astrocytes express two distinct subtypes of P2 receptor, P2Y and P2U. A main goal of the present study was to determine the intracellular signalling pathways mediating the Ca2+ responses produced by stimulating these receptors. Experiments were done using cultured astrocytes from rat dorsal spinal cord. Ca2+ responses were evoked by 2-methylthio-ATP or UTP, nucleotides previously shown to selectively activate P2Y and P2U receptors, respectively, in these cells. P2Y- and P2U-evoked Ca2+ responses were found not to depend upon extracellular Ca2+ and were blocked by thapsigargin, a Ca2+-ATPase inhibitor known to deplete inositol 1,4,5-triphosphate-sensitive Ca2+ stores. Intracellular application of the inositol 1,4,5-triphosphate-sensitive receptor antagonist, heparin, or of the G-protein inhibitor guanosine 5'-O-(2-thiodiphosphate), blocked the P2Y- and P2U-evoked Ca2+ responses. Moreover, the responses were prevented by the phospholipase C inhibitor, U-73122, but were unaffected by the inactive analogue, U-73343. These results indicate that P2Y and P2U receptors on dorsal spinal astrocytes are linked via G-protein coupling to release of intracellular Ca2+ via the phospholipase C/inositol 1,4,5-triphosphate pathway. When we assessed the releasable pools of intracellular Ca2+, by repeated agonist applications in zero extracellular Ca2+, we found that the pool accessed by activating P2U receptors was only a subpool of that accessed by activating P2Y receptors. This implies that there are separable inositol 1,4,5-triphosphate-releasable pools of Ca2+ in dorsal spinal astrocytes and that these may be differentially released by activating distinct metabotropic P2 receptors. This differential release of Ca2+ may be important for physiological as well as pathophysiological events occurring within the spinal cord.