Biomedical subjects
Publications and source records attributed to A B MacDermott.
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.
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.
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.
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.
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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.
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.
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 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.
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).
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 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.
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.
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.
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.
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.
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)
More than 90% of dorsal horn neurons from embryonic day 15-16 rats responded to the inhibitory amino acids GABA and glycine by a transient elevation of intracellular Ca2+ concentration ([Ca2+]i) when maintained in culture for < 1 week. This [Ca2+]i response has previously been shown to be due to depolarization and subsequent Ca2+ entry through voltage-gated Ca2+ channels following activation of bicuculline-sensitive GABAA receptors and strychnine-sensitive glycine receptors. Both the number of cells responding to GABA and glycine and the amplitude of the [Ca2+]i response diminished over time in culture. By 30 days in culture, none of the cells responded to GABA, muscimol or glycine by elevation of [Ca2+]i. The loss of the [Ca2+]i response was not due to a change in the abundance or the properties of voltage-gated Ca2+ channels, since over the same period of time dorsal horn neurons showed a large increase in the amplitude of the [Ca2+]i transient in response to 30 mM K+. Nor was the loss of the [Ca2+]i response due to a loss of GABA and glycine receptors. Instead, the decrease in the [Ca2+]i response over time paralleled a similar change in the electrophysiological responses. More than 90% of the neurons tested were depolarized in response to inhibitory amino acids during the first week in culture. After 30 days, all neurons tested responded to GABA and glycine with a hyperpolarization. These observations add support to the suggestion that GABA and glycine may excite dorsal horn neurons early in development and play a role in postmitotic differentiation.