Locally released small (non-protein) ninhydrin-reacting molecules underlie developmental differences of cultured medullary versus spinal dorsal horn neurons.

Neurons located in the trigeminal subnucleus caudalis (Vc) play crucial roles in pain and sensorimotor functions in the orofacial region. Because of many anatomical and functional similarities with the spinal dorsal horn (SDH), Vc has been termed the medullary dorsal horn--analogous to the SDH. Here, we report that when compared with embryonic SDH neurons in culture, neurons isolated from the Vc region showed significantly slower growth, lower glutamate receptor activity, and more cells undergoing cell death. SDH neuron development was inhibited in co-cultures of SDH and Vc tissues while Vc neuron development was promoted by co-culture with SDH tissues. Furthermore, we identified that small (non-protein) ninhydrin-reacting molecules purified from either embryonic or post-natal Vc-conditioned medium inhibited neuronal growth whereas ninhydrin-reacting molecules from SDH-conditioned medium promoted neuronal growth. These findings suggest the involvement of locally released factors in the region-specific regulation of neuronal development in Vc and SDH, central nervous system regions playing critical roles in pain, and point to novel avenues for investigating central nervous system regionalization and for designing therapeutic approaches to manage neurodegenerative diseases and pain.

A functional interaction of sodium and calcium in the regulation of NMDA receptor activity by remote NMDA receptors.

The NMDA receptor is an important subtype glutamate receptor that acts as a nonselective cation channel highly permeable to both calcium (Ca2+) and sodium (Na+). The activation of NMDA receptors produces prolonged increases of intracellular Ca2+ concentration ([Ca2+]i) and thereby triggers downstream signaling pathways involved in the regulation of many physiological and pathophysiological processes. Previous studies have focused on how Ca2+ or Na+ affects NMDA receptor activity in isolation. Specifically, [Ca2+]i increase may downregulate NMDA channels and thus is considered an important negative feedback mechanism controlling NMDA receptor activity, whereas an increase in intracellular Na+ concentration ([Na+]i) may upregulate NMDA channel activity. Thus so that the activity-dependent regulation of NMDA receptors and neuroplasticity may be further understood, a critical question that has to be answered is how an individual NMDA receptor may be regulated when both of these ionic species flow into neurons during the same time period via neighboring activated NMDA receptors. Here we report that the gating of a NMDA channel is regulated by the activation of remote NMDA receptors via a functional Na+-Ca2+ interaction and that during the activation of NMDA receptors Na+ influx potentiates Ca2+ influx on one hand and overcomes Ca2+-induced inhibition of NMDA channel gating on the other hand. Furthermore, we have identified that a critical increase (5 +/- 1 mM) in [Na+]i is required to mask the effects of Ca2+ on NMDA channel gating in cultured hippocampal neurons. Thus cross talk between NMDA receptors mediated by a functional Na+-Ca2+ interaction is a novel mechanism regulating NMDA receptor activity.

Characteristics of rectal perception are altered in irritable bowel syndrome.

A hallmark symptom of irritable bowel syndrome (IBS) is a lower pain threshold during rectal distension, but the mechanism underlying this disorder remains unclear. Examining the relationship between physiological and perceptual responses to rectal distension can provide insight into the underlying peripheral or central dysfunction in IBS. Therefore, we carried out a study of the rectal sensations of urge to defecate, pain and unpleasantness in relation to the varying states of the rectum. Ten IBS patients and 13 healthy controls underwent six sets of isobaric rectal distensions. The first set was ascending stepwise distensions terminating upon report of moderate pain where verbal ratings of urge, pain, and unpleasantness were acquired. The remaining sets were phasic or tonic distensions at a single pressure eliciting either moderate urge or moderate pain intensity where subjects gave continuous ratings of urge or pain percepts. The McGill Pain Questionnaire (MPQ) was used to assess different qualities of the pain experience during single pressure distensions. Abnormalities in IBS patients included: (1) higher incidence of distensions where unpleasantness is greater than pain intensity, (2) significantly longer persistence of ratings after stimulus termination during phasic distensions eliciting either moderate urge or moderate pain, (3) significantly smaller ratings fluctuations during tonic distensions, and (4) significantly higher MPQ scores for painful tonic distensions. Our study demonstrates that IBS patients have abnormal temporal and intensity properties of rectal sensation. These can be accounted for by either altered peripheral neuromuscular processing and/or processing of ascending rectal input in the central nervous system.

The relationship between rectal pain, unpleasantness, and urge to defecate in normal subjects.

Rectal stimulation under normal or pathological conditions evokes numerous sensations. Previous studies have examined rectal stimulation-evoked pain and urge to defecate, but discrepancies in the findings remain because of the different methodologies used in each study and the reporting of sensations only at the end of or after the applied stimuli. Therefore, we conducted a psychophysical study of various aspects of rectal sensation in normal subjects using a variety of distension stimuli and continuous on-line rating of sensation. Ten normal healthy subjects (eight female and two male) were given rectal distension stimuli delivered by a computer-driven barostat. For some experiments, sensation was continuously monitored and rated on a visual analog scale. Subjects first underwent an ascending series of phasic (30 s) distensions to determine how urge, unpleasantness, and pain intensity varied and interrelated as rectal volume and pressure changed. A second series of distensions examined rectal physiology and perception during short phasic (30 s) or long (300 s) distensions at pressures that elicit either moderate urge or moderate pain and while continuously rating these sensations. The McGill Pain Questionnaire was used to assess the multidimensional aspects of rectal pain with each type of distension. The results of the ascending series revealed significant relationships between sensations as pressure and volume increased. The ratings of urge were double that of pain and unpleasantness, whereas unpleasantness and pain ratings were comparable. Isobaric phasic and tonic distensions were associated with an increase in volume (i.e. accommodation) with time. The magnitude of urge with repetitive short isobaric (30 s) distensions was overall not related to the slight increase in rectal volume, while phasic distensions at moderate pain intensity revealed a significant overall relationship between rectal volume and both unpleasantness and pain intensity. Long isobaric distensions evoked sensations that varied over time despite progressive increases in volume, but less variation in sensation was observed during short phasic distensions which also demonstrated a similar increase in rectal volume. Differences in temporal characteristics of sensations evoked by low-pressure distensions eliciting moderate urge versus high-pressure distensions eliciting moderate pain were illustrated by a significantly longer delay to the diminution of non-painful urge versus pain. Therefore, we conclude (1) Differences in the discrimination and the temporal characteristics of urge at subpainful rectal pressures and of pain at noxious pressures suggest that noxious and non-noxious stimuli are processed differently. (2) The overall unpleasantness and pain correlate with rectal volume during accommodation. However, instantaneous evoked sensations can vary independent of volume changes during constant pressure distension. (3) The reported sensation-related responses to tension and stretch will likely be different depending on the degree of accommodation that is occurring. Moreover, the peripheral receptor mechanisms which contribute to controlling this accommodation will also affect the perception of rectal stimuli. (4) Continuous ratings of rectal sensations are valuable in investigating rectal physiology and the multidimensional nature of rectal symptoms.

An fMRI study of the anterior cingulate cortex and surrounding medial wall activations evoked by noxious cutaneous heat and cold stimuli.

The anterior cingulate cortex (ACC) and adjacent regions in the medial wall have been implicated in sensory, motor and cognitive processes, including pain. Our previous functional magnetic resonance imaging (fMRI) studies have demonstrated pain-related activation of the posterior portion of the ACC during transcutaneous electrical nerve stimulation (TENS) and variable patterns of cortical activation with innocuous and noxious thermal stimuli in individual subjects. The present study represents the companion paper to our recent study of pain- and thermal-related cortical activations with the aim to use fMRI to delineate the activations in the ACC and surrounding regions of the medial wall during application of innocuous and noxious thermal stimuli as well as during performance of a motor task in individual subjects. Ten normal subjects were imaged on a conventional 1.5 T GE 'echospeed' system. Functional images were obtained from sagittal sections through each hemisphere centered at approximately 3-5 and 7-9 mm from midline. Each subject was imaged during innocuous (cool, warm) and noxious thermal (cold, hot) stimulation of the thenar eminence, and execution of a motor (sequential finger-thumb opposition) task. Task-related activations were mostly confined to contralateral and medial ipsilateral images. Although the present results demonstrate intersubject variability in the task-related activations, some general modality-specific patterns were apparent: (i) innocuous thermal-related activations were located mainly in the anterior ACC; (ii) noxious thermal-related activations were primarily located in the anterior ACC, the ventral portion of the posterior ACC, and the supplementary motor area (SMA); (iii) motor-related activations were primarily located in the SMA and dorsal portion of the posterior ACC. These results indicate that specific spatial patterns of activation exist within the ACC and surrounding regions of the medial wall for innocuous and noxious thermal stimuli, and that noxious thermal- and motor-related activations appear to be segregated within the ACC. Therefore, we propose a segregation of the ACC into an anterior non-specific attention/arousal system and a posterior pain system.

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 Ca(2+), 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 Ca(2+) 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.

The removal of extracellular calcium: a novel mechanism underlying the recruitment of N-methyl-D-aspartate (NMDA) receptors in neurotoxicity.

The involvement of NMDA-type glutamate receptor in neuronal injury established in experimental stroke and neurotrauma models has been recently challenged by failures in treatment of stroke/neurotrauma patients with NMDA receptor antagonists. NMDA receptor activity is known to be essential for mediating a multitude of physiological functions. However, how NMDA receptors are recruited to cause neuronal injury remains unclear. Here we report that the time period during which initial NMDA receptor up-regulation occurs is critical for the recruitment of NMDA receptors causing neuronal injury during extracellular calcium (Ca2+) reperfusion in cultured hippocampal neurons, and represents the key period for neuronal protection by NMDA receptor antagonists. Furthermore, we identified that via intracellular sodium (Na+), extracellular Ca2+ depletion induces the up-regulation of NMDA receptor gating. Taken together, our study provides direct experimental evidence suggesting that determination of when and how NMDA receptors are recruited to cause neurotoxicity is essential for guiding treatment via antagonism of NMDA receptor functions.

Gain control of N-methyl-D-aspartate receptor activity by receptor-like protein tyrosine phosphatase alpha.

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 alpha (PTPalpha) binds to the PDZ2 domain of post-synaptic density 95 (PSD95). Thus, Src kinase, its activator (PTPalpha) and substrate (NMDA receptors) are linked by the same scaffold protein, PSD95. Removal of PTPalpha 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 PTPalpha catalytic domains (D1 + D2) into neurones enhances NMDA receptor-mediated synaptic responses. Conversely, the blockade of endogenous PTPalpha inhibits NMDA receptor activity and the induction of long-term potentiation in hippocampal neurones. Thus, PTPalpha is a novel up-regulator of synaptic strength in the CNS.