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ATP-sensitive potassium channels (K(ATP) channels) are important sensors of cellular metabolic state that link metabolism and excitability in neuroendocrine cells, but their role in nonglucosensing central neurons is less well understood. To examine a possible role for K(ATP) channels in modulating excitability in hippocampal circuits, we recorded the activity of single K(ATP) channels in cell-attached patches of granule cells in the mouse dentate gyrus during bursts of action potentials generated by antidromic stimulation of the mossy fibers. Ensemble averages of the open probability (p(open)) of single K(ATP) channels over repeated trials of stimulated spike activity showed a transient increase in p(open) in response to action potential firing. Channel currents were identified as K(ATP) channels through blockade with glibenclamide and by comparison with recordings from Kir6.2 knock-out mice. The transient elevation in K(ATP) p(open) may arise from submembrane ATP depletion by the Na(+)-K(+) ATPase, as the pump blocker strophanthidin reduced the magnitude of the elevation. Both the steady-state and stimulus-elevated p(open) of the recorded channels were higher in the presence of the ketone body R-β-hydroxybutyrate, consistent with earlier findings that ketone bodies can affect K(ATP) activity. Using perforated-patch recording, we also found that K(ATP) channels contribute to the slow afterhyperpolarization following an evoked burst of action potentials. We propose that activity-dependent opening of K(ATP) channels may help granule cells act as a seizure gate in the hippocampus and that ketone-body-mediated augmentation of the activity-dependent opening could in part explain the effect of the ketogenic diet in reducing epileptic seizures.

The cytoplasmic Ca2+clearance rate affects neuronal excitability, plasticity, and synaptic transmission. Here, we examined the modulation of the plasma membrane Ca2+ATPase (PMCA) by tyrosine kinases. In rat sensory neurons grown in culture, the PMCA was under tonic inhibition by a member of the Src family of tyrosine kinases (SFKs). Ca2+clearance accelerated in the presence of selective tyrosine kinase inhibitors. Tonic inhibition of the PMCA was attenuated in cells expressing a dominant-negative construct or shRNA directed to message for the SFKs Lck or Fyn, but not Src. SFKs did not appear to phosphorylate the PMCA directly but instead activated focal adhesion kinase (FAK). Expression of constitutively active FAK enhanced and dominant-negative or shRNA knockdown of FAK attenuated tonic inhibition. Antisense knockdown of PMCA isoform 4 removed tonic inhibition of Ca2+clearance, indicating that FAK acts on PMCA4. The hyaluronan receptor CD44 activates SFK-FAK signaling cascades and is expressed in sensory neurons. Treating neurons with a CD44-blocking antibody or short hyaluronan oligosaccharides, which are produced during injury and displace macromolecular hyaluronan from CD44, attenuated tonic PMCA inhibition. Ca2+-activated K+channels mediate a slow afterhyperpolarization in sensory neurons that was inhibited by tyrosine kinase inhibitors and enhanced by knockdown of PMCA4. Thus, we describe a novel kinase cascade in sensory neurons that enables the extracellular matrix to alter Ca2+signals by modulating PMCA-mediated Ca2+clearance. This signaling pathway may influence the excitability of sensory neurons following injury.

Premature and uncompensated loss of ovarian hormones following ovariectomy (OVX) elevates the risks of cognitive impairment and dementia. These risks are prevented with estrogen (E2)-containing hormone replacement therapy initiated shortly following OVX but not after substantial delay. Currently, the cellular bases underlying these clinical findings are unknown. At the cellular level, intrinsic membrane properties regulate the efficiency of synaptic inputs to initiate output action potentials (APs), thereby affecting neuronal communication, hence cognitive processing. This study tested the hypothesis that in CA1 pyramidal neurons, intrinsic membrane properties and their acute regulation by E2require ovarian hormones for maintenance. Whole-cell current-clamp recordings were performed on neurons from ∼7-month-old OVX rats that experienced either short-term (10 d, control OVX) or long-term (5 months, OVXLT) ovarian hormone deficiency. The results reveal that long-term hormone deficiency reduced intrinsic membrane excitability (IE) as measured by the number of evoked APs and firing duration for a given current injection. This was accompanied by AP broadening, an increased slow afterhyperpolarization (sAHP), and faster accumulation of NaVchannel inactivation during repetitive firing. In the control OVX neurons, E2acutely increased IE and reduced the sAHP. In contrast, acute regulation of IE by E2was absent in the OVXLTneurons. Since the degree of IE of hippocampal pyramidal neurons is positively related with hippocampus-dependent learning ability, and modulation of IE is observed following successful learning, these findings provide a framework for understanding hormone deficiency-related cognitive impairment and the critical window for therapy initiation.

The basolateral amygdala (BLA) assigns emotional significance to sensory stimuli. This association results in a change in the output (action potentials) of BLA projection neurons in response to the stimulus. Neuronal output is controlled by the intrinsic excitability of the neuron. A major determinant of intrinsic excitability in these neurons is the slow afterhyperpolarization (sAHP) that follows action potential (AP) trains and produces spike-frequency adaptation. The sAHP is mediated by a slow calcium-activated potassium current (sIAHP), but little is known about the channels that underlie this current. Here, using whole-cell patch-clamp recordings and high-speed calcium imaging from rat BLA projection neurons, we examined the location and function of these channels. We determined the location of the sIAHPby applying a hyperpolarizing voltage step during the sIAHPand measuring the time needed for the current to adapt to the new command potential, a function of its electrotonic distance from the somatic recording electrode. Channel location was also probed by focally uncaging calcium using a UV laser. Both methodologies indicated that, in BLA neurons, the sIAHPis primarily located in the dendritic tree. EPSPs recorded at the soma were smaller, decayed faster, and showed less summation during the sAHP. Adrenergic stimulation and buffering calcium reduced the sAHP and the attenuation of the EPSP during the sAHP. The sAHP also modulated the AP in the dendrite, reducing the calcium response evoked by a single AP. Thus, in addition to mediating spike-frequency adaptation, the sIAHPmodulates communication between the soma and the dendrite.

Ablation of the distal end of the short arm of chromosome 1 [1p36 deletion syndrome (1p36DS)] is one of the most commonly occurring terminal deletion syndromes in humans, occurring in ∼1 in 5000 newborns. Subjects with 1p36DS manifest a wide range of clinical features including growth delay, congenital heart defects, and craniofacial dysmorphism. In addition, individuals with 1p36DS often exhibit some form of neurological abnormality and are typically cognitively impaired. Although there is significant variability with regard to the extent of the deletion, several genes have been mapped to region 1p36 that are known to regulate neuronal function. One such gene—KCNAB2—encodes the potassium channel auxiliary subunit Kvβ2, which has been previously shown to modulate voltage-gated potassium currents in heterologous expression systems. Here, we present experiments characterizing mice in which the ortholog ofKCNAB2was deleted. We find that deletion ofKcnab2in mice leads to deficits in associative learning and memory. In addition, using whole-cell current-clamp, we find that deletion ofKcnab2leads to a reduction in the slow afterhyperpolarization following a burst of action potentials and a concomitant increase in neuronal excitability in projection neurons in the lateral nucleus of the amygdala. Our results suggest that loss of Kvβ2 likely contributes to the cognitive and neurological impairments observed in 1p36DS patients.

Neuronal firing is known to depend on the variance of synaptic input as well as the mean input current. Several studies suggest that input variance, or “noise,” has a divisive effect, reducing the slope or gain of the firing frequency–current (f–I) relationship. We measured the effects of current noise onf–Irelationships in pyramidal neurons and fast-spiking (FS) interneurons in slices of rat sensorimotor cortex. In most pyramidal neurons, noise had a multiplicative effect on the steady-statef–Irelationship, increasing gain. In contrast, noise reduced gain in FS interneurons. Gain enhancement in pyramidal neurons increased with stimulus duration and was correlated with the amplitude of the slow afterhyperpolarization (sAHP), a major mechanism of spike-frequency adaptation. The 5-HT2receptor agonist α-methyl-5-HT reduced the sAHP and eliminated gain increases, whereas augmenting the sAHP conductance by spike-triggered dynamic-current clamp enhanced the gain increase. These results indicate that the effects of noise differ fundamentally between classes of neocortical neurons, depending on specific biophysical properties including the sAHP conductance. Thus, noise from background synaptic input may enhance network excitability by increasing gain in pyramidal neurons with large sAHPs and reducing gain in inhibitory FS interneurons.

Intense synaptic activity may alter the response properties of neurons in highly interconnected networks. Here we investigate whether the excitability and the intrinsic response properties of neurons in the spinal cord are affected by the increased synaptic conductance during functional network activity. Scratch episodes were induced by mechanical stimulation in the isolated carapace-spinal cord preparation from the adult turtle. Intracellular recordings revealed a dramatic increase in synaptic activity in interneurons and motoneurons during scratch activity. Superimposed slow depolarizing waves were phase-related to the rhythmic bouts of spike activity in the hip flexor nerve. The increase in synaptic conductance in interneurons and motoneurons varied with the scratch rhythm. During individual episodes, the conductance shifted smoothly with the scratch rhythm from near-resting levels to levels two to four times higher. In slice experiments, we found that even moderate increases in the conductance of motoneurons suppressed the slow afterhyperpolarization and the plateau potentials. We conclude that the excitability and the intrinsic response properties of spinal neurons are periodically quenched by high synaptic conductance during functional network activity.

A slow posttrain afterhyperpolarization (sAHP) was studied in rat magnocellular neurosecretory cells (MNCs)in vitro. The sAHP was isolated from other afterpotentials by blocking the depolarizing afterpotential (DAP) with Cs+and the medium afterhyperpolarization (mAHP) with apamin. The sAHP amplitude increased logarithmically with activity (∼3 mV pere-fold increase in number of impulses) and, when firing stopped, decayed exponentially with a time constant of 2 sec. The sAHP was associated with increased membrane conductance, and its amplitude varied linearly with voltage, reversing at the K+equilibrium potential. The sAHP was blocked by Cd2+but not by charybdotoxin or iberiotoxin, blockers of intermediate- and big-conductance-type Ca2+-dependent K+(KCa) channels. The sAHP was reversibly inhibited by muscarine, an effect antagonized by atropine, indicating involvement of muscarinic cholinergic receptors. Muscarine did not affect Ca2+-dependent features of action potentials, DAPs, or the mAHP in MNCs, indicating selective modulation of KCachannels causing the sAHP. Muscarinic inhibition of the sAHP enhanced plateau potentials and increased the mean firing rate and duration of afterdischarges that followed spike trains evoked from voltages near threshold. Similarly, the frequency and duration of the spontaneous phasic bursts that characterize physiologically activated vasopressin-releasing MNCs were enhanced by muscarine. MNCs thus express apamin- and voltage-insensitive KCachannels that mediate an sAHP. The activity dependence and kinetics of the sAHP cause it to mask DAPs in a manner that attenuates the amplitude of plateau potentials. Muscarinic inhibition of the sAHP provides an effective mechanism for promoting phasic firing in MNCs.

In hippocampal neurons, the firing of a train of action potentials is terminated by generation of the slow afterhyperpolarization (AHP). Recordings from hippocampal slices have shown that the slow AHP likely results from the activation of small-conductance calcium-activated potassium (SK) channels by calcium (Ca(2+)) entry through L-type Ca(2+) channels. However, the relative localization of these two channel subtypes is not known. The cloning and characterization of three subtypes of SK channel has suggested that SK1 may underlie generation of the slow AHP. Using a novel antibody directed against rat SK1 (rSK1), it has been determined that the rSK1 channel is primarily in the soma of hippocampal CA1 neurons. In conjunction with antibodies directed against C (Ca(v)1.2) and D (Ca(v)1.3) class L-type Ca(2+) channel alpha1 subunits, it was observed that rSK1 channels were selectively colocalized with D class L-type channels. This colocalization supports the functional coupling of L-type and SK channels previously observed in cell-attached patches from hippocampal neurons. However, it appears contrary to the slow rise and decay of the slow AHP. Induction of delayed facilitation of L-type Ca(2+) channels in cell-attached patches from hippocampal neurons evoked delayed opening of coupled SK channels. Generation of ensemble currents produced waveforms identical to the ionic current underlying the slow AHP (I(sAHP)). Therefore, these data indicate that the slow AHP is somatic in origin, resulting from delayed facilitation of D class L-type Ca(2+) channels colocalized with rSK1 channels.

The identity of the potassium channel underlying the slow, apamin-insensitive component of the afterhyperpolarization current (sIAHP) remains unknown. We studied sIAHPin CA1 pyramidal neurons using simultaneous whole-cell recording, calcium fluorescence imaging, and flash photolysis of caged compounds. Intracellular calcium concentration ([Ca2+]i) peaked earlier and decayed more rapidly than sIAHP. Loading cells with low concentrations of the calcium chelator EGTA slowed the activation and decay of sIAHP. In the presence of EGTA, intracellular calcium decayed with two time constants. When [Ca2+]iwas increased rapidly after photolysis of DM-Nitrophen, both apamin-sensitive and apamin-insensitive outward currents were activated. The apamin-sensitive current activated rapidly (IAHPchannels. When [Ca2+]iwas decreased rapidly via photolysis of diazo-2, the decay of sIAHPwas similar to control (1.7 sec). All results could be reproduced by a model potassium channel gated by calcium, suggesting that the channels underlying sIAHPhave intrinsically slow kinetics because of their high affinity for calcium.

Dopaminergic interplexiform amacrine cells were labeled in transgenic mice with human placental alkaline phosphatase and could therefore be identified after dissociation of the retina and used for whole-cell current and voltage clamp. In absence of synaptic inputs, dopaminergic amacrines spontaneously fired action potentials in a rhythmic pattern. This activity was remarkably robust in the face of inhibition of various voltage-dependent ion channels. It was minimally affected by external cesium or cobalt, suggesting no involvement of either the hyperpolarization-activated cation currentI(h) or voltage-dependent calcium channels. Inhibiting calcium-activated potassium channels by charybdotoxin or tetraethylammonium slowed the repolarizing phase of the action potentials and eliminated a slow afterhyperpolarization but had a scarce effect on the frequency of spontaneous firing. Voltage-clamp experiments showed that the interspike depolarization leading to threshold results from tetrodotoxin-sensitive sodium channels active at the interspike voltages of −60 to −40 mV. Because dopamine acts on distant targets in the retina, the pacemaker activity of dopaminergic amacrines may be necessary to ensure a tonic release of the modulator from their dendritic tree. Pacemaking is a property that this type of retinal amacrine cell shares with the dopaminergic mesencephalic neurons, but the ionic mechanisms responsible for the spontaneous firing are apparently different.

L-type calcium channels are abundant in hippocampal pyramidal neurons and are highly clustered at the base of the major dendrites. However, little is known of their function in these neurons. Single-channel recording using a low concentration of permeant ion reveals a long-lasting facilitation of L-type channel activity that is induced by a depolarizing prepulse or a train of action potential waveforms. This facilitation exhibits a slow rise, peaking 0.5–1 sec after the train and decaying over several seconds. We have termed this behavior “delayed facilitation,” because of the slow onset. Delayed facilitation results from an increase in opening frequency and the recruitment of longer duration openings. This behavior is observed at all membrane potentials between −20 and −60 mV, with the induction and magnitude of facilitation being insensitive to voltage. β-Adrenergic receptor activation blocks induction of delayed facilitation but does not significantly affect normal L-type channel activity. Delayed facilitation of L-type calcium channels provides a prolonged source of calcium entry at negative membrane potentials. This behavior may underlie calcium-dependent events that are inhibited by β-adrenergic receptor activation, such as the slow afterhyperpolarization in hippocampal neurons.