Adrenergic responses in silent and putative inhibitory pacemaker-like neurons of the rat rostral ventrolateral medulla in vitro

Noradrenaline and adrenergic agonists were tested on pacemaker-like and silent neurons of the rat rostral ventrolateral medulla using intracellular recording in coronal brainstem slices as well as in punches containing only the rostral ventrolateral medullary region. Noradrenaline (1-100 microM) depolarized or increased the frequency of discharge of all cells tested in a dose-dependent manner. The noradrenaline-induced depolarization was associated with an apparent increase in cell input resistance at low concentrations and a decrease or no significant change at higher concentrations. Moreover, it was voltage dependent and its amplitude decreased with membrane potential hyperpolarization. Noradrenaline caused a dose-related increase in the frequency and amplitude of spontaneous inhibitory postsynaptic potentials. The alpha 1-adrenoceptor antagonist prazosin (0.5 microM) abolished the noradrenaline depolarizing response as well as-the noradrenaline-evoked increase in synaptic activity and unmasked an underlying noradrenaline dose-dependent hyperpolarizing response associated with a decrease in cell input resistance and sensitive to the alpha 2-adrenoceptor/antagonist yohimbine (0.5 microM). The alpha 1-adrenoceptor agonist phenylephrine (10 microM) mimicked the noradrenaline depolarizing response associated with an increase in membrane resistance as well as the noradrenaline-induced increase in synaptic activity. The alpha 2-adrenoceptor agonists UK-14,304 (1-3 microM) and clonidine (10-30 microM) produced only a small hyperpolarizing response, whereas the beta-adrenoceptor agonist isoproterenol (10-30 microM) had no effect. Baseline spontaneous postsynaptic potentials were abolished by strychnine (1 microM), bicuculline (30 microM) or both. However, only the strychnine-sensitive postsynaptic potentials had their frequency increased by noradrenaline or phenylephrine and they usually occurred with a regular pattern. Tetrodotoxin (1 microM) eliminated 80-95% of baseline spontaneous postsynaptic potentials and prevented the increase in synaptic activity evoked by noradrenaline and phenylephrine. Similar results were obtained in rostral ventrolateral medulla neurons impaled in both coronal slices and punches of the rostral ventrolateral medulla. It is concluded that noradrenaline could play an important inhibitory role in the rostral ventrolateral medulla via at least two mechanisms: an alpha 2-adrenoceptor-mediated hyperpolarization and an enhancement of inhibitory synaptic transmission through activation of alpha 1-adrenoceptors located on the somatic membrane of glycinergic interneurons. Some of these interneurons exhibit a regular discharge similar to the pacemaker-like neurons and might, at least in part, constitute a central inhibitory link in the baroreceptor-vasomotor reflex pathway.     

Bursting and tonic discharges in two classes of reticular thalamic neurons

1. Two types of cat reticular (RE) thalamic cells were disclosed by means of intracellular recordings under urethan anesthesia. The RE neurons were identified by their typical depolarizing spindle oscillations in response to synchronous stimulation of the internal capsule. 2. In type I neurons (n = 41), depolarizing current pulses induced tonic firing at the resting or slightly depolarized membrane potential (Vm) and triggered high-frequency spike bursts at a Vm more negative than -75 mV. As well, these cells discharged rebound bursts at the break of a hyperpolarizing current pulse. Internal capsule stimulation elicited spindle sequences made off by depolarizing waves giving rise to spike bursts. 3. Type II cells (n = 9) did not discharge spike bursts to large depolarizing current pulses even when the Vm reached -100 mV, nor did they fire rebound bursts after long-lasting hyperpolarizing current pulses or spike bursts riding on the rhythmic depolarizing components of spindle sequences. 4. Compared with type I cells, type II cells showed less frequency accommodation during tonic firing. The latter neuronal class discharged at high frequencies (40 Hz) with slight DC depolarization, approximately 8-10 Hz at the resting Vm, and no underlying synaptic or subthreshold oscillatory events could be detected when the firing was blocked by DC hyperpolarization. 5. The presence of two cell classes in the RE nucleus challenges the common view that this nucleus consists of a single neuronal class. We suggest that a different set of conductances is present in type II RE neurons, thus preventing the low-threshold Ca2+ current from dominating the behavior of these cells.     

Electrophysiological properties and their modulation by norepinephrine in the ambiguus neurons of the guinea pig

Electrophysiological properties of guinea pig ambiguus (AMB) neurons were studied in a brainstem slice preparation. During subthreshold depolarization AMB neurons displayed an early slow depolarization and a late outward rectification both of which were blocked by replacing Ca2+ with Co2+ in the extracellular solution. AMB neurons showed hyperpolarizing inward rectification which was blocked by extracellular Cs+ and is likely caused by the activation of Ih: In 58% (n = 49) of AMB neurons spike firing was restricted to the early phase of a long-lasting depolarizing current injection (phasic firing). The remaining AMB neurons showed repetitive firing throughout the depolarization (tonic firing). A Ca(2+)-mediated K+ current (IK(Ca)) caused an afterhyperpolarization that followed both single and repetitive spike firing. IK(Ca) also controlled the firing pattern in both types of firing, especially in the phasic firing. Norepinephrine (NE) blocked both the hyperpolarizing inward rectification and the Ca(2+)-dependent AHP. These effects of NE were antagonized by propranolol. It is proposed that the blockade of IK(Ca) and Ih contribute to the improvement of the 'signal-to-noise ratio' by NE in AMB neurons.     

Profiles of the fatty acids in the plasma membrane of human brain tumors

The ionic channels and signal transduction pathways underlying the 5-hydroxytryptamine (5-HT)-induced hyperpolarization in neurons of the rat dorsolateral septal nucleus (DLSN) were examined by using intracellular and voltage-clamp recording techniques. Application of 5-HT (1-50 microM) caused a hyperpolarizing response associated with a decreased membrane resistance in DLSN neurons. The hyperpolarization induced by 5-HT was blocked by Ba2+ (1 mM) but not by tetraethylammonium (TEA, 3 mM), glibenclamide (100 microM) and extracellular Cs+ (2 mM). 8-Hydroxy-di-n-propylamino tetralin (8-OH-DPAT; 3 microM), a selective agonist for the 5-HT1A receptor, mimicked 5-HT in producing the hyperpolarization. The 5-HT hyperpolarization was blocked by NAN-190 (5 microM), a 5-HT1A receptor antagonist. CP93129 (100 microM), a 5-HT1B receptor agonist, and L-694-247 (100 microM), a 5-HT1B/1D receptor agonist, also produced hyperpolarizing responses. The order of agonist potency was 8-OH-DPAT > CP93129 > or = L-694-247. (+/-)-2,5-Dimethoxy-4-iodoamphetamine hydrochloride (DOI, 100 microM), a 5-HT2 receptor agonist, and RS67333 (100 microM), a 5-HT4 receptor agonist, caused no hyperpolarizing response. The voltage-clamp study showed that 5-HT caused an outward current (I5-HT) in a concentration-dependent manner. I5-HT was associated with an increased membrane conductance. I5-HT reversed the polarity at the equilibrium potential for K+ calculated by the Nernst equation. I5-HT showed inward rectification at membrane potentials more negative than-70 mV. Ba2+ (100 microM) blocked the inward rectifier K+ current induced by 5-HT. I5-HT was irreversibly depressed by intracellular application of guanosine 5'-O-(3-thiotriphosphate)(GTP-gamma S) but not by guanosine 5'-O-(2-thiodiphosphate) (GDP beta S). These results suggest that in rat DLSN neurons activation of 5-HT1A receptors causes a hyperpolarizing response by activating mainly the inward rectifier K+ channels through a GTP-binding protein.     

Functional expression of sperm angiotensin II type I receptor in Xenopus oocyte: modulation of a sperm Ca2+-activated K+ channel

gamma-Hydroxybutyric acid (GHB) is an abused substance that occurs naturally in the basal ganglia. Electrophysiological recordings of membrane voltage and current were made to characterize the effects of GHB on dopamine neurons in the ventral tegmental area of the rat midbrain slice. Perfusate containing GHB caused a concentration-dependent membrane hyperpolarization (EC50 = 0.88 +/- 0.21 mM) and a reduction in input resistance (EC50 = 0.74 +/- 0.21 mM). The highest concentration of GHB studied (10 mM) hyperpolarized neurons by 20 +/- 3 mV and reduced input resistance by 58% +/- 9%. Changes in membrane potential and input resistance were blocked by the gamma-aminobutyric acid antagonist CGP-35348 (300 microM), but neither bicuculline (30 microM) nor strychnine (10 microM) was an effective antagonist. Voltage-clamp recordings demonstrated that GHB (1 mM) evoked 80 +/- 6 pA of outward current (at -60 mV) that reversed at -110 mV (in 2.5 mM K+). Increasing concentrations of extracellular K+ progressively shifted the reversal to more depolarized potentials. In tetrodotoxin (0.3 microM) and tetraethylammonium (10 mM), depolarizing voltage steps (to -30 mV) evoked calcium-dependent current spikes that were completely blocked by GHB (1 mM). These data suggest that GHB is an agonist at gamma-aminobutyric acid receptors and would be expected to inhibit DA release by causing K+-dependent membrane hyperpolarization.     

Inhibition of a hyperpolarization-activated current by clonidine in rat dorsal root ganglion neurons

Whole cell voltage- and current-clamp recordings were carried out to investigate the effects of clonidine, an alpha 2-adrenoceptor agonist, in L4 and L5 dorsal root ganglion (DRG) neurons of the rat. In voltage-clamp mode, application of 20 microM clonidine reversibly reduced the inward current evoked by hyperpolarizing voltage steps. The "clonidine-sensitive current" was obtained by subtracting the current during clonidine application from the control current, and its properties were as follows. 1) It was a slowly activating inward current evoked by hyperpolarization. 2) The reversal potential in the standard extracellular solution ([K+]o = 5 mM, [Na+]o = 151 mM) was -38.3 mV, and reduction of [Na+]o shifted it to a more negative potential, whereas an increase of [K+]o shifted it to a more positive potential, indicating that the current was carried by Na+ and K+ (PNa/PK = 0.22). 3) The relationship between the chord conductance underlying the clonidine-sensitive current and voltage could be fitted by a Boltzmann equation. These results indicate that the clonidine-sensitive current corresponds to a hyperpolarization-activated current (Ih), i.e., clonidine inhibits Ih in rat DRG neurons. DRG neurons were classified as small (15.9-32.9 microns diam), medium-sized (33-42.9 microns), and large (43-63.6 microns), and 7 of 19, 24 of 25, and 22 of 22 of these types exhibited Ih with mean +/- SE clonidine-induced inhibition values of 36.1 +/- 3.5% (n = 7), 43.1 +/- 3.7% (n = 24), and 35.1 +/- 2.7% (n = 22), respectively. Clonidine application to L4 and L5 DRG neurons excised from rats the sciatic nerves of which had been transected 14-35 days previously (transected DRG neurons) also reduced Ih. In current-clamp mode, 9 of 13 intact and 4 of 6 transected medium-sized DRG neurons that exhibited Ih responded to clonidine with hyperpolarization (> 2 mV). Some medium-sized DRG neurons exhibited repetitive action potentials in response to a depolarizing current pulse, and clonidine reduced the firing discharge frequencies in 8 of 11 intact and 3 of 4 transected neurons tested. Injection of a hyperpolarizing current pulse produced time-dependent rectification in DRG neurons that exhibited Ih, and clonidine blocked this rectification in all intact and transected neurons tested. These results suggest that inhibition of Ih due to alpha 2-adrenoceptor activation contributes to modulation of DRG neuronal activity in rats. On the basis of our findings, we discuss the possible mechanisms whereby sympathetically released norepinephrine modulates the abnormal activity of DRG neuronal cell bodies after nerve injury.     

The American Journal of Ophthalmology 1862-1864

A hyperpolarization-activated current (termed I[h]) is believed to provide a pacemaker depolarization in sinoatrial node cells and in some central and peripheral neurons. In the present study, we examined if such an inward cation current exists in primary auditory neurons using the whole-cell patch-clamp technique. A large inward, non-inactivating current was seen during hyperpolarizing steps negative to the resting potential. A depolarizing sag occurred during hyperpolarizing current injection, and upon termination of the current injection there was an overshoot, or a rebound firing. A low concentration of Cs+, but not Ba2+, reversibly blocked the inward current and depolarizing sag. The activation of the current showed voltage dependence with half-activation occurring at -101 +/- 1 mV. The time course of I(h) activation was fitted by double exponential function and was voltage-dependent (time constants: tau1 and tau2 = 480 and 3125 ms at -100 mV, and 66 and 404 ms at -160 mV). The reversal potential of the current was -36 mV measured from tail currents. The conductance of the current was decreased in Na+-free solution, and increased in high K+ solution. Increases in the levels of intracellular cAMP or cGMP enhanced the current. The results suggest that there exists a hyperpolarization-activated inward cation current in mammalian primary auditory neurons. This current may provide a depolarizing current during the membrane hyperpolarization following each firing of the primary auditory nerve.     

Tetanic stimulation induces short-term potentiation of inhibitory synaptic activity in the rostral nucleus of the solitary tract

Whole cell recordings from neurons in the rostral nucleus of the solitary tract (rNST) were made to explore the effect of high-frequency tetanic stimulation on inhibitory postsynaptic potentials (IPSPs). IPSPs were elicited in the rNST by local electrical stimulation after pharmacological blockade of excitatory synaptic transmission. Tetanic stimulation at frequencies of 10-30 Hz resulted in sustained hyperpolarizing IPSPs that had a mean amplitude of -68 mV. The hyperpolarization resulted in a decrease in neuronal input resistance and was blocked by the gamma-aminobutyric acid-A (GABAA) antagonist bicuculline. For most of the neurons (n = 87/102), tetanic stimulation resulted in a maximum hyperpolarization immediately after initiation of the tetanic stimulation, but for some neurons the maximum was achieved after three or more consecutive shock stimuli in the tetanic train of stimuli. When the extracellular Ca2+ concentration was reduced, the maximum IPSP amplitude was reached after several consecutive shock stimuli in the tetanic train for all neurons. Tetanic stimulation at frequencies of 30 Hz and higher resulted in IPSPs that were not sustained but decayed to a more positive level of hyperpolarization. In some neurons the decay was sufficient to become depolarizing and resulted in a biphasic IPSP. It was possible to evoke this biphasic IPSP in all the neurons tested if the cells were hyperpolarized to -75 to -85 mV. The ionic mechanism of the depolarizing IPSPs was examined and was found to be due to an elevation of the extracellular K+ concentration and accumulation of intracellular Cl-. Tetanic stimulation increased the mean 80-ms decay time constant of a single shock-evoked IPSP up to 8 s. The length of the IPSP decay time constant was dependent on the duration and frequency of the tetanic stimulation as well as the extracellular Ca2+ concentration. Afferent sensory input to the rNST consists of trains of relatively high-frequency spike discharges similar to the tetanic stimulation frequencies used to elicit the IPSPs in the brain slices. Thus the short-term changes in inhibitory synaptic activity in the slice preparation probably occur in vivo and may play a key role in taste processing by facilitating synaptic integration.     

Electroresponsive properties and membrane potential trajectories of three types of inspiratory neurons in the newborn mouse brain stem in vitro

1. The electrophysiological properties of inspiratory neurons were studied in a rhythmically active thick-slice preparation of the newborn mouse brain stem maintained in vitro. Whole cell patch recordings were performed from 60 inspiratory neurons within the rostral ventrolateral part of the slice with the aim of extending the classification of inspiratory neurons to include analysis of active membrane properties. 2. The slice generated a regular rhythmic motor output recorded as burst of action potentials on a XII nerve root with a peak to peak time of 11.5 +/- 3.4 s and a duration of 483 +/- 54 ms (means +/- SD, n = 50). Based on the electroresponsive properties and membrane potential trajectories throughout the respiratory cycle, three types of inspiratory neurons could be distinguished. 3. Type-1 neurons were spiking in the interval between the inspiratory potentials (n = 9) or silent with a resting membrane potential of -48.6 +/- 10.1 mV and an input resistance of 306 +/- 130 M omega (n = 15). The spike activity between the inspiratory potentials was burst-like with spikes riding on top of an underlying depolarization (n = 11) or regular with no evidence of bursting (n = 12). Hyperpolarization of the neurons below threshold for spike initiation did not reveal any underlying phasic synaptic activity, that could explain the bursting behavior. 4. Type-1 neurons showed delayed excitation after hyperpolarizing square current pulses or when the neurons were depolarized from a hyperpolarized level. This membrane behavior resembles the response seen in other CNS neurons expressing an IA. The response to 1-s long depolarizing pulses with a large current strength showed signs of activation of an active depolarizing membrane response leading to a transient reduction in the spike amplitude. The relationship between the membrane potential and the amplitude of square current pulses (Vm-I) showed a small upward rectification below -70 mV, and spike adaptation throughout a 1-s pulse had a largely linear time course. 5. Type-1 neurons depolarized and started to fire spikes 398 +/- 102 ms (n = 20) before the upstroke of the integrated XII nerve discharge. The inspiratory potential was followed by fast hyperpolarization, a short fast-repolarizing phase (1,040 +/- 102 ms, n = 5) and a longer slow-repolarizing phase (lasting until the next inspiratory discharge). 6. Type-2 neurons were spiking in the interval between the inspiratory potentials with no evidence of bursting behavior and had an input resistance of 296 +/- 212 M omega (n = 26). The response to hyperpolarizing pulses revealed an initial sag and postinhibitory rebound depolarization. This membrane behavior resembles the response seen in other CNS neurons expressing an Ih. The Vm-I relationship was linear at depolarized potentials and showed a marked upward rectification below -60 mV. Spike trains elicited by 1-s long pulses showed a pronounced early and late adaptation. 7. Type-2 neurons depolarized and started to fire spikes 171 +/- 87 ms (n = 23) before the upstroke of the integrated XII nerve discharge. The inspiratory potential had a variable amplitude from cell to cell and was followed by a short hyperpolarization in the cells displaying a large amplitude inspiratory potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrical activity of the proventriculus of the polychaete worm Syllis spongiphila

The straited myoepithelial cells of the proventriculus of Syllis spongiphila are composed of only one or two sarcomeres that may reach 40 mum in length. Experiments were performed to study some of their electrophysiological properties and their synaptic control. The mean resting potentials recorded in two different bathing media were 59-1 +/- 5-5 mV (S.D., n=91) and 62-5 +/- 6-3 mV (S.D., n=98). At rest the membrane potential is determined largely by permeability of the membrane to K+ ions, but the membrane is also permeable to other ions. On a semilogarithmic plot of membrane potential v. [K]o the mean slope of the data points from 9 to 90 mM-[K]o was 48 +/- 3 mV for a 10-fold change in [K]o. The anterior end of the animal was stimulated with a suction electrode to elicit activity of nerve fibres that innervate the proventriculus. Single indirect stimuli usually evoked hyperpolarizing or biphasic responses, and occasionally depolarizing responses, from the myoepithelial cells. The depolarizing synaptic potentials exhibited a faster time course than the hyperpolarizing ones. The rise time to peak ranged from 20 to 35 ms for simple depolarizations (n=32) and 25-75 ms for simple hyperpolarizations (n=103). Time to decay to half amplitude ranged from 20 to 55 ms for depolarizations (n=29) and 62-135 ms for hyperpolarizations (n=87). Low frequency (is less than or equal to 4 Hz) trains of indirectly applied stimuli elicited mainly hyperpolarizing responses; higher frequency (5-40 Hz) trains elicited complex responses composed of hyperpolarizations and depolarizations. Hyperpolarizations were selectively and reversibly abolished in chloride-free solutions. The reversal potential of the hyperpolarizing synaptic potential was -104 +/- 3 mV (S.D., n=8, 2 preparations). In calcium-free solution both hyperpolarizations and depolarizations were almost completely abolished. 4 mM-Mn2+ added to the bath almost completely abolished the depolarization but not the hyperpolarization. It was not clear whether Mn2+ acted at the presynaptic membrane, the postsynaptic membrane or both. The myoepithelial cells are electrically coupled. The mean space constant of five preparations was 0-52 mm (range 0-40-0-66 mm).     

Bactericidal effect of the Nd:YAG laser in in vitro root canals

The spatio-temporal patterns of neural activity evoked by electrical stimuli to the antennal nerve (AN) in male cockroach antennal lobes (ALs) in vivo were analyzed by optical imaging using a voltage-sensitive dye. The response pattern was initially a depolarization on the AN and subsequently a depolarization followed by a hyperpolarization on the whole area of macroglomerulus (MG) and a part of ordinary glomerulus (OG). It was suggested by the pharmacological results that the depolarizing responses on the AL consist of both a presynaptic response, representing synchronous compound action potentials from the AN, and a postsynaptic response, representing synchronous compound excitatory postsynaptic potentials and action potentials from neurites of AL neurons, and that the inhibitory responses of GABAergic local interneurons in the AN are different in time course from that in the AL.     

The K+/Cl- co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation

GABA (gamma-aminobutyric acid) is the main inhibitory transmitter in the adult brain, and it exerts its fast hyperpolarizing effect through activation of anion (predominantly Cl-)-permeant GABA(A) receptors. However, during early neuronal development, GABA(A)-receptor-mediated responses are often depolarizing, which may be a key factor in the control of several Ca2+-dependent developmental phenomena, including neuronal proliferation, migration and targeting. To date, however, the molecular mechanism underlying this shift in neuronal electrophysiological phenotype is unknown. Here we show that, in pyramidal neurons of the rat hippocampus, the ontogenetic change in GABA(A)-mediated responses from depolarizing to hyperpolarizing is coupled to a developmental induction of the expression of the neuronal (Cl-)-extruding K+/Cl- co-transporter, KCC2. Antisense oligonucleotide inhibition of KCC2 expression produces a marked positive shift in the reversal potential of GABAA responses in functionally mature hippocampal pyramidal neurons. These data support the conclusion that KCC2 is the main Cl- extruder to promote fast hyperpolarizing postsynaptic inhibition in the brain.     

MSX-1 gene expression and regulation in embryonic palatal tissue

1. During cardiac surgery, the heart is arrested and protected by hyperkalaemic cardioplegia. The coronary endothelium may be damaged by ischaemia-reperfusion and cardioplegia. Subsequently, this may affect cardiac function immediately after cardiac surgery and cause mortality and morbidity. 2. We investigated coronary endothelium-smooth muscle interaction after exposure to depolarizing (hyperkalaemic; K+ 20 or 50 mmol/L) and hyperpolarizing (the K+ channel opener aprikalim) cardioplegia and organ preservation solution (University of Wisconsin (UW) solution). Endothelium-dependent relaxation and hyperpolarization of the coronary smooth muscle were studied in the porcine and human large conductance and micro-coronary arteries. Intracellular free calcium concentration in endothelial cells was also measured. 3. The endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation to A23187, bradykinin, and substance P in arteries contracted by either U46619 (10 nmol/L) or K+ (25 mmol/L) was reduced after exposure to either high K+ or UW solution, but was maximally preserved after exposure to aprikalim. The hyperpolarization of the membrane potential in response to the above endothelium-derived relaxing factor stimuli was also reduced by exposure to depolarizing cardioplegia. Studies in microcoronary arteries are in accordance with findings in large arteries. The intracellular free calcium concentration remained unchanged after exposure to hyperkalaemia. 4. We concluded that: (i) during cardiac surgery, the function of coronary circulation may be changed due to exposure to depolarizing cardioplegia or preservation solutions; (ii) the functional change in the coronary circulation is related to the altered interaction between the endothelium and smooth muscle; (iii) depolarizing (hyperkalaemia) cardioplegia or hyperkalaemic organ preservation solutions affect endothelium-smooth muscle interaction through the EDHF pathway; (iv) EDHF relaxes the porcine large and microcoronary arteries through multiple K+ channels; and (v) that hyperpolarizing vasodilators (K+ channel openers) may protect EDHF-mediated endothelial function when used as cardioplegia.     

Mediation by intracellular calcium-dependent signals of hypoxic hyperpolarization in rat hippocampal CA1 neurons in vitro

In response to oxygen deprivation, CA1 pyramidal neurons show a hyperpolarization (hypoxic hyperpolarization), which is associated with a reduction in neuronal input resistance. The role of extra- and intracellular Ca2+ ions in hypoxic hyperpolarization was investigated. The hypoxic hyperpolarization was significantly depressed by tolbutamide (100 microM); moreover, the response was reversed in its polarity in medium containing tolbutamide (100 microM), low Ca2+ (0.25 mM), and Co2+ (2 mM), suggesting that the hypoxic hyperpolarization is mediated by activation of both ATP-sensitive K+ (KATP) channels and Ca(2+)-dependent K+ channels. The hypoxic depolarization in medium containing tolbutamide, low Ca2+, and Co2+ is probably due to inhibition of the electrogenic Na(+)-K+ pump and concomitant accumulation of interstitial K+. Hypoxic hyperpolarizations were depressed in either low Ca2+ (0.25 or 1.25 mM) or high Ca2+ (5 or 7.5 mM) medium (control: 2.5 mM), indicating that there is an optimal extracellular Ca2+ concentration required to produce the hypoxic hyperpolarization. Bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-AM (50-100 microM), procaine (300 microM), or ryanodine (10 microM) significantly depressed the hypoxic hyperpolarization, suggesting that Ca2+ released from intracellular Ca+ stores may have an important role in the generation of hypoxic hyperpolarization. The high-affinity calmodulin inhibitor N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonomide hydrochloride (W-7) (5 microM) completely blocked, whereas the low-affinity calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonomide hydrochloride (W-5) (50 microM) did not affect, the hypoxic hyperpolarization. The calmodulin inhibitor trifluoperazine (50 microM) also suppressed the hypoxic hyperpolarization. In addition, calcium/ calmodulin kinase II inhibitor 1-[N,O-bis (1,5-isoquinol-inesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-pip erazine (KN-62) (10 microM) markedly depressed the amplitude and net outward current of the hypoxic hyperpolarization without affecting the reversal potential. In contrast, neither the myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexa-hydro-1,4-diazepin hydrochloride (ML-7) (10 microM) nor the protein kinase A inhibitor N-[2-(p-bromocinnamyl-amino) ethyl]-5-isoquinolinesulfonamide (H-89) (1 microM) significantly altered the hypoxic hyperpolarization. These results suggest that calmodulin kinase II, which is activated by calmodulin, may contribute to the generation of the hypoxic hyperpolarization. In conclusion, the present study indicates that, in the majority of hippocampal CA1 neurons, the hypoxic hyperpolarization is due to activation of both KATP channels and Ca(2+)-dependent K+ channels.     

Developmental influence of glycinergic transmission: regulation of NMDA receptor-mediated EPSPs

The influence of excitatory transmission on postsynaptic structure is well established in developing animals, but little is known about the role of synaptic inhibition. We addressed this issue in developing gerbils with two manipulations designed to decrease glycinergic transmission in an auditory nucleus, the lateral superior olive (LSO), before the onset of sound-evoked activity. First, contralateral cochlear ablation functionally denervated the glycinergic pathway from the medial nucleus of the trapezoid body (MNTB) to the LSO, while leaving the excitatory pathway intact. Second, continuous release of a glycine receptor antagonist, strychnine (SN), was used to decrease transmission. The strength of excitatory and inhibitory synapses was examined with whole-cell recordings from LSO neurons in a brain-slice preparation. The percentage of LSO neurons exhibiting MNTB-evoked IPSPs was reduced in both ablated and SN-treated animals. In those neurons displaying IPSPs, the amplitude was significantly reduced. This decrease was accompanied by an 8 mV depolarization in the IPSP equilibrium potential. In contrast, the ipsilaterally evoked EPSPs were of unusually long duration in experimental animals. These long-duration EPSPs were significantly shortened by hyperpolarizing the neuron to -90 mV or exposing them to aminophosphonopentanoic acid (AP-5), an NMDA receptor antagonist. Membrane hyperpolarization and AP-5 had little effect in control neurons. In addition, LSO neurons from ablated or SN-treated animals displayed broad rebound depolarizations after membrane hyperpolarization, and these were abolished in the presence of Ni2+. Because both cochlear ablation and SN-rearing were initiated before the onset of sound-evoked activity, the results suggest that spontaneous glycinergic transmission influences the development of postsynaptic properties, including the IPSP reversal potential, NMDA receptor function, and a Ca2+ conductance.     

Endoscopic features of the columnar-lined esophagus

Electrophysiological characterization of neurons within the rat subiculum was carried out with intracellular recordings in an in vitro slice preparation. Subicular neurons responded to threshold pulses of depolarizing current delivered at a resting membrane potential (RMP) of 45.7+/-5.8 mV (mean+/-SD, n=85) with an initial burst of three to five fast action potentials that rode on a depolarizing envelope and was terminated by an afterhyperpolarization (burst AHP) (duration 113+/-35 ms; peak amplitude 2.7+/-0.6 mV, n=10). Tonic firing replaced the bursting mode at membrane potential less negative than -55 mV. Suprathreshold depolarizing pulses evoked at RMP both an initial burst and successive tonic firing. Intracellular staining with biocytin showed morphological features typical of pyramidal cells (n=8). The relationship between frequency of repetitive firing and injected current (f-I) revealed that the burst firing frequency (250-300 Hz) was only slightly influenced by the amount of injected current. By contrast, the f-I curve of the tonic firing phase depended upon current intensity: it displayed an initial segment that increased at first linearly and then turned into a plateau for both the early and the late inter-spike intervals. The frequency of the tonic firing declined only slightly with time, thus suggesting a lack of adaptation. During tonic firing, each single action potential was followed by a fast AHP and a depolarizing afterpotential. Termination of repetitive firing was followed by an AHP (spike-train AHP; duration 223+/-101 ms, peak amplitude 5.6+/-2.4 mV, n=17). Fast spike-train and burst AHPs were reduced by bath application of the Ca2+-channel blockers Co2+ (2 mM) and Cd2+ (1 mM) (n=8), thus suggesting the participation of Ca2+-dependent K+ conductances in these AHPs. Subicular bursting neurons generated persistent, subthreshold voltage oscillations at 5.3+/-1 Hz (n=20) during steady depolarization positive to -60 mV; at values positive to -55 mV, the oscillatory activity could trigger clusters of single action potentials with a periodicity of 0.9-2 Hz. Oscillations were not prevented by application of excitatory amino acid receptor and GABA(A) receptor antagonists (n=5), Ca2+-channel blockers (n=5), or Cs+ (3 mM; n=4), but were abolished by the Na+-channel blocker tetrodotoxin (1 microM; n=6). Our findings demonstrate that pyramidal-like subicular neurons generate both bursting and non-adapting tonic firing, depending upon their membrane potential. These neurons also display oscillatory activity in the range of theta frequency that depends on the activation of a voltage-gated Na+ conductance. These electrophysiological properties may play a role in the process of signals arising from the hippocampal formation before being funnelled towards other limbic structures.     

A family of hyperpolarization-activated mammalian cation channels

Pacemaker activity of spontaneously active neurons and heart cells is controlled by a depolarizing, mixed Na+/K+ current, named Ih (or I(f) in the sinoatrial node of the heart). This current is activated on hyperpolarization of the plasma membrane. In addition to depolarizing pacemaker cells, Ih is involved in determining the resting membrane potential of neurons and provides a mechanism to limit hyperpolarizing currents in these cells. Hormones and neurotransmitters that induce a rise in cyclic AMP levels increase Ih by a mechanism that is independent of protein phosphorylation, and which involves direct binding of the cyclic nucleotide to the channel that mediates Ih. Here we report the molecular cloning and functional expression of the gene encoding a hyperpolarization-activated cation channel (HAC1) that is present in brain and heart. This channel exhibits the general properties of Ih channels. We have also identified full-length sequences of two related channels, HAC2 and HAC3, that are specifically expressed in the brain, indicating the existence of a family of hyperpolarization-activated cation channels.     

Bioethical concepts of health in medicine

The effects of both activation and blockade of dopamine (DA) D1 receptors on long-term depression (LTD) of synaptic transmission were examined in CA1 neurons of rat hippocampal slices. Low frequency stimulation (LFS) consisting of 450 pulses at 1 Hz induced LTD (-14.3%, mean, n = 10) in the slope of the field excitatory postsynaptic potential. SKF-38393 (3-10 microM), an agonist of DA D1 receptors, significantly enhanced LFS-induced LTD (-31.1%, n = 11). SCH-23390 (2 microM), an antagonist of DA D1 receptors, blocked the induction of LTD by LFS (2.5%, n = 6). These results indicate that DA D1 receptors play an important role in the modulation of LFS-induced LTD in rat hippocampal CA1 neurons.     

Radiation therapy of esophageal carcinoma: a report on 180 cases]

To determine whether the charybdotoxin-sensitive subtypes of voltage-gated K+ channels (Kv1.2 and Kv1.3) exist in inhibitory pre-synaptic terminals, effects of K+ channel blockers including TEA, charybdotoxin (ChTX), iberiotoxin (IbTX), kaliotoxin (KTX) and margatoxin (MgTX) on the inhibitory transmission were examined with cultured rat hippocampal neurons. Monosynaptic inhibitory postsynaptic currents (IPSCs) evoked by electrical stimulation of single presynaptic neurons were recorded from the whole-cell clamped postsynaptic neurons. In the presence of TEA, application of ChTX greatly increased the amplitude of IPSCs. A specific maxi-K+ channel blocker IbTX failed to augment IPSCs. KTX and MgTX, both of which block Kv1.3 but not Kv1.2, mimicked the facilitating effect of ChTX. In the absence of TEA, application of ChTX increased the IPSC amplitude significantly, while IbTX was without effect. These results indicate that the ChTX-sensitive subtypes of voltage-gated K+ channels, most likely Kv1.3, contribute to the repolarization of action potentials at presynaptic terminals of hippocampal inhibitory neurons, and that the ChTX-induced facilitation of the transmission can be explained by its effects on the Kv channels rather than maxi-K+ channels.     

Tonoplast action potential in Nitella in relation to vacuolar chloride concentration

The action potential of Nitella internode was studied in relation to K+ and C1- concentrations in the vacuole. When the vacuole of Nitella pulchella was filled with an artificial solution with extremely low C1- concentration, a diphasic action potential (DAP) was observed. T he first phase consists of a rapid depolarization followed by a relatively rapid repolarization, and the second one consists of a strong hyperpolarization followed by a gradual return to the resting potential. When the cell was stimulated immediately after the generation of DAP, a monophasic action potential which resembles an action potential of the natural cell was observed, indicating that the DAP consists of two components with different refractory periods. The refractory period of the component responsible for the depolarizing is shorter than that of a component responsible for the hyperpolarizing phase. Measuring the plasmalemma potential and vascuolar potential separately, it was demonstrated that the hyperpolarizing component of DAP originates from the tonoplast. The action potential of the tonoplast, in contrast with that of the plasmalemma, could be generated independently of concentration of K+ in the vasuole. Since the maximum amplitude of hyperpolarization decreased significantly by increasing C1- concentration of the vacuole, it is concluded that the tonoplast is very sensitive to C1- during excitation.