Differential pH and capsaicin responses of Griffonia simplicifolia IB4 (IB4)-positive and IB4-negative small sensory neurons

Protons play a key role in nociception caused by inflammation and ischaemia, but little is known about the relative sensitivities of different dorsal root ganglion (DRG) neurons. We have therefore examined the responses in vitro of rat DRG cells classified according to whether or not they bind Griffonia simplicifolia IB4 (IB4), a lectin which is widely used to distinguish between two major populations of small diameter neurons. Under voltage-clamp conditions, proton-activated inward currents were found in approximately 90% of small DRG neurons and showed one of three waveforms: transient, sustained or mixed. The majority of IB4-positive (IB4+) neurons (63%) gave rise to sustained inward currents that were sensitive to capsazepine. In contrast, the most prevalent waveform in small IB4-negative (IB4-) neurons (69%) was a mixed response containing transient and sustained components. The transient component was inhibited by amiloride whilst the sustained component showed a variable sensitivity to capsazepine. We also found that more IB4+ cells responded to capsaicin and, on average, gave rise to a larger magnitude of response than small IB4- neurons, consistent with their higher prevalence and greater amplitude of vanilloid receptor 1 (TRPV1)-like acid responses. The increase in intracellular Ca(2+) induced by capsaicin was also slightly greater in IB4+ neurons and in these cells its magnitude correlated with the level of TRPV1 immunoreactivity. Our data suggest that acid-sensing ion channels (ASICs) and TRPV1 are the major acid-sensitive receptors in small IB4- neurons, whilst TRPV1 is the predominant one in IB4+ neurons. Because ASIC-like responses were approximately 10-fold more sensitive to changes in H(+) than TRPV1-like responses, we speculate that small IB4- rather than IB4+ neurons play an essential role in sensing acid. Our results also highlight differences in capsaicin responses between IB4+ and IB4- small neurons and reveal the close link between capsaicin responses and levels of TRPV1 expression.     

Differential slow inactivation and use-dependent inhibition of Nav1.8 channels contribute to distinct firing properties in IB4+ and IB4- DRG neurons

Nociceptive dorsal root ganglion (DRG) neurons can be classified into nonpeptidergic IB(4)(+) and peptidergic IB(4)(-) subtypes, which terminate in different layers in dorsal horn and transmit pain along different ascending pathways, and display different firing properties. Voltage-gated, tetrodotoxin-resistant (TTX-R) Na(v)1.8 channels are expressed in both IB(4)(+) and IB(4)(-) cells and produce most of the current underlying the depolarizing phase of action potential (AP). Slow inactivation of TTX-R channels has been shown to regulate repetitive DRG neuron firing behavior. We show in this study that use-dependent reduction of Na(v)1.8 current in IB(4)(+) neurons is significantly stronger than that in IB(4)(-) neurons, although voltage dependency of activation and steady-state inactivation are not different. The time constant for entry of Na(v)1.8 into slow inactivation in IB(4)(+) neurons is significantly faster and more Na(v)1.8 enter the slow inactivation state than in IB(4)(-) neurons. In addition, recovery from slow inactivation of Na(v)1.8 in IB(4)(+) neurons is slower than that in IB(4)(-) neurons. Using current-clamp recording, we demonstrate a significantly higher current threshold for generation of APs and a longer latency to onset of firing in IB(4)(+), compared with those of IB(4)(-) neurons. In response to a ramp stimulus, IB(4)(+) neurons produce fewer APs and display stronger adaptation, with a faster decline of AP peak than IB(4)(-) neurons. Our data suggest that differential use-dependent reduction of Na(v)1.8 current in these two DRG subpopulations, which results from their different rate of entry into and recovery from the slow inactivation state, contributes to functional differences between these two neuronal populations.     

Differential response properties of IB(4)-positive and -negative unmyelinated sensory neurons to protons and capsaicin

Activation of unmyelinated (C-fiber) nociceptors by noxious chemicals plays a critical role in the initiation and maintenance of injury-induced pain. C-fiber nociceptors can be divided into two groups in which one class depends on nerve growth factor during postnatal development and contains neuropeptides, and the second class depends on glial cell line-derived neurotrophic factor during postnatal development and contains few neuropeptides but binds isolectin B(4) (IB(4)). We determined the sensitivity of these two populations to protons and capsaicin using whole cell recordings of dorsal root ganglion neurons from adult mouse. IB(4)-negative unmyelinated neurons were significantly more responsive to protons than IB(4)-positive neurons in a concentration-dependent manner. Approximately 86% of IB(4)-negative neurons responded to pH 5.0 with an inward current compared with only 33% of IB(4)-positive neurons. The subtypes of proton-evoked currents in IB(4)-negative unmyelinated neurons were also more diverse. Many IB(4)-negative neurons exhibited transient, rapidly inactivating proton currents as well as sustained proton currents. In contrast, IB(4)-positive neurons never displayed transient proton currents and responded to protons only with sustained, slowly inactivating inward currents. The two classes of neurons also responded differently to capsaicin. Twice as many na?ve IB(4)-negative unmyelinated neurons responded to 1 microM capsaicin as IB(4)-positive neurons, and the capsaicin-evoked currents in IB(4)-negative neurons were approximately fourfold larger than those in IB(4)-positive neurons. Interestingly, proton exposure altered the capsaicin responsiveness of the two classes of neurons in opposite ways. Brief preexposure to protons increased the number of capsaicin-responsive IB(4)-positive neurons by twofold and increased the capsaicin-evoked currents by threefold. Conversely, proton exposure decreased the number of capsaicin-responsive IB(4)-negative neurons by 50%. These data suggest that IB(4)-negative unmyelinated nociceptors are initially the primary responders to both protons and capsaicin, but IB(4)-positive nociceptors have a unique capacity to be sensitized by protons to capsaicin-receptor agonists.     

Valproate and sodium currents in cultured hippocampal neurons

Cultured rat hippocampal neurons with short processes were investigated using the whole cell voltage clamp under conditions appropriate for isolating Na⁺ currents. After incubation of the neuron culture for a period of 15–30 min in 1 mM sodium valproate, several parameters of the Na⁺ current were changed. The peak Na⁺ conductance g _p, measured using hyperpolarizing prepulses, was reduced by valproate in a voltage-dependent manner. In the membrane voltage range from -30 to +20 mV, this reduction showed a linear dependence on voltage, increasing from about zero to approximately 30% of g _p, the maximum peak Na⁺ conductance of the neuron. At the holding voltage of -70 mV, the inactivation parameter h _t8 decreased from 0.88 in the control to 0.64 in the valproate solution. This reduction originated mainly from a 10 mV shift in the sigmoid relation between h _t8 and membrane voltage along the voltage axis to hyperpolarizing potentials. The decay of the maximum peak Na⁺ current (inactivation) could be fitted by a biexponential function. Time constants of the fast and slow component at -20 mV decreased in valproate by about 50%. Valproate also retarded the recovery from inactivation, as determined at the holding voltage. The sigmoid recovery from inactivation could reasonably be described by an exponential function with time constant _r and delay time t. Both _r and At increased more than 200% in valproate. Our results indicate that valproate affected the Na⁺ current in hippocampal neurons in a way that contributed to a considerable depression of Na⁺ reactivation. This explains the frequency-dependent inhibition of action potentials as observed in mammalian central nervous tissue and may be the principal action of the anticonvulsant.     

Effects of ganglionic satellite cells and NGF on the expression of nicotinic acetylcholine currents by rat sensory neurons.

1. We have investigated two factors that affect the expression of nicotinic acetylcholine (ACh) currents on neonatal rat sensory neurons: an influence derived from ganglionic satellite cells, and nerve growth factor (NGF). 2. With the use of whole-cell patch-clamp techniques on rat nodose neurons, we have measured the proportion of neurons sensitive to ACh and have quantified their ACh current densities. The majority (60%) of nodose neurons from neonatal animals do not express nicotinic acetylcholine receptors (nAChRs); the remaining 40% had ACh current densities that ranged from 0.4 to 93 pA/pF. Furthermore, neither the proportion nor the ACh current densities change over the first two postnatal weeks in vivo. 3. The expression of ACh currents by these neurons in vivo is controlled, in part, by an influence from the ganglionic satellite cells: culturing neurons in the absence of other cell types results in an increase in the proportion of ACh-sensitive neurons, whereas coculturing neurons with their satellite cells maintains functional nAChR expression in its in vivo state. Furthermore, satellite cells are not required continually, as a brief exposure to this influence, either in vivo or in culture, is sufficient to exert its effect on functional nAChR expression. 4. On removal of this satellite cell influence, the neurons respond to NGF treatment by increasing their ACh current densities: the median ACh current density for neurons grown for 2-3 wk with NGF was 32.5 pA/pF, whereas, the median ACh current density for neurons cultured without NGF for the same time was 4.5 pA/pF.(ABSTRACT TRUNCATED AT 250 WORDS)     

A-type voltage-gated K+ currents influence firing properties of isolectin B4-positive but not isolectin B4-negative primary sensory neurons

Voltage-gated K+ channels (Kv) in primary sensory neurons are important for regulation of neuronal excitability. The dorsal root ganglion (DRG) neurons are heterogeneous, and the types of native Kv currents in different groups of nociceptive DRG neurons are not fully known. In this study, we determined the difference in the A-type Kv current and its influence on the firing properties between isolectin B4 (IB4)-positive and -negative DRG neurons. Whole cell voltage- and current-clamp recordings were performed on acutely dissociated small DRG neurons of rats. The total Kv current density was significantly higher in IB+-positive than that in IB(4)-negative neurons. Also, 4-aminopyridine (4-AP) produced a significantly greater reduction in Kv currents in IB4-positive than in IB4-negative neurons. In contrast, IB4-negative neurons exhibited a larger proportion of tetraethylammonium-sensitive Kv currents. Furthermore, IB4-positive neurons showed a longer latency of firing and required a significantly larger amount of current injection to evoke action potentials. 4-AP significantly decreased the latency of firing and increased the firing frequency in IB4-positive but not in IB4-negative neurons. Additionally, IB4-positive neurons are immunoreactive to Kv1.4 but not to Kv1.1 and Kv1.2 subunits. Collectively, this study provides new information that 4-AP-sensitive A-type Kv currents are mainly present in IB4-positive DRG neurons and preferentially dampen the initiation of action potentials of this subpopulation of nociceptors. The difference in the density of A-type Kv currents contributes to the distinct electrophysiological properties of IB4-positive and -negative DRG neurons.     

Effect of clonidine on potential-dependent sodium currents in sensory neurons

In experiments on rat pups we studied the effect of clonidine on potential-dependent Na⁺ currents in dorsal root ganglia by the voltage clamp method. Clonidine decreased the amplitude of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium currents. The range of acting concentrations and the absence of modulatory effect of norepinephrine on the efficiency of clonidine-induced blockade of sodium currents suggest that this blockade results from a direct interaction of clonidine with sodium channels.__________This revised version was published online in August 2005 with the addition of the issue titleTranslated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 1, pp. 44–48, January, 2005     

Calmodulin regulates current density and frequency-dependent inhibition of sodium channel Nav1.8 in DRG neurons

Sodium channel Nav1.8 produces a slowly inactivating, tetrodotoxin-resistant current, characterized by recovery from inactivation with fast and slow components, and contributes a substantial fraction of the current underlying the depolarizing phase of the action potential of dorsal root ganglion (DRG) neurons. Nav1.8 C-terminus carries a conserved calmodulin-binding isoleucine-glutamine (IQ) motif. We show here that calmodulin coimmunoprecipitates with endogenous Nav1.8 channels from native DRG, suggesting that the two proteins can interact in vivo. Treatment of native DRG neurons with a calmodulin-binding peptide (CBP) reduced the current density of Nav1.8 by nearly 65%, without changing voltage dependency of activation or steady-state inactivation. To investigate the functional role of CaM binding to the IQ motif in the Nav1.8 C-terminus, the IQ dipeptide was substituted by DE; we show that this impairs the binding of CaM to the IQ motif. Mutant Nav1.8IQ/DE channels produce currents with roughly 50% amplitude, but with unchanged voltage dependency of activation and inactivation when expressed in DRG neurons from Nav1.8-null mice. We also show that blocking the interaction of CaM and Nav1.8 using CBP or the IQ/DE substitution causes a buildup of inactivated channels and, in the case of the IQ/DE mutation, stimulation even at a low frequency of 0.1 Hz significantly enhances the frequency-dependent inhibition of the Nav1.8 current. This study presents, for the first time, evidence that calmodulin associates with a sodium channel, Nav1.8, in native neurons, and demonstrates a regulation of Nav1.8 currents that can significantly affect electrogenesis of DRG neurons in which Nav1.8 is normally expressed.     

High voltage-activated Ca(2+) channel currents in isolectin B(4)-positive and -negative small dorsal root ganglion neurons of rats

Voltage-gated Ca(2+) channels in the primary sensory neurons are important for neurotransmitter release and regulation of nociceptive transmission. Although multiple classes of Ca(2+) channels are expressed in the dorsal root ganglion (DRG) neurons, little is known about the difference in the specific channel subtypes among the different types of DRG neurons. In this study, we determined the possible difference in high voltage-activated Ca(2+) channel currents between isolectin B(4) (IB(4))-positive and IB(4)-negative small-sized (15-30 microm) DRG neurons. Rat DRG neurons were acutely isolated and labeled with IB(4) conjugated to a fluorescent dye. Whole-cell patch clamp recordings of barium currents flowing through calcium channels were performed on neurons with and without IB(4). The peak current density of voltage-gated Ca(2+) currents was not significantly different between IB(4)-positive and IB(4)-negative neurons. Also, both nimodipine and omega-agatoxin IVA produced similar inhibitory effects on Ca(2+) currents in these two types of neurons. However, block of N-type Ca(2+) channels with omega-conotoxin GVIA produced a significantly greater reduction of Ca(2+) currents in IB(4)-positive than IB(4)-negative neurons. Furthermore, the IB(4)-positive neurons had a significantly smaller residual Ca(2+) currents than IB(4)-negative neurons. These data suggest that a higher density of N-type Ca(2+) channels is present in IB(4)-positive than IB(4)-negative small-sized DRG neurons. This differential expression of the subtypes of high voltage-activated Ca(2+) channels may contribute to the different function of these two classes of nociceptive neurons.     

Hypoxic augmentation of fast-inactivating and persistent sodium currents in rat caudal hypothalamic neurons

Previous work from this laboratory has indicated that TTX-sensitive sodium channels are involved in the hypoxia-induced inward current response of caudal hypothalamic neurons. Since this inward current underlies the depolarization and increased firing frequency observed in these cells during hypoxia, the present study utilized more detailed biophysical methods to specifically determine which sodium currents are responsible for this hypoxic activation. Caudal hypothalamic neurons from approximately 3-wk-old Sprague-Dawley rats were acutely dissociated and patch-clamped in the voltage-clamp mode to obtain recordings from fast-inactivating and persistent (noninactivating) whole cell sodium currents. Using computer-generated activation and inactivation voltage protocols, rapidly inactivating sodium currents were analyzed during normal conditions and during a brief (3-6 min) period of severe hypoxia. In addition, voltage-ramp and extended-voltage-activation protocols were used to analyze persistent sodium currents during normal conditions and during hypoxia. A polarographic oxygen electrode determined that the level of oxygen in this preparation quickly dropped to 10 Torr within 2 min of initiation of hypoxia and stabilized at <0.5 Torr within 4 min. During hypoxia, the peak fast-inactivating sodium current was significantly increased throughout the entire activation range, and both the activation and inactivation values (V(1/2)) were negatively shifted. Furthermore both the voltage-ramp and extended-activation protocols demonstrated a significant increase in the persistent sodium current during hypoxia when compared with normoxia. These results demonstrate that both rapidly inactivating and persistent sodium currents are significantly enhanced by a brief hypoxic stimulus. Furthermore the hypoxic-induced increase in these currents most likely is the primary mechanism for the depolarization and increased firing frequency observed in caudal hypothalamic neurons during hypoxia. Since these neurons are important in modulating cardiorespiratory activity, the oxygen responsiveness of these sodium currents may play a significant role in the centrally mediated cardiorespiratory response to hypoxia.     

Action potentials and sodium inward currents of developing neurons in Xenopus nerve-muscle cultures

Action potentials and voltage-gated Na+ inward currents from cultured embryonic neurons of Xenopus laevis were recorded using the patch-clamp technique in the whole cell configuration. Neurons together with muscle cells were dissociated from embryos shortly after completion of gastrulation. Under the voltage-clamp condition the voltage-gated Na+ inward current was isolated from other currents by pharmacological means and by ion substitution. A small Na+ current was observed in round cells without neurites (presumptive neurons). The mean amplitude of the peak Na+ current was 2.5 times larger in neurons with short processes than in presumptive neurons. As they developed further by extending longer processes, the maximum amplitude of the Na+ inward current recorded at the soma decreased. In varicosities, the Na+ inward current density was greater than that at the soma of neurons with extended neurites but kinetic properties and voltage-dependency were similar.     

Molecular and functional differences in voltage-activated sodium currents between GABA projection neurons and dopamine neurons in the substantia nigra

GABA projection neurons (GABA neurons) in the substantia nigra pars reticulata (SNr) and dopamine projection neurons (DA neurons) in substantia nigra pars compacta (SNc) have strikingly different firing properties. SNc DA neurons fire low-frequency, long-duration spikes, whereas SNr GABA neurons fire high-frequency, short-duration spikes. Since voltage-activated sodium (Na(V)) channels are critical to spike generation, the different firing properties raise the possibility that, compared with DA neurons, Na(V) channels in SNr GABA neurons have higher density, faster kinetics, and less cumulative inactivation. Our quantitative RT-PCR analysis on immunohistochemically identified nigral neurons indicated that mRNAs for pore-forming Na(V)1.1 and Na(V)1.6 subunits and regulatory Na(V)β1 and Na(v)β4 subunits are more abundant in SNr GABA neurons than SNc DA neurons. These α-subunits and β-subunits are key subunits for forming Na(V) channels conducting the transient Na(V) current (I(NaT)), persistent Na current (I(NaP)), and resurgent Na current (I(NaR)). Nucleated patch-clamp recordings showed that I(NaT) had a higher density, a steeper voltage-dependent activation, and a faster deactivation in SNr GABA neurons than in SNc DA neurons. I(NaT) also recovered more quickly from inactivation and had less cumulative inactivation in SNr GABA neurons than in SNc DA neurons. Furthermore, compared with nigral DA neurons, SNr GABA neurons had a larger I(NaR) and I(NaP). Blockade of I(NaP) induced a larger hyperpolarization in SNr GABA neurons than in SNc DA neurons. Taken together, these results indicate that Na(V) channels expressed in fast-spiking SNr GABA neurons and slow-spiking SNc DA neurons are tailored to support their different spiking capabilities.     

Voltage-dependent sodium and calcium currents in acutely isolated adult rat trigeminal root ganglion neurons

Voltage-dependent sodium (INa) and calcium (ICa) currents in small (<30 microM) neurons from adult rat trigeminal root ganglia were characterized with a standard whole cell patch-clamp technique. Two types of INa showing different sensitivity to tetrodotoxin (TTX) were recorded, which showed marked differences in their activating and inactivating time courses. The activation and the steady-state inactivation kinetics of TTX-resistant INa were more depolarized by about +20 and +30 mV, respectively, than those of TTX-sensitive INa. Voltage-dependent ICa was recorded under the condition that suppressed sodium and potassium currents with 10 mM Ca2+ as a charge carrier. Depolarizing step pulses from a holding potential of -80 mV evoked two distinct inward ICa, low-voltage activated (LVA) and high-voltage activated (HVA) ICa. LVA ICa was first observed at -60 to -50 mV and reached a peak at about -30 mV. Amiloride (0.5 mM) suppressed approximately 60% of the LVA ICa, whereas approximately 10% of HVA ICa was inhibited by the same concentration of the amiloride. LVA ICa was far less affected by the presence of external Cd2+ or the replacement of Ca2+ by 10 Ba2+ than HVA ICa. The omega-conotoxin GVIA (omega-CgTx), an N-type ICa blocker, suppressed approximately 65% of the whole cell HVA ICa at the concentration of 1 microM. The omega-CgTx-resistant HVA ICa was sensitive to nifedipine (10 microM), a dihydropyridine (DHP) calcium channel antagonist, which produced an additional blockade by approximately 25% of the drug-free control ( approximately 70% of the omega-CgTx-resistant ICa). The combination of 10 microM nifedipine and 1 microM omega-CgTx left approximately 13% of the drug-free control ICa unblocked. The DHP agonist S(-)-BayK8644 (5 microM) shifted the activation of the HVA ICa to more negative potentials and increased its maximal amplitude. Additionally, S(-)-BayK8644 caused the appearance of a slowed component of the tail current. These results clearly demonstrate that the presence of two types of sodium channels, TTX sensitive and resistant, and three types of calcium channels, T, L, and N type, in the small-sized adult rat trigeminal ganglion neurons.     

Ileitis modulates potassium and sodium currents in guinea pig dorsal root ganglia sensory neurons

Intestinal inflammation induces hyperexcitability of dorsal root ganglia sensory neurons, which has been implicated in increased pain sensation. This study examined whether alteration of sodium (Na⁺) and/ or potassium (K⁺) currents underlies this hyperexcitability. Ileitis was induced in guinea pig ileum with trinitrobenzene sulphonic acid (TBNS) and dorsal root ganglion neurons innervating the site of inflammation were identified by Fast Blue or DiI fluorescence labelling. Whole cell recordings were made from acutely dissociated small-sized neurons at 7–10 days. Neurons exhibited transient A-type and sustained outward rectifier K⁺ currents. Compared to control, both A-type and sustained K⁺ current densities were significantly reduced (42 and 34 %, respectively;   P < 0.05  ) in labelled neurons from the inflamed intestine but not in non-labelled neurons. A-type current voltage dependence of inactivation was negatively shifted in labelled inflamed intestine neurons. Neurons also exhibited tetrodotoxin-sensitive and resistant Na⁺ currents. Tetrodotoxin-resistant sodium currents were increased by 37 % in labelled neurons from the inflamed intestine compared to control (   P < 0.01  ), whereas unlabelled neurons were unaffected. The activation and inactivation curves of these currents were unchanged by inflammation. These data suggest ileitis increases excitability of intestinal sensory neurons by modulating multiple ionic channels. The lack of effect in non-labelled neurons suggests signalling originated at the nerve terminal rather than through circulating mediators and, given that Na⁺ currents are enhanced whereas K⁺ currents are suppressed, one or more signalling pathways may be involved.     

In vivo expression of major histocompatibility complex molecules on oligodendrocytes and neurons during viral infection

Demyelination in multiple sclerosis and in animal models is associated with infiltrating CD8+ and CD4+ T cells. Although oligodendrocytes and axons are damaged in these diseases, the roles T cells play in the demyelination process are not completely understood. Antigen-specific CD8+ T cell lysis of target cells is dependent on interactions between the T cell receptor and major histocompatibility complex (MHC) class I-peptide complexes on the target cell. In the normal central nervous system, expression of MHC molecules is very low but often increases during inflammation. We set out to precisely define which central nervous system cells express MHC molecules in vivo during infection with a strain of murine hepatitis virus that causes a chronic, inflammatory demyelinating disease. Using double immunofluorescence labeling, we show that during acute infection with murine hepatitis virus, MHC class I is expressed in vivo by oligodendrocytes, neurons, microglia, and endothelia, and MHC class II is expressed only by microglia. These data indicate that oligodendrocytes and neurons have the potential to present antigen to T cells and thus be damaged by direct antigen-specific interactions with CD8+ T lymphocytes.     

Effect of allapinine on sodium currents in single trigeminal neurons and cardiomyocytes of rats

A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Chemistry of Plant Compounds, Academy of Sciences of the Uzbek SSR, Tashkent. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Romodanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 111, No. 4, pp. 388–390, April, 1991.     

Evans blue reduces macroscopic desensitization of non-NMDA receptor mediated currents and prolongs excitatory postsynaptic currents in cultured rat thalamic neurons.

Fast application of L-glutamate, AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) or kainate to cultured rat thalamic neurons revealed properties of non-NMDA (N-methyl-D-aspartate) receptors similar to those described in hippocampal neurons. The kinetics of non-NMDA receptor-mediated currents were altered by the addition of the dye Evans Blue (EB). Macroscopic desensitization was reduced and activation and deactivation kinetics were slowed. Delayed addition of EB, after desensitization of non-NMDA receptors, resulted in reactivation of desensitized receptors. Thus, both ion channel gating and entry into the desensitized state were affected. Evans blue also slowed the activation and the decay of glutamatergic miniature EPSCs (excitatory postsynaptic currents), demonstrating that receptor kinetics determine the time course of the synaptic response.