RESUMEN
As part of the road widening scheme between London and Dover, Oxford Archaeology South uncovered a large boundary ditch of Iron Age origin that contained Iron Age and Roman inhumations, adjacent to which was a small mid-late Roman cemetery, interpreted as a rural cemetery for Romano-British farmers. Grave goods in the cemetery were restricted to a few individuals with hobnailed boots. Bulk bone collagen isotopic analysis of 11 skeletons of Iron Age and Roman date gave a typical C(3) terrestrial signal (average δ(13) C = -19.8, δ(15) N = 9.3), but also revealed one (SK12671) with a diet which included a substantial C(4) component (δ(13) C = -15.2, δ(15) N = 11.2). This is only the second such diet reported in Roman Britain. Subsequent δ(18) O(c) and (87) Sr/(86) Sr measurements on the dental enamel in this individual were, however, consistent with a "local" origin, indicating that either C(4) protein was consumed in Late Roman Britain, or that he came from somewhere else, but where conditions gave rise to similar isotopic values. If we accept the latter, then it indicates that using oxygen and strontium isotopes alone to identify "incomers" may be problematic. The provision of hobnailed boots for the dead appears to have had a strong symbolic element in Late Roman Britain. We suggest that in this case the boots may be significant, in that he was being equipped for the long march home.
Asunto(s)
Arqueología/métodos , Entierro/historia , Isótopos/análisis , Mundo Romano/historia , Zapatos , Adolescente , Adulto , Aminoácidos/análisis , Niño , Colágeno Tipo I/química , Esmalte Dental/química , Dieta , Inglaterra , Femenino , Historia Antigua , Humanos , Lactante , Masculino , Espectrometría de Masas , Proteínas de PlantasRESUMEN
The expression of ATP-sensitive K(+) (K(ATP)) channels by magnocellular cholinergic basal forebrain (BF) neurones was investigated in thin brain slice and dissociated cell culture preparations using a combination of whole-cell, perforated-patch and single-channel recording techniques. Greater than 95% of BF neurones expressed functional K(ATP) channels whose activation resulted in membrane hyperpolarization and a profound fall in excitability. The whole-cell K(ATP) conductance was 14.0 +/- 1.5 nS and had a reversal potential of -91.4 +/- 0.9 mV that shifted by 59.6 mV with a tenfold increase in [K(+)](o). I(KATP) was inhibited reversibly by tolbutamide (IC(50) of 34.1 microM) and irreversibly by glibenclamide (0.3-3 nM) and had a low affinity for [ATP](i) (67% reduction with 6 mm[MgATP](i)). Using perforated-patch recording, a small proportion of the conductance was found to be tonically active. This was weakly potentiated by diazoxide (0.1 mm extracellular glucose) but insensitive to pinacidil (< or =500 microM). Single-channel K(ATP) currents recorded in symmetrical 140 mm K(+)-containing solutions exhibited weak inward rectification with a mean conductance of 66.2 +/- 1.9 pS. Channel activity was inhibited by MgATP (>50 microM) and activated by MgADP (200 microM). The K(+) channels opener diazoxide (200-500 microM) increased channel opening probability (NP(o)) by 486 +/- 120% whereas pinacidil (500 microM) had no effect. In conclusion, the characteristics of the K(ATP) channels expressed by BF neurones are very similar to channels composed of SUR1 and Kir6.2 subunits. In the native cell, their affinity for ATP is close to the resting [ATP](i), potentially allowing them to be modulated by physiologically relevant changes in [ATP](i). The effect of these channels on the level of ascending cholinergic excitation of the cortex and hippocampus is discussed.
Asunto(s)
Potenciales de Acción/fisiología , Adenosina Trifosfato/fisiología , Fibras Colinérgicas/fisiología , Proteínas de la Membrana/fisiología , Prosencéfalo/fisiología , Potenciales de Acción/efectos de los fármacos , Adenosina Trifosfato/farmacología , Animales , Células Cultivadas , Fibras Colinérgicas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Neuronas/efectos de los fármacos , Neuronas/fisiología , Canales de Potasio , Prosencéfalo/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
1. The Ca2+ channel subtypes controlling ACh release from basal forebrain neurones and the ionic basis underlying muscarinic receptor-mediated autoinhibition were studied using skeletal myoballs to detect ACh release from individual rat basal forebrain neurones in culture. 2. Somatic Ca2+ currents evoked using a simulated action potential waveform revealed that Ca2+ entry was primarily through N-, Q- and to a lesser extent R-, T- and L-type Ca2+ channels. 3. Muscarine (10 microM) inhibited N- and Q- but not R-, T- or L-type somatic Ca2+ channels. Agonist inhibition was totally blocked by pre-treatment with pertussis toxin (500 ng ml-1). 4. ACh release from discrete sites along basal forebrain neurites (1. 2 mM extracellular Ca2+) could be largely abolished by blocking Ca2+ entry through either N-type or Q-type Ca2+ channels. Inhibition of Ca2+ entry through L- or T-type channels had no effect upon release. Following inhibition of either N- or Q-type Ca2+ channels, release could be restored to near control levels by raising [Ca2+]o. After selectively blocking N-, Q-, L- and T-type channels, low levels of release could still be evoked as a result of Ca2+ entry through R-type Ca2+ channels. 5. Muscarinic receptor activation reversibly inhibited ACh release due to Ca2+ entry through N-, Q- and R-type Ca2+ channels. In contrast, inhibition of inwardly rectifying K+ channels using Ba2+ (3-10 microM) or substance P (0.03-0.1 microM), or block of SK or BK Ca2+-activated K+ channels with apamin (100 nM) or charbydotoxin (100 nM) respectively, had no effect upon either ACh release or its modulation by muscarinic agonists. 6. These results show that ACh release from individual release sites on basal forebrain neurones is controlled by multiple Ca2+ channel subtypes with overlapping Ca2+ microdomains and that autoinhibition of release results from M2 muscarinic receptor-mediated inhibition of these presynaptic Ca2+ channels rather than as a consequence of K+ channel activation.
Asunto(s)
Acetilcolina/metabolismo , Canales de Calcio Tipo N , Canales de Calcio/fisiología , Neuronas/metabolismo , Prosencéfalo/citología , Prosencéfalo/metabolismo , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Estimulación Eléctrica , Electrofisiología , Retroalimentación/fisiología , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/fisiología , Agonistas Muscarínicos/farmacología , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio , Canales de Potasio/agonistas , Canales de Potasio/metabolismo , Ratas , Receptores Muscarínicos/metabolismoRESUMEN
Morphological and electrophysiological characteristics of magnocellular neurons from basal forebrain nuclei of postnatal rats (11-14 days old) were examined in dissociated cell culture. Neurons were maintained in culture for periods of 5-27 days, and 95% of magnocellular (>23 micron diam) neurons stained positive with acetylcholinesterase histochemistry. With the use of phase contrast microscopy, four morphological subtypes of magnocellular neurons could be distinguished according to the shape of their soma and pattern of dendritic branching. Corresponding passive and active membrane properties were investigated with the use of whole cell configuration of the patch-clamp technique. Neurons of all cell types displayed a prominent (6-39 mV; 6.7-50 ms duration) spike afterdepolarization (ADP), which in some cells reached firing threshold. The ADP was voltage dependent, increasing in amplitude and decreasing in duration with membrane hyperpolarization with an apparent reversal potential of -59 +/- 2.3 (SE) mV. Elevating [Ca2+]o (2.5-5.0 mM) or prolonging spike repolarization with 10 mM tetraethylammonium (TEA) or 1 mM 4-aminopyridine (4-AP), potentiated the ADP while it was inhibited by reducing [Ca2+]o (2.5-1 mM) or superfusion with Cd2+ (100 microM). The ADP was selectively inhibited by amiloride (0.1-0.3 mM or Ni2+ 10 microM) but unaffected by nifedipine (3 microM), omega-conotoxin GVIA (100 nM) or omega-agatoxin IVA (200 nM), indicating that Ca2+ entry was through T-type Ca2+ channels. After inhibition of the ADP with amiloride (300 microM), depolarization to less than -65 mV revealed a spike afterhyperpolarization (AHP) with both fast and slow components that could be inhibited by 4-AP (1 mM) and Cd2+ (100 microM), respectively. In all cell types, current-voltage relationships exhibited inward rectification at hyperpolarized potentials >/=EK (approximately -90 mV). Application of Cs+ (0.1-1 mM) or Ba2+ (1-10 microM) selectively inhibited inward rectification but had no effect on resting potential or cell excitability. At higher concentrations, Ba2+ (>10 microM) also inhibited an outward current tonically active at resting potential (VH -70 mV), which under current-clamp conditions resulted in small membrane depolarization (3-10 mV) and an increase in cell excitability. Depolarizing voltage commands from prepulse potential of -90 mV (VH -70 mV) in the presence of tetrodotoxin (0.5 microM) and Cd2+ (100 microM) to potentials between -40 and +40 mV cause voltage activation of both transient A-type and sustained delayed rectifier-type outward currents, which could be selectively inhibited by 4-AP (0.3-3 mM) and TEA (1-3 mM), respectively. These results show that, although acetylcholinesterase-positive magnocellular basal forebrain neurons exhibit considerable morphological heterogeneity, they have very similar and characteristic electrophysiological properties.
Asunto(s)
Neuronas/citología , Neuronas/fisiología , Prosencéfalo/citología , Prosencéfalo/fisiología , Potenciales de Acción/fisiología , Animales , Bario/farmacología , Calcio/fisiología , Células Cultivadas , Cesio/farmacología , Neuronas/clasificación , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-DawleyRESUMEN
1. Ionotropic glutamate receptor-mediated responses were recorded from rat magnocellular basal forebrain neurones under voltage clamp from a somatically located patch-clamp pipette. Currents were recorded from both acutely dissociated neurones and neurones maintained in culture for up to 6 weeks. 2. Non-NMDA and NMDA receptor-mediated events could be distinguished pharmacologically using the selective agonists (S)-alpha-amino-3-hydroxy-5-methyl-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA), and antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D(-)-2-amino-5-phosphonopentanoic acid (AP5). 3. Responses to rapid application of AMPA displayed pronounced and rapid desensitization. Responses to kainate showed no desensitization. Steady-state EC50 values for AMPA and kainate were 2.7 +/- 0.4 microM (n = 5) and 138 +/- 25 microM (n = 10), respectively. Cyclothiazide markedly increased current amplitude of responses to both agonists, whereas concanavalin A had no clear effect on either response. The selective AMPA receptor antagonist GYKI 53655 inhibited responses to kainate with an IC50 of 1.2 +/- 0.08 microM (n = 5) at -70 mV. These data strongly suggest that AMPA receptors are the predominant non-NMDA receptors expressed by basal forebrain neurones. 4. At -70 mV, approximately 6 % of control current amplitude remained, at a maximally effective concentration of GYKI 53655. This residual response displayed desensitization, was insensitive to cyclothiazide and was potentiated by concanavalin A, suggesting that it was mediated by a kainate receptor. 5. Current-voltage relationships for non-NMDA receptor-mediated currents were obtained from both nucleated patches pulled from neurones in culture and from acutely dissociated neurones. With 30 microM spermine in the recording pipette, currents frequently displayed double-rectification characteristic of non-NMDA receptors with high Ca2+ permeabilities. Ca2+ permeability, relative to Na+ and Cs+, was investigated using constant field theory. The measured Ca2+ to Na+ permeability coefficient ratio was 0.26-3.6; median, 1.27 (n = 15). 6. Current flow through non-NMDA receptors was inhibited by Ca2+, Cd2+ and Co2+ ions. At a holding potential of -70 mV, a maximally effective concentration of Cd2+ (> 30 mM) reduced current amplitude by approximately 90 %, with an IC50 of 44 microM. In six out of seven cells tested, block by Cd2+ was voltage sensitive. 7. Ca2+ permeability of many of the non-NMDA receptors expressed by magnocellular basal forebrain neurones may underlie the unusual sensitivity of cholinergic basal forebrain neurones to non-NMDA receptor-mediated excitotoxicity.
Asunto(s)
Ganglios Basales/metabolismo , Calcio/metabolismo , Neuronas/metabolismo , Prosencéfalo/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Ganglios Basales/citología , Ganglios Basales/efectos de los fármacos , Benzodiazepinas/farmacología , Benzotiadiazinas/farmacología , Cationes Bivalentes/metabolismo , Cationes Bivalentes/farmacología , Células Cultivadas , Concanavalina A/farmacología , Diuréticos , Electrofisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Prosencéfalo/citología , Prosencéfalo/efectos de los fármacos , Ratas , Receptores AMPA/agonistas , Receptores AMPA/antagonistas & inhibidores , Receptores AMPA/metabolismo , Receptores de Ácido Kaínico/agonistas , Receptores de Ácido Kaínico/antagonistas & inhibidores , Receptores de Ácido Kaínico/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Inhibidores de los Simportadores del Cloruro de Sodio/farmacologíaRESUMEN
A wide variety of techniques have been employed for the detection and measurement of neurotransmitter release from biological preparations. Whilst many of these methods offer impressive levels of sensitivity, few are able to combine sensitivity with the necessary temporal and spatial resolution required to study quantal release from single cells. One detection method that is seeing a revival of interest and has the potential to fill this niche is the so-called 'sniffer-patch' technique. In this article, specific examples of the practical aspects of using this technique are discussed along with the procedures involved in calibrating these biosensors to extend their applications to provide quantitative, in addition to simple qualitative, measurements of quantal transmitter release.
Asunto(s)
Técnicas Biosensibles , Neurociencias/métodos , Neurotransmisores/metabolismo , Animales , Calibración , Humanos , Factores de TiempoRESUMEN
The receptor subtype and transduction mechanisms involved in the regulation of various neuronal ionic currents are reviewed, with some recent observations on sympathetic neurons, hippocampal cell membranes and basal forebrain cells.
Asunto(s)
Canales de Calcio/fisiología , Neuronas/fisiología , Canales de Potasio/fisiología , Receptores Muscarínicos/fisiología , Animales , Proteínas de Unión al GTP/fisiología , HumanosRESUMEN
1. Nicotinic acetylcholine (ACh) receptor-rich patches prepared from rat myotubes were used as focal ACh detectors to record the release of ACh from magnocellular basal forebrain (MBF) neurones from 11- to 14-day-old postnatal rats maintained in dissociated cell culture. 2. An action potential generated by intracellularly stimulating the MBF cell soma through a patch electrode induced a brief (mean tau(decay), 6.3 ms) short latency (1.35-5.1 ms; median 3.1 ms) burst of nicotinic channel openings in the detector patch when the latter was positioned at discrete loci along the MBF neurites. Detected ACh concentrations ranged from approximately 480 nM to > 50 microM. Concentrations increased markedly during the first 14 days in vitro and were inversely related to response latency. 3. Sites of release were generally confined to the more proximal neurites within 100 microm of the cell body and were invariably associated with the presence of small (2-3 microm diameter) phase-dark puncta located at discrete intervals along the length of the neurites or at points where short collaterals branched from the main process. Release was never detected from the cell soma except under extreme non-physiological conditions but could occasionally be elicited from growth cones at the ends of the shorter thicker neurites in the absence of a target cell. 4. Evoked release was abolished by tetrodotoxin (0.5 microM) and by superfusing with low Ca(2+)-high Mg(2+)-containing solutions (0.25 mM Ca(2+), 5 mM Mg(2+)). Myotube patch responses were antagonized by d-tubocurarine (3 microM). 5. Muscarine (10 microM) inhibited release by 70 +/- 3% (n = 12 cells). This effect was antagonized by 100 nM methoctramine but not by 100 nM pirenzepine, indicating that it was mediated by M(2) muscarinic ACh receptors. 6. These results indicate that ACh release from the processes of magnocellular cholinergic basal forebrain neurones arises from highly specialized and discrete sites, and that it can be inhibited through activation of muscarinic receptors. It is suggested that the latter results from inhibition of presynaptic Ca(2+) channels and that it might be responsible for feedback autoinhibition of ACh release from cortical afferents of nucleus basalis neurones in vivo.
Asunto(s)
Acetilcolina/metabolismo , Neuritas/metabolismo , Prosencéfalo/metabolismo , Animales , Animales Recién Nacidos , Autorreceptores/fisiología , Células Cultivadas , Estimulación Eléctrica , Electrofisiología , Prosencéfalo/citología , Prosencéfalo/fisiología , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción , Receptores Muscarínicos/fisiologíaRESUMEN
1. The actions of muscarinic agonists and antagonists upon the Ca2+ current (ICa) in acutely dissociated magnocellular cholinergic basal forebrain neurones from 11 to 14-day-old postnatal rats were studied using the whole-cell patch-clamp technique. 2. In all cells studied, muscarinic agonists inhibited a transient component of high-voltage-activated (HVA) current, but had no effect upon the low-voltage-activated (LVA) current. The mean IC50 values for ACh and oxotremorine methiodide (oxo-M), obtained from non-cumulative dose-response curves, were 204 and 363 nM respectively. Superfusion with the K+ channel blocker, tetraethylammonium chloride (TEA; 30 mM) shifted the ACh dose-response curve to the right giving an IC50 value of 22:9 microM. 3. Pirenzepine (0.1-1 microM) and methoctramine (0.03-0.3 microM) produced parallel shifts to the right in the agonist dose-response curves. Schild analysis of the agonist dose ratios yielded pA2 (negative log of the apparent dissociation constant KB) values for pirenzepine and methoctramine of 6.8 and 8.2 respectively, indicating the involvement of an M2 receptor subtype. 4. In the presence of GTP-gamma-S (10-500 microM) in the patch pipette, the agonist-induced inhibition of ICa became irreversible. Dialysis with GDP-beta-S (1 mM) abolished all agonist-induced inhibition of the Ca2+ current. The agonist-induced inhibition of ICa was totally blocked by pretreatment with pertussis toxin (500 ng ml-1) but unaffected by preincubation with cholera toxin (500 ng ml-1). Superfusion with 8-bromo cAMP (0.5-1 mM) did not mimic or prevent the effect of agonist application. 5. Inhibition of the Ca2+ current by muscarinic agonists was only partially blocked by omega-conotoxin GVIA (omega-CgTX GVIA), with approximately 46% of the agonist-sensitive current being resistant to omega-CgTX GVIA. Both the agonist- and omega-CgTX GVIA-sensitive components of the current were abolished following maximal inhibition of ICa by GTP-gamma-S. 6. These results indicate that inhibition of the Ca2+ current by muscarinic agonists is mediated via an M2 muscarinic receptor coupled to a pertussis toxin-sensitive G-protein. The possible modulation of multiple HVA Ca2+ channels by muscarinic agonists and the role that these receptors may play in presynaptic modulation of neurotransmitter release are discussed.
Asunto(s)
Canales de Calcio/metabolismo , Prosencéfalo/metabolismo , Receptores Muscarínicos/metabolismo , Acetilcolina/farmacología , Animales , Bloqueadores de los Canales de Calcio , Canales de Calcio/efectos de los fármacos , Toxina del Cólera/farmacología , Fibras Colinérgicas/efectos de los fármacos , Fibras Colinérgicas/metabolismo , Diaminas/farmacología , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Técnicas In Vitro , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Oxotremorina/farmacología , Parasimpatolíticos/farmacología , Péptidos/farmacología , Toxina del Pertussis , Pirenzepina/farmacología , Ratas , Receptores Muscarínicos/clasificación , Receptores Muscarínicos/efectos de los fármacos , Factores de Virulencia de Bordetella/farmacología , omega-Conotoxina GVIARESUMEN
1. The electrophysiological and pharmacological characteristics of the calcium current (ICa) in acutely dissociated magnocellular cholinergic basal forebrain neurones from 11- to 14-day-old post-natal rats were studied using the whole-cell patch-clamp technique. 2. All cells exhibited a small transient low-voltage-activated (LVA) current with half-activation and half-inactivation potentials of -40.2 and -49.3 mV and slope factors for activation and inactivation of 4.82 and 3.85 mV per e-fold change in membrane potential (Vm) respectively. Activation and inactivation rates for the LVA current were highly voltage dependent. For test potential changes from -50 to -20 mV, the time-to-peak of the current decreased from 39.1 to 6.4 ms, and the time constant of current decay decreased from 81.7 to 15.5 ms. 3. A high-voltage-activated (HVA) component of ICa could be elicited at threshold voltages between -46 and -30 mV from a holding potential (VH) of -80 mV. The HVA current peaked around 0 mV; a 10-fold increase in [Ca2+]o produced a 13 mV positive shift in the peak, whilst the amplitude of the current showed an approximately hyperbolic relationship to [Ca2+]o with half-saturation at 2.5 mM. The transient phase of the HVA current could be described by two exponential functions with time constants tau fast and tau slow of 16.2 and 301 ms. Steady-state inactivation of the transient and extrapolated true sustained (pedestal) components of HVA current were described by Boltzmann equations, with half-inactivation potentials (slope factors) of -47.3 mV, (9.04) and -29.2 mV (11.8) respectively. 4. omega-Conotoxin (omega-CgTX; 100 nM) irreversibly inhibited a kinetically distinct component of HVA current but had no effect upon the transient LVA current. The omega-CgTX-sensitive current could not be distinguished from the control HVA current by the voltage dependence of its activation or inactivation rates. 5. Low concentrations of amiloride (< or = 300 microM) or Ni2+ (< or = 5 microM) selectively inhibited the transient LVA current, with IC50 values of 97 and 5 microM respectively. Cd2+ (< or = 1 microM) selectively blocked a component of HVA current. At higher concentrations, Cd2+ and Ni2+ were non-selective and totally blocked all components of ICa. 6. The lanthanide ions Gd3+ and La3+ produced saturable incomplete block of the HVA current. Maximally effective concentrations of Gd3+ (100 microM) or La3+ (30 microM) inhibited 76.5 and 41.2% respectively of the sustained component of HVA current with corresponding IC50 values of 2.2 and 1.1 microM.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
ATPasas Transportadoras de Calcio/fisiología , Fibras Colinérgicas/metabolismo , Conotoxinas , Prosencéfalo/metabolismo , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Amilorida/farmacología , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Fibras Colinérgicas/efectos de los fármacos , Fibras Colinérgicas/ultraestructura , Iones , Cinética , Potenciales de la Membrana/fisiología , Níquel/farmacología , Nifedipino/farmacología , Péptidos Cíclicos/farmacología , Ratas , Ratas Sprague-DawleyAsunto(s)
Ganglios Autónomos/citología , Plexo Mientérico/citología , Plexo Submucoso/citología , Animales , Bronquios/inervación , Técnicas de Cultivo/métodos , Electrofisiología , Corazón/inervación , Intestinos/inervación , Neuroglía/química , Neurotransmisores/análisis , Receptores de Superficie Celular/análisis , Vejiga Urinaria/inervaciónRESUMEN
1. The actions of gamma-aminobutyric acid (GABA) on the intramural neurones of 14-18 day old rats were studied in situ by use of intracellular current- and voltage-clamp techniques. The ionic conductance changes and the effects of various GABA-receptor agonists and antagonists on these neurones were also investigated. 2. Prolonged application of GABA either by ionophoresis (10 pC-10 nC) or superfusion (10-100 microM), evoked a biphasic membrane depolarization in over 90% of all paratracheal neurones studied. Typically, the response consisted of an initial rapid depolarization (18-45 ms) that subsequently faded over a period of 15-25 s to reveal a second smaller depolarization which was maintained for the duration of GABA application. Both components of the evoked response resulted in an increase in membrane conductance and an inward flow of current. 3. The amplitude of the transient inward current, recorded during the initial phase of the response, was linearly related to the membrane potential at which it was elicited and reversed symmetrically at a membrane potential of -32.7 mV. The underlying increase in conductance was largely independent of membrane potential. The equilibrium potential for the sustained inward current was -38.7 mV. Replacement of extracellular chloride with gluconate ions initially enhanced the GABA-evoked inward current. With successive applications of GABA in low chloride, the evoked current and conductance changes declined markedly. 4. Muscimol superfusion (1-10 microM) or ionophoresis (10 pC-10 nC) mimicked both the initial and late phases of the GABA-induced conductance change and inward current. Baclofen (1-100 microM) had no effect upon either resting membrane potential or conductance in any of the cells tested. 5. The large transient initial phase of the GABA-evoked inward current and depolarization were potently inhibited by picrotoxin (1-50 microM), whereas the smaller sustained inward current was largely resistant to picrotoxin. 6. All of the observed actions of GABA and muscimol were antagonized by bicuculline (0.1-10 microM) in an apparently competitive manner. 7. It is concluded that GABA acts via GABAA receptors present on the soma of paratracheal neurones to produce an increase in membrane chloride conductance. Prolonged application of GABA results in a decline in the observed current due to a combination of two processes: receptor desensitization and shifts in the chloride equilibrium potential. The possible roles for GABA in neural regulation of airway excitability are discussed.
Asunto(s)
Membrana Celular/metabolismo , Cloruros/metabolismo , Neuronas/metabolismo , Receptores de GABA-A/metabolismo , Tráquea/inervación , Animales , Bicuculina/farmacología , Electrofisiología , Potenciales Evocados/efectos de los fármacos , Antagonistas del GABA , Técnicas In Vitro , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Muscimol/farmacología , Picrotoxina/farmacología , Ratas , Ratas Endogámicas , Receptores de GABA-A/efectos de los fármacos , Tetrodotoxina/farmacología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
1. The actions of adenosine 5'-triphosphate (ATP) and related nucleotides and nucleosides on the membrane ion conductances of M and AH type intracardiac neurones cultured from ganglia within the atria and interatrial septum of newborn guinea-pig heart were studied with intracellular current- and voltage-clamp techniques. 2. Approximately 74% (120 out of 161) of AH type cells and 41% (5 out of 12) M cells responded to direct application of ATP (500 microM) onto their soma. 3. In 41% of M and 43% of AH type cells, focal application of ATP (500 microM) evoked rapid depolarization with an increase in conductance which frequently elicited action potential discharge. The underlying inward current had a null potential of -11.2 mV and was reduced in solutions containing low extracellular sodium and calcium but unaffected by reduced chloride-containing solutions. 4. In a further 31% of AH type cells, ATP evoked a multi-component response consisting of an initial depolarization followed by a hyperpolarization and a slow prolonged depolarization. The current underlying the initial depolarization resulted from an increase in conductance and had a null potential of -19.1 mV. The current was increased in low chloride-containing solutions and was only slightly reduced in low sodium- and calcium-containing solutions. The subsequent hyperpolarization and outward current resulted from an increase in membrane conductance and had a null potential of -88.5 mV, which was close to the potassium equilibrium potential in these cells. The slow depolarization and inward current was not associated with change in membrane conductance. 5. In less than 2% of AH cells, ATP evoked a second type of slow depolarization. This was associated with a fall in conductance and had a null potential of -90.7 mV. 6. In 40% of AH cells, adenosine (10-100 microM) inhibited the calcium-sensitive potassium current responsible for the after-hyperpolarization. The action of adenosine was antagonized by the P1-purinoceptor antagonist 8-phenyltheophylline (1-10 microM). 7. The potency order of agonists for all of the ATP-evoked responses, except the slow depolarization associated with a fall in conductance was ATP > ADP with AMP and adenosine being ineffective. 8. Responses to ATP were only weakly desensitized by alpha,,Beta-methylene ATP (3 x 10-6 M) and the potency order of analogues was 2-methylthio ATP > ATP > alpha,beta-methylene ATP, indicating the involvement of receptors similar to P2Y purinoceptors.
Asunto(s)
Adenosina Trifosfato/farmacología , Corazón/inervación , Neuronas/efectos de los fármacos , Adenosina/farmacología , Adenosina Difosfato/farmacología , Adenosina Monofosfato/farmacología , Adenosina Trifosfato/análogos & derivados , Animales , Animales Recién Nacidos/fisiología , Células Cultivadas , Electrodos , Cobayas , Potenciales de la Membrana/efectos de los fármacos , Nucleótidos/farmacología , Canales de Potasio/efectos de los fármacosRESUMEN
1. The electrophysiological characteristics of intramural neurones from the paratracheal ganglia of 14- to 18-day-old rats were studied in vitro using intracellular, single-electrode current- and voltage-clamp techniques. 2. Resting membrane potentials ranged between -50 and -73 mV. In 50-60% of all neurones, random and occasionally patterned bursts of spontaneous, fast synaptic potentials were observed. In all cases, superfusion with either hexamethonium (100 microM), or Ca2(+)-free, high-magnesium-containing solutions abolished all synaptic activity. 3. Two distinct patterns of spike discharge were observed in response to prolonged intrasomal current injection. Most cells (65-75%) fired rhythmic, high-frequency (50-90 Hz) bursts of action potentials, with interburst intervals of between 300 and 500 ms, throughout the period of current stimulation. A further 10-15% of cells fired tonically at low frequencies (10-15 Hz) for the duration of the applied stimulus. In both cell types, trains of action potentials were followed by a pronounced calcium-dependent after-hyperpolarization which persisted for up to 3 s. The magnitude of the after-hyperpolarization following a single spike in tonic-firing cells was considerably larger than in burst-firing cells. Both the action potential and the after-hyperpolarization in all cells displayed a calcium-dependent, tetrodotoxin-resistant component which was abolished by the removal of the extracellular calcium. 4. The spike after-hyperpolarization resulted from activation of an outward calcium-dependent potassium current which reversed at -86.5 mV. This value was shifted by 63.6 mV for a 10-fold increase in extracellular potassium concentration. 5. All of the cells studied exhibited marked outward rectification when depolarized. This resulted from activation of a time- and voltage-dependent M-current. The slow inward current relaxations associated with the M-current became faster at more negative potentials and reversed around -85 mV. Raising the extracellular potassium concentration shifted the reversal potential for the current relaxations to more depolarized potentials in a manner predicted by the Nernst equation for a current carried by potassium ions. Both the outward current at depolarized potentials and the slow current relaxations were potently inhibited by extracellular BaCl2 (1 mM) but were unaffected by CsCl (1-3 mM). 6. Inward rectification at hyperpolarized potentials was a characteristic of all cells. Membrane hyperpolarization revealed inward rectification in the 'instantaneous' current-voltage relationship at membrane potentials greater than -80 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Tráquea/inervación , Potenciales de Acción/fisiología , Animales , Cationes , Estimulación Eléctrica , Electrofisiología , Potenciales Evocados/fisiología , Femenino , Ganglios/fisiología , Técnicas In Vitro , Masculino , Potenciales de la Membrana/fisiología , Neuronas/fisiología , Ratas , Ratas Endogámicas , Sinapsis/fisiologíaRESUMEN
1. The effects of muscarine upon intracardiac neurones cultured from ganglia within the atria and interatrial septum of the newborn guinea-pig heart were studied using intracellular recording techniques. 2. Muscarine applied to the neuronal soma typically produced a biphasic change in membrane potential which consisted of a small hyperpolarization followed by a depolarization. In addition, muscarine (0.01-10 microM) inhibited the calcium-dependent, after-hyperpolarization (AHP) and greatly increased the number of action potentials that could be evoked by a given depolarizing current. 3. The hyperpolarization was associated with a decrease in input resistance and it reversed to become a depolarization at a potential of -86.5 mV. This response was antagonized by 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP; 100 nM) and AF-DX 116 (500 nM), but was unaffected by pirenzepine (0.1-5 microM). 4. Two types of slow depolarization were observed in the presence of muscarine. The most common was associated with an increase in input resistance in the potential range -70 to -40 mV. Pirenzepine (100 nM) selectively antagonized this response, 4-DAMP (100 nM) similarly antagonized the response, but was non-selective. AF-DX 116 (0.5-5 microM) showed no antagonist effect. The less common depolarization (5% of cells) had a long latency and was associated with a decrease in input resistance. 5. Muscarine reduced the duration of the action potential and inhibited the AHP. Cadmium chloride (100 microM) mimicked these actions of muscarine. Application of muscarine immediately following a train of action potentials did not inhibit the AHP, suggesting that muscarine did not directly inhibit the calcium-activated potassium current (IK(Ca)). Muscarine-induced depression of the slow AHP was antagonized by 4-DAMP (100 nM) but was not antagonized by either pirenzepine (0.1-0.5 microM) or AF-DX 116 (0.5-5 microM). 6. It is concluded that the muscarine-induced depolarization of guinea-pig intracardiac neurones results from reduction of a potassium conductance similar to the M-conductance, through activation of M1 muscarinic receptors. The hyperpolarization results from an increase in potassium conductance, through activation of M2 muscarinic receptors.
Asunto(s)
Ganglios/fisiología , Corazón/inervación , Inhibición Neural/fisiología , Neuronas/fisiología , Receptores Muscarínicos/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Células Cultivadas , Cobayas , Corazón/embriología , Potenciales de la Membrana/efectos de los fármacos , Muscarina/antagonistas & inhibidores , Muscarina/farmacología , Parasimpatolíticos/farmacología , Piperidinas/farmacología , Pirenzepina/análogos & derivados , Pirenzepina/farmacologíaRESUMEN
Cell and tissue culture preparations have a number of general advantages for the study of biological processes: cells are more accessible for study, diffusion delays and barriers to applied substances are minimised, the humoral and cellular components of the culture environment can be controlled and progressive changes in intracellular and intercellular events can be directly monitored. These significant advantages mean that culture preparations can provide unique opportunities for investigation of the properties and functions of regulatory peptides. Culture preparations also have disadvantages and not all cultures are suitable for use in all types of experiments; therefore, the choice of preparation must be made accordingly. Here we describe different types of culture preparation and give examples where cultures have been used to examine peptide synthesis, storage, secretion and receptor localisation, as well as the short-term and trophic actions of regulatory peptides.
Asunto(s)
Células Cultivadas , Técnicas de Cultivo/métodos , Hormonas/fisiología , Neuropéptidos/fisiología , Péptidos/fisiología , Animales , Electrofisiología , Hormonas/biosíntesis , Neuropéptidos/biosíntesis , Biosíntesis de PéptidosRESUMEN
Although our knowledge of the intrinsic innervation of the airways is at present rather limited, it would appear that these neurones have several features in common with other intramural ganglia. They show a level of electrophysiologic diversity that might permit considerable integration and modulation to take place. Furthermore, the localization of a wide variety of neuropeptides in the neurones and in fibers within the paratracheal ganglia indicates that they may also possess the neurochemical specialization necessary to allow these ganglia to act as sites of complex local regulation.
Asunto(s)
Neuronas/fisiología , Neuropéptidos/fisiología , Tráquea/inervación , Animales , Sistema Nervioso Autónomo/fisiología , Células Cultivadas , Electrofisiología , Hurones , Neurotransmisores/fisiología , RatasRESUMEN
1. The electrophysiological properties of intracardiac neurones cultured from ganglia within the atria and interatrial septum of the new-born guinea-pig heart were studied using intracellular micro-electrodes. 2. Three types of neurones with resting membrane potentials in the range -45 to -76 mV were detected. The first type, AHs cells, had high (15-28 mV) firing thresholds, pronounced slow post-spike after-hyperpolarizations and fired only once to prolonged intrasomal current injection. The second type, AHm cells, were similar to AHs cells, except that they could fire short bursts of spikes (100-400 ms) at the onset of current injection. The third type, M cells, had low firing thresholds (10-15 mV), no slow after-hyperpolarizations and produced non-adapting trains of action potentials in response to depolarizing current injection. 3. The generation of action potentials in M cells was prevented by tetrodotoxin (TTX; 0.3 microM), whereas in AHs and AHm cells action potentials displayed a channel blockade using solutions containing the divalent cations cadmium, cobalt or manganese (0.02-1 mM). 4. The post-spike after-hyperpolarization in AHs and AHm cells was abolished by the removal of extracellular calcium, shortened in solutions containing the calcium entry blockers CdCl2, MnSO4 and CoCl2 (0.02-1 mM) and prolonged by the addition of calcium (5.0 mM), tetraethylammonium (1-3 mM), 4-aminopyridine (1-3 mM), cyanide (10 microM) or caffeine (100 microM) to the perfusate. 5. The reversal potential of the post-spike after-hyperpolarization was -89.1 mV. This value changed by 62.9 mV for a 10-fold increase in extracellular potassium concentration. 6. The peak conductance change during the post-spike after-hyperpolarization (gK,Ca), was largely independent of membrane potential between -50 and -110 mV. The peak increase in gK,Ca and the duration of the after-hyperpolarization increased with the number of spikes preceding it. 7. It is concluded that calcium entry during the action potential is responsible for the activation of an outward potassium current in the two types of AH cells; the roles played by intracellular calcium extrusion as well as sequestration mechanisms in the generation of the response are discussed.
Asunto(s)
Corazón/inervación , Neuronas/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Calcio/farmacología , Calcio/fisiología , Células Cultivadas , Cobayas , Potasio/farmacología , Sodio/fisiología , Tetrodotoxina/farmacología , Factores de TiempoRESUMEN
The physiological effects of Semliki Forest virus (SFV) induced demyelination on the visual system of mice have been examined by recording electroretinograms (ERGs) and the spike activity in the retinal ganglion cell axons in control and SFV-infected mice. The amplitudes of ERGs evoked by a single flash in the dark-adapted eyes of the SFV infected mice were abnormally enhanced, whereas flicker ERGs were slightly depressed. This hyperactivity was also seen in the ganglion cell axonal discharge. Both visually evoked and spontaneous activity recorded from the optic tract fibres of the infected mice were characterized by rhythmic oscillatory bursts of firing. Similar patterns were seen only very rarely in the evoked activity recorded from control mice. We suggest that this oscillatory firing might be a functional correlate of the types of 'positive symptoms' reported by multiple sclerosis patients with optic neuritis.