Modulation of gamma-aminobutyric acid-mediated inhibitory synaptic currents in dissociated cortical cell cultures.

Inhibitory gamma-aminobutyric acid-mediated synaptic currents were studied in dissociated primary cultures of neonatal rat cortex with the whole-cell patch-clamp technique. Immunocytochemical staining of the cultures showed the presence of a large number of glutamic acid decarboxylase-containing neurons, and electrical stimulation of randomly selected neurons produced in many cases chloride-mediated and bicuculline-sensitive inhibitory synaptic currents in postsynaptic cells. The amplitude and decay time of the inhibitory synaptic currents were increased by flunitrazepam and decreased by the beta-carboline derivative methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, two high-affinity ligands for the allosteric regulatory sites of gamma-aminobutyric acid receptors. The imidazobenzodiazepine Ro 15-1788, another high-affinity ligand of the gamma-aminobutyric acid receptor regulatory sites that has negligible intrinsic activity, blocked the action of flunitrazepam and beta-carboline. However, Ro 15-1788 also increased the decay rate of the inhibitory synaptic currents. This might suggest that an endogenous ligand for the benzodiazepine-beta-carboline binding site is operative in gamma-aminobutyric acid-mediated synaptic transmission.     

Synaptic currents in cerebellar Purkinje cells.

Cerebellar Purkinje cells are known to receive strong excitatory input from two major pathways originating outside the cerebellum and inhibitory input from two types of neurons in the cerebellar cortex. The functions and synaptic strengths of these pathways are only partially known. We have used the patch-clamp technique applied to Purkinje cells in thin slices of rat cerebellum to measure directly the postsynaptic currents arising from the two major excitatory pathways and one of the inhibitory inputs. Inhibitory synaptic currents occur spontaneously with high frequency and are variable in amplitude, ranging, in our recording conditions with high internal Cl-, from less than 100 pA to more than 1 nA. These currents are blocked by the gamma-aminobutyrate type A antagonist bicuculline. One of the excitatory inputs is all or none. For threshold stimulation, the synaptic current is either full amplitude, when the presynaptic fiber is successfully stimulated, or completely absent. This synaptic current is often larger than 1 nA and is virtually eliminated by 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione, a blocking agent thought to be specific for glutamate receptors that are not of the N-methyl-D-aspartate type. Its all-or-none character identifies it as arising from a climbing-fiber synapse. The other excitatory input produces a synaptic current that is smoothly graded as a function of stimulus intensity. This response we believe arises from the stimulation of mossy fibers or granule cells. The synaptic current associated with this input is also largely eliminated by 2 microM 6-cyano-7-nitroquinoxaline-2,3-dione.     

Voltage-gated currents of tilapia prolactin cells

The first recordings of neuron-like electrical activity from endocrine cells were made from fish pituitary cells. However, patch-clamping studies have predominantly utilized mammalian preparations. This study used whole-cell patch-clamping to characterize voltage-gated ionic currents of anterior pituitary cells of Oreochromis mossambicus in primary culture. Due to their importance for control of hormone secretion we emphasize analysis of calcium currents (I(Ca)), including using peptide toxins diagnostic for mammalian neuronal Ca(2+) channel types. These appear not to have been previously tested on fish endocrine cells. In balanced salines, inward currents consisted of a rapid TTX-sensitive sodium current and a smaller, slower I(Ca); there followed outward potassium currents dominated by delayed, sustained TEA-sensitive K(+) current. About half of cells tested from a holding potential (V(h)) of -90 mV showed early transient K(+) current; most cells showed a small Ca(2+)-mediated outward current. I-V plots of isolated I(Ca) with 15 mM [Ca(2+)](o) showed peak currents (up to 20 pA/pF from V(h) -90 mV) at approximately +10 mV, with approximately 60% I(Ca) for V(h) -50 mV and approximately 30% remaining at V(h) -30 mV. Plots of normalized conductance vs. voltage at several V(h)s were nearly superimposable. Well-sustained I(Ca) with predominantly Ca(2+)-dependent inactivation and inhibition of approximately 30% of total I(Ca) by nifedipine or nimodipine suggests participation of L-type channels. Each of the peptide toxins (omega-conotoxin GVIA, omega-agatoxin IVA, SNX482) alone blocked 36-54% of I(Ca). Inhibition by any of these toxins was additive to inhibition by nifedipine. Combinations of the toxins failed to produce additive effects. I(Ca) of up to 30% of total remained with any combination of inhibitors, but 0.1mM cadmium blocked all I(Ca) rapidly and reversibly. We did not find differences among cells of differing size and hormone content. Thus, I(Ca) is carried by high voltage-activated Ca(2+) channels of at least three types, but the molecular types may differ from those characterized from mammalian neurons.     

Glycinergic synaptic currents in Golgi cells of the rat cerebellum.

Recordings were made from Golgi cells in slices from rat cerebellar cortex using whole-cell and outside-out configurations of the patch-clamp technique. Exogenous glycine and gamma-aminobutyric acid (GABA) both activated chloride currents, which could be differentially blocked by strychnine and SR95531, respectively. Inhibitory synaptic currents occurred spontaneously in all Golgi cells. Some were blocked by strychnine while the others were blocked by SR95531. The single channel events occurring during the decay of these two types of inhibitory postsynaptic currents had different amplitudes, which matched the main conductance states of the channels gated by glycine and GABA in outside-out patches. It was concluded that Golgi cells receive both glycinergic and GABAergic synaptic inputs.     

Synaptic transmission between rat cerebellar granule and Purkinje cells in dissociated cell culture: effects of excitatory-amino acid transmitter antagonists.

Monosynaptic excitatory connections between cerebellar granule and Purkinje cells were studied in dissociated cell cultures, and identification of the transmitter and the postsynaptic receptor at this synapse was pharmacologically investigated. The presynaptic granule cell and the postsynaptic Purkinje cell were voltage- or current-clamped simultaneously, and the excitatory postsynaptic current induced by the granule cell was examined. The neurons and monosynaptic excitatory connections were identified as in our earlier study. Several pairs of granule and Purkinje cells were stained with Lucifer yellow and propidium iodide, respectively, and their morphology was examined after electrophysiological recording. The monosynaptic excitatory postsynaptic current was suppressed by 1 mM kynurenate, an antagonist for excitatory-amino acid receptors, but was little affected by 0.2 mM DL-2-amino-5-phosphonovalerate, a selective antagonist of N-methyl-D-aspartate receptors. Glutamate and aspartate induced inward current in the Purkinje cells. These currents were suppressed by kynurenate at 1 mM. DL-2-Amino-5-phosphonovalerate at 0.2 mM suppressed the inward current induced by 100 microM aspartate but did not affect the inward current induced by 10 microM glutamate. These results are consistent with the idea that glutamate, or a glutamate-like substance, but not aspartate is the transmitter released at the synapse between granule and Purkinje cells and that non-N-methyl-D-aspartate receptor channels are functioning in the postsynaptic membrane.     

Iontophoresis of Li+ antagonizes noradrenergic synaptic inhibition of rat cerebellar Purkinje cells.

Li salts provide effective therapy for manic-depressive psychosis, but the site and mechanism of this effect are not known. We have tested the ability of Li, applied by microiontophoresis, to modify the responsiveness of rat cerebellar Purkinje neurons to iontophoretic applications of norepinephrine and gamma-aminobutyrate and to the inhibition produced by stimulation of the noradrenergic ceruleo-cerebellar pathway. As previously reported for rat hippocampal neurons, acute exposure to Li produces selective, reversible antagonism of the effects of norepinephrine and the noradrenergic pathway but does not affect inhibitory actions of gamma-aminobutyrate. Collectively, these selective antagonisms of noradrenergic sympatic inhibitions in the cerebellum and hippocampus may indicate a general effect of Li suitable for extended observations in rats exposed to Li for the chronic periods needed to achieve therapeutic effects in man.     

Cerebellar granule cells in culture: monosynaptic connections with Purkinje cells and ionic currents.

Electrophysiological properties of cerebellar granule cells and synapses between granule and Purkinje cells were studied in dissociated cultures. Electrophysiological properties of neurons and synapses in the mammalian central nervous system are best studied in dissociated cell cultures because of good target cell visibility, control over the contents of the extracellular solution, and the feasibility of whole-cell patch electrode recording, which has been a powerful technique in analyzing biophysical properties of ionic channels in small cells. We have applied this whole-cell recording technique to cultured cerebellar granule cells whose electrophysiological properties have been almost unknown because of their small cell size. In this study, simultaneous intracellular recordings from presynaptic granule and postsynaptic Purkinje cells demonstrated that granule cells made functional monosynaptic connections with Purkinje cells in dissociated cell cultures. Further, the existence of Na, Ca, and K channels in granule cells is demonstrated by external ion substitutions.     

Mifepristone (RU486) protects Purkinje cells from cell death in organotypic slice cultures of postnatal rat and mouse cerebellum

Mifepristone (RU486), which binds with high affinity to both progesterone and glucocorticosteroid receptors (PR and GR), is well known for its use in the termination of unwanted pregnancy, but other activities including neuroprotection have been suggested. Cerebellar organotypic cultures from 3 to 7 postnatal day rat (P3-P7) were studied to examine the neuroprotective potential of RU486. In such cultures, Purkinje cells enter a process of apoptosis with a maximum at P3. This study shows that RU486 (20 microM) can protect Purkinje cells from this apoptotic process. The neuroprotective effect did involve neither PR nor GR, because it could not be mimicked or inhibited by other ligands of these receptors, and because it still took place in PR mutant (PR-KO) mice and in brain-specific GR mutant mice (GRNes/Cre). Potent antioxidant agents did not prevent Purkinje cells from this developmental cell death. The neuroprotective effect of RU486 could also be observed in pathological Purkinje cell death. Indeed, this steroid is able to prevent Purkinje cells from death in organotypic cultures of cerebellar slices from Purkinje cell degeneration (pcd) mutant mice, a murine model of hereditary neurodegenerative ataxia. In P0 cerebellar slices treated with RU486 for 6 days and further kept in culture up to 21 days, the synthetic steroid increased by 16.2-fold the survival of pcd/pcd Purkinje cells. Our results show that RU486 may act through a new mechanism, not yet elucidated, to protect Purkinje cells from death.     

Identification of beta-cells in dissociated rat pancreatic cell suspensions.

beta-Cells may be demonstrated in pancreas sections as well as in monolayer cultures. Until now, however, it has been impossible to differentiate quickly among individual cell types in freshly dissociated cell suspensions prepared for pancreatic monolayer cultures. Rapid identification of endocrine cells within the total cell population can be achieved by means of vital staining with neutral red. Moreover, the direct observation of unfixed and unstained cell suspensions under dark-field illumination allows an immediate identification of granulated beta-cells.     

Calcium dependent forms of synaptic plasticity in cerebellar Purkinje cells

Elevation of intracellular calcium concentration ([Ca(2+)](i)) induces several forms of long-term synaptic plasticity in cerebellar Purkinje cells (PCs). These include (1) long-term depression (LTD) at parallel fiber (PF) to PC synapses, (2) LTD at climbing fiber (CF) to PC synapses, (3) long-term potentiation (LTP) at synapses from inhibitory interneurons (rebound potentiation). The current knowledge about calcium dependency for these forms of synaptic plasticity is described in this chapter. (1) Induction of PF-LTD is dependent on elevation of [Ca(2+)](i), that derives from two distinct sources. One is through voltage-dependent calcium channel (VDCC). A CF stimulation leads to elevation of [Ca(2+)](i) due to activation of VDCCs. The other is from the internal calcium store. PFs stimulation activates metabotropic glutamate receptor subtype 1 (mGluR1). It leads to production of inositol-1,4,5-triphosphate (IP(3)) and diacylglycerol (DG) which cause calcium release from internal stores and activation of protein kinase C, respectively. The conjunctive activation of PF and CF inputs is necessary for PF-LTD. (2) LTD at CF to PC synapses (CF-LTD) is induced by the mechanisms similar to those involved in PF-LTD. CF-LTD requires elevation of [Ca(2+)](i) and activation of the mGluR1 to PKC cascade. (3) Rebound potentiation is induced by transient elevation of [Ca(2+)](i) due to activation of VDCCs or IP3-mediated calcium release from internal stores. Elevation of [Ca(2+)](i) activates calcium/ calmodulin-dependent protein kinase II and leads to persistent up-regulation of postsynaptic GABAA receptor function. At the three types of synapses described above, elevation of [Ca(2+)](i) also causes short-term depression of neurotransmitter release from presynaptic terminals. Recent studies demonstrate that transient elevation of [Ca(2+)](i) produces endocannabinoids in PCs that act retrogradely onto presynaptic terminals and suppress neurotransmitter release.     

Brief dendritic calcium signals initiate long-lasting synaptic depression in cerebellar Purkinje cells.

We have performed experiments designed to test the hypothesis that long-term depression (LTD) of excitatory synaptic transmission in the cerebellar cortex is caused by a rise in postsynaptic Ca concentration. These experiments combined measurements of synaptic efficacy, performed with the thin slice patch clamp technique, with fura-2 measurements of intracellular Ca concentration ([Ca]i) in single cerebellar Purkinje cells. Simultaneous activation of the climbing fiber and parallel fibers innervating single Purkinje cells caused a LTD of transmission of the parallel fiber-Purkinje cell excitatory synapse. This LTD was associated with large and transient rises in [Ca]i in the Purkinje cell and apparently was due to Ca entry through voltage-gated Ca channels in the Purkinje cell dendrites. The rise in [Ca]i produced by climbing fiber activity was necessary for LTD, because addition of the Ca chelator bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) to the interior of the Purkinje cell blocked LTD. Further, elevation of [Ca]i, produced by depolarizing pulses delivered in conjunction with parallel fiber activation, induced a depression of synaptic activity that closely resembled LTD in both time course and magnitude. Thus, a rise in [Ca]i appears to be sufficient to initiate LTD. From these results, we conclude that LTD of the parallel fiber-Purkinje cell synapse is initiated by a brief, climbing fiber-mediated rise in postsynaptic [Ca]i and that LTD is maintained by other, longer-lived processes that are triggered by the rise in postsynaptic [Ca]i.     

NMDARs in granule cells contribute to parallel fiber–Purkinje cell synaptic plasticity and motor learning

Long-term synaptic plasticity is believed to be the cellular substrate of learning and memory. Synaptic plasticity rules are defined by the specific complement of receptors at the synapse and the associated downstream signaling mechanisms. In young rodents, at the cerebellar synapse between granule cells (GC) and Purkinje cells (PC), bidirectional plasticity is shaped by the balance between transcellular nitric oxide (NO) driven by presynaptic N-methyl-D-aspartate receptor (NMDAR) activation and postsynaptic calcium dynamics. However, the role and the location of NMDAR activation in these pathways is still debated in mature animals. Here, we show in adult rodents that NMDARs are present and functional in presynaptic terminals where their activation triggers NO signaling. In addition, we find that selective genetic deletion of presynaptic, but not postsynaptic, NMDARs prevents synaptic plasticity at parallel fiber-PC (PF-PC) synapses. Consistent with this finding, the selective deletion of GC NMDARs affects adaptation of the vestibulo-ocular reflex. Thus, NMDARs presynaptic to PCs are required for bidirectional synaptic plasticity and cerebellar motor learning.

The ability of an organism to adjust its behavior to environmental demands depends on its capacity to learn and execute coordinated movements. The cerebellum plays a central role in this process by optimizing motor programs through trial-and-error learning (1). Within the cerebellum, the synaptic output from granule cells (GCs) to Purkinje cells (PCs) shapes computational operations during basal motor function and serves as a substrate for motor learning (2). Several forms of motor learning depend on changes in the strength of the parallel fiber (PF), the axon of GCs, to the PC synapse (3, 4).In the mammalian forebrain, synaptic plasticity typically relies on postsynaptic N-methyl-D-aspartate receptor (NMDAR) activation, which alters AMPA receptor (AMPAR) turnover at the postsynaptic site (5). However, this may not extend to the cerebellar synapse between GCs and PCs, since no functional postsynaptic NMDARs have been identified in young or adult rodents (6, 7). Pharmacological approaches, however, have shown that both long-term depression (LTD) and long-term potentiation (LTP) induction depend on NMDAR activation at the PF-PC synapse in young rodents (8–12). Hence, the alternative mechanisms for NMDAR-dependent synaptic modulation may involve presynaptic NMDARs activation [(12–15); for review: refs. 16 and 17]. Indeed, cell-specific deletion of NMDARs in GCs abolishes LTP in young rodents (12). In addition to NMDARs, PF-PC synaptic plasticity also requires nitric-oxide (NO) signaling (18–20). As nitric-oxide synthase (NOS) is expressed in GCs, but not in PCs (21), the activation of presynaptic NMDARs might allow Ca²⁺ influx that activates NO synthesis, which in turn may act upon the PCs. However, in the mature cerebellum, the existence of presynaptic NMDARs on PFs and the role of NO in PF-PC plasticity remains a matter of debate. Previously, we have proposed that the activation of putatively presynaptic NMDARs in young rodents is necessary for inducing PF-PC synaptic plasticity without affecting transmitter release (8, 9, 11, 12). More recently, it has been shown that a subset of PFs express presynaptic NMDARs containing GluN2A subunits and that these receptors are functional (11, 12). Thus, in contrast to their role at other synapses, at least in young rodent, presynaptic NMDARs as part of the PF-PC synapses might act via the production of NO to induce postsynaptic plasticity, without altering neurotransmitter release (9, 11, 12, 18–22). However, a causal link between NMDARs activation in PFs, NO synthesis, and synaptic plasticity induction is still missing.In the cerebral cortex, the expression of presynaptic NMDARs is developmentally regulated (23, 24). However, little is known about the presence and function of presynaptic NMDARs in adult tissue. In the adult cerebellum, PCs only express postsynaptic NMDARs at their synapse with climbing fibers (CFs) (25). It has been proposed that the activation of these receptors could have heterosynaptic effects during PF-PC LTD. This mechanism would explain why LTD in adults depends on NMDARs. According to this model, presynaptic NMDARs would be a transient feature of developing tissue and not necessary for induction of synaptic plasticity and motor learning in adult animals (25).Here, we combine electron microscopy, two-photon calcium imaging, synaptic plasticity experiments, and behavioral measurements to show that presynaptic NMDARs are not developmentally regulated but are required for cerebellar motor learning in adults. We demonstrate that presynaptic NMDARs are present and functional in PFs of mature rodents. By specifically deleting the NMDAR subunit GluN1 either in the post- (PC) or the presynaptic cells (GCs), we demonstrate that NMDAR activation in GCs plays a key role in bidirectional synaptic plasticity and in vestibulo-ocular reflex (VOR) adaptation, an important paradigm for testing cerebellar motor learning (26–28). In contrast, NMDARs in PCs are neither involved in PF-PC synaptic plasticity nor required for cerebellar motor learning.     

Simulated responses of cerebellar Purkinje cells are independent of the dendritic location of granule cell synaptic inputs.

Cerebellar Purkinje cell responses to granule cell synaptic inputs were examined with a computer model including active dendritic conductances. Dendritic P-type Ca2+ channels amplified postsynaptic responses when the model was firing at a physiological rate. Small synchronous excitatory inputs applied distally on the large dendritic tree resulted in somatic responses of similar size to those generated by more proximal inputs. In contrast, in a passive model the somatic postsynaptic potentials to distal inputs were 76% smaller. The model predicts that the somatic firing response of Purkinje cells is relatively insensitive to the exact dendritic location of synaptic inputs. We describe a mechanism of Ca2+-mediated synaptic amplification, based on the subspiking threshold recruitment of P-type Ca2+ channels in the dendritic branches surrounding the input site.     

Dendritic spikes mediate negative synaptic gain control in cerebellar Purkinje cells

Dendritic spikes appear to be a ubiquitous feature of dendritic excitability. In cortical pyramidal neurons, dendritic spikes increase the efficacy of distal synapses, providing additional inward current to enhance axonal action potential (AP) output, thus increasing synaptic gain. In cerebellar Purkinje cells, dendritic spikes can trigger synaptic plasticity, but their influence on axonal output is not well understood. We have used simultaneous somatic and dendritic patch-clamp recordings to directly assess the impact of dendritic calcium spikes on axonal AP output of Purkinje cells. Dendritic spikes evoked by parallel fiber input triggered brief bursts of somatic APs, followed by pauses in spiking, which cancelled out the extra spikes in the burst. As a result, average output firing rates during trains of input remained independent of the input strength, thus flattening synaptic gain. We demonstrate that this "clamping" of AP output by the pause following dendritic spikes is due to activation of high conductance calcium-dependent potassium channels by dendritic spikes. Dendritic spikes in Purkinje cells, in contrast to pyramidal cells, thus have differential effects on temporally coded and rate coded information: increasing the impact of transient parallel fiber input, while depressing synaptic gain for sustained parallel fiber inputs.     

Purkinje cell dysfunction and alteration of long-term synaptic plasticity in fetal alcohol syndrome

In cerebellum and other brain regions, neuronal cell death because of ethanol consumption by the mother is thought to be the leading cause of neurological deficits in the offspring. However, little is known about how surviving cells function. We studied cerebellar Purkinje cells in vivo and in vitro to determine whether function of these cells was altered after prenatal ethanol exposure. We observed that Purkinje cells that were prenatally exposed to ethanol presented decreased voltage-gated calcium currents because of a decreased expression of the gamma-isoform of protein kinase C. Long-term depression at the parallel fiber-Purkinje cell synapse in the cerebellum was converted into long-term potentiation. This likely explains the dramatic increase in Purkinje cell firing and the rapid oscillations of local field potential observed in alert fetal alcohol syndrome mice. Our data strongly suggest that reversal of long-term synaptic plasticity and increased firing rates of Purkinje cells in vivo are major contributors to the ataxia and motor learning deficits observed in fetal alcohol syndrome. Our results show that calcium-related neuronal dysfunction is central to the pathogenesis of the neurological manifestations of fetal alcohol syndrome and suggest new methods for treatment of this disorder.     

Multiple types of Ca2+ currents in single canine Purkinje cells

Whole-cell Ca2+ channel currents were recorded from isolated single canine Purkinje and ventricular cells to determine whether there were multiple types of Ca2+ channels in these two cell types, as in many other excitable tissues. The experimental conditions were such that currents other than Ca2+ channel currents were largely suppressed. The charge carrier was either Ca2+ or Ba2+ (5mM). In every canine Purkinje cell studied (n = 36), we saw T and L Ca2+ channel currents that are similar to their counterparts in other tissues. Neither current was affected by tetrodotoxin (30 microM), but both were reduced by Mn2+ (5mM). Ni2+ (50 microM) blocked T more than L current. Nisoldipine (1 microM) apparently abolished the L current but also decreased the T current by 50%. Substitution of Ba2+ for Ca2+ augmented and prolonged L current but did not affect T current significantly. At 36 degrees C and with 5 mM [Ca2+]o, T current inactivated over a voltage range from -70 to -30 mV whereas L current inactivated between -30 and +20 mV. T current was detectable in only some of the ventricular cells studied (8 out of 12). In these cells the ratio of maximal T current to maximal L current (0.2 +/- 0.1, n = 8) was lower than the T/L ratio in Purkinje cells (0.6 +/- 0.2, n = 6). The density of peak L current in ventricular cells (7.5 +/- 1.7 pA/pF, n = 8) was higher than that in Purkinje cells (4.4 +/- 3.4 pA/pF, n = 6). Therefore, in ventricular cells the L current is the main Ca2+ current whereas in Purkinje cells, the T current also contributes significantly to membrane electrical activity. In Purkinje cells, beta-adrenoceptor stimulation by isoproterenol (1 microM) increased L current but did not affect T current. On the other hand, in 70% (7 out of 10) of the Purkinje cells, alpha-adrenoceptor stimulation by 10 microM norepinephrine (in the presence of 2 microM propranolol) increased the T current. Our observations show that the distribution of the two types of Ca2+ channels in canine ventricle is heterogeneous and that the two types of Ca2+ channels are modulated by catecholamines by different receptors.     

Comparison of K+ currents in cardiac Purkinje cells isolated from rabbit and dog

The repolarization reserve determines the ability of drugs to prolong the cardiac action potential duration. Differences in K(+) currents between rabbit and dog cardiac Purkinje cells were studied by recording the transient outward K(+) current (I(to)) as well as the delayed rectifier K(+) currents (I(Ks) and I(Kr)) during repolarization. Purkinje fibers were dissected from dog and rabbit hearts and exposed to enzymatic digestion until isolated cells were obtained. Whole cell voltage clamp methods were used to measure K(+) currents in both cell types. Action potential (AP) recordings from Purkinje cells displayed a rapid phase 1 repolarization due to a prominent I(to) with densities of 13.3+/-2.3 and 9.6+/-0.6 pA/pF at +40 mV in dog and rabbit respectively. I(Ks) tail currents were significantly larger in dog Purkinje cells. I(Kr) tail current densities were comparable in Purkinje cell from both species. Rabbit ventricular and Purkinje cell AP waveforms were used for action potential clamp experiments in TSA201 cells expressing human ether a go-go related gene (HERG). HERG currents elicited by the ventricular waveform reached its maximum amplitude during phase 3 repolarization. In contrast, Purkinje cell AP waveform elicited markedly smaller HERG currents even though the action potential duration was longer. The observations suggest that the fast phase 1 and negative plateau of the Purkinje cell AP limits the contribution of I(Kr) to repolarization. These results provide evidence that rabbit Purkinje cells have a smaller repolarization reserve and provide a biophysical explanation for a previously observed higher sensitivity to QT prolonging drugs in rabbit than dog Purkinje fibers.