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A series of novel aromatic tritylamino heterocycles has been synthesized and the compounds have been tested in comparison with clotrimazole for their ability to inhibit the slow afterhyperpolarization current (sI (AHP)) in cultured rat hippocampal pyramidal neurones. Some analogues of the clotrimazole metabolite, 2-chlorophenyl-diphenyl methanol, having different chlorination substitution in the triphenyl group have also been examined. Two compounds in particular, 3-[(2-chlorophenyl)-diphenylmethylamino] pyridine (3a, UCL 1880) and 2-tritylaminothiazole (6, UCL 2027), are of special interest; they are effective blockers of the sI (AHP) (IC (50) = 1.1-1.2 microM) and are much more selective than clotrimazole since they have less effect on the high voltage-activated Ca2+ current.

Locus coeruleus neurons recorded intracellularly in rat brainstem slices exhibited spontaneous activity and a marked afterhyperpolarization following a burst of spikes. This afterhyperpolarization was associated with an increase in membrane conductance and resulted in a marked postactivation inhibition of spontaneous activity. Since the reversal potential of the afterhyperpolarization was found to be virtually identical when recorded with KCl or potassium acetate-filled electrodes and shifted in the hyperpolarizing and depolarizing direction with decreases and increases in extracellular potassium concentrations, respectively, the afterhyperpolarization seen following a burst of spikes in this cell group appears to be mediated by an increase in potassium conductance. The afterhyperpolarization and postactivation inhibition were markedly attenuated by reducing calcium influx by either omitting extracellular calcium in the bathing medium or blocking calcium channels with manganese or cadmium. Thus, the afterhyperpolarization and the resulting postactivation inhibition appear to be largely mediated by the activation of a calcium-dependent potassium conductance. Previous reports in vivo have suggested that activation of alpha 2-adrenoceptors by norepinephrine release from recurrent axons or dendrites may mediate self-inhibition in the locus coeruleus. In this study, we examined the effect of blocking alpha 2-adrenoceptors on the afterhyperpolarization and postactivation inhibition. Administration of the alpha 2- adrenoceptor antagonist piperoxane failed to produce any changes in either of these parameters, suggesting that at least in vitro the afterhyperpolarization and postactivation inhibition seen in locus coeruleus neurons do not result from the activation of alpha 2- adrenoceptors.

Experiments using intracellular recording techniques were performed on rat hippocampal neurones in vitro, to study the discharge properties of these cells. When CA 1 pyramidal cells were excited by injecting long depolarizing current pulses (approximately 600-800 ms), they responded with an initial rapid action potential discharge which slowed, or accommodated, and then stopped after 200-300 ms. The train of action potentials was followed by a hyperpolarization which was due primarily to calcium-activated potassium conductance (GK(Ca]. The amplitude of this hyperpolarization increased with an increasing number of action potentials in the initial discharge. Blocking the calcium-activated potassium conductance, by injecting EGTA into the cell, by bathing the cell in cadmium, a calcium channel blocker, or by bathing the cell in calcium-free medium, reduced the after-hyperpolarization (a.h.p.) and accommodation such that the frequency of action potential discharge increased and the duration of this discharge was prolonged. Blocking the calcium-activated potassium conductance had a greater effect on discharge frequency later in the action potential train, as late interspike intervals were shortened more than early ones by the application of cadmium or of calcium-free medium. This was presumably because the calcium-activated potassium conductance was more developed later in the train. Accommodation was not completely abolished in the absence of calcium and presence of cadmium, suggesting that other factors, in addition to calcium-activated potassium conductance, contributed to this process. This remaining accommodation was reduced by low doses of carbachol, suggesting that the M-current also plays a role in accommodation. We conclude that accommodation of the action potential discharge of hippocampal pyramidal cells may be regulated by at least two potassium currents: the calcium-activated potassium current and the M-current. Both of these currents are turned on during excitation of the neurone and act in an inhibitory manner on that neurone to limit further action potential discharge.

The effects of serotonin (5-HT) on dentate granule (DG) neurons in hippocampal slices taken from young mature (6-8 months) and old (25-29 months) rats were compared. Intracellular measurements of membrane potential, cell input resistance and slow postspike afterhyperpolarization did not differ significantly between young and old neurons. Neurons recorded in slices taken from old animals responded with less hyperpolarization to increasing doses of the drug, and their responses were significantly reduced after repeated applications of 5-HT. Serotonin-mediated reduction of the slow afterhyperpolarization in young DG neurons was less prominent or totally absent in the old cells. It is concluded that serotonergic postsynaptic actions are impaired in old age.

1. Depolarization-induced voltage and conductance changes were studied in frog montoneurones in isolated, perfused spinal cord slices. Two types of afterhyperpolarization are observed following action potentials in normal Ringer, a fast afterhyperpolarization lastin 5-10 msec and a slow afterhyperpolarization lasting 60-200 msec. Both afterhyperpolarizations are mediated by an increased K+ conductance. 2. The slow afterhyperpolarization and conductance increase underlying it are selectively and reversibly inhibited by perfusion with solutions containing low [Ca2+] (less than or equal to 0-2 nM) or the Ca2+ antagonists Mn2+ (1mM) or Co2+ (5 mM), and are enhanced by perfusion with high [Ca2+]. 3. Addition of 2-5 mM tetraethylammonium ion (TEA+) to the perfusing solution prolongs the falling phase of the action potential and abolishes the fast afterhyperpolarization, but does not inhibit the slow afterhyperolarization. 4. When the voltage-dependent Na+ current is blocked by perfusion with TTX (10-5 M), intracellularly applied depolarizing current steps evoke fast and slow hyperpolarizations with kinetics and pharmacological sensitivities similar to those of the fast and slow afterhyperpolarizations, respectively. The fast hyperpolarization is maximally activated by brief, intense depolarizations, the slow hyperpolarization by prolonged, less intense depolarizations. 5. These pharmacological and kinetic data demonstrate that in frog motoneurones the repolarization-fast afterhyperpolarization sequence and the slow afterhyperpolarization are produced by different K+ conductance systems. The fast K+ conductance activates rapidly on depolarization, decays rapidly on repolarization, and is TEA+ sensitive, while the slow K+ conducatance activates and decays more slowly and is Ca2+-dependent. 6. Motoneurones perfused with TEA+ and TEA often show a slow, regenerative depolarizing response to applied depolarizing currents. These regenerative depolarizations are probably produced by an influx of Ca2+, because they persist in isotonic CaCl2 and are blocked by Mn2+ or low [Ca2+]. The Ca2+-dependence of the slow afterhyperpolarization and the increase in slow afterhyperpolarization magnitude observed following the slow Ca2+ potentials suggest that a depolarization-evoked Ca2+ influx activates the K+ conductance underlying the slow afterhyperpolarization. 7. Motoneurones in which the slow Ca2+ and K+ conductance systems have been enhanced by high [Ca2+] or blocked by Mn2+ show altered discharge patterns in response to intracellularly applied depolarizing current steps. Perfusion with twice normal [Ca2+] (4 mM) causes montoneurones to discharge more slowly at all current intensities, and reduces the slope of the 'steady-state' frequency-current relationship. Mn2+-perfused motoneurones exhibit fairly normal high-frequency discharge at the onset of the current step, but unlike normal motoneurones, do not discharge at frequencies below 60/sec...

1. A long-lasting afterhyperpolarization (AHP) follows current-induced repetitive firing in hippocampal CA1 neurons studied in vitro. A 10-25% increase in membrane slope conductance occurs during the AHP, suggesting that it may be mediated by an increased conductance to either K+ or Cl-. 2. Intracellular Cl- iontophoresis does not alter the AHP but does attenuate the IPSP. In contrast Ba2+, a cation that can decrease K+ conductance, eliminates the AHP but not the IPSP. These findings suggest the AHP is produced by a long-lasting increased conductance to K+, and is distinct from the IPSP. 3. Mn2+, a Ca2+-channel blocker, eliminates the AHP. In comparison, the AHP persists in the presence of the Na+-channel blocker, tetrodotoxin (TTX), and appears to be temporally associated with TTX-resistant "Ca2+ spikes." It is concluded that AHP is probably activated by Ca2+ influx. 4. These observations indicate that the AHP may be produced by a Ca2+ activated K+ current. A balance between cellular depolarization produced by Ca2+ entry and repolarization generated by a Ca2+-activated K+ current appears to operate to control excitability in some mammalian cortical neurons as it does in molluscan neurons. Disruption of this balance by Ba2+ produces spontaneous membrane-potential oscillations and recurrent burst firing in hippocampal neurons. Increases in the magnitude and duration of Ca2+ depolarization and/or decreases in the Ca2+-activated, K+-mediated repolarization may be mechanisms that lead to spontaneous, epileptiform bursting in mammalian cortical neurons.

The origin of Ca2+ that activates the Ca2+-dependent K+ conductance which is responsible for the slow afterhyperpolarization (a.h.p.) following an action potential was studied in bullfrog sympathetic ganglia. The decay phase of the a.h.p. was a graded function of the extracellular Ca2+, and showed a voltage sensitivity opposite to that of the Ca2+-dependent K+-current reported previously (Pallotta et al. 1981), indicating that it reflected the time course of an increase in intracellular free Ca2+. An a.h.p. of longer duration was generated in cells which showed more pronounced rhythmic hyperpolarizations induced by intracellular Ca2+ release. The duration of the a.h.p. recorded with electrodes filled with K3-citrate [a.h.p. (citrate)], which favors Ca2+ release, was longer than the a.h.p. recorded with KCl-filled electrodes [a.h.p. (C1)]. D-600 (50-100 microM) drastically reduced the a.h.p. (C1), but had less effect on the a.h.p. (citrate). Caffeine which facilitates Ca2+ release prolonged the a.h.p. (C1), but had less effect on the a.h.p. (citrate). The a.h.p. (citrate) showed a greater sensitivity to a low temperature than the a.h.p. (C1). Mn2+ (1-3 mM) depressed both types of a.h.ps to the same extent. These results suggest that the origin of intracellular Ca2+ for a.h.p. (C1) is mainly Ca2+ influx during an action potential, while that for the a.h.p. (citrate) is both Ca2+ entry and intracellular Ca2+ release, although the effect of Mn2+ is difficult to explain fully.

Features of the spike afterhyperpolarization (AHP) recorded intracellularly have been analyzed in fast pyramidal tract neurons of cats. Cell input conductance increases during the AHP, possibly because of a change in potassium conductance, as suggested by an AHP equilibrium potential 10–15 mV negative to the resting membrane potential. When more spikes are evoked in succession, AHPs following the first one are strongly reduced in amplitude. The effect is virtually maximal (30–50% of the control) after a single spike and fades out by 200–400 ms after the last spike. At short interspike intervals the initial time course of the depression is hidden by summation occurring between consecutive AHPs.

Intracellular recordings were made from pyramidal CA1-neurones of the hippocampal slice preparation. Bath application of a wide variety of neuroleptics was found to depress the slow afterhyperpolarization, which is mediated in these neurons by a calcium-dependent potassium conductance occurring following a burst of spikes. The depression of this conductance took place in the presence of calcium spikes of normal amplitude and duration, and except in the case of trifluoperazine, without alteration in resting membrane potential or input resistance.

Activity of CA1 neurons was recorded in rat hippocampal slices with a lidocaine-derived QX-572-filled micropipette. The QX compound abolished Na action potentials as reported earlier. In addition it reduced markedly the burst slow afterhyperpolarization seen in these neurons in response to a depolarizing current pulse. It also modified responses to neurotransmitter substances associated with changes in K currents, acetylcholine and serotonin. QX-filled micropipettes can therefore provide some insight into mechanisms of action of certain neurotransmitters in the brain but they cannot be used as selective blockers of Na currents.

Adrenal steroids bind specifically to hippocampal neurons under normal conditions and may contribute to hippocampal cell loss during aging, but little is known about the neurophysiological mechanisms by which they may change hippocampal cell functions. In the present studies, adrenal steroids have been shown to modulate a well-defined membrane conductance in hippocampal pyramidal cells. The calcium-dependent slow afterhyperpolarization is reduced in hippocampal slices from adrenalectomized rats, and it is increased after in vivo or in vitro administration of the adrenal steroid, corticosterone. Calcium action potentials are also reduced in adrenalectomized animals, indicating that the primary effect of corticosteroids may be on calcium conductance. The afterhyperpolarization component reduced by adrenalectomy is greater in aged rats than in young rats, suggesting that, with aging, there is an increased effect of corticosteroids on some calcium-mediated brain processes. Because elevated concentrations of intracellular calcium can be cytotoxic, these observations may increase the understanding of glucocorticoid involvement in brain aging as well as of the normal functions of these steroids in the brain.

1. In hippocampal pyramidal cells, action potentials are followed by three after-hyperpolarizations (AHPs): a fast AHP (fAHP) lasting 2-5 ms, a medium AHP (mAHP) lasting 50-100 ms, and a slow AHP (sAHP) lasting more than 1 s. The mechanism underlying the mAHP was studied in CA1 cells (n = 46) in rat hippocampal slices, using injection of depolarizing current to elicit discharge. 2. The current underlying the mAHP was studied by single-electrode voltage clamp in two ways. Either the voltage clamp was activated following a burst of spikes, thus recording the early tail current underlying the mAHP (hybrid clamp), or, after blocking the spikes with tetrodotoxin, the early tail current following a depolarizing voltage clamp command (to -20 to -45 mV for 100-400 ms) was measured. In both cases, the early tail current (measured at -60 mV) showed the following characteristics: (a) it decayed exponentially with a time constant of about 50 ms; (b) it was substantially reduced by the muscarinic agonist carbachol (40-50 microM); (c) it was moderately reduced (by 20% or less) by Ca2+-free medium and Ca2+ channel blockers (Cd2+, Mn2+), which abolished the fAHP and the sAHP; (d) it was partly blocked by tetraethylammonium (TEA, 1-10 mM) both before and during Ca2+ channel blockade; (e) it was resistant to noradrenaline (5-10 microM), which blocked the sAHP, and to apamin (100 nM). 3. The mAHP itself, recorded under current clamp, showed properties corresponding to those of the early tail current. 4. Unlike the current underlying the sAHP, which was reduced and reversed by hyperpolarization, the early tail current appeared to be reduced only at potentials down to -80 mV, and to increase at more negative potentials. The early tail current and mAHP-like undershoot at hyperpolarized potentials was blocked by external Cs+, but not by carbachol, in contrast to the early tail current and mAHP at -60 mV. 5. It was concluded that two currents contribute to the mAHP: IM (a voltage-gated muscarine-sensitive K+ current) and IC (a Ca2+-dependent TEA-sensitive K+ current). TEA reduced both the IM (5 mM) and the IC (1 mM) component of the mAHP. When the cell is hyperpolarized, a third current, IQ (a Ca+-sensitive mixed Na+-K+ inward current activated by hyperpolarization), masks the reversal of the mAHP by causing a depolarizing sag which resembles the decay of the mAHP.

The effect of dopamine (DA) on the calcium-activated potassium conductance underlying the slow afterhyperpolarization (AHP) which follows a train of action potentials in hippocampal pyramidal cells was studied utilizing the in vitro hippocampal slice preparation. Bath-applied DA (1-100 microM) significantly reduced the AHP in a reversible, dose-dependent manner. Neither the amount of current injected to elicit the AHP nor its initial amplitude had an effect on the reduction of the AHP by DA. DA did not depress calcium spikes, suggesting that the blockade of the AHP likely occurs at a step subsequent to the entry of calcium. Since DA's actions on the AHP closely mimicked those of norepinephrine, we examined the effect of beta-adrenergic antagonists on DA's actions. At concentrations which in other systems have been shown not to block DA stimulated adenylate cyclase, beta-adrenergic antagonists completely inhibited the reduction of the AHP by DA. In some cells DA also elicited small hyperpolarizations which were not blocked by application of dopamine receptor antagonists. These findings strongly suggest that a major electrophysiological action of DA in the hippocampus (i.e. blockade of the AHP) is due to its cross reactivity with beta-adrenergic receptors and that rigid pharmacologic criteria must be used before attributing an action of DA unambiguously to its interaction with DA receptors.

Summary In the brain, calcium influx following a train of action potentials activates potassium channels that mediate a slow afterhyperpolarization current (I sAHP ). The key steps between calcium influx and potassium channel activation remain unknown. Here we report that the key intermediate between calcium and the sAHP channels is the diffusible calcium sensor hippocalcin. Brief depolarizations sufficient to activate the I sAHP in wild-type mice do not elicit the I sAHP in hippocalcin knockout mice. Introduction of hippocalcin in cultured hippocampal neurons leads to a pronounced I sAHP , while neurons expressing a hippocalcin mutant lacking N-terminal myristoylation exhibit a small I sAHP that is similar to that recorded in uninfected neurons. This implies that hippocalcin must bind to the plasma membrane to mediate its effects. These findings support a model in which the calcium sensor for the sAHP channels is not preassociated with the channel complex.

The slow afterhyperpolarization in hippocampal pyramidal neurons is mediated by a calcium-activated potassium current (IAHP) and is a target for a variety of different neurotransmitters. The characteristics of the channels underlying I AHP and how they are modulated by neurotransmitters are, however, unknown. In this study, we have examined the properties of the channels underlying IAHP using fluctuation analysis of the macroscopic current. Our results indicate that this channel has a unitary conductance of 2–5 pS and a mean open time of about 2 ms. When the peak amplitude of IAHP was maximal, these channels have an open probability of 0.4. Noradrenaline and carbachol reduced I AHP amplitude by lowering open channel probability. These results indicate that a novel calcium-activated potassium channel underlies I AHP . This channel is modulated in a similar fashion by two different transmitter systems that utilize distinct protein kinases.

Quinine (100 microM) abolished the slow calcium-dependent afterhyperpolarization which occurs after an action potential in some neurones of the guinea-pig myenteric and submucous plexus. This occurred without any effect on the amplitude or time course of the action potential itself, or on the faster calcium-independent afterhyperpolarization. Tetraethylammonium did not reduce the slow afterhyperpolarization. Quinine also abolished the hyperpolarization which was evoked by intracellular injection of calcium ions.

Intracellular recordings were made from neurons in bullfrog sympathetic ganglia maintained in vitro. The action potential afterhyperpolarization was comprised of two components, namely an initial, shorter-lasting (up to 200ms) event and a later, longer-lasting (up to 2500ms) one, both of which resulted from an opening of potassium channels. Both of these components reversed their polarities at between-80 and -100mV. The initial part of the afterhyperpolarization, however, always reversed its polarity at a less negative potential than the latter part. Mathematical analysis, by means of a hypothetical membrane equivalent circuit, revealed that this difference in the reversal potentials could be explained by a simple potassium-channel opening hypothesis without considering any additional current other than potassium-current for either component of the spike afterhyperpolarization.

The hippocampus, as well as a variety of other brain regions, is known to receive a diffuse projection of noradrenaline (NA) containing fibres which originates in the brain stem1–4. Although there is considerable evidence for the involvement of this system in a variety of behaviours5–7, the precise cellular actions of NA are poorly understood. Early studies emphasized the direct inhibitory effects of NA8–12; more recent experiments have shown that at several sites, NA, or stimulation of NA-containing afferents, can also facilitate excitatory synaptic responses13–18. This has led to the concept that NA increases the ‘signal-to-noise’ ratio of neurones13, acting as an ‘enabling’ device4 which allows cells to respond more briskly to conventional synaptic excitation. In the olfactory bulb, NA reduces inhibitory postsyn-aptic potentials by a presynaptic action19, which could contribute to enhanced excitatory synaptic responses. However, in other systems, NA has been reported to enhance excitatory responses to iontophoretically applied transmitters, and it was proposed that NA increases the sensitivity of the neurone to these excitatory transmitters13–15. We report here experiments that could explain such direct effects. We have found that NA and cyclic AMP block the Ca2+-activated K+ conductance in hippocampal pyramidal cells and that this blockade occurs at a step subsequent to the entry of Ca2+ into the neurone. As a consequence, the spike frequency adaptation or accommodation which normally occurs with depolarizing stimuli is severely reduced. Thus, NA greatly increases the number of spikes elicited by a depolarizing stimulus.

Dopamine was found to have two electrophysiological effects on CA1 pyramidal cells in rat hippocampal slices. It increased the slow afterhyperpolarisation caused by a slow Ca2+-activated K+ conductance and it had an effect on action potentials that is postulated to be due to an increase in a fast Ca2+-activated K+ conductance. A given CA1 cell showed either one or both of the two responses to dopamine, or no response.

1. Extracellular iontophoretic applications of DNP lead to an increase in the membrane conductance of cat spinal motoneurones, manifested by a rise in input conductance, a slower rate of rise and fall of action potentials, and occlusion of the afterhyperpolarization. 2. There is also some hyperpolarization, but the reversal potential for the action of DNP is only about 12 mV more negative than the resting potential. 3. These effect of DNP can be abolished or significantly reduced by intracellular injections of EGTA. On the other hand, DNP can partly reverse the decreased conductance and the depression of the slow afterhyperpolarization caused by EGTA. 4. Intracellular injections of DNP also induce a rise in input conductance; when repeated, they tend to have a depolarizing effect, mainly irreversible. 5. It is concluded that DNP acts principally inside the motoneurone, by liberating bound internal Ca, the free Ca ions then raising membrane conductance, especially GK.

1. The effects of norepinephrine (NE) and related agonists and antagonists were examined on large neurons from layer V of cat sensorimotor cortex ("Betz cells") were examined in a brain slice preparation using intracellular recording, constant current stimulation and single microelectrode voltage clamp. 2. Application of NE (0.1-100 microM) usually caused a small depolarization from resting potential; hyperpolarizations were rare. Application of NE reversibly reduced rheobase and both the Ca2+- and Na+-dependent portions of the slow afterhyperpolarization (sAHP) that followed sustained firing evoked by constant current injection. The faster Ca2+-dependent medium afterhyperpolarization (mAHP), the fast afterhyperpolarization (fAHP), the action potential, and input resistance were unaffected. 3. The changes in excitability produced by NE application were most apparent during prolonged stimulation. The cells exhibited steady repetitive firing to currents that were formerly ineffective. The slow phase of spike frequency adaptation was reduced selectively and less habituation occurred during repeated long-lasting stimuli. The relation between firing rate and injected current became steeper if firing rate was averaged over several hundred milliseconds. 4. During voltage clamp in TTX, NE application selectively reduced the slow component of Ca2+-mediated K+ current. The faster Ca2+-mediated K+ current was unaffected, as were two voltage-dependent, transient K+ currents, the anomalous rectifier and leakage conductance measured at resting potential. Depolarizing voltage steps in the presence of Cd2+ revealed an apparent time- and voltage-dependent increase of the persistent Na+ current after NE application. The voltage-clamp results suggested ionic mechanisms for all effects seen during constant current stimulation except the depolarization from resting potential. The latter was insensitive to Cd2+ and TTX and occurred without a detectable change in membrane conductance. 5. NE application did not alter Ca2+ spikes evoked in the presence of TTX and 10 mM TEA. Inward Ca2+ currents examined during voltage clamp in TTX (with K+ currents reduced) became slightly larger after NE application. We conclude that NEs reduction of the slow Ca2+-mediated K+ current is not caused by reduction of Ca2+ influx. 6. Effects on membrane potential, rheobase, and the sAHP were mimicked by the beta-adrenergic agonist isoproterenol, but not by the alpha-adrenergic agonists clonidine or phenylephrine at higher concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of prolonged periods (up to 4 h) of perfusion with verapamil (100 μM) or D600 (100 μM) on synaptically and directly evoked responses of rat CA1 pyramidal cells were determined in vitro. The slow depolarization underlying burst generation and the slow afterhyperpolarization following directly evoked repetitive firing were blocked, but spike frequency accomodation was not. There was an increase in threshold for evoking synaptic responses and the amplitude of evoked inhibitory postsynaptic potentials (IPSPs) was decreased slightly. The results suggest that verapamil can partially block voltage dependent Ca influx into CA1 cells and that currents underlying accomodation and the slow afterhyperpolarization (AHP) may differ.

Intracellular recordings were made from visceral sensory neurons in vitro. Bradykinin (BK) depressed a slow Ca2+-activated K+-dependent (KCa) spike afterhyperpolarization (AHPs) in the range of 0.2–1 nM. BK did not affect the fast KCa spike afterhyperpolarization preceding the AHPs or the action potential waveform. Indomethacin pretreatment abolished the BK effect. These results indicate that BK can influence the excitability of vagal afferent neurons.

The blockade of a slow Ca2+-activated K+-dependent afterhyperpolarization (AHPs) in rabbit visceral sensory neurons by the prostaglandins, PGE1 and PGD2, was investigated to determine whether the blockade was indirectly due to a reduction in Ca2+ influx. The prostaglandins (PGs) could block the AHPs in the absence of any change in Ca2+-dependent spikes elicited in the presence of tetrodotoxin and tetraethylammonium bromide. A PG-induced decrease in Ca2+-dependent spike width observed in some neurons was temporally dissociated from the PG-induced block of the AHPs. In addition, a slow afterhyperpolarization produced by the application of the Ca2+ ionophore, A23187, was blocked by the PGs. It is concluded that a reduction in Ca2+ influx is not responsible for the PG-induced blockade of the AHPs.

1. Intracellular recording from hippocampal CA1 pyramidal cells in the slice preparation was used to analyse the pharmacological sensitivity of action potential repolarization and the hyperpolarizations that follow the action potential. The Ca2+-activated after-hyperpolarizations (a.h.p.s) could be divided into a fast a.h.p. with a time course of milliseconds, and a slow a.h.p. which lasted for a few seconds at a temperature of 30 degrees C. 2. The repolarization of the action potential is sensitive to the Ca2+ channel blocker Cd2+. This effect is simultaneous with a block of the fast a.h.p. which follows immediately upon the repolarization of the action potential. The slow a.h.p. was also blocked by Cd2+. 3. Low concentrations of the K+ channel blocker, tetraethylammonium (TEA; 200-500 microM), block the fast a.h.p. and slow down action potential repolarization. The slow a.h.p. was not affected by low concentrations of TEA. 4. The action potential repolarization and the fast a.h.p. are also reversibly sensitive to charybdotoxin. This agent had no effect on the slow a.h.p. 5. When EGTA or BAPTA were added to the normal recording electrolyte (KMeSO4), the generation of slow a.h.p.s was prevented. In addition, cells impaled with BAPTA-containing electrodes displayed broader action potentials and much reduced fast a.h.p.s compared to recordings made with electrodes containing KMeSO4 alone or with EGTA. 6. The slow a.h.p. can be eliminated by noradrenaline, 8-bromocyclic AMP or carbachol. Under these conditions there are no effects on the fast a.h.p. or on action potential duration. 7. Block of the fast a.h.p. with TEA or CTX (charybdotoxin) is associated with an increased frequency of the first few action potentials during a depolarization. This is a quite distinct effect from the greatly increased number of action potentials which results from block of the slow a.h.p. 8. The results support a conclusion that the fast a.h.p. is generated by the TEA- and voltage-sensitive Ca2+-activated K+ current, IC. This current is involved in spike repolarization and turns off upon the return to resting potential. Thus block of IC has no effect on the slow a.h.p. which is caused by a separate membrane current.

Intracellular recordings were made from red nucleus (RN) neurons in guinea-pig slice preparations. The slow afterhyperpolarization (AHP) following an action potential was reversibly abolished by Co2+ or Mn2+. Its amplitude was dependent on the extracellular K+ concentration. When tetraethylammonium was added to the perfusing solution, a tetrodotoxin-resistant regenerative depolarization was evoked which was blocked by Co2+ or Mn2+. There results suggest that the slow AHP is produced by an increase in Ca2+-dependent K+ conductance and that RN neurons have a voltage-dependent Ca2+ conductance.

Electrophysiologic effects of intracellularly injected apamin, a Ca 2+ -dependent K + channel blocker, were investigated in neurons of the motor cortex of awake cats. Single-electrode voltage clamp techniques were used to measure changes in membrane currents including those that were synaptically activated. All changes occured within 2–4 min after pressure injection of apamin with partial recovery observed within 8–15 min. Apamin selectively abolished an outward current that mediated a slow afterhyperpolarization (AHP) following intracellular depolarizing current pulses and action potentials without influencing the time course of the action potentials or an associated fast AHP component. In addition apamin increased the number and frequency of spike discharges evoked by the depolarizing current pulses and produced a small increase in the rate of background firing activity. The baseline resting potential and input resistance were essentially unchanged by apamin. Apamin also diminished a late, slowly decaying component of inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) elicited by stimulation of the ventrolateral thalamus or the pyramidal tract. The apamin-induced changes were concomitant with a decrease of the decay time constant of both IPSPs and IPSCs and a positive shift in their reversal potential. The results suggest that the late, slowly decaying component of these inhibitory postsynaptic responses is generated by an apamin-sensitive Ca 2+ -dependent K + conductance which is also responsible for the slow AHP.

Imipramine, a tricyclic antidepressant, acts acutely to block the reuptake of serotonin (5-HT) and norepinephrine (NE). However, imipramine's action as an antidepressant takes several weeks to develop. This study investigated acute and chronic effects of imipramine on intracellularly-recorded responses mediated by 5-HT and beta-adrenergic receptors on pyramidal cells from area CA1 of rat hippocampal slices maintained in vitro. Addition of 10 microM imipramine in the perfusion medium sinistrally shifted the 5-HT1A concentration-response curve for membrane hyperpolarization and the 5-HT concentration-response curve for the reduction in the amplitude of the slow afterhyperpolarization (AHP) elicited by a train of action potentials. After two weeks of treatment with imipramine (10 mg/kg daily i.p. injections or s.c. osmotic mini-pumps) the responses to 5-HT were not altered. In contrast the concentration-response curve for the beta-adrenergic mediated reduction in AHP amplitude was significantly altered; there was a reduction in Emax and a log unit dextral shift in EC50. There was no change in the concentration-response curve for the beta-adrenergic mediated depolarization. These data are in agreement with previous biochemical results reporting a decrease in beta-adrenergic receptor mediated stimulation in adenylyl cyclase and down-regulation of beta-receptor in cortex and hippocampus. These findings suggest that a consequence of long-term imipramine treatment is a decrease in the augmentation of cell excitation normally produced by beta-adrenergic receptor stimulation.

Ca2+-sensitive K+ currents involved in the slow afterhyperpolarization (a.h.p.) of an action potential of bullfrog sympathetic neurones were studied with a single-electrode voltage clamp method. The outward tail current (IAH) generated after the end of a depolarizing command pulse (from the holding potential of -60 mV to 0 mV, 5-20 ms in duration), mimicking an action potential, was separated into at least two exponential components (IAHf and IAHs). They were identified as K+ currents, since their reversal potentials were close to the K+ equilibrium potential and they were sensitive to external K+. The time constant of IAHf (tf; 44 ms at -60 mV) was decreased by membrane hyperpolarization from -40 to -80 mV, while that of IAHs (ts; 213 ms) remained constant. Removal of external Ca2+ or addition of Cd2+ significantly decreased the IAHs amplitude (As) and tf without a change in ts and the IAHf amplitude (Af). On the other hand, increasing Ca2+ influx by applying repetitive command pulses enhanced both Af and As with negligible effects on tf and ts, and produced a much slower component. Intracellular injection of EGTA reduced Af with no effect on tf, and increased As with a decreased ts. Both muscarine and (+/-)-tubocurarine, which reduced IAHs, hardly affected IAHf. These results indicate that a.h.p. is induced by the activation of two distinct Ca2+-dependent K+ channels, which differ in voltage sensitivity, Ca2+-dependence and pharmacology.

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)

1. Intracellular recordings were made from guinea-pig myenteric neurones in vitro. 2. From one to sixty action potentials were followed by an afterhyperpolarization, the amplitude and duration of which increased with the number of preceding action potentials. 3. The afterhyperpolarization reversed its polarity at a membrane potential of -91 mV. This value changed by 58 mV when the potassium concentration of the perfusing solution was changed ten-fold. 4. The afterhyperpolarization was abolished in calcium-free solutions. It was shortened in low calcium (1·2 mM) solutions and prolonged in solutions which contained high (5·0 mM) calcium concentrations, TEA (1 mM) or caffeine (1 μM). 5. The conductance increase during the afterhyperpolarization (gK, Ca) was calculated from the amplitude of electrotonic potentials, taking advantage of the lack of membrane rectification in the range -60 to -90 mV. Peak gK, Ca increased as the number of action potentials was increased, but was relatively independent of membrane potential in this range. 6. gK, Ca declined with a time course which was single exponential (time constant 1·5-5 s) following one to six action potentials, and double exponential (time constants about 3 and 12 s) following fifteen to sixty action potentials. 7. It is concluded that the calcium which enters the neurone during the action potential elevates the membrane potassium conductance. The time course of this conductance increase probably reflects the free intracellular calcium concentration, and therefore describes the calcium sequestration or extrusion process.

Synaptic excitation of hippocampal cells during blockade of synaptic inhibition results in an epileptiform "burst" potential followed by a prolonged afterhyperpolarization. This afterhyperpolarization resembles the one that is seen after the epileptic interictal spike and that is considered of critical importance in preventing seizure development. The afterhyperpolarization produced in the presence of y-aminobutyric acid antagonists is associated with a conductance increase and is inhibitory. It can occur in an all-or-none fashion after a burst, is independent of chloride, and is depressed by barium. The afterhyperpolarization has a reversal potential of (-86) millivolts, and the reversal potential is strongly dependent on the extracellular concentration of potassium. The afterhyperpolarization appears to be an intrinsic, inhibitory potassium potential mediated by calcium. This finding has implications for understanding the cellular mechanisms of epilepsy.

(1) The effect of active and inactive phorbol esters on synaptic transmission and on membrane properties of CA1 pyramidal cells in hippocampus have been analyzed by intracellular recording. (2) 4 beta-phorbol-12,13 dibutyrate (beta PDBu), but not the alpha-isomer, increased the firing probability, reduced the spike latency and enhanced the EPSP amplitude in response to synaptic activation. The effect was similar to the changes seen in long term potentiation. After alpha PDBu addition it was possible to elicit further enhancement by tetanization, but not after beta PDBu administration. (3) A slowly developing hyperpolarization was seen after active phorbol ester application without apparent changes in the soma input resistance. (4) Active phorbol esters reduced the slow afterhyperpolarization (AHP) in these cells without affecting the intermediate AHP.

The input/output relationship in primary visual cortex neurons is influenced by the history of the preceding activity. To understand the impact that membrane potential trajectory and firing pattern has on the activation of slow conductances in cortical neurons we compared the afterpotentials that followed responses to different stimuli evoking similar numbers of action potentials. In particular, we compared afterpotentials following the intracellular injection of either square or sinusoidal currents lasting 20 seconds. Both stimuli were intracellular surrogates of different neuronal responses to prolonged visual stimulation. Recordings from 99 neurons in slices of visual cortex revealed that for stimuli evoking an equivalent number of spikes, sinusoidal current injection activated a slow afterhyperpolarization of significantly larger amplitude (8.5±3.3 mV) and duration (33±17 s) than that evoked by a square pulse (6.4±3.7 mV, 28±17 s; p, Vanessa F. Descalzo was supported by a FPI fellowship (Spanish government).

Principal neurons in the lateral nucleus of the amygdala (LA) exhibit a continuum of firing properties in response to prolonged current injections ranging from those that accommodate fully to those that fire repetitively. In most cells, trains of action potentials are followed by a slow afterhyperpolarization (AHP) lasting several seconds. Reducing calcium influx either by lowering concentrations of extracellular calcium or by applying nickel abolished the AHP, confirming it is mediated by calcium influx. Blockade of large conductance calcium-activated potassium channel (BK) channels with paxilline, iberiotoxin, or TEA revealed that BK channels are involved in action potential repolarization but only make a small contribution to the fast AHP that follows action potentials. The fast AHP was, however, markedly reduced by low concentrations of 4-aminopyridine and alpha-dendrotoxin, indicating the involvement of voltage-gated potassium channels in the fast AHP. The medium AHP was blocked by apamin and UCL1848, indicating it was mediated by small conductance calcium-activated potassium channel (SK) channels. Blockade of these channels had no effect on instantaneous firing. However, enhancement of the SK-mediated current by 1-ethyl-2-benzimidazolinone or paxilline increased the early interspike interval, showing that under physiological conditions activation of SK channels is insufficient to control firing frequency. The slow AHP, mediated by non-SK BK channels, was apamin-insensitive but was modulated by carbachol and noradrenaline. Tetanic stimulation of cholinergic afferents to the LA depressed the slow AHP and led to an increase in firing. These results show that BK, SK, and non-BK SK-mediated calcium-activated potassium currents are present in principal LA neurons and play distinct physiological roles.

The cellular mechanisms of spike-frequency adaptation during prolonged discharges and of the slow afterhyperpolarization (AHP) that follows, as occur in vivo with contrast adaptation, were investigated with intracellular recordings of cortical neurons in slices of ferret primary visual cortex. Intracellular injection of 2 Hz sinusoidal or constant currents for 20 sec resulted in a slow (τ = 1–10 sec) spike-frequency adaptation, the degree of which varied widely among neurons. Reducing either [Ca2+]o or [Na+]o reduced the rate of spike-frequency adaptation. After the prolonged discharge was a slow (12–75 sec) AHP that was associated with an increase in membrane conductance and a rightward shift in the discharge frequency versus injected current relationship. The reversal potential of the slow AHP was sensitive to changes in [K+]o, indicating that it was mediated by a K+ current. Blockade of transmembrane Ca2+ conductances did not reduce the slow AHP. In contrast, reductions of [Na+]o reduced the slow AHP, even in the presence of pronounced Ca2+ spikes. We suggest that the activation of Na+-activated and Ca2+-activated K+ currents plays an important role in prolonged spike-frequency adaptation and therefore may contribute to contrast adaptation and other forms of adaptation in the visual system in vivo., This work was supported by grants from National Science Foundation and the National Institute of Health.

A single-electrode voltage-clamp technique was employed on in vitro hippocampal slices to examine the membrane current responsible for the slow afterhyperpolarization (AHP) in CA1 pyramidal cells. This was achieved by using conventional procedures to evoke an AHP in current clamp, followed rapidly by a switch into voltage clamp (hybrid clamp). The AHP current showed a dependence on extracellular K+, which was close to that predicted for a K+ current by the Nernst equation. The AHP current could be blocked by Cd2+ or norepinephrine. Although the AHP current showed a requirement for voltage-dependent Ca2+ entry, the current did not show any clear intrinsic voltage dependence. Once activated, AHP current is not turned off by hyperpolarizing the membrane potential. The effects of norepinephrine, Cd2+, and tetraethylammonium (TEA) were used to identify an AHP current component to the outward current evoked by depolarizing voltage commands from holding potentials that approximate to the resting potential for these cells. The AHP current can contribute significantly to the outward current during the depolarizing command. Upon repolarization it is evident as a slow outward tail current. This slow tail current had the same time constant as AHP currents evoked by hybrid clamp. Fast components to the tail currents were also observed. These were sensitive to Cd2+ and TEA. They probably represent a voltage-sensitive gKCa, sometimes termed C-current. The strong sensitivity to voltage and TEA displayed by the conventionally described gKCa (IC) are properties inconsistent with the AHP. It seems likely that the AHP current (IAHP) represents a Ca2+-activated K+ current separate from IC and that these two currents coexist in the same cell.

Hippocalcin is a neuronal calcium sensor protein previously implicated in regulating neuronal viability and plasticity. Hippocalcin is the most highly expressed neuronal calcium sensor in the medium spiny striatal output neurons that degenerate selectively in Huntington's disease (HD). We have previously shown that decreased hippocalcin expression occurs in parallel with the onset of disease phenotype in mouse models of HD. Here we show by in situ hybridization histochemistry that hippocalcin RNA is also diminished by 63% in human HD brain. These findings lead us to hypothesize that diminished hippocalcin expression might contribute to striatal neurodegeneration in HD. We tested this hypothesis by assessing whether restoration of hippocalcin expression would decrease striatal neurodegeneration in cellular models of HD comprising primary striatal neurons exposed to mutant huntingtin, the mitochondrial toxin 3-nitropropionic acid or an excitotoxic concentration of glutamate. Counter to our hypothesis, hippocalcin expression did not improve the survival of striatal neurons under these conditions. Likewise, expression of hippocalcin together with interactor proteins including the neuronal apoptosis inhibitory protein did not increase the survival of striatal cells in cellular models of HD. These results indicate that diminished hippocalcin expression does not contribute to HD-related neurodegeneration.

The hippocampus is among the regions in the brain richest in M1 cholinergic receptors. Topical application of acetylcholine (ACh) onto hippocampal slices produces a characteristic complex response consisting of a depolarization, an increase in input resistance especially upon depolarization and a blockade of a slow afterhyperpolarization (AHP). The first two of these responses can be recorded also in non-cholinergic septal neurons in an area which contains about 8% of the M1 muscarinic receptors found in the hippocampus. The responses of hippocampal but not septal neurons to ACh involve an increase in the spontaneous synaptic activity and a decrease in evoked responses to afferent stimulation. The dissociated hippocampal culture was used to study these presynaptic effects. The neurons in culture possess muscarinic receptors which develop gradually over a period of several weeks after plating. ACh rarely depolarizes hippocampal neurons in culture. Instead, it causes an increase in spontaneous discharge of small postsynaptic currents (PSC’s) and a marked decrease of large, evoked PSC’s. In some cultured hippocampal cells ACh reduced ICa without affecting any of several outward K currents studied. It is suggested that ACh reduces evoked activity by reducing Ca currents at presynaptic terminals.

Trains of action potentials in hippocampal pyramidal neurons are followed by a prolonged afterhyperpolarization (AHP) lasting several seconds, which is attributable to the activation of a slow calcium-activated potassium current (sIAHP). Here we examine the location of sIAHPon CA1 pyramidal neurons by comparing it with two GABAergic inhibitory postsynaptic currents (IPSCs) with known somatic and dendritic locations. Whole-cell patch-clamp recordings were made from CA1 pyramidal neurons in acute hippocampal slices. Stepping the membrane potential at the peak of sIAHPproduced a relaxation (“switchoff”) of the AHP current with a time constant of 7.4 ± 0.4 msec (mean ± SEM). The switchoff time constants for somatic and dendritic GABAAIPSCs were 3.5 ± 0.5 msec and 8.8 ± 0.3 msec, respectively. This data, together with cable modeling, indicates that active sIAHPchannels are distributed over the proximal dendrites within ∼200 μm of the soma. Excitatory postsynaptic potentials (EPSPs) evoked in stratum (s.) radiatum had their amplitudes shunted more by the AHP than did EPSPs evoked in s. oriens, suggesting that active AHP channels are restricted to the apical dendritic tree. Blockade of the AHP during a tetanus, which in control conditions elicited a decremental short-term potentiation (STP), converted STP to long-term potentiation (LTP). Thus, activation of the AHP increases the threshold for induction of LTP. These results suggest that in addition to its established role in spike frequency adaptation, the AHP works as an adjustable gain control, variably hyperpolarizing and shunting synaptic potentials arising in the apical dendrites.