Capacity for consciousness under ketamine anaesthesia is selectively associated with activity in posteromedial cortex in rats.

It remains unclear how specific cortical regions contribute to the brain's overall capacity for consciousness. Clarifying this could help distinguish between theories of consciousness. Here, we investigate the association between markers of regionally specific (de)activation and the brain's overall capacity for consciousness. We recorded electroencephalographic responses to cortical electrical stimulation in six rats and computed Perturbational Complexity Index state-transition (PCIST), which has been extensively validated as an index of the capacity for consciousness in humans. We also estimated the balance between activation and inhibition of specific cortical areas with the ratio between high and low frequency power from spontaneous electroencephalographic activity at each electrode. We repeated these measurements during wakefulness, and during two levels of ketamine anaesthesia: with the minimal dose needed to induce behavioural unresponsiveness and twice this dose. We found that PCIST was only slightly reduced from wakefulness to light ketamine anaesthesia, but dropped significantly with deeper anaesthesia. The high-dose effect was selectively associated with reduced high frequency/low frequency ratio in the posteromedial cortex, which strongly correlated with PCIST. Conversely, behavioural unresponsiveness induced by light ketamine anaesthesia was associated with similar spectral changes in frontal, but not posterior cortical regions. Thus, activity in the posteromedial cortex correlates with the capacity for consciousness, as assessed by PCIST, during different depths of ketamine anaesthesia, in rats, independently of behaviour. These results are discussed in relation to different theories of consciousness.

General Anesthesia Disrupts Complex Cortical Dynamics in Response to Intracranial Electrical Stimulation in Rats.

The capacity of human brain to sustain complex cortical dynamics appears to be strongly associated with conscious experience and consistently drops when consciousness fades. For example, several recent studies in humans found a remarkable reduction of the spatiotemporal complexity of cortical responses to local stimulation during dreamless sleep, general anesthesia, and coma. However, this perturbational complexity has never been directly estimated in non-human animals in vivo previously, and the mechanisms that prevent neocortical neurons to engage in complex interactions are still unclear. Here, we quantify the complexity of electroencephalographic (EEG) responses to intracranial electrical stimulation in rats, comparing wakefulness to propofol, sevoflurane, and ketamine anesthesia. The evoked activity changed from highly complex in wakefulness to far simpler with propofol and sevoflurane. The reduced complexity was associated with a suppression of high frequencies that preceded a reduced phase-locking, and disruption of functional connectivity and pattern diversity. We then showed how these parameters dissociate with ketamine and depend on intensity and site of stimulation. Our results support the idea that brief periods of activity-dependent neuronal silence can interrupt complex interactions in neocortical circuits, and open the way for further mechanistic investigations of the neuronal basis for consciousness and loss of consciousness across species.

Two distinct pools of large-conductance calcium-activated potassium channels in the somatic plasma membrane of central principal neurons.

Although nerve cell membranes are often assumed to be uniform with respect to electrical properties, there is increasing evidence for compartmentalization into subdomains with heterogeneous impacts on the overall cell function. Such microdomains are characterized by specific sets of proteins determining their functional properties. Recently, clustering of large-conductance calcium-activated potassium (BK(Ca)) channels was shown at sites of subsurface membrane cisterns in cerebellar Purkinje cells (PC), where they likely participate in building a subcellular signaling unit, the 'PLasmERosome'. By applying SDS-digested freeze-fracture replica labeling (SDS-FRL) and postembedding immunogold electron microscopy, we have now studied the spatial organization of somatic BK(Ca) channels in neocortical layer 5 pyramidal neurons, principal neurons of the central and basolateral amygdaloid nuclei, hippocampal pyramidal neurons and dentate gyrus (DG) granule cells to establish whether there is a common organizational principle in the distribution of BK(Ca) channels in central principal neurons. In all cell types analyzed, somatic BK(Ca) channels were found to be non-homogenously distributed in the plasma membrane, forming two pools of channels with one pool consisting of clustered channels and the other of scattered channels in the extrasynaptic membrane. Quantitative analysis by means of SDS-FRL revealed that about two-thirds of BK(Ca) channels belong to the scattered pool and about one-third to the clustered pool in principal cell somata. Overall densities of channels in both pools differed in the different cell types analyzed, although being considerably lower compared to cerebellar PC. Postembedding immunogold labeling revealed association of clustered channels with subsurface membrane cisterns and confirmed extrasynaptic localization of scattered channels. This study indicates a common organizational principle for somatic BK(Ca) channels in central principal neurons with the formation of a clustered and a scattered pool of channels, and a cell-type specific density of this channel type.

Kv7/KCNQ/M-channels in rat glutamatergic hippocampal axons and their role in regulation of excitability and transmitter release.

M-current (I(M)) plays a key role in regulating neuronal excitability. Mutations in Kv7/KCNQ subunits, the molecular correlates of I(M), are associated with a familial human epilepsy syndrome. Kv7/KCNQ subunits are widely expressed, and I(M) has been recorded in somata of several types of neurons, but the subcellular distribution of M-channels remains elusive. By combining field-potential, whole-cell and intracellular recordings from area CA1 in rat hippocampal slices, and computational modelling, we provide evidence for functional M-channels in unmyelinated axons in the brain. Our data indicate that presynaptic M-channels can regulate axonal excitability and synaptic transmission, provided the axons are depolarized into the I(M) activation range (beyond approximately -65 mV). Here, such depolarization was achieved by increasing the extracellular K(+) concentration ([K(+)](o)). Extracellular recordings in the presence of moderately elevated [K(+)](o) (7-11 mm), showed that the specific M-channel blocker XE991 reduced the amplitude of the presynaptic fibre volley and the field EPSP in a [K(+)](o)-dependent manner, both in stratum radiatum and in stratum lacknosum moleculare. The M-channel opener, retigabine, had opposite effects. The higher the [K(+)](o), the greater the effects of XE991 and retigabine. Similar pharmacological modulation of EPSPs recorded intracellularly from CA1 pyramidal neurons, while blocking postsynaptic K(+) channels with intracellular Cs(+), confirmed that active M-channels are located presynaptically. Computational analysis with an axon model showed that presynaptic I(M) can control Na(+) channel inactivation and thereby affect the presynaptic action potential amplitude and Ca(2+) influx, provided the axonal membrane potential is sufficiently depolarized. Finally, we compared the effects of blocking I(M) on the spike after-depolarization and bursting in CA3 pyramidal neuron somata versus their axons. In standard [K(+)](o) (2.5 mm), XE991 increased the ADP and promoted burst firing at the soma, but not in the axons. However, I(M) contributed to the refractory period in the axons when spikes were broadened by a low dose 4-aminopyridine (200 microm). Our results indicate that functional Kv7/KCNQ/M-channels are present in unmyelinated axons in the brain, and that these channels may have contrasting effects on excitability depending on their subcellular localization.

Cerebellar ataxia and Purkinje cell dysfunction caused by Ca2+-activated K+ channel deficiency.

Malfunctions of potassium channels are increasingly implicated as causes of neurological disorders. However, the functional roles of the large-conductance voltage- and Ca(2+)-activated K(+) channel (BK channel), a unique calcium, and voltage-activated potassium channel type have remained elusive. Here we report that mice lacking BK channels (BK(-/-)) show cerebellar dysfunction in the form of abnormal conditioned eye-blink reflex, abnormal locomotion and pronounced deficiency in motor coordination, which are likely consequences of cerebellar learning deficiency. At the cellular level, the BK(-/-) mice showed a dramatic reduction in spontaneous activity of the BK(-/-) cerebellar Purkinje neurons, which generate the sole output of the cerebellar cortex and, in addition, enhanced short-term depression at the only output synapses of the cerebellar cortex, in the deep cerebellar nuclei. The impairing cellular effects caused by the lack of postsynaptic BK channels were found to be due to depolarization-induced inactivation of the action potential mechanism. These results identify previously unknown roles of potassium channels in mammalian cerebellar function and motor control. In addition, they provide a previously undescribed animal model of cerebellar ataxia.

BK channel activity determines the extent of cell degeneration after oxygen and glucose deprivation: a study in organotypical hippocampal slice cultures.

BK channels are voltage- and calcium-dependent potassium channels whose activation tends to reduce cellular excitability. In hippocampal pyramidal cells, BK channels repolarize somatic action potentials, and recent immunogold and electrophysiological analyses have revealed a presynaptic pool of BK channels that can regulate glutamate release. Agents that modulate BK channel activity would therefore be expected to affect cell excitability and neurotransmitter release also under pathological conditions. We have investigated the role of BK potassium channels in a model of ischemia-induced nerve cell degeneration. Organotypical slice cultures of rat hippocampus were exposed to oxygen and glucose deprivation (OGD), and cell death was assessed by the fluorescent dye propidium iodide. OGD induced cell death in the CA1 region and to a lesser extent in CA3. Treatment with the BK channel blockers, paxilline and iberiotoxin, during and after OGD induced increased cell death in CA1 and CA3. Both BK channel blockers also sensitized the relatively resistant granule cells in fascia dentata to OGD. The effect of paxilline and iberiotoxin was evident from 3 h after OGD, indicating a role of BK channels early in the post-ischemic phase or during OGD itself. The BK channel opener, NS1619, turned out to be gliotoxic, and this effect was not counteracted by paxilline and iberiotoxin. Our data show that blockade of BK channels aggravates OGD-induced cell damage and suggest that BK channels act as a kind of 'emergency brake' during and/or after ischemia. Accordingly, the BK channel is a potential molecular target for neuroprotective therapy in stroke.

"Agriculture at risk: A report to the nation"--a historical review, critical analysis, and implications for future planning.

"Agricultural at Risk: A Report to the Nation" is a proceedings document of a three-year (1987-1990) policy development process entitled "Agricultural Occupational and Environmental Health: Policy Strategies for the Future." That process culminated in the emergence of occupational health and safety in agriculture as a public health policy issue in the U.S. Several agricultural health and safety programs evolved as direct or indirect consequences of this process, including, but not limited to, the NIOSH agricultural occupational health programs, The Kellogg Foundation agricultural health grants programs, and the prospective chronic health studies of pesticides (EPA-NlH funded). The Agriculture at Risk report resulted in 86 specific recommendations. The authors of this article reviewed each of these recommendations and rated them on a subjective scale as to their degree of attainment. They found that 44% of the recommendations had received moderate to substantial action. Most of the positive action was in the areas of research and coalition building. A noticeable lack of action was in the areas of standards and regulation, occupational health service delivery, and education categories. This article concludes with an analysis of the limitations of the AAR report, changes in exposed populations over the past decade, and specific recommendations on future actions to address the issues.

Presynaptic Ca2+-activated K+ channels in glutamatergic hippocampal terminals and their role in spike repolarization and regulation of transmitter release.

Large-conductance Ca(2+)-activated K(+) channels (BK, also called Maxi-K or Slo channels) are widespread in the vertebrate nervous system, but their functional roles in synaptic transmission in the mammalian brain are largely unknown. By combining electrophysiology and immunogold cytochemistry, we demonstrate the existence of functional BK channels in presynaptic terminals in the hippocampus and compare their functional roles in somata and terminals of CA3 pyramidal cells. Double-labeling immunogold analysis with BK channel and glutamate receptor antibodies indicated that BK channels are targeted to the presynaptic membrane facing the synaptic cleft in terminals of Schaffer collaterals in stratum radiatum. Whole-cell, intracellular, and field-potential recordings from CA1 pyramidal cells showed that the presynaptic BK channels are activated by calcium influx and can contribute to repolarization of the presynaptic action potential (AP) and negative feedback control of Ca(2+) influx and transmitter release. This was observed in the presence of 4-aminopyridine (4-AP, 40-100 microm), which broadened the presynaptic compound action potential. In contrast, the presynaptic BK channels did not contribute significantly to regulation of action potentials or transmitter release under basal experimental conditions, i.e., without 4-AP, even at high stimulation frequencies. This is unlike the situation in the parent cell bodies (CA3 pyramidal cells), where BK channels contribute strongly to action potential repolarization. These results indicate that the functional role of BK channels depends on their subcellular localization.

Interaction between synaptic excitation and slow afterhyperpolarization current in rat hippocampal pyramidal cells.

1. Whole cell recordings from CA1 pyramidal cells were performed to investigate the interaction between excitatory postsynaptic potentials (EPSPs) or currents (EPSCs), and the slow Ca(2+)-dependent K(+) current, I(sAHP). Blockers of the slow afterhyperpolarization (sAHP) such as isoprenaline (ISO) or noradrenaline (NA) reduced the hyperpolarization that followed a short train of EPSPs, and slowed the decay of summated EPSPs or EPSCs. 2. ISO/NA action on synaptic responses was observed in the absence of action potentials, but was curtailed by Ca(2+) chelation (10 mM EGTA in the electrode) and was not observed with a caesium-based recording solution. This suggests the involvement of an ISO/NA-sensitive Ca(2+)-dependent K(+) current without a requirement for regenerative spiking. 3. An ISO/NA-sensitive sAHP was observed following both NMDA and non-NMDA receptor-mediated EPSP trains in nominally zero Mg(2+) medium. Isoprenaline sensitivity was blocked by hyperpolarization during EPSPs or by isradipine, suggesting a requirement for voltage-dependent Ca(2+) influx during EPSPs. The data indicate that bursts of EPSPs can activate voltage-gated Ca(2+) channels, which trigger I(sAHP) during synaptic responses. 4. A decrease in EPSP temporal summation occurred during both spike-evoked sAHPs and persistent activation of sAHP conductance following internal dialysis with diazo-2 (2 mM). At constant membrane potential, diazo-2 caused a decrease in membrane time constant and input resistance and accelerated the rate of EPSP decay. Photolysis of diazo-2 or application of NA reduced the resting sAHP conductance, causing an increased membrane time constant and input resistance in association with an increase in EPSP half-width. 5. These results indicate that short bursts of EPSPs can activate a Ca(2+)-dependent K(+) current resembling I(sAHP), and that activation of this current reduces the postsynaptic response to high-frequency synaptic input. The findings imply that modulation of I(sAHP) can regulate synaptic efficacy and may influence the threshold for tetanus-induced synaptic plasticity.

Protein kinase A mediates the modulation of the slow Ca(2+)-dependent K(+) current, I(sAHP), by the neuropeptides CRF, VIP, and CGRP in hippocampal pyramidal neurons.

We have studied modulation of the slow Ca(2+)-activated K(+) current (I(sAHP)) in CA1 hippocampal pyramidal neurons by three peptide transmitters: corticotropin releasing factor (CRF, also called corticotropin releasing hormone, CRH), vasoactive intestinal peptide (VIP), and calcitonin gene-related peptide (CGRP). These peptides are known to be expressed in interneurons. Using whole cell voltage clamp in hippocampal slices from young rats, in the presence of tetrodotoxin (TTX, 0.5 microM) and tetraethylammonium (TEA, 5 mM), I(sAHP) was measured after a brief depolarizing voltage step eliciting inward Ca(2+) current. Each of the peptides CRF (100-250 nM), VIP (400 nM), and CGRP (1 microM) significantly reduced the amplitude of I(sAHP). Thus the I(sAHP) amplitude was reduced to 22% by 100 nM CRF, to 17% by 250 nM CRF, to 22% by 400 nM VIP, and to 40% by 1 microM CGRP. We found no consistent concomitant changes in the Ca(2+) current or in the time course of I(sAHP) for any of the three peptides, suggesting that the suppression of I(sAHP) was not secondary to a general suppression of Ca(2+) channel activity. Because each of these peptides is known to activate the cyclic AMP (cAMP) cascade in various cell types, and I(sAHP) is known to be suppressed by cAMP via the cAMP-dependent protein kinase (PKA), we tested whether the effects on I(sAHP) by CRF, VIP, and CGRP are mediated by PKA. Intracellular application of the PKA-inhibitor Rp-cAMPS significantly reduced the suppression of I(sAHP) by CRF, VIP, and CGRP. Thus with 1 mM Rp-cAMPS in the recording pipette, the average suppression of I(sAHP) was reduced from 78 to 26% for 100 nM CRF, from 83 to 32% for 250 nM CRF, from 78 to 30% for 400 nM VIP, and from 60 to 7% for 1 microM CGRP. We conclude that CRF, VIP, and CGRP suppress the slow Ca(2+)-activated K(+) current, I(sAHP), in CA1 hippocampal pyramidal neurons by activating the cAMP-dependent protein kinase, PKA. Together with the monoamine transmitters norepinephrine, serotonin, histamine, and dopamine, these peptide transmitters all converge on the cAMP cascade modulating I(sAHP).

The role of BK-type Ca2+-dependent K+ channels in spike broadening during repetitive firing in rat hippocampal pyramidal cells.

1. The role of large-conductance Ca2+-dependent K+ channels (BK-channels; also known as maxi-K- or slo-channels) in spike broadening during repetitive firing was studied in CA1 pyramidal cells, using sharp electrode intracellular recordings in rat hippocampal slices, and computer modelling. 2. Trains of action potentials elicited by depolarizing current pulses showed a progressive, frequency-dependent spike broadening, reflecting a reduced rate of repolarization. During a 50 ms long 5 spike train, the spike duration increased by 63.6 +/- 3.4 % from the 1st to the 3rd spike. The amplitude of the fast after-hyperpolarization (fAHP) also rapidly declined during each train. 3. Suppression of BK-channel activity with (a) the selective BK-channel blocker iberiotoxin (IbTX, 60 nM), (b) the non-peptidergic BK-channel blocker paxilline (2-10 microM), or (c) calcium-free medium, broadened the 1st spike to a similar degree ( approximately 60 %). BK-channel suppression also caused a similar change in spike waveform as observed during repetitive firing, and eliminated (occluded) most of the spike broadening during repetitive firing. 4. Computer simulations using a reduced compartmental model with transient BK-channel current and 10 other active ionic currents, produced an activity-dependent spike broadening that was strongly reduced when the BK-channel inactivation mechanism was removed. 5. These results, which are supported by recent voltage-clamp data, strongly suggest that in CA1 pyramidal cells, fast inactivation of a transient BK-channel current (ICT), substantially contributes to frequency-dependent spike broadening during repetitive firing.

Functional and molecular aspects of voltage-gated K+ channel beta subunits.

Voltage-gated potassium channels (Kv) of the Shaker-related superfamily are assembled from membrane-integrated alpha subunits and auxiliary beta subunits. The beta subunits may increase Kv channel surface expression and/or confer A-type behavior to noninactivating Kv channels in heterologous expression systems. The interaction of Kv alpha and Kv beta subunits depends on the presence or absence of several domains including the amino-terminal N-type inactivating and NIP domains and the Kv alpha and Kv beta binding domains. Loss of function of Kv beta 1.1 subunits leads to a reduction of A-type Kv channel activity in hippocampal and striatal neurons of knock-out mice. This reduction may be correlated with altered cognition and motor control in the knock-out mice.

Modulation of the Ca2+-activated K+ current sIAHP by a phosphatase-kinase balance under basal conditions in rat CA1 pyramidal neurons.

The slow Ca2+-activated K+ current, sIAHP, underlying spike frequency adaptation, was recorded with the whole cell patch-clamp technique in CA1 pyramidal neurons in rat hippocampal slices. Inhibitors of serine/threonine protein phosphatases (microcystin, calyculin A, cantharidic acid) caused a gradual decrease of sIAHP amplitude, suggesting the presence of a basal phosphorylation-dephosphorylation turnover regulating sIAHP. Because selective calcineurin (PP-2B) inhibitors did not affect the amplitude of sIAHP, protein phosphatase 1 (PP-1) or 2A (PP-2A) are most likely involved in the basal regulation of this current. The ATP analogue, ATP-gamma-S, caused a gradual decrease in the sIAHP amplitude, supporting a role of protein phosphorylation in the basal modulation of sIAHP. When the protein kinase A (PKA) inhibitor adenosine-3', 5'-monophosphorothioate, Rp-isomer (Rp-cAMPS) was coapplied with the phosphatase inhibitor microcystin, it prevented the decrease in the sIAHP amplitude that was observed when microcystin alone was applied. Furthermore, inhibition of PKA by Rp-cAMPS led to an increase in the sIAHP amplitude. Finally, an adenylyl cyclase inhibitor (SQ22, 536) and adenosine 3',5'-cyclic monophosphate-specific type IV phosphodiesterase inhibitors (Ro 20-1724 and rolipram) led to an increase or a decrease in the sIAHP amplitude, respectively. These findings suggest that a balance between basally active PKA and a phosphatase (PP-1 or PP-2A) is responsible for the tonic modulation of sIAHP, resulting in a continuous modulation of excitability and firing properties of hippocampal pyramidal neurons.

Reduced K+ channel inactivation, spike broadening, and after-hyperpolarization in Kvbeta1.1-deficient mice with impaired learning.

A-type K+ channels are known to regulate neuronal firing, but their role in repetitive firing and learning in mammals is not well characterized. To determine the contribution of the auxiliary K+ channel subunit Kvbeta1.1 to A-type K+ currents and to study the physiological role of A-type K+ channels in repetitive firing and learning, we deleted the Kvbeta1.1 gene in mice. The loss of Kvbeta1.1 resulted in a reduced K+ current inactivation in hippocampal CA1 pyramidal neurons. Furthermore, in the mutant neurons, frequency-dependent spike broadening and the slow afterhyperpolarization (sAHP) were reduced. This suggests that Kvbeta1.1-dependent A-type K+ channels contribute to frequency-dependent spike broadening and may regulate the sAHP by controlling Ca2+ influx during action potentials. The Kvbeta1.1-deficient mice showed normal synaptic plasticity but were impaired in the learning of a water maze test and in the social transmission of food preference task, indicating that the Kvbeta1.1 subunit contributes to certain types of learning and memory.

Interaction between alpha- and beta-adrenergic receptor agonists modulating the slow Ca(2+)-activated K+ current IAHP in hippocampal neurons.

Noradrenaline inhibits the Ca(2+)-activated K+ current IAHP, which underlies the slow afterhyperpolarization and spike frequency adaptation in hippocampal and neocortical neurons. The resulting increase in excitability probably contributes to the state control of the forebrain during arousal and attention. The modulation of IAHP by noradrenaline has previously been shown to be mediated by beta 1 receptors, cyclic AMP and protein kinase A, but not by alpha receptors. We have now tested the possibility that alpha receptors also contribute to IAHP modulation through interaction with beta receptors, by the use of whole-cell recordings in CA1 pyramidal cells of rat hippocampal slices. The alpha-receptor agonist 6-fluoro-noradrenaline strongly potentiated the effect of isoproterenol on IAHP. The synergistic effect of 6-fluoro-noradrenaline and isoproterenol was blocked by the beta-receptor antagonist timolol, but the receptor type mediating the effect of 6-fluoro-noradrenaline could not be unequivocally identified by using alpha-receptor antagonists. The effect of high concentrations of noradrenaline on IAHP was only partly blocked by the beta-receptor antagonist timolol, and was further reduced by blocking alpha receptors, again suggesting a contribution from alpha receptors. In contrast, the effect of low concentrations of noradrenaline seemed to be potentiated by the alpha-receptor antagonist phentolamine in 57% of the cells, suggesting concentration-dependent antagonistic interaction between alpha and beta receptors. Further tests indicated that the cross-talk between 6-fluoro-noradrenaline and isoproterenol occurs upstream from cyclic AMP production, and that protein kinase A serves as a final common path for the modulation of IAHP by noradrenaline, and by the combination of 6-fluoro-noradrenaline and isoproterenol.

Evidence that Ca/calmodulin-dependent protein kinase mediates the modulation of the Ca2+-dependent K+ current, IAHP, by acetylcholine, but not by glutamate, in hippocampal neurons.

Muscarinic and metabotropic glutamate receptor agonists increase the excitability of hippocampal and other cortical neurons by suppressing the Ca2+-activated K+current, IAHP, which underlies the slow afterhyperpolarization (AHP) and spike frequency adaptation. We have examined the mechanism of action of a muscarinic agonist (carbachol) and a metabotropic glutamate receptor agonist (1-Aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) on IAHP in hippocampal CA1 neurons in slices, by using highly specific protein kinase inhibitors. We found that inhibition of protein kinase A (PKA) with the adenosine 3',5'-cyclic monophosphate (cAMP) analogue Rp-adenosine-3',5'-cyclic phosphorothioate Rp-cAMPS, did not prevent the muscarinic and glutamatergic suppression of IAHP. In contrast, two specific peptide inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaM-K II), each partially blocked the effect of carbachol, but not the effect of t-ACPD on IAHP. We conclude that CaM-K II, but not PKA, is involved in mediating the muscarinic suppression of IAHP, although other pathways may also contribute. In contrast, neither CaM-K II nor PKA seems to mediate the metabotropic glutamate receptor action on IAHP.

Cholinergic modulation of the action potential in rat hippocampal neurons.

The cholinergic input to the hippocampus from the medial septum is important for modulating hippocampal activity and functions, including theta rhythm and spatial learning. Neuromodulation by transmitters in central nervous system neurons usually affects cell excitability by modifying the membrane potential, discharge pattern and spike frequency. Here we describe another type of neuromodulation: changing the action potential waveform. During intracellular recordings from CA1 pyramidal cells in hippocampal slices from rats, the cholinergic agonist carbachol caused several reversible changes in the action potential: low doses (2 microM) caused an increase in spike duration; high doses (10-40 microM) or long-lasting applications also reduced the spike amplitude and rate of rise, and raised the spike threshold. These effects are similar to those of metabotropic glutamate receptor agonists or phorbol esters, both of which activate protein kinase C. The effects were blocked by the muscarinic antagonist atropine, and were prevented by Ca(2+)-free medium and by Ca(2+)-channel blockers. However, the cholinergic spike modulation was not occluded or mimicked by blocking the Ca(2+)-dependent K+ currents IC or IAHP, suggesting that these K+ currents are not involved in the modulation. We conclude that muscarinic receptor activation modulates the action potential in CA1 pyramidal cells via a Ca(2+)-dependent mechanism, possibly involving protein kinase C. This modulation and the similar effects mediated by metabotropic glutamate receptors to our knowledge provide the only examples of neuromodulation of the action potential in the vertebrate central nervous system-a form of modulation known to regulate Ca2+ influx and transmitter release, and to mediate synaptic plasticity and learning in invertebrates.

Protein kinase A-independent modulation of ion channels in the brain by cyclic AMP.

Ion channels underlying the electrical activity of neurons can be regulated by neurotransmitters via two basic mechanisms: ligand binding and covalent modification. Whereas neurotransmitters often act by binding directly to ion channels, the intracellular messenger cyclic AMP is thought usually to act indirectly, by activating protein kinase A, which in turn can phosphorylate channel proteins. Here we show that cyclic AMP, and transmitters acting via cyclic AMP, can act in a protein kinase A-independent manner in the brain. In hippocampal pyramidal cells, cyclic AMP and norepinephrine were found to cause a depolarization by enhancing the hyperpolarization-activated mixed cation current, IQ (also called Ih). This effect persisted even after protein kinase A activity was blocked, thus strongly suggesting a kinase-independent action of cyclic AMP. The modulation of this current by ascending monoaminergic fibers from the brainstem is likely to be a widespread mechanism, participating in the state control of the brain during arousal and attention.

Dopamine modulates the slow Ca(2+)-activated K+ current IAHP via cyclic AMP-dependent protein kinase in hippocampal neurons.

1. The effects of dopamine on the slow Ca(2+)-dependent K+ current (IAHP; AHP, afterhyperpolarization) and spike frequency adaptation were studied by whole cell voltage-clamp and sharp microelectrode current-clamp recordings in rat CA1 pyramidal neurons in rat hippocampal slices. 2. Dopamine suppressed IAHP in a dose-dependent manner, under whole cell voltage-clamp conditions. Similarly, under current-clamp conditions, dopamine inhibited spike frequency adaptation and suppressed the slow afterhyperpolarization. 3. The effect of dopamine on IAHP was mimicked by a D1 receptor agonist and blocked by dopamine receptor antagonists only in a minority of the cells. 4. Dopamine suppressed IAHP after blocking or desensitizing the beta-adrenergic receptors and, hence, did not act by cross-reacting with this receptor type. 5. The effects of dopamine on IAHP and spike frequency adaptation were suppressed by blocking the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent kinase (PKA) with Rp-cAMPS and, hence, are probably mediated by the activation of this kinase. 6. We conclude that dopamine increases hippocampal neuron excitability, like other monoamine neurotransmitters, by suppressing IAHP and spike frequency adaptation, via cAMP and protein kinase A. The receptor type mediating this effect of dopamine remains to be defined.