Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors modulate release of inhibitory amino acids in rat spinal cord dorsal horn.

Local inhibition within the spinal cord dorsal horn is mediated by the neurotransmitters GABA and glycine and strongly influences nociceptive and temperature signaling. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are expressed by inhibitory interneurons and have been shown to modulate GABA release in other regions of the CNS. In the spinal cord, there is morphological evidence for presynaptic AMPA receptor subunits in GABAergic dorsal horn neurons, but functional data are lacking. To determine if AMPA receptors are indeed functional at presynaptic terminals of inhibitory neurons, we recorded evoked and miniature inhibitory postsynaptic currents (mIPSPs) in the superficial dorsal horn of the rat spinal cord. We show that AMPA receptor activation enhances spontaneous release of inhibitory amino acids in the presence of tetrodotoxin onto both lamina II neurons and NK1 receptor-expressing (NK1R+) lamina I neurons. This effect is sensitive to the concentration of extracellular Ca2+, yet is not fully blocked in most neurons in the presence of Cd2+, suggesting possible Ca2+ entry through AMPA receptors. Postsynaptic Ca2+ elevation is not required for these changes. AMPA-induced increases in mIPSP frequency are also seen in more mature dorsal horn neurons, indicating that these receptors may play a role in nociceptive processing in the adult. In addition, we have observed AMPA-induced depression of evoked release of GABA and glycine onto lamina I NK1R+ neurons. Taken together these data support a role for presynaptic AMPA receptors in modulating release of GABA and glycine in the superficial dorsal horn. Because inhibition in the dorsal horn is important for controlling pain signaling, presynaptic AMPA receptors acting to modulate the inhibitory inputs onto dorsal horn neurons would be expected to impact upon pain signaling in the spinal cord dorsal horn.

Functional similarities and differences of AMPA and kainate receptors expressed by cultured rat sensory neurons.

Dorsal root ganglion neurons express functional AMPA and kainate receptors near their central terminals. Activation of these receptors causes a decrease in glutamate release during action potential evoked synaptic transmission. Due to differences in kinetic properties and expression patterns of these two families of glutamate receptors in subpopulations of sensory neurons, AMPA and kainate receptors are expected to function differently. We used embryonic dorsal root ganglion (DRG) neurons maintained in culture to compare functional properties of kainate and AMPA receptors. Most DRG neurons in culture expressed kainate receptors and about half also expressed AMPA receptors. Most AMPA and kainate receptor-expressing DRG neurons were sensitive to capsaicin, suggesting involvement of these glutamate receptors in nociception. When activated by kainate, AMPA receptors were capable of driving a sustained train of action potentials while kainate receptors tended to activate action potential firing more transiently. Glutamate elicited more action potentials and a larger steady-state depolarization in neurons expressing both AMPA and kainate receptors than in neurons expressing only kainate receptors. Adding to their more potent activation properties, AMPA receptors recovered from desensitization much more quickly than kainate receptors. Activation of presynaptic receptors by low concentrations of kainate, but not ATPA, caused a tetrodotoxin-sensitive increase in the frequency of spontaneous EPSCs recorded in dorsal horn neurons. By recording synaptic pairs of DRG and dorsal horn neurons, we found that activation of presynaptic kainate and AMPA receptors decreased evoked glutamate release from terminals of DRG neurons in culture. Our data suggest that the endogenous ligand, glutamate, will cause a different physiological impact when activating these two types of non-NMDA glutamate receptors at central or peripheral nerve endings of sensory neurons.

Glutamate and GABA: a painful combination.

Regulation of release of inhibitory neurotransmitter is a key element of plasticity in dorsal horn function. In this issue of Neuron, Kerchner et al. report that neurotransmitter release from inhibitory dorsal horn neurons is affected by activation of presynaptic kainate-type glutamate receptors.

Kainate receptors expressed by a subpopulation of developing nociceptors rapidly switch from high to low Ca2+ permeability.

Dorsal root ganglion (DRG) neurons first express kainate receptor subunits, predominantly GluR5, during embryonic development. In the DRG and throughout the nervous system, substantial editing of GluR5 mRNA occurs with developmental maturation (Bernard et al., 1999). The accompanying change in Ca(2+) permeability of functional kainate receptors that is the predicted outcome of this developmental regulation of mRNA editing has not been investigated. Here we report that kainate receptors on DRG neurons from late embryonic and newborn rats are predominantly Ca(2+) permeable but then become fully Ca(2+) impermeable later in the first postnatal week. Using multiple markers for nociceptor subpopulations, we show that this switch in Ca(2+) permeability is not caused by the appearance of a new subpopulation of nociceptors with different receptor properties. Instead, the change in Ca(2+) permeability matches the time course of post-transcriptional RNA editing of GluR5 at the Q/R site within the pore of the channel, indicating that the change is probably caused by developmentally regulated RNA editing. We also report that, on the basis of the strong correlation of receptor expression with expression of the surface markers LA4, isolectin B4, and LD2, kainate receptors are present on C-fiber-type neurons projecting to lamina II of spinal cord dorsal horn. These results raise the possibility that kainate receptors in their Ca(2+)-permeable form serve a developmental role in synapse formation between this population of C-fibers and their targets in the spinal cord dorsal horn. Thereafter, the receptors may serve a new function that does not require Ca(2+) permeability.

Adenosine triphosphate-evoked currents in cultured dorsal root ganglion neurons obtained from rat embryos: desensitization kinetics and modulation of glutamate release.

Sensory neurons express purinergic P2X receptors on their central and peripheral terminals as well as their cell bodies. ATP activation of these receptors drives action potential firing and glutamate release with potentially important consequences for sensory function. Here we show ATP-gated currents activated in cultured embryonic dorsal root ganglion neurons have heterogeneity of time-courses comparable to those observed in different subpopulations of acutely dissociated adult dorsal root ganglion neurons. The distribution of time-courses across the population of cultured neurons is strongly influenced by culture conditions. Heterogeneity in ATP current kinetics occurs even though immunocytochemical staining reveals a relatively homogeneous and widespread expression of the P2X2 and P2X3 subunits. We show that the time-courses of ATP-gated currents recorded at the cell bodies are mirrored by the time-courses of transmitter release from the dorsal root ganglion nerve terminals, indicating similar P2X receptor properties on the soma and their associated terminals. Our results illustrate a functional heterogeneity of P2X receptor-mediated currents that is strongly influenced by external factors. This heterogeneity in current kinetics may have implications for neuronal function as it constrains the time-course of ATP-mediated modulation of neurotransmitter release at sensory nerve terminals.

Activation of NMDA receptors drives action potentials in superficial dorsal horn from neonatal rats.

We have investigated the role of NMDA receptors in mediating synaptic transmission in spinal cord lamina II over the first 2 weeks of postnatal development. High intensity root stimulation evoked D-APV-sensitive slow synaptic activity in lamina II neurons that drove action potential firing. This NMDA receptor-mediated activity was enhanced when bicuculline and strychnine were used to block synaptic inhibition. When activated by repetitive focal stimulation, synaptic activity mediated by NMDA receptors alone drove action potential firing. NMDA receptors were also able to drive action potential firing at synapses where AMPA receptors were present but blocked. Our data show that in lamina II of the dorsal horn, NMDA receptors significantly affect neuronal excitability even in the absence of co-activation of AMPA receptors.

Subpopulations of GABAergic and non-GABAergic rat dorsal horn neurons express Ca2+-permeable AMPA receptors.

Subpopulations of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors that are either permeable or impermeable to Ca2+ are expressed on dorsal horn neurons in culture. While both mediate synaptic transmission, the Ca2+ -permeable AMPA receptors provide a Ca2+ signal that may result in a transient change in synaptic strength [Gu, J.G., Albuquerque, C., Lee, C.J. & MacDermott, A.B. (1996) Nature, 381, 793]. To appreciate the relevance of these receptors to dorsal horn physiology, we have investigated whether they show selective expression in identified subpopulations of dorsal horn neurons. Expression of Ca2+-permeable AMPA receptors was assayed using the kainate-induced cobalt loading technique first developed by Pruss et al. [Pruss, R.M., Akeson, R.L., Racke, M.M. & Wilburn, J.L. (1991) Neuron, 7, 509]. Subpopulations of dorsal horn neurons were identified using immunocytochemistry for gamma-aminobutyric acid (GABA), glycine, substance P receptor (NK1 receptor) and the Ca2+-binding proteins, calretinin and calbindin D28K. We demonstrate that, in dorsal horn neurons in culture, kainate-induced cobalt uptake is selectively mediated by Ca2+-permeable AMPA receptors, and that a majority of GABA and NK1 receptor-expressing neurons express Ca2+-permeable AMPA receptors. GABAergic dorsal horn neurons are important in local inhibition as well as in the regulation of transmitter release from primary afferent terminals. NK1 receptor-expressing dorsal horn neurons include many of the projection neurons in the nociceptive spino-thalamic pathway. Thus, we have identified two populations of dorsal horn neurons representing important components of dorsal horn function that express Ca2+-permeable AMPA receptors. Furthermore, we show that several subpopulations of putative excitatory interneurons defined by calretinin and calbindin expression do not express Ca2+-permeable AMPA receptors.

Presynaptic ionotropic receptors and the control of transmitter release.

The quantity of neurotransmitter released into the synaptic cleft, the reliability with which it is released, and the response of the postsynaptic cell to that transmitter all contribute to the strength of a synaptic connection. The presynaptic nerve terminal is a major regulatory site for activity-dependent changes in synaptic function. Ionotropic receptors for the inhibitory amino acid GABA, expressed on the presynaptic terminals of crustacean motor axons and vertebrate sensory neurons, were the first well-defined mechanism for the heterosynaptic transmitter-mediated regulation of transmitter release. Recently, presynaptic ionotropic receptors for a large range of transmitters have been found to be widespread throughout the central and peripheral nervous systems. In this review, we first consider some general theoretical issues regarding whether and how presynaptic ionotropic receptors are important regulators of presynaptic function. We consider the criteria that should be met to identify a presynaptic ionotropic receptor and its regulatory function and review several examples of presynaptic receptors that meet at least some of those criteria. We summarize the classic studies of presynaptic inhibition mediated by GABA-gated Cl channels and then focus on presynaptic nicotinic ACh receptors and presynaptic glutamate receptors. Finally, we briefly discuss evidence for other types of presynaptic ionotropic receptors.

The distribution of neurons expressing calcium-permeable AMPA receptors in the superficial laminae of the spinal cord dorsal horn.

The superficial dorsal horn is a major site of termination of nociceptive primary afferents. Fast excitatory synaptic transmission in this region is mediated mainly by release of glutamate onto postsynaptic AMPA and NMDA receptors. NMDA receptors are known to be Ca2+-permeable and to provide synaptically localized Ca2+ signals that mediate short-term and long-term changes in synaptic strength. Less well known is a subpopulation of AMPA receptors that is Ca2+-permeable and has been shown to be synaptically localized on dorsal horn neurons in culture (Gu et al., 1996) and expressed by dorsal horn neurons in situ (Nagy et al., 1994; Engelman et al., 1997). We used kainate-induced cobalt uptake as a functional marker of neurons expressing Ca2+-permeable AMPA receptors and combined this with markers of nociceptive primary afferents in the postnatal rat dorsal horn. We have shown that cobalt-positive neurons are located in lamina I and outer lamina II, a region strongly innervated by nociceptors. These cobalt-positive neurons colocalize with afferents labeled by LD2, and with the most dorsal region of capsaicin-sensitive and IB4- and LA4-positive afferents. In contrast, inner lamina II has a sparser distribution of cobalt-positive neurons. Some lamina I neurons expressing the NK1 receptor, the receptor for substance P, are also cobalt positive. These neurons are likely to be projection neurons in the nociceptive pathway. On the basis of all of these observations, we propose that Ca2+-permeable AMPA receptors are localized to mediate transmission of nociceptive information.

Effects of the P2-purinoceptor antagonists suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid on glutamatergic synaptic transmission in rat dorsal horn neurons of the spinal cord.

The effects of suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) on glutamatergic synaptic transmission were studied on dorsal horn lamina II neurons of rat spinal cord slice preparation and cultured dorsal horn neurons. Suramin at 100 microM significantly suppressed the amplitude of the evoked excitatory postsynaptic currents (EPSCs) by 33%, miniature EPSC (mEPSC) amplitude was decreased by 46% and the mEPSC frequency also decreased by 41%. PPADS at 50 microM had little effect on either the evoked EPSCs or mEPSCs. The lack of effect of PPADS on glutamatergic synaptic transmission suggests that the effect of suramin is less likely to be mediated by P2x receptors. When whole-cell (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) currents evoked by glutamate were examined, both suramin and PPADS showed no inhibition of peak amplitude. However, the onset of glutamate-evoked whole-cell currents became significantly slowed by suramin but not by PPADS. The suppression of synaptic transmission by suramin may be due, in part, to the slowed onset of glutamate-evoked AMPA currents. These results suggest that the analgesic effects of suramin shown in cancer patients and animal pain models may not be solely due to its antagonism to purinoceptors. PPADS is probably a more suitable antagonist for the study of synaptic P2x receptor function at excitatory synapses mediated by AMPA receptors.

NMDA EPSCs at glutamatergic synapses in the spinal cord dorsal horn of the postnatal rat.

In rat dorsal horn, little is known about the properties of synaptic NMDA receptors during the first two postnatal weeks, a period of intense synaptogenesis. Using transverse spinal cord slices from postnatal day 0-15 rats, we show that 20% of glutamatergic synapses tested at low-stimulation intensity in spinal cord laminae I and II were mediated exclusively by NMDA receptors. Essentially all of the remaining glutamatergic EPSCs studied were attributable to the activation of both NMDA and AMPA receptors. Synaptic NMDA receptors at pure and mixed synapses showed similar sensitivity to membrane potential, independent of age, indicating similar Mg2+ sensitivity. Kinetic properties of NMDA EPSCs from pure and mixed synapses were measured at +50 mV. The 10-90% rise times of the pure NMDA EPSCs were slower (16 vs 10 msec), and the decay tau values were faster (tau1, 24 vs 42 msec; tau2, 267 vs 357 msec) than NMDA EPSCs at mixed synapses. Our results indicate that NMDA receptors are expressed at glutamatergic synapses at a high frequency, either alone or together with AMPA receptors, consistent with the prominent role of NMDA receptors in central sensitization (McMahon et al., 1993).

Activation of ATP P2X receptors elicits glutamate release from sensory neuron synapses.

Painful stimuli to the skin initiate action potentials in the peripheral terminals of dorsal root ganglion (DRG) neurons. These action potentials propagate to DRG central terminals in the dorsal horn of the spinal cord, evoking release of excitatory transmitters such as glutamate onto postsynaptic dorsal horn neurons. P2X receptors, a family of ligand-gated ion channels activated by the endogenous ligand ATP, are highly expressed by DRG neurons. Immunoreactivity to P2X receptors has been identified in the dorsal horn superficial laminae associated with nociceptive DRG central terminals, suggesting the presence of presynaptic P2X receptors. Here we have used a DRG-dorsal horn co-culture system to show that P2X receptors are localized at presynaptic sites on DRG neurons; that activation of these receptors results in increased frequency of spontaneous glutamate release; and that activation of P2X receptors at or near presynaptic DRG nerve terminals elicits action potentials that cause evoked glutamate release. Thus activation of P2X receptors at DRG central terminals can modify sensory signal throughput, and might even initiate sensory signals at central synapses without direct peripheral input. This putative central modulation and generation of sensory signals may be associated with physiological and pathological pain sensation, making presynaptic P2X receptors a possible target for pain therapy.

ATP P2X receptors mediate fast synaptic transmission in the dorsal horn of the rat spinal cord.

ATP has been proposed to mediate synaptic transmission in the spinal cord dorsal horn, particularly in the pathway carrying nociceptive information. Using transverse spinal cord slices from postnatal rats, we show that EPSCs mediated by P2X receptors, and presumably activated by synaptically released ATP, are evoked in a subpopulation of spinal cord lamina II neurons, a region known to receive strong input from nociceptive primary afferents. The P2X receptors on acutely dissociated dorsal horn neurons are nondesensitizing, insensitive to alphabeta methylene ATP, and show strong but variable sensitivity to the antagonists suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). These characteristics are consistent with a heterogeneous population of P2X receptors, the composition of which includes P2X2, P2X4, and P2X6 receptor subtypes. Our results suggest that ATP-activated P2X receptors in lamina II of the rat spinal cord may play a role in transmitting or modulating nociceptive information.

Ca(2+)-dependent inactivation of NMDA receptors: fast kinetics and high Ca2+ sensitivity in rat dorsal horn neurons.

1. Ca(2+)-dependent inactivation (CDI) of NMDA receptors was studied using both cultured embryonic rat dorsal horn neurons and acutely dissociated postnatal rat dorsal horn neurons. The perforated patch recording method was employed in order to preserve intracellular Ca2+ buffers and other cellular constituents. In this way, the kinetics of intracellular Ca2+ concentration ([Ca2+]i) transients and other second messenger signalling systems were maintained in a relatively normal condition. 2. Continuous application of 30 microM NMDA to cultured dorsal horn neurons voltage clamped at -70 mV evoked currents that inactivated to about 40% of the peak value with time constants between 200 and 600 ms. CDI with similar kinetics was also observed in acutely dissociated postnatal rat dorsal horn neurons. 3. When NMDA was applied in a low (20 microM) Ca2+ bath or when dorsal horn neurons were held at +70 mV, inactivation was either very weak or absent. The peaks of NMDA currents were significantly suppressed when preceded by voltage steps to 0 mV or by evoked action potentials. The suppression was dependent on the presence of Ca2+ in the extracellular solution. Voltage steps to +100 mV were ineffective in suppressing NMDA responses. Therefore, the observed inactivation was caused by an increase in [Ca2+]i following Ca2+ entry through NMDA channels or through voltage gated Ca2+ channels. 4. Caffeine application reduced currents evoked by subsequent NMDA applications. This reduction was not dependent on the presence of extracellular Ca2+ but was abolished after incubation of the cells with ryanodine, suggesting that Ca2+ release from intracellular stores also induced CDI. 5. Simultaneous measurements of somal [Ca2+]i and of currents evoked by somal NMDA applications showed that the magnitude of CDI was correlated with [Ca2+]i levels and that [Ca2+]i elevations of 100-300 nM were usually sufficient to inactivate NMDA currents by more than 30%. 6. Dose-response curves of non inactivated and inactivated NMDA responses showed that the apparent receptor affinity for NMDA is not different under the two conditions. CDI is caused instead by non-competitive inhibition of NMDA receptors. CDI was not overcome by increasing glycine concentration, suggesting that it is not mediated by glycine dissociation from the receptor. 7. These results show that, with an intact intracellular environment, CDI in dorsal horn neurons constitutes a potent, inhibitory control of NMDA currents with a faster onset than previously demonstrated. CDI is induced by a variety of [Ca2+]i-elevating stimuli of physiological relevance including Ca2+ entry through ligand- and voltage-gated channels and Ca2+ release from intracellular stores. Our demonstration that CDI is strongly expressed in neurons maturing in vivo supports the hypothesis that CDI may regulate, in part, the postsynaptic integration of excitatory input in the mature or maturing nervous system.

Synaptic strengthening through activation of Ca2+-permeable AMPA receptors.

Postsynaptic Ca2+ elevation during synaptic transmission is an important trigger for short- and long-term changes in synaptic strength in the vertebrate central nervous system. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate) receptors, a subfamily of glutamate receptors, mediate much of the excitatory synaptic transmission in the brain and spinal cord. It has been shown that a subtype of the AMPA receptor is Ca2+-permeable and is present in the subpopulations of neurons. When synaptically localized, these receptors should mediate postsynaptic Ca2+ influx, providing a trigger for changes in synaptic strength. Here we show that Ca2+-permeable AMPA receptors are synaptically localized on a subpopulation of dorsal horn neurons, and that they provide a synaptically gated route of Ca2+ entry, and that activation of these receptors strengthens synaptic transmission mediated by AMPA receptors. This pathway for postsynaptic Ca2+ influx may provide a new form of activity-dependent modulation of synaptic strength.

NMDA receptor-mediated calcium entry in the absence of AMPA receptor activation in rat dorsal horn neurons.

This study tested quantitatively the degree to which selective activation of NMDA receptors increases the intracellular concentration of free Ca2+ ions ([Ca2+]i) in neurons isolated from the rat spinal cord dorsal horn and grown in culture. The results show that activation of NMDA receptors, without additional depolarizing stimuli, produce [Ca2+]i increases in the neuronal cell bodies in the presence of physiological concentrations of Mg2+. Furthermore, this phenomenon occurs during synaptic activation of NMDA receptors. Thus, it cannot be assumed that in physiological extracellular [Mg2+], there is an absolute requirement for additional membrane depolarization before NMDA receptor activation stimulates significant increases in [Ca2+]i.

Variable distributions of Ca(2+)-permeable and Ca(2+)-impermeable AMPA receptors on embryonic rat dorsal horn neurons.

1. By measuring the apparent reversal potential (aErev) of kainate- and alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA)-evoked currents associated with changes in extracellular Ca2+ concentration ([Ca2+]e), we have been able to identify embryonic dorsal horn neurons grown in tissue culture that express Ca(2+)-permeable non-N-methyl-D-aspartic acid (NMDA) receptors. 2. The relative expression of Ca(2+)-permeable and Ca(2+)-impermeable non-NMDA receptors varies from cell to cell. This was evident from the range of a ErevS observed for kainate-evoked currents in a 0 mM [Na+]e, 10 mM [Ca2+]e bath. Under these conditions, aErev ranged from -96 to -21 mV, suggesting that the percentage of the non-NMDA receptors on each neuron that are Ca(2+)-permeable is variable. 3. To determine the extent to which the variability in aErev is due to variable receptor expression rather than experimental variability, we compared the effects of changes in [Ca2+]e on kainate-evoked currents and NMDA-evoked currents on the same cells. Assuming that all of the NMDA receptors on each neuron have a similar Ca2+ permeability, this approach provides an index of the sensitivity of our assay system. The reversal potential of NMDA-evoked currents in 10 mM [Ca2+]e ranged from -30 to -7 mV, whereas on the same population of neurons, the aErev of kainate-evoked currents ranged from -92 to -40 mV. 4. The rectification properties of the non-NMDA currents were generally linear or outwardly rectifying in normal bath solution. When the PCa/PCs ratio in 0 mM [Na+]e, 10 mM [Ca2+]e bath solution was assessed as a function of the rectification index in standard bath, a poor correlation was found between Ca2+ permeability and the rectification index. 5. The aErev of kainate-evoked currents was similar to that of cyclothiazide-enhanced AMPA-evoked currents observed on the same cells (-66.5 +/- 18.4 and -64.0 +/- 13.9 mV, mean +/- SD, respectively). This suggests that kainate is primarily activating the AMPA receptor and that the majority of non-NMDA receptors on embryonic dorsal horn neurons in culture are high-affinity AMPA receptors. 6. Immunocytochemical evidence suggests that the AMPA receptor subunits GluR1-4 are expressed to a variable degree from cell to cell in our cultures. We found evidence for low levels of expression of the kainate receptor subunits GluR5-7. The immunocytochemical observations support the physiological data indicating that much of the kainate-evoked current recorded in our experiments can be accounted for by kainate activation of AMPA receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium entry through a subpopulation of AMPA receptors desensitized neighbouring NMDA receptors in rat dorsal horn neurons.

1. A Ca(2+)-dependent interaction between non-NMDA and NMDA receptors was studied in embryonic rat dorsal horn neurons grown in tissue culture using perforated-patch recording. Specifically, non-NMDA receptors were found to induce reversible inhibition of NMDA receptors in a manner dependent on the presence of extracellular Ca2+. 2. Non-NMDA receptor-induced inhibition of NMDA receptors was mediated by the elevation of intracellular Ca2+ concentration produced by Ca2+ entry through a subpopulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) non-NMDA receptors. Furthermore, Ca2+ entry through the AMPA channels alone is sufficient for desensitization of NMDA channels to occur. 3. Imaging of neuritic sites of Ca2+ revealed that Ca(2+)-permeable AMPA channels are often co-localized with NMDA channels on dorsal horn neurons, indicating that the Ca(2+)-mediated interaction between receptors may occur within small dendritic domains. 4. The ability of Ca(2+)-permeable AMPA channels to inhibit adjacent NMDA channels may contribute to the postsynaptic integration of excitatory input.

Substance P elevates intracellular calcium in both neurons and glial cells from the dorsal horn of the spinal cord.

1. We used microfluorimetric measurement of [Ca2+]i to identify substance P-sensitive cells acutely isolated from the dorsal horn of neonatal rats. We then used morphological, physiological, and immunocytochemical criteria to delineate two distinct populations of substance P-sensitive dorsal horn cells. 2. One population of cells with small-diameter cell bodies and many fine processes responds to substance P by releasing Ca2+ from internal stores. Many of these cells express the O4 surface antigen, and are thus likely to be glial cells, probably from the oligodendrocyte lineage. None of the cells with glial attributes respond to N-methyl-D-aspartate (NMDA), providing further evidence that they are nonneuronal. 3. In a second population of dorsal horn cells, substance P elevates [Ca2+]i by promoting Ca2+ entry. This class of cells is morphologically distinct from substance P-sensitive glial cells in that it exhibits large-diameter cell bodies, has smooth tapering processes, and is sensitive to NMDA. This second class of cells is therefore likely to consist of neurons. 4. Consistent with the identification of different mechanisms of Ca2+ elevation in the two cell types, the kinetics of the substance P-evoked release of Ca2+ in glial cells is very different than the kinetics of the Ca(2+)-entry response evoked in neurons. The glial cell response had a rapid average rate of rise (mean = 260 +/- 105 nM/s) and relatively brief duration (mean = 7.6 +/- 2.2 s) whereas the neuronal response had a much slower rate of rise (mean = 10 +/- 9 nM/s) with a much longer duration.(ABSTRACT TRUNCATED AT 250 WORDS)