Modulation of mechanosensory threshold in Aplysia by serotonin, small cardioactive peptideB (SCPB), FMRFamide, acetylcholine, and dopamine

Mechanosensory threshold of tail sensory neurons in Aplysia was tested after injecting the tail with neuromodulators known to affect defensive behavior in this animal. Serotonin (5-HT) and small cardioactive peptide (SCPB) reduced peripheral threshold, while FMRFamide, acetylcholine (ACh), and dopamine increased threshold. FMRFamide and ACh also reduced spontaneous activity of sensory neurons. Glutamate and taurine had no effect. SCPB effects persisted for 15-30 min after washout. Functional similarities between peripheral and central effects of these neuromodulators support the hypothesis of coordinate modulation of both regions of the sensory neuron by the same set of modulators following noxious stimulation.     

Acetylcholine modulates respiratory pattern: effects mediated by M3-like receptors in preBötzinger complex inspiratory neurons

Perturbations of cholinergic neurotransmission in the brain stem affect respiratory motor pattern both in vivo and in vitro; the underlying cellular mechanisms are unclear. Using a medullary slice preparation from neonatal rat that spontaneously generates respiratory rhythm, we patch-clamped inspiratory neurons in the preB?tzinger complex (preB?tC), the hypothesized site for respiratory rhythm generation, and simultaneously recorded respiratory-related motor output from the hypoglossal nerve (XIIn). Most (88%) of the inspiratory neurons tested responded to local application of acetylcholine (ACh) or carbachol (CCh) or bath application of muscarine. Bath application of 50 microM muscarine increased the frequency, amplitude, and duration of XIIn inspiratory bursts. At the cellular level, muscarine induced a tonic inward current, increased the duration, and decreased the amplitude of the phasic inspiratory inward currents in preB?tC inspiratory neurons recorded under voltage clamp at -60 mV. Muscarine also induced seizure-like activity evident during expiratory periods in XIIn activity; these effects were blocked by atropine. In the presence of tetrodotoxin (TTX), local ejection of 2 mM CCh or ACh onto preB?tC inspiratory neurons induced an inward current along with an increase in membrane conductance under voltage clamp and induced a depolarization under current clamp. This response was blocked by atropine in a concentration-dependent manner. Bath application of 1 microM pirenzepine, 10 microM gallamine, or 10 microM himbacine had little effect on the CCh-induced current, whereas 10 microM 4-diphenylacetoxy-N-methylpiperidine methiodide blocked the current. The current-voltage (I-V) relationship of the CCh-induced response was linear in the range of -110 to -20 mV and reversed at -11.4 mV. Similar responses were found in both pacemaker and nonpacemaker inspiratory neurons. The response to CCh was unaffected when patch electrodes contained a high concentration of EGTA (11 mM) or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (10 mM). The response to CCh was reduced greatly by substitution of 128 mM Tris-Cl for NaCl in the bath solution; the I-V curve shifted to the left and the reversal potential shifted to -47 mV. Lowering extracellular Cl(-) concentration from 140 to 70 mM had no effect on the reversal potential. These results suggest that in preB?tC inspiratory neurons, ACh acts on M3-like ACh receptors on the postsynaptic neurons to open a channel permeable to Na(+) and K(+) that is not Ca(2+) dependent. This inward cation current plays a major role in depolarizing preB?tC inspiratory neurons, including pacemakers, that may account for the ACh-induced increase in the frequency of respiratory motor output observed at the systems/behavioral level.     

Mechanoafferent neurons innervating tail of Aplysia. II. Modulation by sensitizing stimulation

The tail-withdrawal reflex of Aplysia can be sensitized by weak stimulation of a site outside the site used to test the reflex or by repeatedly stimulating the test site itself. The sensitization of tail-withdrawal responses is associated with enhanced activation of the tail motor neurons and heterosynaptic facilitation of the monosynaptic connections between the tail sensory neurons and tail motor neurons. This synaptic facilitation can occur under conditions in which neither posttetanic potentiation nor generalized changes in postsynaptic input resistance contribute to the facilitation. In addition to producing monosynaptic excitatory postsynaptic potentials (EPSPs), action potentials in tail sensory neurons often recruit longer latency polysynaptic input to the tail motor neurons during sensitization. Strong, noxious tail shock similar in intensity to that used previously for sensitization and aversive classical conditioning of other responses in Aplysia produces more heterosynaptic facilitation than does weak sensitizing stimulation. Heterosynaptic facilitation builds up progressively with multiple trials and lasts for hours. Very strong shocks to the tail can change the response characteristics of tail sensory neurons so that a prolonged, regenerative burst of spikes is elicited by a brief intracellular depolarizing pulse. This bursting response produced by sensitizing stimulation has not been described previously in Aplysia sensory neurons and can greatly amplify the synaptic input to tail motor neurons from the sensory neurons. In addition, strong shocks to the tail increase the duration and magnitude of individual sensory neuron action potentials. Sensitizing tail stimulation usually produces long-lasting depolarization of the tail motor neurons and often long-lasting hyperpolarization of the tail sensory neurons. The tail motor and sensory neurons show both increases and decreases of input resistance following sensitizing stimulation. However, the small, occasional increases in input resistance of the motor neuron are insufficient to explain the heterosynaptic facilitation produced by sensitizing stimulation. Serotonin (5-HT) application can mimic many of the effects of sensitizing tail shock, including facilitation of both tail withdrawal and the monosynaptic connections between tail sensory and motor neurons, hyperpolarizing and depolarizing responses in the tail sensory neurons, and an increase in the duration and magnitude of the sensory neuron action potential. In the nearly isolated sensory neuron soma, 5-HT usually produces a slow, decreased conductance depolarizing response, suggesting that the 5-HT-induced hyperpolarizing response see     

Long-lasting reduction of excitability by a sodium-dependent potassium current in cat neocortical neurons

1. The function and ionic mechanism of a slow outward current were studied in large layer V neurons of cat sensorimotor cortex using an in vitro slice preparation and single microelectrode voltage clamp. 2. With Ca2+ influx blocked, a slow relaxation ("tail") of outward current followed either (1) repetitive firing evoked for 1 s or (2) a small 1-s depolarizing voltage clamp step that activated the persistent Na+ current of neocortical neurons, INaP. When a depolarization that activated INaP was maintained, an outward current gradually developed and increased in amplitude over a period of tens of seconds to several minutes. An outward tail current of similar duration followed repolarization. The slow outward current was abolished by TTX, indicating it depended on Na+ influx. 3. With Ca2+ influx blocked, the onset of the slow Na+-dependent outward current caused spike frequency adaptation during current-evoked repetitive firing. Following the firing, the decay of the Na+-dependent current caused a slow afterhyperpolarization (sAHP) and a long-lasting reduction of excitability. It also was responsible for habituation of the response to repeated identical current pulses. 4. The Na+-dependent tail current had properties expected of a K+ current. Membrane chord conductance increased during the tail, and tail amplitude was reduced or reversed by membrane potential hyperpolarization and raised extracellular K+ concentration [( K+]0). 5. The current tail was reduced reversibly by the K+ channel blockers TEA (5-10 mM), muscarine (5-20 microM), and norepinephrine (100 microM). These agents also resulted in a larger, more sustained inward current during the preceding step depolarization. Comparison of current time course before and after the application of blocking agents suggested that, in spite of its capability for slow buildup and decay, the onset of the Na+-dependent outward current occurs within 100 ms of an adequate step depolarization. 6. With Ca2+ influx blocked, extracellular application of dantrolene sodium (30 microM) had no clear effect on the current tail or the corresponding sAHP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chick spinal accessory lobes contain functional neurons expressing voltagegated sodium channels generating action potentials

Ten pairs of protrusions, called accessory lobes (ALs), exist at the lateral sides of avian lumbosacral spinal cords. Histological evidence has shown that neurons are present in AL and behavioral evidence suggests that AL acts as a sensory organ of equilibrium during bipedal walking. However, there is little functional evidence to indicate that cells in AL have neuronal functions. To elucidate this point, we developed a method to dissociate cells from chick AL and made electrophysiological recordings with the whole-cell patch clamp technique. Cells dissociated by enzymatic digestion from chick AL contained two major types of cells. One was round with clear cytosol and the other had a round cell body, rich cytosolic structures and some processes. Rapidly activating inward currents and slowly activating outward currents were recorded in response to depolarizing pulses to -10 mV under the voltage clamp configuration only from the latter type of cells. TTX at 100 nM inhibited the inward current by 85%, indicating the functional expression of TTX-sensitive voltage-gated Na(+) channel (VGSC). Activation and inactivation kinetics of the inward currents in AL cells were similar to those of mammalian VGSC. The VGSC-expressing AL cells generated action potentials in response to depolarization under the current clamp configuration. These results clearly indicate that functional neurons expressing fast inactivating and TTXsensitive VGSC which generate action potentials exist in the AL of the chick. These lines of cellular evidence clearly indicate that functional neurons exist in ALs and further support the proposal that the chick ALs function as the sensory organ of equilibrium.     

Fulfenamic acid sensitive,Ca(2+)-dependent inward current induced by nicotinic acetylcholine receptors in dopamine neurons

Nicotinic acetylcholine receptors (nAChRs) exhibit high Ca(2+) permeabilities and the Ca(2+)-influx through the nAChRs may be involved in regulation of a variety of signal processing in the postsynaptic neurons. The mesencephalic dopamine (DA) neurons receive cholinergic inputs from the brainstem and express abundant nAChRs. Here we report that the Ca(2+)-influx induced by a transient pressure application of ACh activates an inward current mediated by nAChRs and subsequently an inward current component that is sensitive to fulfenamic acid (FFA) and phenytoin, presumably a Ca(2+)-activated nonselective cation current in the DA neurons in the midbrain slices of the rat. The FFA- and phenytoin-sensitive current exhibits a negative slope conductance below -40 mV, suggesting its role in significant enhancement of depolarizing responses. In the current clamp recordings with perforated patch clamp configuration, bath application of carbachol markedly enhanced the glutamate-induced depolarization, which led to a long-lasting depolarizing hump. Activation of nAChRs is involved in this process, in cooperation with muscarinic receptors that suppress afterhyperpolarization caused by Ca(2+)-activated K(+)-channels. The long-lasting depolarizing hump was suppressed by FFA. All these results suggested a potential role of the FFA-sensitive current triggered by nAChR activation in marked enhancement of the excitatory synaptic response in DA neurons.     

MK-801 blocks nicotinic depolarizations of guinea pig myenteric neurons

Intracellular recordings were made from guinea-pig myenteric neurons. Acetylcholine (ACh), applied by iontophoresis, produced a depolarization that was blocked by hexamethonium (EC50 3.2 microM) but not by scopolamine (1 microM). MK-801 (0.3-30 microM), a non-competitive antagonist at N-methyl-D-aspartate receptors, also blocked the nicotinic depolarization (EC50 = 4.5 microM). Voltage clamp measurements of membrane current showed that MK-801 (1 microM) and hexamethonium (1 microM) both produced a greater inhibition of ACh-induced inward currents at -100 mV than at -40 mV. MK-801 (10 microM) did not change the depolarization evoked by 5-HT (acting at 5-HT3 receptors) in the same neurons in which it reduced the nicotinic response by more than 70%. It is concluded that MK-801 can act as a non-competitive antagonist of nicotinic responses in myenteric neurons.     

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

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

Electrophysiological detection of acetylcholinesterase activity using concentration clamp on physically isolated Aplysia neurons

In this study, we have used electrophysiological techniques to evaluate the acetylcholinesterase (AChE) activity on single neurons physically isolated from the pedal ganglia of Aplysia californica and kurodai. Acetylcholine (ACh) was applied to cells having Na-dependent response using a concentration clamp technique which allows a step-like complete change of external medium around the cell (diameter more than 120 micron) within 30 msec. When the neuron was exposed to ACh (50 microM) initially by rapid and brief (400-800 msec) flow and continuously after stopping the perfusion, the induced current rose to a peak and then decayed in the presence of ACh. However, the current increased again when the rapid flow of the same ACh solution around the cell was resumed. The increase in the current by the resumption of the perfusion was not seen when carbachol was substituted for ACh or when extraordinarily high concentration (10 mM) of ACh was used. Furthermore, this increase in the current was blocked by the antiAChE agent, edrophonium, in a dose-dependent manner. These results suggest that acetylcholinesterase (AChE) causes a local depletion of ACh at the membrane surface where the rate of hydrolysis of ACh exceeds the rate of ACh diffusion from bulk solution and the increase in the current by the resumption of the perfusion resulted from the restoration of ACh concentration at the membrane surface. This electrophysiological indication of AChE activity may be a useful tool for the study of AChE by other than biochemical means.     

Separation of current induced by potassium accumulation from acetylcholine-induced relaxation current in the rabbit S-A node

In a previous analysis on the rabbit S-A node the ACh-induced current was separated from the membrane current by subtracting the control from the current recorded in presence of ACh. In view of a possible interference of K accumulation processes, in the present paper the validity of the subtraction method was tested by studying the direct and indirect effects of ACh on the outward potassium current (iK). The following results were obtained. (1) The ACh-dependent channel activation and the iK-channel activation are different processes. (2) The activation curve of iK and the time constant of decay of iK current on return from a depolarizing clamp pulse were not affected by ACh. (3) In the majority of the experiments the presence of an accumulation component in the extra-current elicited by ACh could not be resolved. In a few cases the amplitude of the tail current was decreased in the presence of ACh. (4) In the case where iK was reduced, the fully-activated current-voltage relationship (i/K) was altered in the same way as that observed when the external K concentration was increased. In this case the difference between the control and the current recorded in the presence of ACh yielded a current component having a time constant similar to that of iK. We concluded that the decrease in the amplitude was due to an increase in K concentration in the clefts between the cells (K accumulation), associated with ACh application. No direct effect of ACh on the iK channel is apparent. (5) Because of the difference in the time constants of the relaxation current and the current change induced by accumulation the two processes could be clearly separated from each other.     

大鼠肾上腺嗜铬细胞瘤细胞的神经型烟碱受体电流(英文)

本实验采用膜片箝技术观察了神经生长因子分化 5～ 10 d的大鼠肾上腺嗜铬细胞瘤 (PC12 )细胞上烟碱受体离子通道电流。在全细胞膜片箝条件下 ,当箝制电压为 -80 m V时 ,灌流乙酰胆碱 (ACh,3 0μmol/ L )诱发一内向电流 ,快速始发并衰减。此乙酰胆碱诱发电流 (IAch)随膜去极化和超级化而分别减小和增大 ,且被筒箭毒所阻断 ,可见此电流是由烟碱受体引起的。全细胞IACh呈较强的内向整流。其衰减相可被一双指数函数拟合 ,时间常数 (τ)分别在秒和分级。τf 对浓度的依赖性较τs强。 PC12细胞表面含有与交感神经元相似的烟碱受体 ,因此提供了一个交感神经元样的同源细胞株。它是研究神经烟碱受体调节的一个很好的模型系统     

Catecholamine and acetylcholine sensitivity of rat lateral hypothalamic neurons related to learning.

1. Unit activity in the rat lateral hypothalamus (LHA) was recorded during discrimination learning of cue tone (CTS) or cue light (CL) stimulation that predicted reward by glucose or intracranial self stimulation (ICSS), or aversion by weak electric shock or tail pinch. Roles of the catecholaminergic and cholinergic systems in the LHA were investigated by electrophoretic application of dopamine (DA), norepinephrine (NE), acetylcholine (ACh), and their antagonists [spiperone (SPP), phenoxybenzamine (PBZ), phentolamine, propranolol, and atropine (Atr)]. 2. Activity of 264 LHA neurons was recorded. Of these, 234 (89%) responded during CTS learning in one or more phases. Of 121 neurons tested by both rewarding and aversive stimuli, 86 (71%) discriminated reward and aversion and their respective CTSs. 3. Effects of DA on 138, NE on 134, and ACh on 73 neurons were tested. Among these, 67 were tested with all three. DA inhibited 40 and excited 14. NE inhibited 74 and excited 10. ACh excited 35 and inhibited 3. DA-sensitive neurons responded to both NE (P less than 0.001) and ACh (P less than 0.05) more often than DA-insensitive neurons. In most cases, the effect of DA was similar to the effect of NE, and opposite to the effect of ACh. The inhibitory effect of DA was blocked by SPP, a D2 antagonist, and the excitatory effect of ACh was blocked by Atr. The inhibitory effect of NE was blocked by the beta-antagonist, propranolol, and enhanced by the alpha-antagonist, phentolamine. 4. DA-sensitive neurons responded to both rewarding and aversive stimuli and respective CTS+ and CTS- more often than DA-insensitive neurons (P less than 0.01). The effect of DA was usually similar to the effect of rewarding stimuli and their predicting CTS+ and was opposite to the effect of aversive stimuli and their predicting CTS-. 5. The proportion of NE-sensitive neurons that responded to rewarding and aversive stimuli was the same as the proportion of NE-insensitive neurons that responded to the same stimuli. NE-sensitive neurons responded to CTS+ and CTS- more often than NE-insensitive neurons (P less than 0.01). The effect of NE was usually similar to the effect of rewarding stimuli and predicting CTS+, and opposite to the effect of aversive stimuli and predicting CTS-. 6. ACh-sensitive neurons responded to aversive stimuli and predicting CTS- more often than ACh-insensitive neurons (P less than 0.01), but the response ratio of ACh-sensitive neurons to rewarding stimuli was similar to that of ACh-sensitive neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of acetylcholine on coding of taste information in the primary gustatory cortex in rats

Acetylcholine (ACh) receptors are widely distributed throughout the cerebral cortex in rats. Recently, cholinergic innervation of the gustatory cortex (GC) was reported to be involved in certain taste learning in rats. Here, the effects of iontophoretic application of ACh on the response properties of GC neurons were studied in urethane-anesthetized rats. ACh affected spontaneous discharges in a small fraction of taste neurons (11 of 86 neurons tested), but influenced taste responses in 27 of 43 neurons tested. No correlations with ACh susceptibility were noted for spontaneous discharges and taste responses. Among the 27 neurons, ACh facilitated taste responses in 13, inhibited taste responses in 13 and either facilitated or inhibited taste responses depending on the stimuli in 1. Furthermore, ACh affected the responses to best stimuli that produced the largest responses among four basic tastants (best responses) in 7 of 27 taste neurons, to non-best responses in 9, and to both best and non-best responses in 11. ACh mostly inhibited the best responses (13 of 18 neurons). Thus, ACh often decreased the response selectivity to the four basic tastants and changed the response profile. Atropine, a general antagonist of muscarinic receptors, antagonized ACh actions on taste responses or displayed the opposite effects on taste responses to ACh actions in two-thirds of the neurons tested. These findings indicate that ACh mostly modulates taste responses through muscarinic receptors, and suggest that ACh shifts the state of the neuron network in the GC, in terms of the response selectivities and response profiles.     

Potassium channels Kv1.1, Kv1.2 and Kv1.6 influence excitability of rat visceral sensory neurons

Voltage-gated potassium channels, Kv1.1, Kv1.2 and Kv1.6, were identified as PCR products from mRNA prepared from nodose ganglia. Immunocytochemical studies demonstrated expression of the proteins in all neurons from ganglia of neonatal animals (postnatal days 0-3) and in 85-90 % of the neurons from older animals (postnatal days 21-60). In voltage clamp studies, α-dendrotoxin (α-DTX), a toxin with high specificity for these members of the Kv1 family, was used to examine their contribution to K⁺ currents of the sensory neurons. α-DTX blocked current in both A- and C-type neurons. The current had characteristics of a delayed rectifier with activation positive to −50 mV and little inactivation during 250 ms pulses. In current-clamp experiments α-DTX, used to eliminate the current, had no effect on resting membrane potential and only small effects on the amplitude and duration of the action potential of A- and C-type neurons. However, there were prominent effects on excitability. α-DTX lowered the threshold for initiation of discharge in response to depolarizing current steps, reduced spike after-hyperpolarization and increased the frequency/pattern of discharge of A- and C-type neurons at membrane potentials above threshold. Model simulations were consistent with these experimental results and demonstrated how the other major K⁺ currents function in response to the loss of the α-DTX-sensitive current to effect these changes in action potential wave shape and discharge.     

Nicotinic acetylcholine receptor subtypes involved in facilitation of GABAergic inhibition in mouse superficial superior colliculus

The superficial superior colliculus (sSC) is a key station in the sensory processing related to visual salience. The sSC receives cholinergic projections from the parabigeminal nucleus, and previous studies have revealed the presence of several different nicotinic acetylcholine receptor (nAChR) subunits in the sSC. In this study, to clarify the role of the cholinergic inputs to the sSC, we examined current responses induced by ACh in GABAergic and non-GABAergic sSC neurons using in vitro slice preparations obtained from glutamate decarboxylase 67-green fluorescent protein (GFP) knock-in mice in which GFP is specifically expressed in GABAergic neurons. Brief air pressure application of acetylcholine (ACh) elicited nicotinic inward current responses in both GABAergic and non-GABAergic neurons. The inward current responses in the GABAergic neurons were highly sensitive to a selective antagonist for alpha3beta2- and alpha6beta2-containing receptors, alpha-conotoxin MII (alphaCtxMII). A subset of these neurons exhibited a faster alpha-bungarotoxin-sensitive inward current component, indicating the expression of alpha7-containing nAChRs. We also found that the activation of presynaptic nAChRs induced release of GABA, which elicited a burst of miniature inhibitory postsynaptic currents mediated by GABA(A) receptors in non-GABAergic neurons. This ACh-induced GABA release was mediated mainly by alphaCtxMII-sensitive nAChRs and resulted from the activation of voltage-dependent calcium channels. Morphological analysis revealed that recorded GFP-positive neurons are interneurons and GFP-negative neurons include projection neurons. These findings suggest that nAChRs are involved in the regulation of GABAergic inhibition and modulate visual processing in the sSC.     

Nicotinic ACh receptors in area postrema neurons of immature rat brain

The electrophysiological properties of nicotinic ACh receptors (nAChR) were investigated in acutely dissociated area postrema (AP) neurons of the immature rat brain using the whole-cell patch-clamp recording method. ACh induced a transient inward current exhibiting a strong inward rectification. The ACh response was mimicked by nicotine and cytisine, and was inhibited by nAChR antagonists, but not by 10(-7) M atropine. Muscarinic AChR agonists did not induce any current. We confirmed the Ca2+ permeability of nAChR. These results indicate the presence of nAChR on AP neurons, and suggest that the activation of nAChR play important roles in cardiovascular functions in rats.     

Cholinergic modulation of non-N-methyl-d-aspartic acid glutamatergic transmission in the chick ventral lateral geniculate nucleus

Neurotransmission between glutamatergic terminals of retinal ganglion cells and principal neurons of the ventral lateral geniculate nucleus (LGNv) was examined with patch clamp recordings in chick brain slices during electrical stimulation of the optic tract. Since muscarinic and nicotinic receptors are present in high densities in LGNv, the present study examined possible roles of both receptors in modulating retinogeniculate transmission. During whole-cell recordings from LGNv neurons, acetylcholine (ACh, 100 μM) caused an initial increase in amplitudes of optic tract-evoked non-N-methyl-d-aspartic acid (NMDA) glutamatergic postsynaptic currents (PSCs). This increase was unchanged when 1 μM atropine was present, indicating that this initial enhancement of PSCs was due entirely to activation of nicotinic receptors. However, during washout of ACh the amplitudes of evoked PSCs became significantly decreased by 40.4±5.0% for several minutes before recovering to their original amplitudes, an effect blocked by 1 μM atropine. Exogenously applied muscarine (10 μM) markedly depressed optic tract-evoked PSCs, and this decrease in amplitude was blocked by atropine. In a second set of experiments, we examined effects of releasing endogenous ACh prior to optic tract stimulation. This was accomplished by stimulation of the lateral portion of LGNv via a separate conditioning electrode. Following a brief train of low intensity conditioning stimuli, non-NMDA glutamatergic PSCs evoked by optic tract stimulation were potentiated. However, at higher conditioning stimulus intensities the PSCs were markedly decreased compared with control, and this decrease was partially blocked by atropine (1 μM). Neither ACh nor muscarine altered amplitudes of PSCs elicited by exogenously applied glutamate. Muscarine significantly reduced the frequency but not the amplitudes of miniature PSCs, consistent with a presynaptic location for muscarinic receptors mediating these effects. Thus while activation of nicotinic receptors potentiates retinogeniculate transmission, activation of muscarinic receptors mediates depression of transmission, demonstrating a complex cholinergic modulation of sensory information in LGNv.     

GABA activated chloride currents in cultured rat hippocampal and septal region neurons can be inhibited by curare and atropine

In order to study the influence of curare and atropine on the gamma-aminobutyric acid (GABA)-evoked chloride current, we have investigated cultures of hippocampal and septal region neurons from embryonic rats (E18). The neurons were cultured under the trophic influence of spatially separated astrocytes in serum-free medium. By means of the patch clamp technique, the excitable cells displayed GABA induced chloride currents within 1-15 days in vitro. In both cultures picrotoxin or bicuculline as well as curare or atropine reversibly inhibited the chloride current in a dose dependent manner. We conclude that curare and atropine are not specifically anti-cholinergic for cultured central neurons. Our results provide evidence for common ligand binding properties shared by GABAA and acetylcholine (ACh) receptors supporting the recent concept of a receptor superfamily.     

Muscarinic receptor activation induces a prolonged post-stimulus afterdepolarization with a conductance decrease in guinea-pig olfactory cortex neurones in vitro.

The persistent excitation of guinea-pig olfactory cortical neurones in vitro by the muscarinic agonist oxotremorine-M (OXO-M) was investigated. In OXO-M (10-20 microM), a slowly-decaying afterdepolarization (sADP) accompanied by sustained repetitive firing was induced following a long depolarizing stimulus. The corresponding slow inward current (IADP) revealed under voltage clamp behaved like a K(+)-mediated tail current, but was associated with a decreased membrane conductance. IADP was insensitive to tetrodotoxin (TTX), Ba2+, Cs+, or 4-aminopyridine (4-AP), but was blocked by 500 microM TEA or TBA (tetrabutylammonium). The OXO-M response and IADP were also reduced by Cd2+ or Ca(2+)-free solution, suggesting a dependence on Ca(2+)-entry. We propose that OXO-M induces a novel outward K+ current that can be slowly de-activated by Ca(2+)-entry during a depolarizing stimulus. Summation of IADP tail currents could contribute to the sustained muscarinic excitation of mammalian cortical neurones.     

Effects of increased intracellular Cl- concentration on membrane responses to acetylcholine in the isolated endothelium of guinea pig mesenteric arteries

ACh-induced membrane responses in vascular endothelial cells that have been reported vary between preparations from a sustained hyperpolarization to a transient hyperpolarization followed by a depolarization; the reason for this variation is unknown. Using the perforated whole-cell clamp technique, we investigated ACh-induced membrane currents in freshly isolated endothelial layers having a resting membrane potential of less negative than -10 mV. A group of cells was electrically isolated using a wide-bore micropipette, and their membrane potential was well controlled. ACh activated K(+) and Cl(-) currents simultaneously. The K(+) current was blocked by a combination of charybdotoxin and apamin and appears to result from the opening of IK(Ca) and SK(Ca) channels. The Cl(-) current was partially blocked by tamoxifen, niflumic acid, or DIDS and appears to be produced by Ca(2+)-activated Cl(-) channels. When the pipettes contained 20 mM Cl(-), the ACh-induced K(+) conductance started decreasing during a 1-min application of ACh while the Cl(-) conductance continued, making the ACh-induced hyperpolarization sustained. When the pipettes contained 150 mM Cl(-), both conductances started decreasing during a 1-min application of ACh, making the ACh-induced hyperpolarization small and transient. [Cl(-)](i) is very likely modified by experimental procedures such as the cell isolation and the intracellular dialysis with the pipette solution. Such a variability in [Cl(-)](i) may be one of the reasons for the variations in the ACh-induced membrane response.