Zayd M. Khaliq

Relative Contributions of Axonal and Somatic Na Channels to Action Potential Initiation in Cerebellar Purkinje Neurons

Neuronal excitability is likely to be regulated by the site of action potential initiation, the location on a neuron that crosses threshold first. Although initiation is axonal in many neurons, in Purkinje cells, somatic conductances can generate spontaneous action potentials, suggesting that the perisomatic region (soma and/or initial segment) contributes to spike initiation and may regulate firing. To identify directly the cellular regions at which Na channel modulation significantly influences firing, we measured spontaneous and evoked action potentials in Purkinje cells in cerebellar slices from postnatal day 14–28 mice while applying drugs locally to either the soma/initial segment or the first node of Ranvier. Na currents were decreased by tetrodotoxin (TTX) or increased by β-pompilidotoxin (β-PMTX). Dual somatic and axonal recordings indicated that spike thresholds and input–output curves were sensitive to TTX or β-PMTX at the perisomatic region but were unchanged by either drug at the first node. When perisomatic Na channel availability was reduced with subsaturating TTX, however, the input–output curve became shallower during additional TTX block of nodal channels, revealing a latent role for nodal Na channels in facilitating firing. In perisomatic TTX, axons failed to generate spontaneous or evoked spike trains. In contrast, choline block of the initial segment alone altered normal input–output curves. The data suggest that, although the first node reliably follows action potentials, spike initiation in Purkinje neurons occurs in the initial segment. Moreover, Purkinje cell output depends on the density, availability, and kinetics of perisomatic Na channels, a characteristic that may distinguish spontaneously firing from quiescent neurons.

Axonal Propagation of Simple and Complex Spikes in Cerebellar Purkinje Neurons

In cerebellar Purkinje neurons, the reliability of propagation of high-frequency simple spikes and spikelets of complex spikes is likely to regulate inhibition of Purkinje target neurons. To test the extent to which a one-to-one correspondence exists between somatic and axonal spikes, we made dual somatic and axonal recordings from Purkinje neurons in mouse cerebellar slices. Somatic action potentials were recorded with a whole-cell pipette, and the corresponding axonal signals were recorded extracellularly with a loose-patch pipette. Propagation of spontaneous and evoked simple spikes was highly reliable. At somatic firing rates of ∼200 spikes/sec, <10% of spikes failed to propagate, with failures becoming more frequent only at maximal somatic firing rates (∼260 spikes/sec). Complex spikes were elicited by climbing fiber stimulation, and their somatic waveforms were modulated by tonic current injection, as well as by paired stimulation to depress the underlying EPSCs. Across conditions, the mean number of propagating action potentials remained just above two spikes per climbing fiber stimulation, but the instantaneous frequency of the propagating spikes changed, from ∼375 Hz during somatic hyperpolarizations that silenced spontaneous firing to ∼150 Hz during spontaneous activity. The probability of propagation of individual spikelets could be described quantitatively as a saturating function of spikelet amplitude, rate of rise, or preceding interspike interval. The results suggest that ion channels of Purkinje axons are adapted to produce extremely short refractory periods and that brief bursts of forward-propagating action potentials generated by complex spikes may contribute transiently to inhibition of postsynaptic neurons.

Pacemaking in Dopaminergic Ventral Tegmental Area Neurons: Depolarizing Drive from Background and Voltage-Dependent Sodium Conductances

Dopaminergic neurons in the ventral tegmental area (VTA) fire spontaneously in a pacemaker-like manner. We analyzed the ionic currents that drive pacemaking in dopaminergic VTA neurons, studied in mouse brain slices. Pacemaking was not inhibited by blocking hyperpolarization-activated cation current (Ih) or blocking all calcium current by Mg2+ replacement of Ca2+. Tetrodotoxin (TTX) stopped spontaneous activity and usually resulted in stable resting potentials near −60 mV to −55 mV, 10–15 mV below the action potential threshold. When external sodium was replaced by N-methyl-d-glucamine (NMDG) with TTX present, cells hyperpolarized by an average of −11 mV, suggesting a significant resting sodium conductance not sensitive to TTX. Voltage-clamp experiments using slow (10 mV/s) ramps showed a steady-state, steeply voltage-dependent current blocked by TTX that activates near −60 mV, as well as a sodium “background” current with little voltage sensitivity, revealed by NMDG replacement for sodium with TTX present. We quantified these two components of sodium current during the pacemaking trajectory using action potential clamp. The initial phase of depolarization, up to approximately −55 mV, is driven mainly by non-voltage-dependent sodium background current. Above −55 mV, TTX-sensitive voltage-dependent “persistent” Na current helps drive the final phase of depolarization to the spike threshold. Voltage-dependent calcium current is small at all subthreshold voltages. The pacemaking mechanism in VTA neurons differs from that in substantia nigra pars compacta (SNc) neurons, where subthreshold calcium current plays a dominant role. In addition, we found that non-voltage-dependent background sodium current is much smaller in SNc neurons than VTA neurons.

A new glucocerebrosidase chaperone reduces α-synuclein and glycolipid levels in iPSC-derived dopaminergic neurons from patients with gaucher disease and parkinsonism

Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.

Dynamic, nonlinear feedback regulation of slow pacemaking by A-type potassium current in ventral tegmental area neurons.

We analyzed ionic currents that regulate pacemaking in dopaminergic neurons of the mouse ventral tegmental area by comparing voltage trajectories during spontaneous firing with ramp-evoked currents in voltage clamp. Most recordings were made in brain slice, with key experiments repeated using acutely dissociated neurons, which gave identical results. During spontaneous firing, net ionic current flowing between spikes was calculated from the time derivative of voltage multiplied by cell capacitance, signal-averaged over many firing cycles to enhance resolution. Net inward interspike current had a distinctive nonmonotonic shape, reaching a minimum (generally <1 pA) between −60 and −55 mV. Under voltage clamp, ramps over subthreshold voltages elicited a time- and voltage-dependent outward current that peaked near −55 mV. This current was undetectable with 5 mV/s ramps and increased steeply with depolarization rate over the range (10–50 mV/s) typical of natural pacemaking. Ramp-evoked subthreshold current was resistant to α-dendrotoxin, paxilline, apamin, and tetraethylammonium but sensitive to 4-aminopyridine and 0.5 mm Ba2+, consistent with A-type potassium current (IA). Same-cell comparison of currents elicited by various ramp speeds with natural spontaneous depolarization showed how the steep dependence of IA on depolarization rate results in small net inward currents during pacemaking. These results reveal a mechanism in which subthreshold IA is near zero at steady state, but is engaged at depolarization rates >10 mV/s to act as a powerful, supralinear feedback element. This feedback mechanism explains how net ionic current can be constrained to <1–2 pA but reliably inward, thus enabling slow, regular firing.

Differential Regulation of Action Potential Shape and Burst-Frequency Firing by BK and Kv2 Channels in Substantia Nigra Dopaminergic Neurons.

Little is known about the voltage-dependent potassium currents underlying spike repolarization in midbrain dopaminergic neurons. Studying mouse substantia nigra pars compacta dopaminergic neurons both in brain slice and after acute dissociation, we found that BK calcium-activated potassium channels and Kv2 channels both make major contributions to the depolarization-activated potassium current. Inhibiting Kv2 or BK channels had very different effects on spike shape and evoked firing. Inhibiting Kv2 channels increased spike width and decreased the afterhyperpolarization, as expected for loss of an action potential-activated potassium conductance. BK inhibition also increased spike width but paradoxically increased the afterhyperpolarization. Kv2 channel inhibition steeply increased the slope of the frequency–current (f–I) relationship, whereas BK channel inhibition had little effect on the f–I slope or decreased it, sometimes resulting in slowed firing. Action potential clamp experiments showed that both BK and Kv2 current flow during spike repolarization but with very different kinetics, with Kv2 current activating later and deactivating more slowly. Further experiments revealed that inhibiting either BK or Kv2 alone leads to recruitment of additional current through the other channel type during the action potential as a consequence of changes in spike shape. Enhancement of slowly deactivating Kv2 current can account for the increased afterhyperpolarization produced by BK inhibition and likely underlies the very different effects on the f–I relationship. The cross-regulation of BK and Kv2 activation illustrates that the functional role of a channel cannot be defined in isolation but depends critically on the context of the other conductances in the cell. SIGNIFICANCE STATEMENT This work shows that BK calcium-activated potassium channels and Kv2 voltage-activated potassium channels both regulate action potentials in dopamine neurons of the substantia nigra pars compacta. Although both channel types participate in action potential repolarization about equally, they have contrasting and partially opposite effects in regulating neuronal firing at frequencies typical of bursting. Our analysis shows that this results from their different kinetic properties, with fast-activating BK channels serving to short-circuit activation of Kv2 channels, which tend to slow firing by producing a deep afterhyperpolarization. The cross-regulation of BK and Kv2 activation illustrates that the functional role of a channel cannot be defined in isolation but depends critically on the context of the other conductances in the cell.

Tonic Firing Rate Controls Dendritic Ca2+ Signaling and Synaptic Gain in Substantia Nigra Dopamine Neurons

Substantia nigra dopamine neurons fire tonically resulting in action potential backpropagation and dendritic Ca2+ influx. Using Ca2+ imaging in acute mouse brain slices, we find a surprisingly steep relationship between tonic firing rate and dendritic Ca2+. Increasing the tonic rate from 1 to 6 Hz generated Ca2+ signals up to fivefold greater than predicted by linear summation of single spike-evoked Ca2+-transients. This “Ca2+ supralinearity” was produced largely by depolarization of the interspike voltage leading to activation of subthreshold Ca2+ channels and was present throughout the proximal and distal dendrites. Two-photon glutamate uncaging experiments show somatic depolarization enhances NMDA receptor-mediated Ca2+ signals >400 μm distal to the soma, due to unusually tight electrotonic coupling of the soma to distal dendrites. Consequently, we find that fast tonic firing intensifies synaptically driven burst firing output in dopamine neurons. These results show that modulation of background firing rate precisely tunes dendritic Ca2+ signaling and provides a simple yet powerful mechanism to dynamically regulate the gain of synaptic input.

Dopamine Inhibition Differentially Controls Excitability of Substantia Nigra Dopamine Neuron Subpopulations through T-Type Calcium Channels

While there is growing appreciation for diversity among ventral tegmental area dopamine neurons, much less is known regarding functional heterogeneity among the substantia nigra pars compacta (SNc) neurons. Here, we show that calbindin-positive dorsal tier and calbindin-negative ventral tier SNc dopaminergic neurons in mice comprise functionally distinct subpopulations distinguished by their dendritic calcium signaling, rebound excitation, and physiological responses to dopamine D2-receptor (D2) autoinhibition. While dopamine is known to inhibit action potential backpropagation, our experiments revealed an unexpected enhancement of excitatory responses and dendritic calcium signals in the presence of D2-receptor inhibition. Specifically, dopamine inhibition and direct hyperpolarization enabled the generation of low-threshold depolarizations that occurred in an all-or-none or graded manner, due to recruitment of T-type calcium channels. Interestingly, these effects occurred selectively in calbindin-negative dopaminergic neurons within the SNc. Thus, calbindin-positive and calbindin-negative SNc neurons differ substantially in their calcium channel composition and efficacy of excitatory inputs in the presence of dopamine inhibition. SIGNIFICANCE STATEMENT Substantia nigra dopaminergic neurons can be divided into two populations: the calbindin-negative ventral tier, which is vulnerable to neurodegeneration in Parkinsons disease, and the calbindin-positive dorsal tier, which is relatively resilient. Although tonic firing is similar in these subpopulations, we find that their responses to dopamine-mediated inhibition are strikingly different. During inhibition, calbindin-negative neurons exhibit increased sensitivity to excitatory inputs, which can then trigger large dendritic calcium transients due to strong expression of T-type calcium channels. Therefore, SNc neurons differ substantially in their calcium channel composition, which may contribute to their differential vulnerability. Furthermore, T-currents increase excitation efficacy onto calbindin-negative cells during dopamine inhibition, suggesting that shared inputs are differentially processed in subpopulations resulting in distinct downstream dopamine signals.

Electrical and Ca2+ signaling in dendritic spines of substantia nigra dopaminergic neurons

Little is known about the density and function of dendritic spines on midbrain dopamine neurons, or the relative contribution of spine and shaft synapses to excitability. Using Ca2+ imaging, glutamate uncaging, fluorescence recovery after photobleaching and transgenic mice expressing labeled PSD-95, we comparatively analyzed electrical and Ca2+ signaling in spines and shaft synapses of dopamine neurons. Dendritic spines were present on dopaminergic neurons at low densities in live and fixed tissue. Uncaging-evoked potential amplitudes correlated inversely with spine length but positively with the presence of PSD-95. Spine Ca2+ signals were less sensitive to hyperpolarization than shaft synapses, suggesting amplification of spine head voltages. Lastly, activating spines during pacemaking, we observed an unexpected enhancement of spine Ca2+ midway throughout the spike cycle, likely involving recruitment of NMDA receptors and voltage-gated conductances. These results demonstrate functionality of spines in dopamine neurons and reveal a novel modulation of spine Ca2+ signaling during pacemaking. DOI: http://dx.doi.org/10.7554/eLife.13905.001

Enhanced sensitivity to hyperpolarizing inhibition in mesoaccumbal relative to nigrostriatal dopamine neuron subpopulations.

Midbrain dopamine neurons recorded in vivo pause their firing in response to reward omission and aversive stimuli. While the initiation of pauses typically involves synaptic or modulatory input, intrinsic membrane properties may also enhance or limit hyperpolarization, raising the question of how intrinsic conductances shape pauses in dopamine neurons. Using retrograde labeling and electrophysiological techniques combined with computational modeling, we examined the intrinsic conductances that shape pauses evoked by current injections and synaptic stimulation in subpopulations of dopamine neurons grouped according to their axonal projections to the nucleus accumbens or dorsal striatum in mice. Testing across a range of conditions and pulse durations, we found that mesoaccumbal and nigrostriatal neurons differ substantially in rebound properties with mesoaccumbal neurons displaying significantly longer delays to spiking following hyperpolarization. The underlying mechanism involves an inactivating potassium (IA) current with decay time constants of up to 225 ms, and small-amplitude hyperpolarization-activated currents (IH), characteristics that were most often observed in mesoaccumbal neurons. Pharmacological block of IA completely abolished rebound delays and, importantly, shortened synaptically evoked inhibitory pauses, thereby demonstrating the involvement of A-type potassium channels in prolonging pauses evoked by GABAergic inhibition. Therefore, these results show that mesoaccumbal and nigrostriatal neurons display differential responses to hyperpolarizing inhibitory stimuli that favors a higher sensitivity to inhibition in mesoaccumbal neurons. These findings may explain, in part, observations from in vivo experiments that ventral tegmental area neurons tend to exhibit longer aversive pauses relative to SNc neurons. SIGNIFICANCE STATEMENT Our study examines rebound, postburst, and synaptically evoked inhibitory pauses in subpopulations of midbrain dopamine neurons. We show that pauses in dopamine neuron firing, evoked by either stimulation of GABAergic inputs or hyperpolarizing current injections, are enhanced by a subclass of potassium conductances that are recruited at voltages below spike threshold. Importantly, A-type potassium currents recorded in mesoaccumbal neurons displayed substantially slower inactivation kinetics, which, combined with weaker expression of hyperpolarization-activated currents, lengthened hyperpolarization-induced delays in spiking relative to nigrostriatal neurons. These results suggest that input integration differs among dopamine neurons favoring higher sensitivity to inhibition in mesoaccumbal neurons and may partially explain in vivo observations that ventral tegmental area neurons exhibit longer aversive pauses relative to SNc neurons.