Jeremy F. Atherton

Ionic Mechanisms Underlying Autonomous Action Potential Generation in the Somata and Dendrites of GABAergic Substantia Nigra Pars Reticulata Neurons In Vitro

Through their repetitive discharge, GABAergic neurons of the substantia nigra pars reticulata (SNr) tonically inhibit the target nuclei of the basal ganglia and the dopamine neurons of the midbrain. As the repetitive firing of SNr neurons persists in vitro, perforated, whole-cell and cell-attached patch-clamp recordings were made from rat brain slices to determine the mechanisms underlying this activity. The spontaneous activity of SNr neurons was not perturbed by the blockade of fast synaptic transmission, demonstrating that it was autonomous in nature. A subthreshold, slowly inactivating, voltage-dependent, tetrodotoxin (TTX)-sensitive Na+ current and a TTX-insensitive inward current that was mediated in part by Na+ were responsible for depolarization to action potential (AP) threshold. An apamin-sensitive spike afterhyperpolarization mediated by small-conductance Ca2+-dependent K+ (SK) channels was critical for the precision of autonomous activity. SK channels were activated, in part, by Ca2+ flowing throughω-conotoxin GVIA-sensitive, class 2.2 voltage-dependent Ca2+ channels. Although Cs+/ZD7288 (4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride)-sensitive hyperpolarization-activated currents were also observed in SNr neurons, they were activated at voltages that were in general more hyperpolarized than those associated with autonomous activity. Simultaneous somatic and dendritic recordings revealed that autonomously generated APs were observed first at the soma before propagating into dendrites up to 120 μm from the somatic recording site. Backpropagation of autonomously generated APs was reliable with no observable incidence of failure. Together, these data suggest that the resting inhibitory output of the basal ganglia relies, in large part, on the intrinsic firing properties of the neurons that convey this signal.

Enhancement of Excitatory Synaptic Integration by GABAergic Inhibition in the Subthalamic Nucleus

The activity patterns of subthalamic nucleus (STN) neurons, which are intimately related to normal movement and abnormal movement in Parkinsons disease (PD), are sculpted by feedback GABAergic inhibition from the reciprocally connected globus pallidus (GP). To understand the principles underlying the integration of GABAergic inputs, we used gramicidin-based patch-clamp recording of STN neurons in rat brain slices. Voltage-dependent Na+ (Nav) channels actively truncated synthetic IPSPs and were required for autonomous activity. In contrast, hyperpolarization-activated cyclic nucleotide-gated and class 3 voltage-dependent Ca2+ channels contributed minimally to the integration of single or low-frequency trains of IPSPs and autonomous activity. Interestingly, IPSPs modified action potentials (APs) in a manner that suggested IPSPs enhanced postsynaptic Nav channel availability. This possibility was confirmed in acutely isolated STN neurons using current-clamp recordings containing IPSPs as voltage-clamp waveforms. Tetrodotoxin-sensitive subthreshold and spike-associated Na+ currents declined during autonomous spiking but were indeed transiently boosted after IPSPs. A functional consequence of inhibition-dependent augmentation of postsynaptic excitability was that EPSP–AP coupling was dramatically improved when IPSPs preceded EPSPs. Because STN neuronal activity exhibits coherence with cortical β-oscillations in PD, we tested how rhythmic sequences of cortical EPSPs were integrated in the absence and presence of feedback inhibition. STN neuronal activity was consistently entrained by EPSPs only in the presence of feedback inhibition. These observations suggest that feedback inhibition from the GP is critical for the emergence of coherent β-oscillations between the cortex and STN in PD.

Cellular principles underlying normal and pathological activity in the subthalamic nucleus.

The motor symptoms of Parkinsons disease are associated with abnormal, correlated, low frequency, rhythmic burst activity in the subthalamic nucleus and connected nuclei. Research into the mechanisms controlling the pattern of subthalamic activity has intensified because therapies that manipulate the pattern of subthalamic activity, such as deep brain stimulation and levodopa administration, improve motor function in Parkinsons disease. Recent findings suggest that dopamine denervation of the striatum and extrastriatal basal ganglia profoundly alters the transmission and integration of glutamatergic cortical and GABAergic pallidal inputs to subthalamic neurons, leading to pathological activity that resonates throughout the basal ganglia and wider motor system.

Cellular Mechanisms Underlying Burst Firing in Substantia Nigra Dopamine Neurons

Burst firing of substantia nigra dopamine (SN DA) neurons is believed to represent an important teaching signal that instructs synaptic plasticity and associative learning. However, the mechanisms through which synaptic excitation overcomes the limiting effects of somatic Ca2+-dependent K+ current to generate burst firing are controversial. Modeling studies suggest that synaptic excitation sufficiently amplifies oscillatory dendritic Ca2+ and Na+ channel currents to lead to the initiation of high-frequency firing in SN DA neuron dendrites. To test this model, visually guided compartment-specific patch-clamp recording and ion channel manipulation were applied to rodent SN DA neurons in vitro. As suggested previously, the axon of SN DA neurons was typically found to originate from a large-diameter dendrite that was proximal to the soma. However, in contrast to the predictions of the model, (1) somatic current injection generated firing that was similar in frequency and form to burst firing in vivo, (2) the efficacy of glutamatergic excitation was inversely related to the distance of excitation from the axon, (3) pharmacological blockade or genetic deletion of Ca2+ channels did not prevent high-frequency firing, (4) action potential bursts were invariably detected first at sites that were proximal to the axon, and (5) pharmacological blockade of Na+ channels in the vicinity of the axon/soma but not dendritic excitation impaired burst firing. Together, these data suggest that SN DA neurons integrate their synaptic input in a more conventional manner than was hypothesized previously.

Sparse but Selective and Potent Synaptic Transmission From the Globus Pallidus to the Subthalamic Nucleus

The reciprocally connected GABAergic globus pallidus (GP)-glutamatergic subthalamic nucleus (STN) network is critical for voluntary movement and an important site of dysfunction in movement disorders such as Parkinsons disease. Although the GP is a key determinant of STN activity, correlated GP-STN activity is rare under normal conditions. Here we define fundamental features of the GP-STN connection that contribute to poorly correlated GP-STN activity. Juxtacellular labeling of single GP neurons in vivo and stereological estimation of the total number of GABAergic GP-STN synapses suggest that the GP-STN connection is surprisingly sparse: single GP neurons maximally contact only 2% of STN neurons and single STN neurons maximally receive input from 2% of GP neurons. However, GP-STN connectivity may be considerably more selective than even these estimates imply. Light and electron microscopic analyses revealed that single GP axons give rise to sparsely distributed terminal clusters, many of which correspond to multiple synapses with individual STN neurons. Application of the minimal stimulation technique in brain slices confirmed that STN neurons receive multisynaptic unitary inputs and that these inputs largely arise from different sets of GABAergic axons. Finally, the dynamic-clamp technique was applied to quantify the impact of GP-STN inputs on STN activity. Small fractions of GP-STN input were sufficiently powerful to inhibit and synchronize the autonomous activity of STN neurons. Together these data are consistent with the conclusion that the rarity of correlated GP-STN activity in vivo is due to the sparsity and selectivity, rather than the potency, of GP-STN synaptic connections.

Selective Participation of Somatodendritic HCN Channels in Inhibitory But Not Excitatory Synaptic Integration in Neurons of the Subthalamic Nucleus

The activity patterns of subthalamic nucleus (STN) neurons are intimately linked to motor function and dysfunction and arise through the complex interaction of intrinsic properties and inhibitory and excitatory synaptic inputs. In many neurons, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play key roles in intrinsic excitability and synaptic integration both under normal conditions and in disease states. However, in STN neurons, which strongly express HCN channels, their roles remain relatively obscure. To address this deficit, complementary molecular and cellular electrophysiological, imaging, and computational approaches were applied to the rat STN. Molecular profiling demonstrated that individual STN neurons express mRNA encoding several HCN subunits, with HCN2 and 3 being the most abundant. Light and electron microscopic analysis showed that HCN2 subunits are strongly expressed and distributed throughout the somatodendritic plasma membrane. Voltage-, current-, and dynamic-clamp analysis, two-photon Ca2+ imaging, and computational modeling revealed that HCN channels are activated by GABAA receptor-mediated inputs and thus limit synaptic hyperpolarization and deinactivation of low-voltage-activated Ca2+ channels. Although HCN channels also limited the temporal summation of EPSPs, generated through two-photon uncaging of glutamate, this action was largely shunted by GABAergic inhibition that was necessary for HCN channel activation. Together the data demonstrate that HCN channels in STN neurons selectively counteract GABAA receptor-mediated inhibition arising from the globus pallidus and thus promote single-spike activity rather than rebound burst firing.

Heterosynaptic regulation of external globus pallidus inputs to the subthalamic nucleus by the motor cortex

The two principal movement-suppressing pathways of the basal ganglia, the so-called hyperdirect and indirect pathways, interact within the subthalamic nucleus (STN). An appropriate level and pattern of hyperdirect pathway cortical excitation and indirect pathway external globus pallidus (GPe) inhibition of the STN are critical for normal movement and are greatly perturbed in Parkinsons disease. Here we demonstrate that motor cortical inputs to the STN heterosynaptically regulate, through activation of postsynaptic NMDA receptors, the number of functional GABAA receptor-mediated GPe-STN inputs. Therefore, a homeostatic mechanism, intrinsic to the STN, balances cortical excitation by adjusting the strength of GPe inhibition. However, following the loss of dopamine, excessive cortical activation of STN NMDA receptors triggers GPe-STN inputs to strengthen abnormally, contributing to the emergence of pathological, correlated activity.

Autonomous initiation and propagation of action potentials in neurons of the subthalamic nucleus

The activity of the subthalamic nucleus (STN) is intimately related to movement and is generated, in part, by voltage‐dependent Na+ (Nav) channels that drive autonomous firing. In order to determine the principles underlying the initiation and propagation of action potentials in STN neurons, 2‐photon laser scanning microscopy was used to guide tight‐seal whole‐cell somatic and loose‐seal cell‐attached axonal/dendritic patch‐clamp recordings and compartment‐selective ion channel manipulation in rat brain slices. Action potentials were first detected in a region that corresponded most closely to the unmyelinated axon initial segment, as defined by Golgi and ankyrin G labelling. Following initiation, action potentials propagated reliably into axonal and somatodendritic compartments with conduction velocities of ∼5 m s−1 and ∼0.7 m s−1, respectively. Action potentials generated by neurons with axons truncated within or beyond the axon initial segment were not significantly different. However, axon initial segment and somatic but not dendritic or more distal axonal application of low [Na+] ACSF or the selective Nav channel blocker tetrodotoxin consistently depolarized action potential threshold. Finally, somatodendritic but not axonal application of GABA evoked large, rapid inhibitory currents in concordance with electron microscopic analyses, which revealed that the somatodendritic compartment was the principal target of putative inhibitory inputs. Together the data are consistent with the conclusions that in STN neurons the axon initial segment and soma express an excess of Nav channels for the generation of autonomous activity, while synaptic activation of somatodendritic GABAA receptors regulates the axonal initiation of action potentials.

Accumulation of cytoplasmic calcium, but not apamin‐sensitive afterhyperpolarization current, during high frequency firing in rat subthalamic nucleus cells

The autonomous firing pattern of neurons in the rat subthalamic nucleus (STN) is shaped by action potential afterhyperpolarization currents. One of these is an apamin‐sensitive calcium‐dependent potassium current (SK). The duration of SK current is usually considered to be limited by the clearance of calcium from the vicinity of the channel. When the cell is driven to fire faster, calcium is expected to accumulate, and this is expected to result in accumulation of calcium‐dependent AHP current. We measured the time course of calcium transients in the soma and proximal dendrites of STN neurons during spontaneous firing and their accumulation during driven firing. We compared these to the time course and accumulation of AHP currents using whole‐cell and perforated patch recordings. During spontaneous firing, a rise in free cytoplasmic calcium was seen after each action potential, and decayed with a time constant of about 200 ms in the soma, and 80 ms in the dendrites. At rates higher than 10 Hz, calcium transients accumulated as predicted. In addition, there was a slow calcium transient not predicted by summation of action potentials that became more pronounced at high firing frequency. Spike AHP currents were measured in voltage clamp as tail currents after 2 ms voltage pulses that triggered action currents. Apamin‐sensitive AHP (SK) current was measured by subtraction of tail currents obtained before and after treatment with apamin. SK current peaked between 10 and 15 ms after an action potential, had a decay time constant of about 30 ms, and showed no accumulation. At frequencies between 5 and 200 spikes s−1, the maximal SK current remained the same as that evoked by a single action potential. AHP current did not have time to decay between action potentials, so at frequencies above 50 spikes s−1 the apamin‐sensitive current was effectively constant. These results are inconsistent with the view that the decay of SK current is governed by calcium dynamics. They suggest that the calcium is present at the SK channel for a very short time after each action potential, and the current decays at a rate set by the deactivation kinetics of the SK channel. At high rates, repetitive firing was governed by a fast apamin‐insensitive AHP current that did not accumulate, but rather showed depression with increases in activation frequency. A slowly accumulating AHP current, also insensitive to apamin, was extremely small at low rates but became significant with higher firing rates.

Death of dopaminergic neurones in the rat substantia nigra can be induced by damage to globus pallidus

Parkinsons disease is a debilitating disorder that results from the death of dopaminergic neurones in the substantia nigra. Subthalamic nucleus neurones use glutamate as their neurotransmitter and send excitatory projections to the substantia nigra. Changes in both the mean firing rate and firing pattern of neurones of the subthalamic nucleus have been found in patients with this disease. This has led to the suggestion that hyperactivity of the subthalamic nucleus may be involved in the pathology of the dopaminergic neurones. Subthalamic nucleus lesions or treatment with glutamatergic antagonists can be neuroprotective in animal models of Parkinsons disease but until now there has been no direct evidence that hyperactivity of subthalamic nucleus neurones can lead to downstream cell death. Here we show that lesions of the rat globus pallidus (a treatment that has been shown to increase subthalamic nucleus neuronal activity) result in a significant reduction of the number of dopaminergic neurones in the substantia nigra.