Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Quartier Hôpital, 














University
of Liege

The nervous system adapts to experience by changes in synaptic strength. The mechanisms of synaptic plasticity include changes in the probability of transmitter release and in postsynaptic responsiveness. Experimental and neuropharmacological evidence points toward a third variable in synaptic efficacy: changes in presynaptic transmitter concentration. Several groups, including our own, have reported changes in the amplitude and frequency of postsynaptic (miniature) events indicating that alterations in transmitter content cause alterations in vesicular transmitter content and vesicle dynamics. It is, however, not a priori clear how transmitter metabolism will affect vesicular transmitter content and how this in turn will affect pre- and postsynaptic functions. We therefore have constructed a model of the presynaptic terminal incorporating vesicular transmitter loading and the presynaptic vesicle cycle. We hypothesize that the experimentally observed synaptic plasticity after changes in transmitter metabolism puts predictable restrictions on vesicle loading, cytoplasmic-vesicular transmitter concentration gradient, and on vesicular cycling or release. The results of our model depend on the specific mechanism linking presynaptic transmitter concentration to vesicular dynamics, that is, alteration of vesicle maturation or alteration of release. It also makes a difference whether differentially filled vesicles are detected and differentially processed within the terminal or whether vesicle filling acts back onto the terminal by presynaptic autoreceptors. Therefore, the model allows one to decide, at a given synapse, how transmitter metabolism is linked to presynaptic function and efficacy.

Transmitter metabolism as a mechanism of synaptic plasticity: a modeling study.

Action potentials in central neurons are initiated near the axon initial segment, propagate into the axon, and finally invade the presynaptic terminals, where they trigger transmitter release. Voltage-gated Na(+) channels are key determinants of excitability, but Na(+) channel density and properties in axons and presynaptic terminals of cortical neurons have not been examined yet. In hippocampal mossy fiber boutons, which emerge from parent axons en passant, Na(+) channels are very abundant, with an estimated number of approximately 2000 channels per bouton. Presynaptic Na(+) channels show faster inactivation kinetics than somatic channels, suggesting differences between subcellular compartments of the same cell. Computational analysis of action potential propagation in axon-multibouton structures reveals that Na(+) channels in boutons preferentially amplify the presynaptic action potential and enhance Ca(2+) inflow, whereas Na(+) channels in axons control the reliability and speed of propagation. Thus, presynaptic and axonal Na(+) channels contribute differentially to mossy fiber synaptic transmission.

Presynaptic action potential amplification by voltage-gated Na+ channels in hippocampal mossy fiber boutons.

It is widely accepted that the hippocampus plays a major role in learning and memory. The mossy fiber synapse between granule cells in the dentate gyrus and pyramidal neurons in the CA3 region is a key component of the hippocampal trisynaptic circuit. Recent work, partially based on direct presynaptic patch-clamp recordings from hippocampal mossy fiber boutons, sheds light on the mechanisms of synaptic transmission and plasticity at mossy fiber synapses. A high Na(+) channel density in mossy fiber boutons leads to a large amplitude of the presynaptic action potential. Together with the fast gating of presynaptic Ca(2+) channels, this generates a large and brief presynaptic Ca(2+) influx, which can trigger transmitter release with high efficiency and temporal precision. The large number of release sites, the large size of the releasable pool of vesicles, and the huge extent of presynaptic plasticity confer unique strength to this synapse, suggesting a large impact onto the CA3 pyramidal cell network under specific behavioral conditions. The characteristic properties of the hippocampal mossy fiber synapse may be important for pattern separation and information storage in the dentate gyrus-CA3 cell network.

Timing and efficacy of transmitter release at mossy fiber synapses in the hippocampal network.

Rigorous analysis of synaptic transmission in the central nervous system requires access to presynaptic terminals. However, cortical terminals have been largely inaccessible to presynaptic patch-clamp recording, due to their small size. Using improved patch-clamp techniques in brain slices, we recorded from mossy fiber terminals in the CA3 region of the hippocampus, which have a diameter of 2-5 microm. The major steps of improvement were the enhanced visibility provided by high-numerical aperture objectives and infrared illumination, the development of vibratomes with minimal vertical blade vibrations and the use of sucrose-based solutions for storage and cutting. Based on these improvements, we describe a protocol that allows us to routinely record from hippocampal mossy fiber boutons. Presynaptic recordings can be obtained in slices from both rats and mice. Presynaptic recordings can be also obtained in slices from transgenic mice in which terminals are labeled with enhanced green fluorescent protein.

Patch-clamp recording from mossy fiber terminals in hippocampal slices.

Dopamine (DA) neurons and GABA neurons of the substantia nigra (SN) promote distinct functions in the control of movement and have different firing properties and action potential (AP) waveforms. APs recorded from DA and GABA neurons differed in amplitude, maximal rate of rise, and duration. In addition, the threshold potential for APs was higher in DA neurons than in GABA neurons. The activation of voltage-gated Na(+) channels accounts largely for these differences as the application of a low concentration of the voltage-gated Na(+) channel blocker TTX had an effect on all of these parameters. We have examined functional properties of somatic Na(+) channels in nucleated patches isolated from DA and GABA neurons. Peak amplitudes of macroscopic Na(+) currents were smaller in DA neurons in comparison to those in GABA neurons. The mean peak Na(+) conductance density was 24.5 pS microm(-2) in DA neurons and almost twice as large, 41.6 pS microm(-2), in GABA neurons. The voltage dependence of Na(+) channel activation was not different between the two types of SN neurons. Na(+) channels in DA and GABA neurons, however, differed in the voltage dependence of inactivation, the mean mid-point potential of steady-state inactivation curve being more positive in DA neurons than in GABA neurons. The results suggest that specific Na(+) channel gating properties and Na(+) conductance densities in the somatic membrane of SN neurons may have consequences on synaptic signal integration in the soma of both types of neurons and on somatodendritic release of dopamine by DA neurons.

Differences in Na+ conductance density and Na+ channel functional properties between dopamine and GABA neurons of the rat substantia nigra.

Zinc actions on synaptic transmission span the modulation of neurotransmitter receptors, transporters, activation of intracellular cascades and alterations in gene expression. Whether and how zinc affects inhibitory synaptic signalling in the dentate gyrus remains largely unexplored. We found that mono- and di-synaptic GABAergic inputs onto dentate granule cells were reversibly depressed by exogenous zinc application and enhanced by zinc chelation. Blocking T-type Ca(2+) channels prevented the effect of zinc chelation. When recording from dentate fast-spiking interneurones, zinc chelation facilitated T-type Ca(2+) currents, increased action potential half-width and decreased spike threshold. It also increased the offset of the input-output relation in a manner consistent with enhanced excitability. In granule cells, chelation of zinc reduced the time window for the integration of glutamatergic inputs originating from perforant path synapses, resulting in reduced spike transfer. Thus, zinc-mediated modulation of dentate interneurone excitability and GABA release regulates information flow to local targets and hippocampal networks.

Endogenous zinc depresses GABAergic transmission via T-type Ca(2+) channels and broadens the time window for integration of glutamatergic inputs in dentate granule cells.

The hyperpolarization-activated cation current Ih is expressed in dopamine neurons of the substantia nigra, but the subcellular distribution of the current and its role in synaptic integration remain unknown. We used cell-attached patch recordings to determine the localization profile of Ih along the somatodendritic axis of nigral dopamine neurons in slices from young rats. Ih density is higher in axon-bearing dendrites, in a membrane area close to the axon origin, than in the soma and axon-lacking dendrites. Dual current-clamp recordings revealed a similar contribution of Ih to the waveform of single excitatory postsynaptic potentials throughout the somatodendritic domain. The Ih blocker ZD 7288 increased the temporal summation in all dendrites with a comparable effect in axon- and non-axon dendrites. The strategic position of Ih in the proximity of the axon may influence importantly transitions between pacemaker and bursting activities and consequently the downstream release of dopamine.

High dendritic expression of Ih in the proximity of the axon origin controls the integrative properties of nigral dopamine neurons.

Although small-conductance Ca(2+)-activated K(+) (SK) channels and various types of voltage-gated Ca(2+) (Cav) channels have been described in midbrain dopaminergic neurons, the nature of their interactions is unclear. More particularly, the role of various Cav channel types in either promoting irregularity of firing (by generating an inward current during SK channel blockade) or promoting regularity of firing (by providing the source of Ca(2+) for the activation of SK channels) has not been systematically explored. We addressed this question using intracellular and extracellular recordings from substantia nigra, pars compacta (SNc), dopaminergic neurons in rat midbrain slices. Neurons were pharmacologically isolated from their differences. When examining the ability of various Cav channel blockers to inhibit the SK-mediated afterhyperpolarization (AHP), we found that only the N-type Cav channel blocker ω-conotoxin-GVIA was able to reduce the apamin-sensitive AHP, but only partially (~40%). Specific blockers of L, P/Q, T or R channels had no effect on this AHP. Combining ω-conotoxin-GVIA and other specific blockers did not yield greater block and even the broad Cav blocker Cd(2+) induced a submaximal (~75%) effect. Extracellular recordings examining firing regularity yielded congruent results: none of the specific blockers was able to increase firing irregularity to the extent that the specific SK blocker apamin did. The irregularity of firing observed with apamin could only be reversed by blocking L-type Ca(2+) channels. Thus various sources of Ca(2+) appear to be required for SK channel activation in SNc neurons (some of them still unidentified), ensuring robustness of pacemaking regularity.

Interactions between calcium channels and SK channels in midbrain dopamine neurons and their impact on pacemaker regularity: Contrasting roles of N- and L-type channels.

Dopaminergic (DA) neurons located in the ventral midbrain continuously generate a slow endogenous pacemaker activity, the mechanism of which is still debated. It has been suggested that, in the substantia nigra pars compacta (SNc), the pacemaking relies more on Ca(2+) channels and that the density of L-type Ca(2+) channels is higher in these DA neurons than in those located in the ventral tegmental area (VTA). This might lead to a higher Ca(2+) load in SNc DA neurons and explain their higher susceptibility to degeneration. However, direct evidence for this hypothesis is lacking. We found that the L-type current and channel density are indeed higher in the somata of rat SNc DA neurons and that this current undergoes less inactivation in this region. Nonstationary fluctuation analysis measurements showed a much higher number of L-type channels in the soma of SNc DA neurons, as well as a smaller single-channel conductance, pointing to a possible different molecular identity of L-type channels in DA neurons from the two areas. A major consequence of this is that pacemaking and, even more so, bursting are associated with a larger Ca(2+) entry through L-type channels in SNc DA neurons than in their VTA counterparts. Our results establish a molecular and functional difference between two populations of midbrain DA neurons that may contribute to their differential sensitivity to neurodegeneration.

Dominique Engel

GIGA-Neurosciences

Quartier Hôpital, University of Liege

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dominique Engel

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Quartier Hôpital, University of Liege

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

GIGA-Neurosciences