Role of the spinal cord NR2B-containing NMDA receptors in the development of neuropathic pain

Activation of N-methyl-d-aspartate (NMDA) receptors in the spinal dorsal horn has been shown to be essential for the initiation of central sensitization and the hyperexcitability of dorsal horn neurons in chronic pain. However, whether the spinal NR2B-containing NMDA (NMDA-2B) receptors are involved still remains largely unclear. Using behavioral test and in vivo extracellular electrophysiological recording in L5 spinal nerve-ligated (SNL) neuropathic rats, we investigate the roles of spinal cord NMDA-2B receptors in the development of neuropathic pain. Our study showed that intrathecal (i.t.) injection of Ro 25-6981, a selective NMDA-2B receptor antagonist, had a dose-dependent anti-allodynic effect without causing motor dysfunction. Furthermore, i.t. application of another NMDA-2B receptor antagonist ifenprodil prior to SNL also significantly inhibited the mechanical allodynia but not the thermal hyperalgesia. These data suggest that NMDA-2B receptors at the spinal cord level play an important role in the development of neuropathic pain, especially at the early stage following nerve injury. In addition, spinal administration of Ro 25-6981 not only had a dose-dependent inhibitory effect on the C-fiber responses of dorsal horn wide dynamic range (WDR) neurons in both normal and SNL rats, but also significantly inhibited the long-term potentiation (LTP) in the C-fiber responses of WDR neurons induced by high-frequency stimulation (HFS) applied to the sciatic nerve. These results indicate that activation of the dorsal horn NMDA-2B receptors may be crucial for the spinal nociceptive synaptic transmission and for the development of long-lasting spinal hyperexcitability following nerve injury. In conclusion, the spinal cord NMDA-2B receptors play a role in the development of central sensitization and neuropathic pain via the induction of LTP in dorsal horn nociceptive synaptic transmission. Therefore, the spinal cord NMDA-2B receptor is likely to be a target for clinical pain therapy.     

NMDA receptor 2B subunit-mediated synaptic transmission in the superficial dorsal horn of peripheral nerve-injured neuropathic mice

Previous research has shown that peripheral inflammation and peripheral nerve injury alter the properties of NMDA receptors in the spinal dorsal horn. However, there is no direct evidence that demonstrates the influence of peripheral nerve injury on NMDA receptor-mediated synaptic transmission in the spinal dorsal horn. Using whole cell tight-seal methods, NMDA receptor-mediated excitatory postsynaptic currents (NMDA EPSCs) were recorded from superficial dorsal horn neurons in adult mouse spinal cord slices. Peripheral nerve injury-induced changes in the pharmacological and electrophysiological properties of synaptic NMDA receptors were studied. The ratio of the amplitude of NMDA EPSCs to that of non-NMDA EPSCs was larger in nerve-ligated neuropathic mice than in sham-operated control mice. The decay phase of the NMDA EPSCs was slower in nerve-ligated neuropathic mice. The NR2B subunit-specific NMDA receptor antagonist ifenprodil (10 microM) reduced the amplitude of the NMDA EPSCs and shortened their decay phase. The sensitivity of NMDA EPSCs to ifenprodil was significantly larger in nerve-ligated neuropathic mice than in sham-operated control mice. Single-cell RT-PCR analysis performed on superficial dorsal horn neurons showed that the incidence of NR2A mRNA-expressing neurons was reduced in nerve-ligated neuropathic mice. This result, together with the electrophysiological findings, suggests that the subunit composition of the subsynaptic NMDA receptors in the superficial dorsal horn was altered by peripheral nerve injury. Pharmacological and electrophysiological changes observed in the present experiments might be the underlying causes of the hyperalgesia and allodynia induced by peripheral nerve injury and inflammation.     

L-type calcium channels and NMDA receptors: a determinant duo for short-term nociceptive plasticity

In the dorsal horn of the spinal cord, pain-transmitting neurons exhibit action potential windup, a form of short-term plasticity, which consists of a progressive increase in neuronal response during repetitive stimulation of nociceptive input fibers. Windup depends on N-methyl-D-aspartate (NMDA) receptor activation, but previous in vitro studies indicated that windup also relies on intrinsic plateau properties of spinal neurons. In the present study, we considered the possible involvement of these properties in windup in vivo. For this purpose, we first studied a nociceptive flexion reflex in the rat. We showed that windup of the reflex is actually suppressed by blockers of L-type calcium current and Ca(2+)-activated non-specific cationic current (Ican), the two main depolarizing conductances of plateau potentials. We further showed that, during windup, NMDA receptors provide a critical excitatory component in a dynamic balance of excitatory and inhibitory inputs which ultimately activates L-type calcium channels. The nociceptive reflex involves at least two neuronal groups, which may express intrinsic amplification properties, motor neurons and dorsal horn neurons. By means of extracellular recordings in the dorsal horn, we showed that windup of dorsal horn neuron discharge was sensitive to the modulators of L-type calcium current. Altogether, our results suggest that, in vivo, windup also depends on the amplification properties of spinal neurons, the triggering of which requires previous activation of NMDA receptors.     

Chronic stress modulation of prefrontal cortical NMDA receptor expression disrupts limbic structure-prefrontal cortex interaction

Chronic stress causes various detrimental effects including cognitive and affective dysfunctions. Given the recent findings emphasizing the importance of information processing between the prefrontal cortex (PFC) and limbic structures on cognitive and affective functions, impairments of these functions caused by chronic stress may be associated with stress-induced adaptive and maladaptive responses in limbic structure-PFC interaction. In this study we have shown that chronic stress disrupts limbic structure-PFC interaction by modulating N-methyl-D-aspartate (NMDA) receptor expression in the PFC. We found that chronic stress decreased expression of NR1, NR2A and NR2B subunits of NMDA receptors in the PFC but not in the motor cortex. However, the reduction in NR2B subunits of NMDA receptors was larger in the dorsal part than the ventral part of PFC. In agreement with this observation, administration of the NMDA antagonist that was more selective for NMDA receptors containing NR2B subunits induced alterations of synchronous local field potentials between the PFC and limbic structures, synaptic plasticity induction in the limbic structure-PFC pathway, and spike firing of PFC neurons that were similar to those observed in the dorsal PFC of rats exposed to chronic stress. In contrast, administration of the NMDA antagonist that was not subunit-selective resulted in electrophysiological alterations resembling to those observed in the ventral PFC of rats exposed to chronic stress. These results suggest that chronic stress disrupts NMDA receptor-dependent limbic structure-PFC information processing.     

Electrophysiological evidence for a presynaptic mechanism of morphine withdrawal in the neonatal rat spinal cord

Dorsal and ventral root depolarizing responses to capsaicin (1 microM) and substance P (SP; 1 microM) were measured from the isolated, hemisected spinal cord of the neonatal rat. Capsaicin depolarized the dorsal and ventral roots. The mechanism of ventral root depolarization was presynaptic; since dorsal root depolarization preceded the ventral, and the ventral depolarization was eliminated when synaptic transmission was blocked in the absence of calcium. SP depolarized the ventral root without affecting the dorsal root. The SP-induced depolarization of the ventral root was reduced but not abolished by blocking synaptic transmission with low calcium, suggesting that SP acted postsynaptically on motoneurons and excitatory interneurons to depolarize the ventral root. Morphine (10 microM) abolished the capsaicin-induced ventral root depolarization, but only slightly suppressed the SP response (30%). The capsaicin-induced depolarization of the ventral root was enhanced greatly (238%) when morphine, which had been in the superfusion for 1 h, was removed and naloxone (1 microM) was added to the superfusion solution, whereas the SP response was not augmented during withdrawal from acute morphine. Furthermore, a putative SP antagonist ([D-Arg1, D-Pro2, D-Tryp7,9, Leu11]-SP) prevented the augmented capsaicin ventral root response during precipitated withdrawal. These data provide electrophysiological evidence for a presynaptic mechanism of acute morphine withdrawal in the neonatal rat spinal cord.     

Spinal entry route for ventral root afferent fibers in the cat

Twelve anesthetized and paralyzed cats were used to study the spinal entry routes of ventral root afferent fibers. In all animals, the spinal cord was transected at two different levels, L5 and S2. The L5 through S2 dorsal roots were cut bilaterally, making spinal cord segments L5-S2 neurally isolated from the body except for the L5-S2 ventral roots. From this preparation, a powerful excitation of the discharge rate of motor neurons and dorsal horn cells within the isolated spinal segments was observed after intraarterial injection of bradykinin (50 micrograms in 0.5 ml saline). This excitation of the spinal neurons can be considered the most convincing evidence of the potential physiologic role of the ventral root afferent fibers entering the spinal cord directly through the ventral root, because the apparent route of neuronal input from the periphery is through the ventral roots. However, additional control experiments conducted in the present study showed that the excitation persisted even after cutting all ventral roots within the isolated spinal segments, indicating that excitation was not mediated by the ventral roots. Furthermore, direct application of bradykinin on the dorsal surface of the spinal cord also increased the motoneuronal discharge rate, suggesting that excitation of spinal neurons produced by intraarterial injection of bradykinin is due to a direct action of bradykinin on the spinal cord. Thus, we provided an alternate explanation for the most convincing evidence indicating that physiologically important ventral root afferent fibers enter the spinal cord directly through the ventral root. Based on existing experimental evidence, it is likely that the majority of physiologically active ventral root afferent fibers travel distally toward the dorsal root ganglion and then enter the spinal cord through the dorsal root.     

Plateau potential-dependent windup of the response to primary afferent stimuli in rat dorsal horn neurons

In the spinal cord, repetitive stimulation of nociceptive afferent fibres induces a progressive build-up of dorsal horn neuron (DHN) responses. This 'action potential windup' is used as a cellular model of central sensitization to pain. It partly relies on synaptic plasticity, being reduced after blocking NMDA and neurokinin receptors. Using intracellular recordings in a slice preparation of the rat spinal cord, we have analysed the implication of an additional non-synaptic component of windup. Primary afferent fibres were electrically stimulated in the dorsal root. Of 47 responding deep DHNs, 17 (36%) produced action potential windup and afterdischarge during consecutive periods of repeated stimuli (0.4-1 Hz) activating high- (n = 13 neurons) and low-threshold (n = 6 neurons) afferent fibres. When the NMDA receptors were blocked, the rate of windup did not change. In all neurons, there was an absolute correlation between expression of windup and the production of calcium-dependent plateau potentials. Sensitization of the DHN response, similar to the synaptically induced windup, was obtained by repetitive intracellular injection of depolarizing current pulses. This intracellularly induced windup had the same pharmacology as the plateau potential. Synaptically induced windup was also abolished by nifedipine, an L-type calcium-channel blocker. Expression of plateau properties in DHNs is therefore a critical component of windup, operating downstream of synaptic processes. Being associated with calcium influx, generation of plateau potentials could be a link between short-term plasticity and the long-term modification of DHN excitability associated with central sensitization.     

Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain

Despite abundant evidence implicating the importance of N-methyl-D-aspartate (NMDA) receptors in the spinal cord for pain transmission, the signal transduction coupled to NMDA receptor activation is largely unknown for the neuropathic pain state that lasts over periods of weeks. To address this, we prepared mice with neuropathic pain by transection of spinal nerve L5. Wild-type, NR2A-deficient, and NR2D-deficient mice developed neuropathic pain; in addition, phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 was observed in the superficial dorsal horn of the spinal cord 1 week after nerve injury. Neuropathic pain and NR2B phosphorylation at Tyr1472 were attenuated by the NR2B-selective antagonist CP-101,606 and disappeared in mice lacking Fyn kinase, a Src-family tyrosine kinase. Concomitant with the NR2B phosphorylation, an increase in neuronal nitric oxide synthase activity was visualized in the superficial dorsal horn of neuropathic pain mice by NADPH diaphorase histochemistry. Electron microscopy showed that the phosphorylated NR2B was localized at the postsynaptic density in the spinal cord of mice with neuropathic pain. Indomethacin, an inhibitor of prostaglandin (PG) synthesis, and PGE receptor subtype EP1-selective antagonist reduced the NR2B phosphorylation in these mice. Conversely, EP1-selective agonist stimulated Fyn kinase-dependent nitric oxide formation in the spinal cord. The present study demonstrates that Tyr1472 phosphorylation of NR2B subunits by Fyn kinase may have dual roles in the retention of NMDA receptors in the postsynaptic density and in activation of nitric oxide synthase, and suggests that PGE2 is involved in the maintenance of neuropathic pain via the EP1 subtype.     

Participation of AMPA- and NMDA-type excitatory amino acid receptors in the spinal reflex transmission,in rat

Classical in vitro and in vivo models and electrophysiological techniques were used to investigate the role of AMPA- and NMDA-type glutamate receptors in various components of spinal segmental reflex potentials. In the rat hemisected spinal cord preparation, the AMPA antagonists NBQX and GYKI 52466 abolished the monosynaptic reflex (MSR) potential but caused only partial inhibition of the motoneuronal population EPSP. NMDA antagonists had no noticeable effect on the MSR in normal medium, but markedly depressed the late part of EPSP. However, an NMDA receptor antagonist sensitive monosynaptic response was recorded in magnesium-free medium at complete blockade of the AMPA receptors. In spinalized rats, the AMPA antagonists completely blocked all components of the dorsal root stimulation evoked potential. MK-801 (2mg/kg, i.v.) reduced monosynaptic responses in a frequency dependent way, with no effect at 0.03 Hz and 22% inhibition at 0.25 Hz. The reduction of the di- and polysynaptic reflex components was about 30% and did not depend on stimulation frequency. Long-latency reflex discharge responses, especially when evoked by train stimulation, were more sensitive to MK-801 than the polysynaptic reflex.These results suggest that glutamate activates MSR pathways through AMPA receptors. However, under certain conditions, NMDA receptors can modulate this transmission through plastic changes in the underlying neuronal circuits. AMPA and NMDA receptors play comparable roles in the mediation of longer latency reflex components.     

Effects of a tachykinin NK3 receptor antagonist, SR 142801, studied in isolated neonatal rat spinal cord

Effects of a nonpeptide tachykinin NK₃ receptor antagonist, SR 142801, were studied in the isolated spinal cord preparation of the neonatal rat. Potential changes were recorded extracellularly from a lumbar ventral root. Bath-application of neurokinin B induced a dose-dependent depolarization of the ventral root. SR 142801 caused rightward shifts of the concentration-response curve for neurokinin B with pA₂ of 6.57, but did not affect the depolarizing responses to other agonists. Stimulation of a dorsal root evoked in the ipsilateral ventral root of the same segment monosynaptic and polysynaptic reflexes of fast time course which were followed by a slow depolarization (ipsilateral slow ventral root potential). SR 142801 depressed the ipsilateral slow ventral root potential. The present results indicate that SR 142801 is a specific antagonist for tachykinin NK₃ receptors in the spinal cord and suggest that NK₃ receptors are involved in primary afferent-evoked nociceptive responses of spinal neurones.     

Induction of the immediate early gene c-jun in human spinal cord in amyotrophic lateral sclerosis with concomitant loss of NMDA receptor NR-1 and glycine transporter mRNA

The aetiology of the sporadic form of amyotrophic lateral sclerosis (ALS) is poorly understood although abnormalities in glutamate and glycine transport have been implicated which both could contribute to a neurodegenerative process mediated through the N-methyl-d-aspartate (NMDA) receptor. In this study we have used in situ hybridization to investigate whether any changes in the expression of NMDA receptors, the glycine transporter or glutamate-mediated injury responses are detectable in ALS. Two immediate early genes were investigated as markers of neuronal injury responses, c-jun and zif-268, both constitutively expressed in the spinal cord. Levels of c-jun mRNA were most abundant in intermediate grey and layer IX of the ventral horn containing motor neurones. This pattern was markedly changed in ALS with large increases (2–3 fold) in c-jun mRNA occurring in dorsal and ventral horn. The marked increase in c-jun mRNA was also substantiated by slot blot analysis of tissue homogenates of spinal cord and a parallel induction of zif-268 mRNA was also seen. NMDA receptor NR-1 mRNA was widely distributed in control spinal cord with the highest concentrations occurring in layers IX, X, intermediate grey and dorsal horn. The ALS cases showed a selective decrease in the level of NR-1 mRNA in the ventral region (50%) whilst no significant decrease was detected in the dorsal region. Quantitation of tissue homogenates with dorsal and ventral regions combined also yielded a significant decrease of 40% which supports the analysis from in situ hybridization densitometry. The distribution of the glycine transporter was characterised with an oligonucleotide probe and showed a specific localisation to motor neurones of the ventral horn, layer II of the dorsal horn and low levels throughout grey matter. In cases of ALS, substantial and selective loss (75%) of the glycine transporter occurred in ventral grey matter (P < 0.001) with little change occurring in dorsal grey matter. This deficit was also s was also substantiated by slot blot analysis of tissue homogenates where a decrease of 59% was obtained (P < 0.005). The induction of c-jun and zif-268 detected in ALS spinal cord in this study indicates the presence of a potential cellular response to neuronal ‘stress’ that may play an important role in subsequent neurodegenerative or reparative mechanisms.     

Upregulation of mRNAs coding for AMPA and NMDA receptor subunits and metabotropic glutamate receptors in the dorsal horn of the spinal cord in a rat model of diabetes mellitus

Recent studies suggest that glutamate plays a pivotal role in the processing of sensory information in the spinal cords of patients with diabetic neuropathy. However, the specific glutamate receptors that that are involved have yet to be determined. We therefore conducted a study to characterize the expression of messenger RNAs (mRNAs) coding for subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors and N-methyl-d-aspartate (NMDA) receptors and for metabotropic glutamate receptors (mGluRs) in the dorsal horn of the lumbar segment of the spinal cord in a rat model (streptozotocin [STZ]-induced) of diabetic neuropathy. The levels of mRNAs coding for AMPA receptor subunits, GluR1, GluR2, and GluR3, were significantly increased in all layers (laminae I-V) of the dorsal horn in diabetic (STZ-injected) rats compared to control (vehicle-injected) rats. The hybridization signals for NR2A mRNA and NR2B mRNA were significantly elevated in the deep layer of the dorsal horn of diabetic rats. In diabetic (STZ-induced) rats, the levels of expression of mGluR1 mRNA and mGluR5 mRNA were significantly increased in all layers of the dorsal horn. These results suggest that abnormal expression of multiple glutamate receptors is involved in the development of diabetic neuropathy and that glutamate receptors are promising targets in the treatment of this disorder.     

Effect of serotonin on the background activity of neurons in the isolated spinal cord semisegment of the rat

The background activity of neurons in the isolated lumbar semisegment of 14-22 days old white rats was investigated before and after bath application of serotonin (5-HT, 1.10(-8)-1.10(-4) mol/l). 50% investigated neurons in dorsal horn and 70.6% interneurons in the ventral horn reacted on 5-HT. Excitation reactions prevailed. In the dorsal horn 5-HT evoked activation in 62.4% of responding interneurons, depression--in 8.4% and mixed reaction--in 29.2%. In the ventral horn 91.6% of all responses were activation reactions and only 8.4%--biphase ones. 5-HT induced discharges of motoneurons in the ventral root and ventral root depolarization. The data obtained have shown mainly the activation effect of 5-HT on the background activity of spinal neurons.     

Substance P release in the dorsal horn assessed by receptor internalization: NMDA receptors counteract a tonic inhibition by GABA(B) receptors

Inhibitory amino acids have antinociceptive actions in the spinal cord that may involve inhibition of neurotransmitter release from primary afferents. Rat spinal cord slices with dorsal roots were used to study the effect of GABA and glycine on substance P release, assessed by the internalization of neurokinin 1 receptors. After electrical stimulation of the dorsal root at 100 Hz, about half of neurokinin 1 receptor-immunoreactive neurons in laminae I-IIo showed internalization. This internalization was inhibited by GABA (100 microM) and the GABA(B) agonist R-baclofen (10 microM), but not by the GABA(A) agonist muscimol (20 microM) or glycine (100 microM). The GABA(B) antagonist 2-hydroxysaclofen (100 microM) reversed the inhibitory effect of GABA, but not the GABA(A) antagonist bicuculline (100 microM). These findings demonstrate that GABA(B) receptors, but not GABA(A) or glycine receptors, inhibit substance P release induced by dorsal root stimulation. In contrast, R-baclofen did not inhibit the internalization produced by NMDA (100 microM), indicating that the stimulatory effect of NMDA receptors on substance P release is able to surmount the inhibitory effect of GABA(B) receptors. In the presence of the GABA(B) antagonist 2-hydroxysaclofen (100 microM), but not in its absence, stimulation of the dorsal root at 1 or 10 Hz was able to elicit internalization, which was not inhibited by the NMDA receptor antagonist AP-5 (50 microM) or the channel blocker MK-801 (10 microM). Therefore, inhibition of substance P release by GABA(B) receptors is tonic, and in its absence SP release no longer requires NMDA receptor activation.     

Spinal ventral root after-discharges as a pain index: involvement of NK-1 and NMDA receptors

Nociceptive signals are transmitted to the spinal dorsal horn via primary afferent fibers, and the signals induce withdrawal reflexes by activating spinal motoneurons in the ventral horn. Therefore, nociceptive stimuli increase motoneuronal firing and ventral root discharges. This study was aimed to develop a method for the study of pain mechanisms and analgesics by recording ventral root discharges. Spinalized rats were laminectomized in the lumbo-sacral region. The fifth lumbar ventral root was sectioned and placed on a pair of wire electrodes. Multi unit efferent discharges from the ventral root were increased by mechanical stimulation using a von Frey hair applied to the plantar surface of the hindpaw. The low-intensity mechanical stimuli increased the discharges during stimulation (during-discharges) without increasing the discharges after cessation of stimulation (after-discharges), and the high-intensity mechanical stimuli increased both during- and after-discharges. Pretreatment with resiniferatoxin, an ultrapotent analogue of capsaicin, halved during-discharges and eliminated after-discharges, suggesting that after-discharges are generated by heat- and mechanosensitive polymodal nociceptors. Ezlopitant, a neurokinin-1 (NK-1) receptor antagonist, but not its inactive enantiomer, selectively reduced the after-discharges. Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, preferentially reduced the after-discharges, demonstrating that NK-1 and NMDA receptors mediate the after-discharges. Morphine reduced the after-discharges without affecting during-discharges. By contrast, mephenesin, a centrally acting muscle relaxant, reduced both during- and after-discharges. There results suggest that simultaneous recordings of during- and after-discharges are useful to study pain mechanisms and analgesics as well as to discriminate the analgesic effects from the side effects such as muscle relaxant effects.     

Effects of M-current modulators on the excitability of immature rat spinal sensory and motor neurones

M-currents have been shown to control neuronal excitability in a variety of central and peripheral neurones. Here we studied the effects of specific M-current modulators on the excitability of spinal neurones and their response to synaptic activation. Experiments were performed in vitro using the hemisected spinal cord from 7- to 11-day-old rats. Intracellular recordings were obtained from lumbar deep dorsal horn and motor neurones. Neuronal excitability was assessed by applying outward current pulses and synaptic responses were elicited by activation of a lumbar dorsal root. The M-current antagonist 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991) and the agonist retigabine were superfused at 10 microM. Retigabine produced hyperpolarization and a large decrease in the excitability of motor (7/7) and dorsal horn neurones (11/12). The effects of retigabine were fully reversed by XE-991. XE-991 induced depolarization of most neurones tested and a large increase in the excitability of motor neurones (7/7) but only a weak increase in the excitability of a proportion of dorsal horn neurones (4/10). The effects of XE-991 were partly reversed by retigabine. Consistent with their effects on neuronal excitability, retigabine showed a general depressant effect on synaptic transmission, whereas XE-991 showed the opposite tendency to potentiate responses to dorsal root stimulation, particularly in motor neurones. The results show that retigabine can depress spinal excitability and the transmission of nociceptive information. Results also indicate a post-synaptic expression of functional M-currents in most motor neurones and a considerable proportion of deep dorsal horn neurones.     

Segmental effect of NMDA block in the dorsal horn on the pressor reflex

The purpose of this study was to examine the influence of NMDA receptor blockade in the dorsal horn of adjacent spinal segments as it pertains to the pressor reflex evoked by static contraction and stretch of skeletal muscle. In this preparation, cats were anesthetized and the afferent fibers mediating the pressor reflex entered the spinal cord via the L₇ dorsal root. Blockade of dorsal horn NMDA receptors at L₆ and L₇ attenuated the pressor reflex evoked by static contraction and muscle stretch. However, NMDA block in the L₆ dorsal horn alone failed to alter the peak increase in MAP produced by static contraction and muscle stretch, but the initial pressor response evoked by static contraction was attenuated. These data support the hypothesis that the pressor reflex is partially mediated by activation of NMDA receptors in the dorsal horn, and this occurs at multiple spinal segments. Further, these data suggest that activation of NMDA receptors plays an important role in initiating the rise in arterial pressure produced by static contraction of skeletal muscle.     

Ionotropic glutamate receptor expression in human spinal cord during first trimester development

Quantitative receptor autoradiography and immunoblotting were used to study the expression and distribution of AMPA, kainate and NMDA receptors in first trimester human spinal cord obtained from elective abortions ranging from 4 to 11.5 weeks of gestational age. Spinal cord tissue sections were processed for receptor autoradiography with the ligands [3H]AMPA, [3H]kainate and [3H]MK-801 and the optical density was measured separately in a dorsal region (alar plate) and ventral region (basal plate) of the autoradiographs. Binding sites for all three ligands were demonstrated already at 4-5.5 weeks of gestation and increased continuously during the first trimester both in the dorsal and ventral regions. [3H]AMPA binding to both high- and low-affinity sites increased from undetectable levels to about 35 and 400 fmol/mg tissue, respectively, during this period. A temporal difference in the distribution of [3H]AMPA binding sites was observed. The early homogeneous pattern of [3H]AMPA binding in both alar and basal plates had changed to a heterogeneous pattern at 11 weeks of gestation with the highest density of [3H]AMPA binding sites in the superficial layers of the immature dorsal horn. [3H]kainate and [3H]MK-801 binding sites were densely and homogeneously distributed already at 4 weeks, and steadily increased six- and two-fold, respectively, to about 100 fmol/mg tissue at 11.5 weeks of gestation. Immunoreactive bands corresponding to the NMDA receptor subunits NR1, NR2A, NR2B, NR2C and NR2D were demonstrated by immunoblotting at the earliest between 4.5 and 7 weeks and increasing concentrations were seen up to 11 weeks of gestation. These results suggest that AMPA, kainate and NMDA receptors are expressed in the human spinal cord early in embryogenesis.     

Substance P receptors in the human spinal cord: decrease in amyotrophic lateral sclerosis

The distribution of substance P receptors was examined by autoradiography at all levels of the human postmortem spinal cord using the ligand [125I]Bolton-Hunter substance P. Adjacent sections were used to localize substance P-like immunoreactivity by a radioimmunohistochemical technique. In the control spinal cord substance P-like immunoreactivity was found to be highly concentrated in the superficial layers of the dorsal horn, intermediolateral cell columns and lamina X, while lower levels of immunoreactivity were observed in other areas of the grey matter of the spinal cord. In contrast, high densities of substance P binding sites were localized not only to the substantia gelatinosa of the dorsal horn but also to other regions of the grey matter of the spinal cord, particularly in the area of the preganglionic sympathetic neurons in the intermediolateral cell column and in the region of the somatic motor neurons of the ventral horn. In 5 cases of amyotrophic lateral sclerosis we found a marked reduction of substance P binding, especially in the ventral horn associated with the loss of motor neurons. These results suggest a postsynaptic localization of substance P receptors to the motor neurons of the ventral horn in the human spinal cord and a role for substance P in the function of motor neurons.     

Spinal terminal fields of dorsal root fibers in the pigeon (Columba livia).

This paper describes the spinal pattern of brachial and lumbosacral dorsal root terminations in the pigeon. These are presented within the framework of the cytoarchitectonic analysis of the preceding report (Leonard and Cohen, '75). At the level of dorsal root section and immediately adjacent sections dense terminal fields were evident throughout the ipsilateral dorsal horn (Layers I-V), including the substantia gelatinosa. However, degeneration in the substantia gelatinosa was prominent only with short survival times. There were mediolateral differences in the density of degenerating material in the dorsal horn, as well as variations between the patterns at the cervical and lumbosacral enlargements. The general pattern of degeneration in the dorsal horn extended approximately three segments rostral and two segments caudal to the level of root section, becoming progressively less dense with distance from the level of rhizotomy. An exception to this was degeneration in the dorsal magnocellular (Clarke's) column where, following section of either brachial or lumbosacral roots, degeneration could be traced into the thoracic spinal cord. However, these ascending and descending projections upon column at thoracic levels overlap minimally if at all. Degeneration was sparse in the intermediate zone and ventral horn (Layers VI-IX). It tended to concentrate centrally in Layer VI at the enlargements, and much of the degeneration ventral to this was interpreted as fibers en passage to the motoneuronal cell groups, Layer IX. Terminal fields in Layer IX were evident at both cervical and lumbosacral enlargements. They were largely restricted to the lateral motoneuronal cell group and were more prominent at cervical levels where degeneration was commonly observed around cell bodies and their proximal dendrites. As with the cytoarchitectonic organization of the spinal gray in the pigeon, the terminal fields of brachial and lumbosacral dorsal root fibers appear to have a pattern similar to that reported in mammalian literature.