Paul H. M. Kullmann

Cav1.3 Channel Voltage Dependence, Not Ca2+ Selectivity, Drives Pacemaker Activity and Amplifies Bursts in Nigral Dopamine Neurons

Cav1.3 (α1D) L-type Ca2+ channels have been implicated in substantia nigra (SN) dopamine (DA) neuron pacemaking and vulnerability to Parkinsons disease. These effects may arise from the depolarizing current and cytoplasmic Ca2+ elevation produced by Cav1.3 channels at subthreshold membrane potentials. However, the assumption that the Ca2+ selectivity of Cav1.3 channels is essential has not been tested. In this study the properties of SN DA neuron L-type Ca2+ channels responsible for driving pacemaker activity in juvenile rat brain slices were probed by replacing native channels blocked with the dihydropyridine nimodipine with virtual channels generated by dynamic clamp. Surprisingly, virtual L-type channels that mimic native and recombinant Cav1.3 channels supported pacemaker activity even though dynamic clamp currents are not carried by Ca2+. This effect is specific because pacemaker activity could not be restored by tonic current injection, virtual nonselective leak channels or virtual NMDA receptors, which share with L-type channels a negative slope conductance region in their current–voltage (I–V) curve. Altering virtual channels showed that the production of pacemaker activity depended on the characteristic voltage dependence of DA neuron L-type channels, while activation kinetics and reversal potential were not critical parameters. Virtual L-type channels also supported slow oscillatory potentials and enhanced firing rate during evoked bursts. Thus, Cav1.3 channel voltage dependence, rather than Ca2+ selectivity, drives pacemaker activity and amplifies bursts in SN DA neurons.

Glycinergic and GABAergic calcium responses in the developing lateral superior olive

The lateral superior olive (LSO), a binaural nucleus involved in sound localization, receives tonotopically organized inhibitory inputs from the medial nucleus of the trapezoid body (MNTB). During development, the tonotopic organization of this glycinergic/GABAergic MNTB–LSO pathway is established by activity‐dependent axonal reorganization. However, the underlying mechanisms by which this reorganization takes place have remained largely unknown. As cytosolic calcium is one of the most important second messengers responsible for inducing synaptic plasticity and reorganization, we examined whether and how activity in the MNTB–LSO pathway changes the intracellular calcium concentration ([Ca2+]i) in developing LSO neurons. By applying calcium imaging techniques to Fura‐2‐labelled slices from neonatal rats and mice, we found that glycine and GABA (γ‐aminobutyric acid) affect [Ca2+]i in LSO neurons in an age‐dependent manner; during the first postnatal week, the period at which glycine and GABA are depolarizing in the LSO, glycine and GABA always increased [Ca2+]i. However, in 2‐week‐old animals, the time around hearing onset when glycine and GABA are hyperpolarizing, glycine and GABA slightly decreased [Ca2+]i. Calcium responses could also be elicited by stimulation of afferent fibres from the MNTB, and these synaptic responses were mediated by glycine and GABAA receptors. Furthermore, GABA, which is a neurotransmitter only in the immature MNTB–LSO pathway, played a major role in generating MNTB‐elicited Ca2+ responses. The direct link of glycinergic/GABAergic synaptic activity to intracellular calcium signalling during the period of inhibitory synaptic plasticity could be one of the mechanisms by which tonotopic MNTB–LSO connections become established.

Glycinergic/GABAergic synapses in the lateral superior olive are excitatory in neonatal C57Bl/6J mice

The lateral superior olive (LSO), a nucleus involved in sound localization, receives tonotopically organized, inhibitory input from the medial nucleus of the trapezoid body (MNTB). To better understand the development of this glycinergic/GABAergic pathway, we used Gramicidin-perforated patch clamp recordings to characterize MNTB-evoked postsynaptic potentials in LSO neurons of neonatal C57Bl/6J mice. We found that during the first postnatal week, MNTB-evoked responses change from being depolarizing to being hyperpolarizing. Most interestingly, depolarizing glycinergic/GABAergic synaptic potentials were able to trigger action potentials, demonstrating that the MNTB-LSO pathway can act as a true excitatory pathway. This transient excitatory action of immature MNTB-LSO synapses might play an important role in activity-dependent sharpening of the tonotopic organization of inhibitory connections in the LSO.

D2 Autoreceptors Chronically Enhance Dopamine Neuron Pacemaker Activity

Activation of D2 autoreceptors on midbrain dopamine neurons has been shown previously to acutely open K+ channels to inhibit intrinsically generated pacemaker activity. Here we report that D2 autoreceptors act chronically to produce an opposite action: to increase the speed and regularity of repetitive action potential firing. Voltage-, current-, and dynamic-clamp experiments, using conventional whole-cell and perforated patch-clamp recording, with cultured rat midbrain dopamine neurons show that a change in the number of functional A-type K+ channels alters firing rate and susceptibility to irregularity produced by other channels. cAMP and protein kinase A mediate the long-term action of D2 receptors in a manner that counters the short-term effect of this signaling pathway on K+ channel gating. We conclude that D2 autoreceptors, in addition to mediating acute negative feedback, are responsible for long-term enhancement of the rate and fidelity of dopamine neuron pacemaker activity.

Synthesis, Photophysical, Photochemical and Biological Properties of Caged GABA, 4-[[(2H-1-Benzopyran-2-one-7-amino-4-methoxy) carbonyl] amino] Butanoic Acid¶

The photorelease of a caged neurotransmitter can be used to investigate the function of neuronal circuits in tissues. We have designed and synthesized a stable, caged γ‐aminobutyric acid (GABA) derivative, 4‐[[(2H‐1‐benzopyran‐2‐one‐7‐amino‐4‐methoxy)carbonyl]amino] butanoic acid (BC204), that releases the neurotransmitter in physiological medium when irradiated with UV light at 300–400 nm in PBS at pH 7.4. The release of GABA occurs with the formation of the major photoproduct, 7‐amino‐4‐(hydroxymethyl)‐2H‐1‐benzopyran‐2‐one, via a solvolytic photodegradation mechanism of the coumarin moiety and was confirmed by electrospray mass spectrometry/mass spectrometry (ESI MS/MS). BC204 is chemically stable and shows no intrinsic activity after many hours under physiological dark conditions. These properties suggest that BC204 is an excellent form of caged GABA that is well suited for long‐term biological studies.

Excitatory action of an immature glycinergic/GABAergic sound localization pathway

Most mammals determine the azimuthal direction of incoming sound using auditory cues arising from differences in interaural sound intensity. The first station in the ascending auditory pathway, which processes interaural intensity differences, is the lateral superior olive (LSO), a binaural nucleus in the auditory brainstem. LSO neurons encode interaural intensity differences by integrating excitatory input from the ipsilateral cochlea and inhibitory input from the contralateral cochlea. Both inputs converge on single neurons in a highly organized, frequency-specific manner. The correct development of the precise arrangement of these inputs and their physiological properties depends on neuronal activity. Previous studies have shown that inhibitory, glycinergic/GABAergic inputs to the LSO are transiently depolarizing, and it has been hypothesized that this depolarizing action enables developing inhibitory inputs to act as excitatory inputs. In support of this hypothesis, we recently demonstrated that depolarizing glycinergic/GABAergic inputs can increase the intracellular calcium concentration in immature LSO neurons and elicit action potentials. These results provide support for the notion that the influence of glycinergic/GABAergic synaptic activity on development of the LSO involves calcium-dependent signaling mechanisms.

Dopamine Neuron Responses Depend Exponentially on Pacemaker Interval

Midbrain dopamine neuron activity results from the integration of the responses to metabo- and ionotropic receptors with the postsynaptic excitability of these intrinsic pacemakers. Interestingly, intrinsic pacemaker rate varies greatly between individual dopamine neurons and is subject to short- and long-term regulation. Here responses of substantia nigra dopamine neurons to defined dynamic-clamp stimuli were measured to quantify the impact of cell-to-cell variation in intrinsic pacemaker rate. Then this approach was repeated in single dopamine neurons in which pacemaker rate was altered by activation of muscarinic receptors or current injection. These experiments revealed a dramatic exponential dependence on pacemaker interval for the responses to voltage-gated A-type K+ channels, voltage-independent cation channels and ionotropic synapses. Likewise, responses to native metabotropic (GABAb and mGluR1) inhibitory synapses depended steeply on pacemaker interval. These results show that observed variations in dopamine neuron pacemaker rate are functionally significant because they produce a >10-fold difference in responses to diverse stimuli. Both the magnitude and the mathematical form of the relationship between pacemaker interval and responses were not previously anticipated.

Dendritic Ca2+ responses in neonatal lateral superior olive neurons elicited by glycinergic/GABAergic synapses and action potentials.

During development, GABA/glycinergic connections from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) gradually change from being depolarizing to being hyperpolarizing. Previous studies have shown that depolarizing MNTB-LSO synapses can trigger action potentials and increase the concentration of intracellular calcium. In the present study we used confocal calcium imaging combined with whole-cell patch clamp recordings to investigate how depolarizing MNTB inputs in neonatal rats and mice increase the calcium concentration in the dendrites of LSO neurons. Our results show that subthreshold synaptic responses can elicit local dendritic calcium responses while suprathreshold responses reliably generate global calcium responses that are observed in all dendritic processes. The amplitude of global dendritic calcium responses increased with distance from the soma. Global calcium responses were blocked by tetrodotoxin and could not be recovered by somatic injection of action potential waveforms indicating that global calcium responses are generated by back-propagating sodium action potentials.

NMDAR-Mediated Calcium Transients Elicited by Glutamate Co-Release at Developing Inhibitory Synapses

Before hearing onset, the topographic organization of the inhibitory sound localization pathway from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) is refined by means of synaptic silencing and strengthening. During this refinement period MNTB-LSO synapses not only release GABA and glycine but also release glutamate. This co-released glutamate can elicit postsynaptic currents that are predominantly mediated by NMDA receptors (NMDARs). To gain a better understanding of how glutamate contributes to synaptic signaling at developing MNTB-LSO inhibitory synapses, we investigated to what degree and under what conditions NMDARs contribute to postsynaptic calcium responses. Our results demonstrate that MNTB-LSO synapses can elicit compartmentalized calcium responses along aspiny LSO dendrites. These responses are significantly attenuated by the NMDAR antagonist APV. APV, however, had no effect on somatically recorded electrical postsynaptic responses, indicating little, if any, contribution of NMDARs to spike generation. NMDAR-mediated calcium responses were decreased when increasing extracellular magnesium concentrations to physiological levels indicating that MNTB-LSO synapses activate magnesium sensitive NMDAR on immature LSO dendrites. In Fura-2 AM loaded neurons, blocking GABAA and glycine receptors increased NMDAR contribution to somatic calcium responses suggesting that GABA and glycine, perhaps by shunting backpropagating action potentials, decrease the level of NMDAR activation under strong stimulus conditions.

Virtual leak channels modulate firing dynamics and synaptic integration in rat sympathetic neurons: implications for ganglionic transmission in vivo

The synaptic organization of paravertebral sympathetic ganglia enables them to relay activity from the spinal cord to the periphery and thereby control autonomic functions, including blood pressure and body temperature. The present experiments were done to reconcile conflicting observations in tissue culture, intact isolated ganglia and living animals. By recording intracellularly from dissociated neurons and intact ganglia, we found that when electrode damage makes cells leaky it could profoundly distort cellular excitability and the integration of synaptic potentials. The experiments relied on the dynamic clamp method, which allows the creation of virtual ion channels by injecting current into a cell based upon a mathematical model and using rapid feedback between the model and cell. The results support the hypothesis that sympathetic ganglia can produce a 2.4‐fold amplification of presynaptic activity. This could aid understanding of the neural hyperactivity that is believed to drive high blood pressure in some patients.