Xian-Min Yu

Dual Regulation of NMDA Receptor Functions by Direct Protein-Protein Interactions with the Dopamine D1 Receptor

Dopamine D1-like receptors, composed of D1 and D5 receptors, have been documented to modulate glutamate-mediated fast excitatory synaptic neurotransmission. Here, we report that dopamine D1 receptors modulate NMDA glutamate receptor-mediated functions through direct protein-protein interactions. Two regions in the D1 receptor carboxyl tail can directly and selectively couple to NMDA glutamate receptor subunits NR1-1a and NR2A. While one interaction is involved in the inhibition of NMDA receptor-gated currents, the other is implicated in the attenuation of NMDA receptor-mediated excitotoxicity through a PI-3 kinase-dependent pathway.

Appearance of new receptive fields in rat dorsal horn neurons following noxious stimulation of skeletal muscle: a model for referral of muscle pain?

To test the hypothesis that painful stimuli to skeletal muscle lead to a widespread unmasking of synaptic connections in dorsal horn neurons, intramuscular injections of bradykinin (BKN) were made outside the receptive fields (RFs) of these cells in the rat. Following BKN injections, new RFs all of which were located in the deep tissues and had high mechanical thresholds appeared in 9 out of 21 (42.8%) nociceptive dorsal horn neurons which originally had a single RF in deep tissues or in the skin. The appearance of new RFs may lead to a mislocation of the source of pain if in fact the impulse activity of a nociceptive dorsal horn neuron contains information on the site of the stimulus.

Effects of inflammatory irritant application to the rat temporomandibular joint on jaw and neck muscle activity

&NA; An electromyographic (EMG) study was carried out in 40 anaesthetized rats to determine if the activity of jaw and neck muscles could be influenced by injection of the small‐fibre excitant and inflammatory irritant mustard oil into the region of the temporomandibular joint (TMJ). Injection of a vehicle (mineral oil, 20 &mgr;l) did not produce any significant change in EMG activity. In contrast, injection of mustard oil (20 &mgr;l, 20%) evoked increases in EMG activity in the jaw muscles but not in the neck muscles. The increased EMG activity evoked by mustard oil was reflected in 1 or 2 phases of increased activity. The early EMG increase occurred soon after the mustard oil injection (mean latency ± SD: 3.5 ± 2.3 sec), peaked within 1 min, and then subsided (mean duration: 7.5 ± 5.2 min). The later EMG increase occurred at 14.6 ± 10.0 min after the mustard oil injection and lasted 14.3 ± 12.3 min. These excitatory effects of mustard oil on the EMG activity of jaw muscles appear to have a reflex basis since they could be abolished by pre‐administration of local anaesthetic into the TMJ region. These results document that TMJ injection of mustard oil results in a sustained and reversible activation of jaw muscles that may be related to the reported clinical occurrence of increased muscle activity associated with trauma to the TMJ.

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.

Involvement of NMDA receptor mechanisms in jaw electromyographic activity and plasma extravasation induced by inflammatory irritant application to temporomandibular joint region of rats

&NA; The aim of this study was to examine the possible role of N‐methyl‐ d‐aspartate (NMDA) receptor mechanisms in responses induced by the small‐fibre excitant and inflammatory irritant mustard oil injected into the temporomandibular joint (TMJ) region of rats. The effects of the non‐competitive NMDA antagonist MK‐801 were tested on the mustard oil‐evoked increases in electromyographic (EMG) activity of the masseter and digastric muscles and Evans Blue plasma extravasation. Five minutes before the mustard oil injection, MK‐801 or its vehicle was administered systemically (i.v.), into the third ventricle (i.c.v.), or locally into the TMJ region. Compared with control animals receiving vehicle, the rats receiving MK‐801 at an i.v. dose of 0.5 mg/kg (n = 5) showed a significant reduction in the incidence and magnitude of EMG responses as well as in the plasma extravasation evoked by mustard oil; MK‐801 at an i.v. dose of 0.1 mg/kg (n = 5) had no significant effect on plasma extravasation or on the incidence and magnitude of EMG responses but did significantly increase the latency of EMG responses. An i.c.v. dose of 0.1 mg/kg (n = 5) or 0.01 mg/kg (n = 5) had no significant effect on plasma extravasation or incidence of EMG responses but did significantly reduce the magnitudes of the masseter EMG response; the 0.01 mg/kg dose also significantly increased the latency of the digastric EMG response. The magnitudes of both the masseter and digastric EMG responses were also significantly reduced by MK‐801 administered into the TMJ region at a dose of 0.1 mg/kg (n = 5) but not by 0.01 mg/kg (n = 5); neither dose significantly affected the incidence of EMG responses or the plasma extravasation. These data suggest that both central and peripheral NMDA receptor mechanisms may play an important role in EMG responses evoked by the small‐fibre excitant and inflammatory irritant mustard oil, but that different neurochemical mechanisms may be involved in the plasma extravasation induced by mustard oil.

Excitatory effects on neck and jaw muscle activity of inflammatory irritant applied to cervical paraspinal tissues

&NA; A study was carried out in 19 anaesthetized rats to determine if the electromyographic (EMG) activity of jaw and neck muscles could be influenced by injection of the inflammatory irritant mustard oil into deep paraspinal tissues surrounding the C1‐3 vertebrae. The EMG activity was recorded ipsilaterally in the digastric, masseter and trapezius muscles and bilaterally in deep neck muscles (rectus capitis posterior). In comparison with control (vehicle) injections, mustard oil (20 &mgr;l, 20%) injected into the deep paraspinal tissues induced significant increases in EMG activity in the neck muscles in all the animals and in the jaw muscles in the majority of the animals; the effects of mustard oil were more prominent in the former. The EMG response evoked by mustard oil injection was frequently reflected in two phases of enhanced activity. The early phase of the increase in EMG activity was usually initiated immediately following mustard oil injection (mean latency: 20.4 ± 17.7 sec) and lasted 1.6 ± 1.1 min. The second phase occurred 11.3 ± 7.6 min later and lasted 11.0 ± 8.1 min. Evans Blue extravasation was apparent in the deep paraspinal tissues surrounding the C1‐3 vertebrae after mustard oil injection, and histological examination showed that mustard oil injection induced an inflammatory reaction in the rectus capitis posterior muscle. These results document that injection of the inflammatory irritant mustard oil into deep paraspinal tissues results in a sustained and reversible activation of both jaw and neck muscles. Such effects may be related to the reported clinical occurrence of increased muscle activity associated with trauma to deep tissues.

Gain control of N-methyl-d-aspartate receptor activity by receptor-like protein tyrosine phosphatase α

Src kinase regulation of N‐methyl‐D‐aspartate (NMDA) subtype glutamate receptors in the central nervous system (CNS) has been found to play an important role in processes related to learning and memory, ethanol sensitivity and epilepsy. However, little is known regarding the mechanisms underlying the regulation of Src family kinase activity in the control of NMDA receptors. Here we report that the distal phosphatase domain (D2) of protein tyrosine phosphatase α (PTPα) binds to the PDZ2 domain of post‐synaptic density 95 (PSD95). Thus, Src kinase, its activator (PTPα) and substrate (NMDA receptors) are linked by the same scaffold protein, PSD95. Removal of PTPα does not affect the association of Src with NMDA receptors, but turns off the constitutive regulation of NMDA receptors by the kinase. Further more, we found that application of the PTPα catalytic domains (D1 + D2) into neurones enhances NMDA receptor‐mediated synaptic responses. Conversely, the blockade of endogenous PTPα inhibits NMDA receptor activity and the induction of long‐term potentiation in hippocampal neurones. Thus, PTPα is a novel up‐regulator of synaptic strength in the CNS.

Involvement of trigeminal subnucleus caudalis (medullary dorsal horn) in craniofacial nociceptive reflex activity

We have previously shown that an increase in electromyographic (EMG) activity of digastric (DIG) and masseter (MASS) muscles can be reflexly evoked by injection into the rats temporomandibular joint (TMJ) region of the small-fibre excitant and inflammatory irritant mustard oil (MO). Since the trigeminal (V) subnucleus caudalis (Vc, i.e. medullary dorsal horn) has traditionally been viewed as an essential brainstem relay site of nociceptive information from craniofacial tissues, an EMG study was carried out in 45 anaesthetized rats to determine if Vc is involved in the MO-evoked increases in jaw muscle EMG activity. The effects of histologically confirmed surgical or chemical lesions of Vc on this evoked EMG activity were tested in different groups of rats. MO injection into the left TMJ region of intact rats evoked bilateral increases in EMG activity of DIG and MASS which could be significantly reduced by surgical transection of the left caudal brainstem at the obex level; MO injection into the right TMJ region in these same rats still readily evoked increases in EMG activity. A sagittal section medial to Vc or transection at the level of the second cervical spinal segment did not produce any significant reduction in the reflexly evoked EMG activity. Neurones in Vc, as opposed to fibres of passage, appear to be important for the MO-evoked EMG activity, since injection into Vc of the neurotoxic chemical ibotenic acid significantly reduced the mustard oil-evoked EMG activity. The Vc also appears to play a role in the activation of contralateral V motoneurons, as evidenced by the activation of the contralateral DIG and MASS muscles by the injection of MO into the left TMJ region of intact rats and by the reduction of this evoked EMG activity in the contralateral DIG and MASS of rats with a surgical transection or ibotenic acid lesion of the left Vc. These findings suggest that Vc may be a critical element in the neural pathways underlying the reflex responses evoked bilaterally in DIG and MASS muscles by noxious stimulation of the TMJ region.

The regulation of N-methyl-D-aspartate receptors by Src kinase.

Src family kinases (SFKs) play critical roles in the regulation of many cellular functions by growth factors, G‐protein‐coupled receptors and ligand‐gated ion channels. Recent data have shown that SFKs serve as a convergent point of multiple signaling pathways regulating N‐methyl‐d‐aspartate (NMDA) receptors in the central nervous system. Multiple SFK molecules, such as Src and Fyn, closely associate with their substrate, NMDA receptors, via indirect and direct binding mechanisms. The NMDA receptor is associated with an SFK signaling complex consisting of SFKs; the SFK‐activating phosphatase, protein tyrosine phosphatase α; and the SFK‐inactivating kinase, C‐terminal Src kinase. Early studies have demonstrated that intramolecular interactions with the SH2 or SH3 domain lock SFKs in a closed conformation. Disruption of the interdomain interactions can induce the activation of SFKs with multiple signaling pathways involved in regulation of this process. The enzyme activity of SFKs appears ‘graded’, exhibiting different levels coinciding with activation states. It has also been proposed that the SH2 and SH3 domains may stimulate catalytic activity of protein tyrosine kinases, such as Abl. Recently, it has been found that the enzyme activity of neuronal Src protein is associated with its stability, and that the SH2 and SH3 domain interactions may act not only to constrain the activation of neuronal Src, but also to regulate the enzyme activity of active neuronal Src. Collectively, these findings demonstrate novel mechanisms underlying the regulation of SFKs.

Control of Excitatory Synaptic Transmission by C-terminal Src Kinase

The induction of long-term potentiation at CA3-CA1 synapses is caused by an N-methyl-d-aspartate (NMDA) receptordependent accumulation of intracellular Ca2+, followed by Src family kinase activation and a positive feedback enhancement of NMDA receptors (NMDARs). Nevertheless, the amplitude of baseline transmission remains remarkably constant even though low frequency stimulation is also associated with an NMDAR-dependent influx of Ca2+ into dendritic spines. We show here that an interaction between C-terminal Src kinase (Csk) and NMDARs controls the Src-dependent regulation of NMDAR activity. Csk associates with the NMDAR signaling complex in the adult brain, inhibiting the Src-dependent potentiation of NMDARs in CA1 neurons and attenuating the Src-dependent induction of long-term potentiation. Csk associates directly with Src-phosphorylated NR2 subunits in vitro. An inhibitory antibody for Csk disrupts this physical association, potentiates NMDAR mediated excitatory postsynaptic currents, and induces long-term potentiation at CA3-CA1 synapses. Thus, Csk serves to maintain the constancy of baseline excitatory synaptic transmission by inhibiting Src kinase-dependent synaptic plasticity in the hippocampus.