RESUMEN
The majority of excitatory neurotransmission in vertebrate CNS is mediated by glutamate binding to different types of receptors. Among them, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and kainite receptors (KAR) are ionotropic receptors playing important pathophysiological roles. A number of small molecules acting as positive allosteric modulators (PAM) of AMPAR have been proposed as drugs for neurological disorders, however, there is no such abundance of ligands capable of modulating KARs activity. We investigated the ability of IDRA21 and of its derivative, compound 2 (c2), to modulate glutamate-evoked currents at native and recombinantly expressed AMPA and KA receptors. By using the patch clamp technique we analyzed the activity of the two compounds in primary cultures of cerebellar granule neurons and in HEK293 cells transiently transfected with KARs and AMPAR subunits. It resulted that both benzothiadiazine derivatives potentiate AMPAR and KAR mediated currents in native and recombinant receptors, c2 being always more potent and efficacious than IDRA21. The potency of both compounds was higher in native receptors than in recombinant receptors. In HEK293 cells transfected with AMPAR subunits, the efficacy of IDRA21 and c2 was much higher in GluA1 than in GluA2 homomeric receptors while their potency did not change. In recombinant KAR, both compounds had a potency in the high micromolar range, while the efficacy reached a maximum in the GluK2 expressing cells. The benzothiadiazine effect, both in native and recombinant receptors, was detected mainly on plateau current, involving a decrease in AMPAR and KAR desensitization. Our study demonstrates for the first time that two positive allosteric modulators of AMPAR, IDRA21 and its derivative, c2, potentiate KAR activity. Furthermore, we highlighted their subunit selectivity that may enable the design of potent and selective PAMs, which could be relevant for the development of new drugs and for a better understanding of KAR functions in the CNS.
Asunto(s)
Benzotiadiazinas , Ácido Glutámico , Receptores de Ácido Kaínico , Benzotiadiazinas/farmacología , Ácido Glutámico/metabolismo , Células HEK293 , Humanos , Neuronas , Técnicas de Placa-Clamp , Receptores de Ácido Kaínico/química , Receptores de Ácido Kaínico/metabolismoRESUMEN
Postnatal development sees a strong synaptogenesis in rat superficial dorsal horn. My studies show that synapses mediated by two excitatory neurotransmitters, glutamate and ATP, are functional since the very first postnatal days. Using an electrophysiological approach, the functional properties of two receptors activated by these neurotransmitters, glutamatergic NMDA and ATP ionotropic receptors, are described.
Asunto(s)
Células del Asta Posterior/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Receptores Purinérgicos P2/fisiología , Sinapsis/fisiología , Animales , Animales Recién Nacidos , Células del Asta Posterior/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Animales Recién Nacidos/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Médula Espinal/fisiología , Potenciales de Acción/fisiología , Animales , Bicuculina/farmacología , Estimulación Eléctrica/métodos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Antagonistas del GABA/farmacología , Glicinérgicos/farmacología , Técnicas In Vitro , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Técnicas de Placa-Clamp , Ratas , Raíces Nerviosas Espinales/fisiología , Estricnina/farmacología , Sinapsis/efectos de los fármacos , Sinapsis/fisiologíaRESUMEN
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.
Asunto(s)
Glutamatos/fisiología , Neuronas/efectos de los fármacos , Fosfato de Piridoxal/análogos & derivados , Médula Espinal/efectos de los fármacos , Suramina/farmacología , Transmisión Sináptica/efectos de los fármacos , Animales , Animales Recién Nacidos , Potenciales Evocados/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Técnicas In Vitro , Neuronas/citología , Antagonistas del Receptor Purinérgico P2 , Fosfato de Piridoxal/farmacología , Ratas , Médula Espinal/citología , Médula Espinal/fisiología , Factores de Tiempo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
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).
Asunto(s)
Animales Recién Nacidos/fisiología , Potenciales Postsinápticos Excitadores/fisiología , N-Metilaspartato/fisiología , Médula Espinal/fisiología , Sinapsis/fisiología , Animales , Electrofisiología , Cinética , Magnesio/farmacología , Ratas , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
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.
Asunto(s)
Adenosina Trifosfato/farmacología , Ganglios Espinales/efectos de los fármacos , Receptores Purinérgicos/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Animales , Técnicas de Placa-Clamp , Ratas , Factores de TiempoRESUMEN
Whole-cell patch clamp recording techniques were applied to periglomerular (PG) cells in slices of the frog olfactory bulb (OB) preparation to study the basic electrical properties of these inhibitory interneurons. The cells were intracellularly stained with Lucifer Yellow for precise identification. Under current-clamp conditions PG cells showed rich spontaneous excitatory synaptic activity at rest, usually leading to overshooting, TTX-sensitive action potentials. The passive cable properties of the cell membrane have been carefully characterised. Depolarisation of this neurone under voltage-clamp conditions activated a complex pattern of current flow, that has been dissected into its main components. The currents have been isolated resorting to their different kinetic and pharmacological properties. Four main voltage dependent ionic currents have been isolated, two inward currents, I(Na) and I(Ca), and two outward currents carried by potassium ions, one fast transient, I(A)-type and another similar to the delayed rectifier type. These currents have been characterised kinetically and pharmacologically. The functional implications of their properties are discussed.
Asunto(s)
Canales de Calcio/fisiología , Interneuronas/fisiología , Bulbo Olfatorio/fisiología , Canales de Sodio/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Canales de Calcio/efectos de los fármacos , Técnicas In Vitro , Interneuronas/efectos de los fármacos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Bulbo Olfatorio/citología , Bulbo Olfatorio/efectos de los fármacos , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Rana esculenta , Canales de Sodio/efectos de los fármacos , Tetrodotoxina/farmacologíaRESUMEN
Whole-cell patch clamp recording techniques were applied to periglomerular (PG) cells in slices of the frog olfactory bulb (OB) to study the properties of the excitatory synapses in the triad formed by the olfactory nerve (ON) and the dendrites of mitral/tufted (MT) cells and PG cells. The postsynaptic response evoked by ON stimulation was glutamatergic and could be dissected into NMDA and non-NMDA components of equivalent amplitudes. The dendro-dendritic synapse between MT and PG cells could be activated following antidromic stimulation of the lateral and medial olfactory tract (LOT and MOT). In this case the postsynaptic potentials had amplitudes and durations comparable to those obtained by ON stimulation, the neurotransmitter was glutamate, but the synapse was largely dominated by the slow NMDA component.
Asunto(s)
Mesangio Glomerular/fisiología , Bulbo Olfatorio/fisiología , Sinapsis/fisiología , Animales , Técnicas In Vitro , Técnicas de Placa-Clamp , Rana esculentaRESUMEN
Voltage-activated currents have been recorded from periglomerular cells in thin slices of frog olfactory bulb. Cells were examined with whole-cell patch clamp methods. The voltage-dependent potassium currents were studied after pharmacological block of inward currents. Depolarising steps from -130 mV gave an early transient, A-type, outward current and a delayed rectifier K+ current (IKV). The two currents could be isolated on the basis of the differences in their kinetic properties. The A-current developed following a third-order kinetics when the membrane was depolarised to potentials more positive than -40 mV after preconditioning to potentials more negative than -60 mV. Once activated (tau a 2.5 ms at 0 mV), IA inactivated following a single exponential (tau ha about 60 ms). IKV activated with a second-order kinetics above -30 mV with a time constant of 4 ms at 0 mV. IA and IKV were sensitive, respectively, to 4-aminopyridine (4-AP) and tetraethylammonium (TEA).
Asunto(s)
Interneuronas/química , Bulbo Olfatorio/fisiología , Canales de Potasio/fisiología , 4-Aminopiridina/farmacología , Animales , Anuros , Cadmio/farmacología , Calcio/metabolismo , Técnicas In Vitro , Interneuronas/fisiología , Potenciales de la Membrana/fisiología , Bulbo Olfatorio/citología , Técnicas de Placa-Clamp , Potasio/metabolismo , Potasio/farmacología , Bloqueadores de los Canales de Potasio , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
Kinetic properties of the sodium current in periglomerular (PG) cells were investigated by applying whole-cell patch-clamp techniques to thin slices of the frog olfactory bulb. Eight of the cells were intracellularly stained with Lucifer Yellow for precise identification. Under current-clamp conditions PG cells showed rich spontaneous activity at rest. Na current was isolated from other current contributions by equimolar substitution of K+ with Cs+ in the intracellular solution to prevent K-currents, and 100 microM Cd2+ in the external solution to block Ca-current. Depolarisations beyond -40 mV activated a fast transient TTX-sensitive inward current. Once activated, INa declined exponentially to zero following a single exponential. The underlying conductance showed a sigmoidal activation between -40 and +30 mV, with half activation at -17.4 mV and a maximal value of 9.7 nS per neurone. The steady-state inactivation was complete at -30 mV and completely removed at -90 mV, with a midpoint at -56 mV. The activation process could be adequately described by third order kinetics, with time constants ranging from 260 microseconds at -20 mV to 70 microseconds at +50 mV.
Asunto(s)
Interneuronas/fisiología , Bulbo Olfatorio/fisiología , Canales de Sodio/fisiología , Transmisión Sináptica/fisiología , Animales , Cadmio/farmacología , Cesio/farmacología , Técnicas In Vitro , Interneuronas/efectos de los fármacos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Bulbo Olfatorio/citología , Bulbo Olfatorio/efectos de los fármacos , Técnicas de Placa-Clamp , Rana esculenta , Canales de Sodio/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Tetrodotoxina/farmacologíaRESUMEN
1. Whole-cell voltage clamp recordings were used to study the action of the transition ion zinc on the A-current kinetics in granule cells from rat cerebellar slices. 2. The effects of zinc have been tested in the concentration range from 1 microM to 1 mM, and fully characterized on all kinetic parameters at 100 and 300 microM. All the effects observed were rapid, concentration dependent and fully reversible. 3. Steady-state inactivation curves are strongly shifted towards depolarized potentials, with activation curves much less so. These shifts lead to an increase of the peak current amplitude around physiological resting membrane potentials and to a decrease at hyperpolarized potentials. 4. The forward 'on' rate constants are slowed by Zn2+ at a concentration of 100-300 microM by a factor from 1.5 to 4. The backward 'off' rate constants are unaffected by Zn2+. 5. The development of IA inactivation, as measured from the current decay, is not affected by Zn2+ up to 1 mM. Removal of inactivation is, on the contrary, significantly slowed. 6. The results are neither compatible with the theory of the surface charge screening effect nor with a mechanism involving channel block. It seems more likely that Zn2+ interferes with the channel gating by binding to a specific domain of the channel protein. 7. After treatment with Hg2+, which is irreversible, Zn2+ still maintains its effects, which suggest that the two divalents act at different sites. 8. In view of the widespread distribution of zinc throughout the brain, its actions on the A-current could play an important role in physiological function.
Asunto(s)
Cerebelo/metabolismo , Canales Iónicos/metabolismo , Neuronas/metabolismo , Zinc/farmacología , Animales , Cerebelo/citología , Cerebelo/efectos de los fármacos , Electrofisiología , Técnicas In Vitro , Canales Iónicos/efectos de los fármacos , Cinética , Mercurio/farmacología , Conformación Molecular , Ratas , Zinc/metabolismoRESUMEN
1. Whole-cell voltage-clamp techniques were used to study voltage-activated transient potassium currents in a large sample (n = 143) of granule cells (GrC) from rat cerebellar slices. Tetrodotoxin (TTX; 0.1 microM) was used to block sodium currents, while calcium current was too small to be seen under ordinary conditions. 2. Depolarizing pulses from -50 mV evoked a slow, sustained outward current, developing with a time constant of 10 ms, inactivating over a time scale of seconds and which could be suppressed by 20 mM tetraethylammonium (TEA). By preventing the Ca2+ inflow, this slow outward current could be further separated into a Ca(2+)-dependent and a Ca(2+)-independent component. 3. After conditioning hyperpolarizations to potentials negative to -60 mV, depolarizations elicited transient outward current, peaking in 1-2 ms and inactivating rapidly (approximately 10 ms at 20 degrees C), showing the overall kinetic characteristics of the A-current (IA). The current activated following third-order kinetics and showed a maximal conductance of 12 nS per cell, corresponding to a normalized conductance of 3.8 nS/pF. 4. IA was insensitive to TEA and to the Ca(2+)-channel blockers. 4-Aminopyridine (4-AP) reduced the A-current amplitude by approximately 20%, and the delayed outward currents by > 80%. 5. Voltage-dependent steady-state inactivation of peak IA was described by a Boltzmann function with a slope factor of 8.4 mV and half-inactivation occurring at -78.8 mV. Activation of IA was characterized by a Boltzmann curve with the midpoint at -46.7 mV and with a slope factor of 19.8 mV. 6. IA activation and inactivation was best fitted by the Hodgkin-Huxley m3h formalism. The rate of activation, tau a, was voltage-dependent, and had values ranging from 0.55 ms at -40 mV to 0.2 ms at +50 mV. Double-pulse experiment showed that development and removal of inactivation followed a single-exponential time course; the inactivation time constant, tau ha, was markedly voltage-dependent and ranged from approximately 10 ms at -40 and -100 mV and 70 ms at -70 mV. 7. A set of continuous equations has been developed describing the voltage-dependence of both the steady-state and time constant of activation and inactivation processes, allowing a satisfactory numerical reconstruction of the A-current over the physiologically significant membrane voltage range.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Cerebelo/metabolismo , Canales de Potasio/metabolismo , 4-Aminopiridina/farmacología , Animales , Bario/farmacología , Cerebelo/citología , Cesio/farmacología , Gránulos Citoplasmáticos/metabolismo , Electrofisiología , Técnicas In Vitro , Cinética , Modelos Neurológicos , Neuronas/metabolismo , Potasio/metabolismo , Ratas , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Tetrodotoxina/farmacologíaRESUMEN
The effect of Zn2+ 100 microM-1 mM has been studied on the kinetics of the A-current in granule cells from rat cerebellar slices using the patch-clamp technique in the whole-cell configuration. Zn2+ induced marked shifts towards positive potentials of both the activation and inactivation steady-state curves, a reduction of maximal amplitude and a slowing of the activation kinetics, leaving unaffected the inactivation time constants. These modifications cannot be explained in terms of the screening of the negative surface charges, but are probably due to a direct action on the A-channel. The alterations observed in the IA kinetics could be of physiological relevance in some neurological disorders for which significant increase of the Zn2+ levels in the cerebrospinal fluid have been described.
Asunto(s)
Corteza Cerebelosa/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Zinc/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Corteza Cerebelosa/citología , Corteza Cerebelosa/fisiología , Espacio Extracelular/fisiología , Potasio/metabolismo , RatasRESUMEN
1. The electrophysiological effects of a pumiliotoxin-B-like alkaloid extracted from the skin of the Australian frog Pseudophryne coriacea (PsC) have been studied in rat superior cervical ganglia at 37 degrees C. 2. PsC (50 mg/ml) elicits a broadening of the evoked compound action potential and, at rest, the appearance of spontaneous spike discharge at 10-20 Hz. Action potentials presumably originate far away from the soma, which is invaded in a typical IS-SD sequence. 3. The toxin effect is not related to any direct action on the preganglionic fibers of the sympathetic trunk, and does not involve synaptic mechanisms. 4. Two-electrode voltage-clamp experiments showed that the main properties of the major voltage-dependent ionic currents are apparently unaffected by the toxin, while the cell input resistance is considerably reduced. 5. The data are consistent with the hypothesis that PsC elicits a cationic permeability increase generating a pacemaker current in a region close to the cell soma.
Asunto(s)
Alcaloides/aislamiento & purificación , Venenos de Anfibios , Anuros , Neurotoxinas/aislamiento & purificación , Neurotoxinas/farmacología , Piel/química , Potenciales de Acción/efectos de los fármacos , Alcaloides/farmacología , Animales , Conductividad Eléctrica , Electrofisiología , Ganglios Simpáticos/efectos de los fármacos , Ganglios Simpáticos/fisiología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Ratas , Ratas Wistar , Sinapsis/efectos de los fármacos , Sinapsis/fisiologíaRESUMEN
Thin slices were prepared from cerebella of 10-24 day old rats and examined with whole-cell patch-clamp methods. Depolarizing steps from holding potentials negative to -60 mV elicited an early transient outward current, identified as IA, and a late outward K+ current. Depolarizations from -50 mV failed to evoke any A current and gave only a slowly rising component similar to the delayed K+ current, which inactivated thereafter with a time constant of 2.5 s at -30 mV. The IA peaked in 1-2 ms, decayed following a double exponential with time constants of 8.1 and 53.2 ms at +20 mV and was half-inactivated at -82.5 mV. 4-AP 4 mM depressed both K+ currents showing little specificity between them, while TEA 20 mM selectively abolished only the delayed K+ current.