Receptor-mediated Component Research Articles

Contractile activity of human greater saphenous vein mediated by P2-receptors.

In vitro experiments were carried out to measure the contractile responses to P(2)-receptor agonists in the greater saphenous vein isolated from patients with obliterating vascular atherosclerosis and varicose veins of the legs. In patients with varicose veins, the contractile responses of the greater saphenous vein to ATP, alpha,beta-methylene-ATP, and UTP were significantly lower than in patients with obliterating atherosclerosis, while the responses to ADP, adenosine, and 2-methylthio-ATP were similar in both groups. These data attest to the presence of P(2)-receptor-mediated contraction component in the greater saphenous vein, which are pronouncedly weakened during varicose disease.

Excitatory synaptic currents in lumbosacral parasympathetic preganglionic neurons evoked by stimulation of the dorsal commissure.

Excitatory pathways from the dorsal commissure (DCM) to L(6)-S(1) parasympathetic preganglionic neurons (PGN) were examined using whole-cell patch-clamp recording techniques in spinal cord slices from neonatal rats. PGN were identified by retrograde axonal transport of a fluorescent dye injected into the intraperitoneal space. Excitatory postsynaptic currents (EPSCs) were evoked in PGN by stimulation of DCM in the presence of bicuculline methiodide (10 microM) and strychnine (1 microM) to block inhibitory pathways. Electrical stimulation of DCM evoked two types of inward currents. In the majority of PGN (n = 66), currents (mean amplitude, 47.9 +/- 4.7 pA) occurred at a short and relatively constant latency (3.8 +/- 0.1 ms) and presumably represent monosynaptic EPSCs (Type 1). However, in other neurons (n = 20), a different type of EPSC (Type 2) was noted, consisting of a fast monosynaptic component followed by a prolonged inward current with superimposed fast transients presumably representing excitatory inputs mediated by polysynaptic pathways. Type 1 EPSCs were pharmacologically dissected into two components. A fast component was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 5 microM) and a slowly decaying component was blocked by 2-amino-5-phosphonovalerate (APV, 50 microM). The fast component of Type 1 EPSCs had a linear current-voltage relationship and reversed at a membrane potential of -7.6 +/- 1.3 mV (n = 5). The fast component of Type 2 EPSCs was also blocked by 5 microM CNQX and the remaining slower component was blocked by 50 microM APV. When the DCM was stimulated in the presence of 50 microM APV, the time to peak and decay time constant in Type 1 EPSCs were 1.9 +/- 0.2 and 4.1 +/- 0.8 ms, respectively. Examination of the NMDA receptor-mediated component of the EPSCs in the presence of 5 microM CNQX revealed a current-voltage relationship that had a region of negative slope conductance (from -20 to -80 mV), which was abolished in Mg(2+)-free external solution. The time to peak and decay time constant of this component were 14.2 +/- 2.0 and 91.0 +/- 12.4 ms, respectively. Type 1 EPSCs in some PGN responded in an all-or-none manner and presumably represented unitary synaptic responses; whereas Type 2 EPSCs always exhibited a graded stimulus intensity-response relationship. Paired-pulse facilitation (50-ms interstimulus intervals; 141 +/- 5.6% increase, n = 8) of EPSCs was observed. These results indicate that PGN receive monosynaptic and polysynaptic glutamatergic excitatory inputs from neurons and/or axonal pathways in the DCM.

Synaptic Reorganization in an Experimental Model of Posttraumatic Epilepsy.

Synaptic Activity in Chronically Injured, Epileptogenic Sensorimotor Neocortex Li H, Prince D J Neurophysiol 2002;88:2–12 We recorded spontaneous and evoked synaptic currents in pyramidal neurons of layer V in chronically injured, epileptogenic neocortex to assess changes in the efficacy of excitatory and inhibitory neurotransmission that might promote cortical hyperexcitability. Partial sensory-motor neocortical isolations with intact blood supply (“undercuts”) were made in 20 rats on postnatal day 21–25 and examined 2–6 weeks later in standard brain slice preparations by using whole-cell patch-clamp techniques. Age-matched, uninjured naive rats (n = 20) were used as controls. Spontaneous and miniature excitatory and inhibitory postsynaptic currents (s- and mEPSCs; s- and mIPSCs) were recorded by using patch-clamp techniques. The average frequency of s- and mEPSCs was significantly higher, whereas that of s- and mIPSCs was significantly lower in neurons of undercuts versus controls. The increased frequency of excitatory events was due to an increase in both s- and mEPSC frequency, suggesting an increased number of excitatory contacts and/or increased release probability at excitatory terminals. No significant difference was observed in 10–90% rise time of these events. The input-output slopes of fast, short-latency, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate (AMPA/KA) receptor-mediated components of evoked EPSCs were steeper in undercuts than in controls. The peak amplitude of the AMPA/KA component of EPSCs evoked by suprathreshold stimuli was significantly greater in the partially isolated neocortex. In contrast, the N-methyl-d-aspartate receptor-mediated component of evoked EPSCs was not significantly different in neurons of injured versus control cortex, suggesting that the increased AMPA/KA component was due to postsynaptic alterations. Results support the conclusion that layer V pyramidal neurons receive increased AMPA/KA receptor-mediated excitatory synaptic drive and decreased γ-aminobutyric acid type A (GABAA) receptor-mediated inhibition in this chronically injured, epileptogenic cortex. This shift in the balance of excitatory and inhibitory synaptic activation of layer V pyramidal cells toward excitation might be maladaptive and play a critical role in epileptogenesis.

Alteration of dorsal root ganglion P2X3 receptor expression and function following spinal nerve ligation in the rat.

One subtype of ATP-gated ion channel, the P2X(3) receptor, is expressed primarily on peripheral sensory neurons. While it is known that P2X(3) receptors can participate in certain forms of nociceptive signaling, their involvement in neuropathic pain transmission is not known. We have examined the expression and function of P2X(3) receptors in a rat spinal nerve ligation model of neuropathic pain. Fourteen days following L5/L6 spinal nerve ligation, the corresponding dorsal root ganglia (DRG) were removed from animals exhibiting mechanical allodynia, and these were studied using immunohistochemical and electrophysiological techniques. Using a polyclonal antibody to label the P2X(3) receptor, a significant reduction in neuronal P2X(3) immunoreactivity was observed in the ipsilateral (injured) L5 and L6 DRG following nerve ligation. In vitro electrophysiological analysis of acutely isolated DRG neurons revealed a similar decrease in functional P2X(3)-containing receptors. In small diameter (22-25 micro m) neurons, a significant reduction in the number of cells exhibiting a response to alpha,beta-meATP was observed. However, a subset of small diameter neurons retained P2X(3) responses of equal amplitude to those recorded from naive and sham control DRG neurons. Interestingly, P2X(3) immunoreactivity and P2X(3)-like responses were also detected in a subset of larger diameter (50 micro m) neurons and the number and amplitude of these responses were unchanged after spinal nerve ligation. These results suggest that, while there appears to be a decrease in fast desensitizing P2X(3) receptors following L5/L6 nerve ligation injury, certain subsets of small and large DRG neurons maintain normal P2X(3) receptor expression and function. These remaining receptors may provide a P2X(3) receptor-mediated component to neuropathic pain.

Differences in ratios of GABA, glycine and glutamate immunoreactivities in nerve terminals on rat hindlimb motoneurons: A possible source of post-synaptic variability

Previous pharmacological and physiological data on GABA and glycine receptor-dependent components of miniature inhibitory post-synaptic currents show that the electrophysiological characteristics of synaptic transmission from inhibitory synapses on spinal motoneurons are highly variable. Although post-synaptic factors are thought to be the major underlying cause of this variability, quantitative immunohistochemical data suggest that the transmitter content of afferents also vary from terminal to terminal. To examine whether ratios of amino acid staining densities vary similar to those of components of post-synaptic currents mediated by the corresponding receptors, we quantified immunogold labeling for GABA, glycine and the major excitatory transmitter, glutamate, in nerve terminals contacting the dendrites of motoneurons retrogradely labeled from the rat hindlimb muscle, biceps femoris. Nearly all terminals (94%) were immunoreactive for at least one amino acid and 64% of these contained two or three amino acids. All possible combinations of GABA, glycine and glutamate labeling were found. Over 70% of the terminals contained glycine, of which 60% also labeled for GABA. Of these GABA/glycine boutons, 40% also had glutamate. Half of all terminals contained GABA, but terminals immunoreactive for GABA alone were extremely rare. Immunoreactivity for glutamate occurred in 48% of all terminals and nearly 60% of these also contained glycine. Labeling densities for GABA, glycine and glutamate varied over a wide range from terminal to terminal. We hypothesize that this diversity in amino acid content may be a major underlying cause of variability in GABA- and glycine receptor-mediated components of miniature inhibitory post-synaptic currents in motoneurons.

Synaptic activity in chronically injured, epileptogenic sensory-motor neocortex.

We recorded spontaneous and evoked synaptic currents in pyramidal neurons of layer V in chronically injured, epileptogenic neocortex to assess changes in the efficacy of excitatory and inhibitory neurotransmission that might promote cortical hyperexcitability. Partial sensory-motor neocortical isolations with intact blood supply ("undercuts") were made in 20 rats on postnatal day 21-25 and examined 2-6 wk later in standard brain slice preparations using whole cell patch-clamp techniques. Age-matched, uninjured naive rats (n = 20) were used as controls. Spontaneous and miniature excitatory and inhibitory postsynaptic currents (s- and mEPSCs; s- and mIPSCs) were recorded using patch-clamp techniques. The average frequency of s- and mEPSCs was significantly higher, while that of s- and mIPSCs was significantly lower in neurons of undercuts versus controls. The increased frequency of excitatory events was due to an increase in both s- and mEPSC frequency, suggesting an increased number of excitatory contacts and/or increased release probability at excitatory terminals. No significant difference was observed in 10-90% rise time of these events. The input-output slopes of fast, short-latency, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate (AMPA/KA) receptor-mediated components of evoked EPSCs were steeper in undercuts than in controls. The peak amplitude of the AMPA/KA component of EPSCs evoked by supra-threshold stimuli was significantly greater in the partially isolated neocortex. In contrast, the N-methyl-D-aspartate receptor-mediated component of evoked EPSCs was not significantly different in neurons of injured versus control cortex, suggesting that the increased AMPA/KA component was due to postsynaptic alterations. Results support the conclusion that layer V pyramidal neurons receive increased AMPA/KA receptor-mediated excitatory synaptic drive and decreased GABA(A) receptor-mediated inhibition in this chronically injured, epileptogenic cortex. This shift in the balance of excitatory and inhibitory synaptic activation of layer V pyramidal cells toward excitation might be maladaptive and play a critical role in epileptogenesis.

Glutamatergic neural transmission in the nucleus tractus solitarius: N-methyl-D-aspartate receptors.

1. The nucleus tractus solitarius (NTS) is the first central site where the reflex control of autonomic, including baroreceptor, reflex function is coordinated. Autonomic signals are transmitted from the first-order visceral afferent fibres to second-order NTS neurons by L-glutamate. It is well established that activation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors, which mediate the fast component of L-glutamate signalling, is required for generating changes in membrane potentials of the second-order NTS neurons. The contribution of the slower-developing, longer-lasting N-methyl-D-aspartate (NMDA) receptor-mediated component of glutamate signalling to synaptic transmission at these synapses is less well understood. 2. The aim of this work is to highlight evidence that functional NMDA receptors exist on second-order NTS neurons in autonomic, including baroreceptor, afferent pathways by determining whether NMDA receptors can be activated by: (i) exogenous application of NMDA; and (ii) endogenous release of L-glutamate from autonomic afferent fibres. Studies were performed on second-order neurons in transverse and horizontal brainstem slices containing the intermediate NTS and the tractus solitarius. Second-order NTS neurons were identified by electrophysiological criteria or by attached fluorescent-labelled aortic depressor nerve (ADN) boutons. 3. N-Methyl-D-aspartate (50 nmol(-2) micromol) dose-dependently evoked excitatory post-synaptic currents in second-order NTS neurons (P = 0.004; n = 4). The NMDA receptor-mediated currents were also synaptically evoked by low-frequency stimulation of the autonomic afferent fibres in the tractus solitarius. The NMDA receptor-mediated currents were blocked by the NMDA receptor antagonist AP5 (n = 7; P = 0.027). 4. The findings suggest that functional NMDA receptors exist on second-order NTS neurons. While the NMDA receptor- mediated currents may not be required for signal transmission when the second-order neurons are at resting membrane potential, their activation may help to modulate autonomic signal transmission in the NTS under conditions in which the membrane is depolarized by high frequency or convergent inputs.

Somatostatin sst(2) receptors inhibit peristalsis in the rat and mouse jejunum.

Somatostatin [somatotropin release-inhibitory factor (SRIF)] has widespread actions throughout the gastrointestinal tract, but the receptor mechanisms involved are not fully characterized. We have examined the effect of selective SRIF-receptor ligands on intestinal peristalsis by studying migrating motor complexes (MMCs) in isolated segments of jejunum from rats, mice, and sst(2)-receptor knockout mice. MMCs were recorded in 4- to 5-cm segments of jejunum mounted horizontally in vitro. MMCs occurred in rat and mouse jejunum with intervals of 104.4 +/- 10 and 131.2 +/- 8 s, respectively. SRIF, octreotide, and BIM-23027 increased the interval between MMCs, an effect fully or partially antagonized by the sst(2)-receptor antagonist Cyanamid154806. A non-sst(2) receptor-mediated component was evident in mouse as confirmed by the observation of an inhibitory action of SRIF in sst(2) knockout tissue. Blocking nitric oxide generation abolished the response to SRIF in rat but not mouse jejunum. sst(2) Receptors mediate inhibition of peristalsis in both rat and mouse jejunum, but a non-sst(2) component also exists in the mouse. Nitrergic mechanisms are differentially involved in rat and mouse jejunum.

Postsynaptic induction and presynaptic expression of group 1 mGluR-dependent LTD in the hippocampal CA1 region.

Activation of metabotropic glutamate receptors (mGluRs) with the group I mGluR selective agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) induces a long-term depression (LTD) of excitatory synaptic transmission in the CA1 region of the hippocampus. Here we investigated the potential roles of pre- and postsynaptic processes in the DHPG-induced LTD at excitatory synapses onto hippocampal pyramidal cells in the mouse hippocampus. Activation of mGluRs with DHPG, but not ACPD, induced LTD at both Schaffer collateral/commissural fiber synapses onto CA1 pyramidal cells and at associational/commissural fiber synapses onto CA3 pyramidal cells. DHPG-induced LTD was blocked when the G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate) was selectively delivered into postsynaptic CA1 pyramidal cells via an intracellular recording electrode, suggesting that DHPG depresses synaptic transmission through a postsynaptic, GTP-dependent signaling pathway. The effects of DHPG were also strongly modulated, however, by experimental manipulations that altered presynaptic calcium influx. In these experiments, we found that elevating extracellular Ca(2+) concentrations ([Ca(2+)](o)) to 6 mM almost completely blocked the effects of DHPG, whereas lowering [Ca(2+)](o) to 1 mM significantly enhanced the ability of DHPG to depress synaptic transmission. Enhancing Ca(2+) influx by prolonging action potential duration with bath applications of the K(+) channel blocker 4-aminopyridine (4-AP) also strongly reduced the effects of DHPG in the presence of normal [Ca(2+)](o) (2 mM). Although these findings indicate that alterations in Ca(2+)-dependent signaling processes strongly regulate the effects of DHPG on synaptic transmission, they do not distinguish between potential pre- versus postsynaptic sites of action. We found, however, that while inhibiting both pre- and postsynaptic K(+) channels with bath-applied 4-AP blocked the effects of DHPG; inhibition of postsynaptic K(+) channels alone with intracellular Cs(+) and TEA had no effect on the ability of DHPG to inhibit synaptic transmission. This suggests that presynaptic changes in transmitter release contribute to the depression of synaptic transmission by DHPG. Consistent with this, DHPG induced a persistent depression of both AMPA and N-methyl-D-aspartate receptor-mediated components of excitatory postsynaptic currents in voltage-clamped pyramidal cells. Together our results suggest that activation of postsynaptic mGluRs suppresses transmission at excitatory synapses onto CA1 pyramidal cells through presynaptic effects on transmitter release.

Modulatory actions of serotonin, norepinephrine, dopamine, and acetylcholine in spinal cord deep dorsal horn neurons.

The deep dorsal horn represents a major site for the integration of spinal sensory information. The bulbospinal monoamine transmitters, released from serotonergic, noradrenergic, and dopaminergic systems, exert modulatory control over spinal sensory systems as does acetylcholine, an intrinsic spinal cord biogenic amine transmitter. Whole cell recordings of deep dorsal horn neurons in the rat spinal cord slice preparation were used to compare the cellular actions of serotonin, norepinephrine, dopamine, and acetylcholine on dorsal root stimulation-evoked afferent input and membrane cellular properties. In the majority of neurons, evoked excitatory postsynaptic potentials were depressed by the bulbospinal transmitters serotonin, norepinephrine, and dopamine. Although, the three descending transmitters could evoke common actions, in some neurons, individual transmitters evoked opposing actions. In comparison, acetylcholine generally facilitated the evoked responses, particularly the late, presumably N-methyl-D-aspartate receptor-mediated component. None of the transmitters modified neuronal passive membrane properties. In contrast, in response to depolarizing current steps, the biogenic amines significantly increased the number of spikes in 14/19 neurons that originally fired phasically (P < 0.01). Together, these results demonstrate that even though the deep dorsal horn contains many functionally distinct subpopulations of neurons, the bulbospinal monoamine transmitters can act at both synaptic and cellular sites to alter neuronal sensory integrative properties in a rather predictable manner, and clearly distinct from the actions of acetylcholine.

Distinct NMDA and AMPA receptor-mediated responses in mouse and human Cajal-Retzius cells.

This study examined glutamate-activated current responses of mouse and human Cajal-Retzius (C-R) cells. Thin cortical slices were prepared from the brains of mice 4-6 days after birth and from those of midgestational human fetuses. Both human and mouse C-R cells displayed glutamate-induced whole-cell current responses that were voltage-dependent and included an N-methyl-D-aspartate (NMDA) receptor-mediated component that was differentially sensitive to blockade by the NMDA receptor antagonists 2-amino-5-phosphonovaleric acid and ifenprodil. alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a non-NMDA glutamate receptor agonist, induced current responses in human but not in mouse C-R cells. These results, taken together, lead us to conclude that human C-R cells express both NMDA and AMPA types of glutamate receptors very early during development of the cortex. In contrast, mouse C-R cells express only the NMDA type of glutamate receptor. Thus we demonstrate a species-dependent sensitivity of C-R cells to glutamate and postulate that this differential sensitivity may account in part for a species-dependent difference in the persistence of C-R cells during cortical development.

Activation of Synaptic NMDA Receptors by Action Potential-Dependent Release of Transmitter during Hypoxia Impairs Recovery of Synaptic Transmission on Reoxygenation

Increased levels of glutamate and the subsequent activation of NMDA receptors are responsible for neuronal damage that occurs after an ischemic or hypoxic episode. In the present work, we investigated the relative contribution of presynaptic and postsynaptic blockade of synaptic transmission, as well as of blockade of NMDA receptors, for the facilitation of recovery of synaptic transmission in the CA1 area of rat hippocampal slices exposed to prolonged (90 min) hypoxia. During hypoxia, there was a complete inhibition of field EPSPs, which was fully reversible if released adenosine was allowed to act. When adenosine A(1) receptors were blocked with the selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), recovery of synaptic transmission from hypoxia was significantly attenuated, and this impairment could be overcome by preventing synaptic transmission during hypoxia either with tetrodotoxin (TTX) or by switching off the afferent stimulation but not by postsynaptic blockade of transmission with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or selective blockade of adenosine A(2A) receptors. When synaptic transmission was allowed to occur during hypoxia, because of the presence of DPCPX, there was an NMDA receptor-mediated component of the EPSCs recorded in CA1 pyramidal neurons, and blockade of NMDA receptors with AP-5 restored recovery of synaptic transmission from hypoxia. It is concluded that impairment of recovery of synaptic transmission after an hypoxic insult results from activation of synaptic NMDA receptors by synaptically released glutamate and that adenosine by preventing this activation efficiently facilitates recovery.

Modulation of excitatory synaptic transmission by GABA(C) receptor-mediated feedback in the mouse inner retina.

In many vertebrate CNS synapses, the neurotransmitter glutamate activates postsynaptic non-N-methyl-D-aspartate (NMDA) and NMDA receptors. Since their biophysical properties are quite different, the time course of excitatory postsynaptic currents (EPSCs) depends largely on the relative contribution of their activation. To investigate whether the activation of the two receptor subtypes is affected by the synaptic interaction in the inner plexiform layer (IPL) of the mouse retina, we analyzed the properties of the light-evoked responses of ON-cone bipolar cells and ON-transient amacrine cells in a retinal slice preparation. ON-transient amacrine cells were whole cell voltage-clamped, and the glutamatergic synaptic input from bipolar cells was isolated by a cocktail of pharmacological agents (bicuculline, strychnine, curare, and atropine). Direct puff application of NMDA revealed the presence of functional NMDA receptors. However, the light-evoked EPSC was not significantly affected by D(-)-2-amino-5-phosphonopentanoic acid (D-AP5), but suppressed by 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) or 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466). These results indicate that the light-evoked EPSC is mediated mainly by AMPA receptors under this condition. Since bipolar cells have GABA(C) receptors at their terminals, it has been suggested that bipolar cells receive feedback inhibition from amacrine cells. Application of (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), a specific blocker of GABA(C) receptors, suppressed both the GABA-induced current and the light-evoked feedback inhibition observed in ON-cone bipolar cells and enhanced the light-evoked EPSC of ON-transient amacrine cells. In the presence of TPMPA, the light-evoked EPSC of amacrine cells was composed of AMPA and NMDA receptor-mediated components. Our results suggest that photoresponses of ON-transient amacrine cells in the mouse retina are modified by the activation of presynaptic GABA(C) receptors, which may control the extent of glutamate spillover.

Glutamate and GABA mediate suprachiasmatic nucleus inputs to spinal-projecting paraventricular neurons.

We used patch-clamp recordings in slice preparations from Sprague-Dawley rats to evaluate responses of 20 spinal-projecting neurons in the dorsal paraventricular nucleus (PVN) to electrical stimulation in suprachiasmatic nucleus (SCN). Neurons containing a retrograde label transported from the thoracic (T(1)-T(4)) intermediolateral column displayed three intrinsic properties that collectively allowed distinction from neighboring parvocellular or magnocellular cells: a low-input resistance, a hyperpolarization-activated time-dependent inward rectification, and a low-threshold calcium conductance. Twelve of fifteen cells tested responded to electrical stimulation in SCN. All of 10 cells tested in media containing 2,3,-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium (5 microM) and D(-)-2-amino-5-phosphonopentanoic acid (20 microM) responded with constant latency (11.4 +/- 0.7 ms) inhibitory postsynaptic potentials, able to follow 20- to 50-Hz stimulation and blockable with bicuculline (20 microM). By contrast, all eight cells tested in the presence of bicuculline demonstrated constant latency (9.8 +/- 0.6 ms) excitatory postsynaptic potentials that followed at 20-50 Hz and featured both non-N-methyl-D-aspartate (NMDA) and NMDA receptor-mediated components. We conclude that both GABAergic and glutamatergic neurons in SCN project directly to spinal-projecting neurons in the dorsal PVN.

Synaptically-released zinc inhibits N-methyl- d-aspartate receptor activation at recurrent mossy fiber synapses

Hippocampal slices from pilocarpine-treated rats were used to explore the effect of zinc released at mossy fiber synapses on dentate granule cells. Chelation of zinc enhanced the N-methyl- d-aspartate (NMDA) receptor-mediated component of the excitatory postsynaptic current (EPSC), but did not affect the AMPA/kainate receptor-mediated component. Its effect was detectable only at negative membrane potentials and was pathway specific. Thus corelease of zinc reduces the ability of glutamate to activate postsynaptic NMDA receptors. Through this action, zinc would be expected to attenuate granule cell epileptiform activity supported by the recurrent mossy fiber pathway.

P38 stress-activated protein kinase inhibitor reverses bradykinin B 1 receptor-mediated component of inflammatory hyperalgesia

The effects of a p38 stress-activated protein kinase inhibitor, 4-(4-fluorophenyl)-2-(-4-methylsulfonylphenyl)-5-(4-pyridynyl) imidazole (SB203580), were evaluated in a rat model of inflammatory hyperalgesia. Oral, but not intrathecal, administration of SB203580 significantly reversed inflammatory mechanical hyperalgesia induced by injection of complete Freund's adjuvant into the hindpaw. SB203580 did not, however, affect the increased levels of interleukin-1β and cyclo-oxygenase 2 protein observed in the hindpaw following complete Freund's adjuvant injection. Intraplantar injection of interleukin-1β into the hindpaw elicited mechanical hyperalgesia in the ipsilateral paw, as well as in the contralateral paw, following intraplantar injection of the bradykinin B 1 receptor agonist des-Arg 9-bradykinin. Oral administration of SB203580 1 h prior to interleukin-1β administration prevented the development of hyperalgesia in the ipslateral paw and the contralateral bradykinin B 1 receptor-mediated hyperalgesia. In addition, following interleukin-1β injection into the ipsilateral paw, co-administration of SB203580 with des-Arg 9-bradykinin into the contralateral paw inhibited the bradykinin B 1 receptor-mediated hyperalgesia. In human embryonic kidney 293 cells expressing the human bradykinin B 1 receptor, its agonist des-Arg 10-kallidin produced a rapid phosphorylation of endogenous p38 stress-activated protein kinase. Our data suggest that p38 stress-activated protein kinase is involved in the development of inflammatory hyperalgesia in the rat, and that its pro-inflammatory effects involve the induction of the bradykinin B 1 receptor as well as functioning as its downstream effector.

Early onset of glutamatergic and GABAergic synaptic activity in the visual layers of the rodent superior colliculus

During postnatal development, the retinocollicular pathway undergoes activity-dependent refinement, resulting in the precise retinotopic map seen in adults. Previous studies established that retinal efferents reach the mouse superior colliculus (SC) by embryonic day 16. Morphologically, synapses were found in the rat SC before birth. As part of an extended project aimed at understanding the development of synaptic transmission in the visual layers of the SC, we report here the presence of functionally active synapses immediately after birth. Circuit activity in mouse SC neurons was detected in horizontal slices of the visual layers using cell-attached voltage clamp. The spontaneous discharge of action potentials was abolished by glutamatergic blockers and facilitated by bicuculline, showing that circuit activity is based on synaptic transmission and that the action of γ-aminobutyric acid is inhibitory. Using whole-cell voltage clamp, spontaneous glutamatergic postsynaptic currents as well as miniature GABAergic postsynaptic currents were recorded on postnatal day 1. Excitatory and inhibitory postsynaptic currents could also be evoked by electrical stimulation. Glutamatergic postsynaptic currents comprised both (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-d-aspartate receptor-mediated components. The early function of glutamatergic and GABAergic synaptic transmission in the visual layers of SC suggests that SC neurons are able to process information originating from retinal axons immediately after birth.

Theta-frequency facilitation of AMPA receptor-mediated synaptic currents in the principal cells of the medial septum.

Recent evidence suggests that Ca(2+)-permeable AMPA receptors display rapid, short-lasting current facilitation. In this study, we investigated the properties of AMPA receptor-mediated synaptic currents in medial septal neurons of the rat in an in vitro slice preparation. Immunocytochemistry with a selective antibody to the GluR2 subunit revealed that both choline acetyltransferase-containing and parvalbumin-containing neurons of the medial septum express no detectable GluR2 subunit immunoreactivity. We used whole cell voltage-clamp recordings to measure synaptically evoked AMPA receptor-mediated currents from medial septal neurons following stimulation of midline afferents. The GYKI 52466 (50 microM)- and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) (20 microM)-sensitive AMPA receptor-mediated component of the synaptic response was isolated by blocking GABA(A)- and N-methyl-D-aspartate receptor-mediated currents with 30 microM bicuculline and 100 microM 2-amino-5-phosphonovaleric acid, respectively. In some cases, patched cells were filled with Lucifer yellow (0.1%) and imaged using 2-photon laser scanning microscopy. AMPA receptor-mediated currents that were observed in large medial septal neurons (20--30 microm) displayed rectification. These currents were sensitive to external application of philanthotoxin-343 (PhTx-343, 50 microM), a potent, high-affinity antagonist of Ca(2+)-permeable, GluR2-lacking AMPA receptors. Rectifying AMPA receptor-mediated currents also displayed a rapid increase in amplitude when evoked five times at low frequency such as 6 Hz. In contrast to currents observed in large medial septal neurons, AMPA-receptor mediated currents evoked in the remaining small (8--11 microm) neurons were nonrectifying and displayed rapid synaptic depression when stimulated five times at 6 Hz. The currents evoked in these cells were unaffected by external application of PhTx-343 and were therefore GluR2-containing AMPA receptors. The results of the present study demonstrate that the principal projection neurons of the medial septum contain PhTx-343-sensitive, GluR2-lacking AMPA receptors that display rapid current facilitation when stimulated at low frequencies.

Electrophysiological and morphological characterization of neurons within neocortical ectopias.

Focal developmental abnormalities in neocortex, including ectopic collections of neurons in layer I (ectopias), have been associated with behavioral and neurological deficits. In this study, we used infrared differential interference contrast microscopy and whole cell patch-clamp to complete the first characterization of neurons within and surrounding neocortical ectopias. Current-clamp recordings revealed that neurons within ectopias display multiple types of action potential firing patterns, and biocytin labeling indicated that approximately 20% of the cells in neocortical ectopias can be classified as nonpyramidal cells and the rest as atypically oriented pyramidal cells. All cells had spontaneous excitatory (glutamatergic) and inhibitory (GABAergic) postsynaptic currents. Exhibitory postsynaptic currents consisted of both N-methyl-D-aspartate (NMDA) receptor-mediated and AMPA/kainate (A/K) receptor-mediated currents. The NMDA receptor-mediated component had decay time constants of 15.35 +/- 2.2 (SE) ms, while the A/K component had faster decay kinetics of 7.6 +/- 1.7 ms at -20 mV. GABA(A) receptor-mediated synaptic currents in ectopic cells reversed at potentials near the Cl- equilibrium potential and had decay kinetics of 16.65 +/- 1.3 ms at 0 mV. Furthermore we show that cells within ectopias receive direct excitatory and inhibitory input from adjacent normatopic cortex and can display a form of epileptiform activity.

P2X receptor expression in mouse urinary bladder and the requirement of P2X(1) receptors for functional P2X receptor responses in the mouse urinary bladder smooth muscle.

1. We have used subtype selective P2X receptor antibodies to determine the expression of P2X(1 - 7) receptor subunits in the mouse urinary bladder. In addition we have compared P2X receptor mediated responses in normal and P2X(1) receptor deficient mice to determine the contribution of the P2X(1) receptor to the mouse bladder smooth muscle P2X receptor phenotype. 2. P2X(1) receptor immunoreactivity was restricted to smooth muscle of the bladder and arteries and was predominantly associated with the extracellular membrane. Diffuse P2X(2) and P2X(4) receptor immunoreactivity not associated with the extracellular membrane was detected in the smooth muscle and epithelial layers. Immunoreactivity for the P2X(7) receptor was associated with the innermost epithelial layers and some diffuse staining was seen in the smooth muscle layer. P2X(3), P2X(5) and P2X(6) receptor immunoreactivity was not detected. 3. P2X receptor mediated inward currents and contractions were abolished in bladder smooth muscle from P2X(1) receptor deficient mice. In normal bladder nerve stimulation evoked contractions with P2X and muscarinic acetylcholine (mACh) receptor mediated components. In bladder from the P2X(1) receptor deficient mouse the contraction was mediated solely by mACh receptors. Contractions to carbachol were unaffected in P2X(1) receptor deficient mice demonstrating that there had been no compensatory effect on mACh receptors. 4. These results indicate that homomeric P2X(1) receptors underlie the bladder smooth muscle P2X receptor phenotype and suggest that mouse bladder from P2X(1) receptor deficient and normal animals may be models of human bladder function in normal and diseased states.