High frequency stimulation of the subthalamic nucleus leads to presynaptic GABA(B)-dependent depression of subthalamo-nigral afferents.

Patients with akinesia benefit from chronic high frequency stimulation (HFS) of the subthalamic nucleus (STN). Among the mechanisms contributing to the therapeutic success of HFS-STN might be a suppression of activity in the output region of the basal ganglia. Indeed, recordings in the substantia nigra pars reticulata (SNr) of fully adult mice revealed that HFS-STN consistently produced a reduction of compound glutamatergic excitatory postsynaptic currents at a time when the tetrodotoxin-sensitive components of the local field potentials had already recovered after the high frequency activation. These observations suggest that HFS-STN not only alters action potential conduction on the way towards the SNr but also modifies synaptic transmission within the SNr. A classical conditioning-test paradigm was then designed to better separate the causes from the indicators of synaptic depression. A bipolar platinum-iridium macroelectrode delivered conditioning HFS trains to a larger group of fibers in the STN, while a separate high-ohmic glass micropipette in the rostral SNr provided test stimuli at minimal intensity to single fibers. The conditioning-test interval was set to 100 ms, i.e. the time required to recover the excitability of subthalamo-nigral axons after HFS-STN. The continuity of STN axons passing from the conditioning to the test sites was examined by an action potential occlusion test. About two thirds of the subthalamo-nigral afferents were occlusion-negative, i.e. they were not among the fibers directly activated by the conditioning STN stimulation. Nonetheless, occlusion-negative afferents exhibited signs of presynaptic depression that could be eliminated by blocking GABA(B) receptors with CGP55845 (1 µM). Further analysis of single fiber-activated responses supported the proposal that the heterosynaptic depression of synaptic glutamate release during and after HFS-STN is mainly caused by the tonic release of GABA from co-activated striato-nigral afferents to the SNr. This mechanism would be consistent with a gain-of-function hypothesis of DBS

Reduced tonic inhibition in striatal output neurons from Huntington mice due to loss of astrocytic GABA release through GAT-3

The extracellular concentration of the two main neurotransmitters glutamate and GABA is low but not negligible which enables a number of tonic actions. The effects of ambient GABA vary in a region-, cell-type and age-dependent manner and can serve as indicators of disease-related alterations. Here we explored the tonic inhibitory actions of GABA in Huntington's disease (HD). HD is a devastating neurodegenerative disorder caused by a mutation in the huntingtin gene. Whole cell patch clamp recordings from striatal output neurons (SONs) in slices from adult wild type mice and two mouse models of HD (Z_Q175_KI homozygotes or R6/2 heterozygotes) revealed an HD-related reduction of the GABA(A) receptor-mediated tonic chloride current (ITonic(GABA)) along with signs of reduced GABA(B) receptor-mediated presynaptic depression of synaptic GABA release. About half of ITonic(GABA) depended on tetrodotoxin-sensitive synaptic GABA release, but the remaining current was still lower in HD. Both in WT and HD, ITonic(GABA) was more prominent during the first four hours after preparing the slices, when astrocytes but not neurons exhibited a transient depolarization. All further tests were performed within 1 to 4 h in vitro. Experiments with SNAP5114, a blocker of the astrocytic GABA transporter GAT-3, suggest that in WT but not HD GAT-3 operated in the releasing mode. Application of a transportable substrate for glutamate transporters (D-aspartate 0.1 - 1 mM) restored the non-synaptic GABA release in slices from HD mice. ITonic(GABA) was also rescued by applying the hyperagonist gaboxadol (0.33 µM). The results lead to the hypothesis that lesion-induced astrocyte depolarization facilitates nonsynaptic release of GABA through GAT-3. However, the capacity of depolarized astrocytes to provide GABA for tonic inhibition is strongly reduced in HD

Hypothetical mechanisms of DBS effects.

<p>A) Simplified scheme of the neural connections presumably activated by HFS-STN in the rodent brain. Note that most of the collaterals and the entopeduncular nucleus were omitted. The subthalamic nucleus and the substantia nigra are shown enlarged. Abbreviations: GP - Globus pallidus, STN - subthalamic nucleus, SNr - Substantia nigra pars reticulata, SNc - Substantia nigra pars compacta. CM - centromedian nucleus, PPTg - Pedunculopontine tegmental nucleus. Ach - acetylcholine, DA - dopamine. For more details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082191#pone.0082191-Parent2" target="_blank">[77]</a>. The equivalents of primate and rodent structures are reviewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082191#pone.0082191-Tepper1" target="_blank">[78]</a>. B) Scheme of modifications presumably initiated by HFS-STN in the normal rodent brain. It is assumed that HFS-STN co-activates at least three types of afferents innervating the SNr: GABAergic afferents of striatal and pallidal origin and glutamatergic afferents of subthalamic origin. The latter is the core element of the presented circuit and exhibits the following four modifications: 1) Suppression of AP generation and conduction (recovery time: <100 ms, B) depletion of transmitter release (recovery time: <5 s), 3) heterosynaptic depression via presynaptic GABA(B) receptors (recovery time: <1 min), 4) depression of the recurrent pathway through the STN (recovery time: >10 min). Mechanism #1 has been studied in more detail by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082191#pone.0082191-Zheng1" target="_blank">[35]</a>. The present experiments focused on the mechanism #2. Details on mechanisms #3 and #4 will be published in a forthcoming paper.</p

Compound glutamate- and GABA-induced ion currents during and after multi-unit HFS-STN.

<p>Responses of SNr neurons to Macrostim in the STN. Note that the driving force for the transmitter-induced currents is 60 mV for glutamate but only 50 mV for GABA. A) Recordings in the presence of gabazine (15 µM) to show compound glutamatergic ion currents. The calibration bars in (B) also apply to (A). Note strong attenuation with train repetition. B) Recordings in the presence of DNQX (10 µM) and APV (50 µM). Note that a fraction of the GABA-induced chloride current remains during and after HFS-STN. C,D) Enlarged individual traces from same experiments as in A,B, respectively. Shown are the last 3 trials of an HFS-STN conditioning session. The glutamatergic responses are strongly attenuated, GABAergic currents persist. E) Plot of current integrals during individual HFS-STN trials against trial number. The responses are normalized to the current integral during the first train (only cells with integral values >25 pA*s during first train included).</p

Local field potentials in the SNr after low and high frequency stimulation of the STN.

<p>A) Scheme of experiment. B) Image of the slice showing the electrode positions for the experiment illustrated in C. C) Individual (grey) and average (black) traces of FVs in gabazine and DNQX+APV. D) Specimen records of FVs in the SNr at various stimulus intensities. E) Dependency of FV amplitude on stimulus intensities. F) Control response before HFS-STN and single conditioning-test trial. Please note that both the conditioning HFS stimuli and the low frequency test stimuli were delivered through the same bipolar macroelectrode in the STN. (a–c) refer to pulses inducing the FV shown in (G). The traces in the bottom of (F) correspond to the first and the last 5 stimuli within an HFS train. In this case traces in black represent responses during the first HFS train, traces in gray - responses during the last HFS train in a series of 60 HFS trials. G) Individual (gray) and averaged (black) FVs recorded before, during and after HFS-STN, as indicated in (F). H) Quantification of results from 9 slices/9 mice. I,K) Dynamics of FV amplitude within one HFS-STN train (I) and during train repetitions at a rate of 6/s (K). Note that the 1st response in each train recovers fully (I) or even displays slight potentiation with train repetition (K) while the last responses only exhibit depression.</p

Effects of multi-unit and single-unit HFS on glutamatergic unitary synaptic responses in the SNr.

<p>Effects of multi-unit and single-unit HFS on glutamatergic unitary synaptic responses in the SNr.</p

Scheme of occlusion test and conditioning experiment.

<p>A) Experimental arrangement showing the involved cellular elements, their connections and sites of stimulation/recording. Green: glutamatergic elements. Red: GABAergic elements. “MacroStim” - bipolar platinum iridium macroelectrode for multi-unit STN activation. “Microstim” - glass pipettes for activation if single axons. B) Image of a sagittal slice comprising the STN and the SNr. Note tip positions of the stimulating and recording pipettes. C,D) Occlusion test to determine whether the unit selected for Microstim belonged to the axonal pool activated by Macrostim. Full algebraic summation of the compound response to Macrostim and the unitary response to Microstim indicates that there is no continuity of the test fiber into the STN (C). Accordingly, full summation is taken as evidence that the fiber selected for Microstim was not occlusion-positive into the STN (D). E) Pulse patterns during conditioning Macrostim in the STN and test Microstim of a single STN fiber in the SNr. Each experimental session consisted of three parts: Control – a pair Microstim pulses at an interstimulus interval of 50 ms, HFS-STN – combination of HFS Macrostim trains followed at an interval of 100 ms by a pair of Microstim pulses, and Recovery – same as control. The repetition rate of the trials was 1/6 s.</p

Two types of unitary IPSCs and their responses to single-unit HFS.

<p>A) Experiment in the presence of DNQX (10 µM) and APV (50 µM). Holding voltage −50 mV, chloride equilibrium potential −100 mV. A,B) Traces of averaged uIPSCs belonging to the more rapidly decaying type with paired pulse facilitation (A) and to the more slowly decaying type with paired pulse depression (B). Black trace: Control, Grey trace: HFS-conditioned. C, D) Single-unit HFS experiment. Traces belong to the same experiment as (A,B), respectively. Note larger integral current in C). E) Plot of normalized uIPSC amplitudes in response to the first test pulse following after HFS. Note persistence of current responses from afferents with slowly decaying uIPSCs. F–H) Characteristics of uIPSCs. Note differences in the time constant of decay (F), paired pulse ratio (G) and the integral current during HFS (H). The broken line in (F) denotes separation threshold. I–M) Quantification of results from HFS-conditioning. The differences between Control and respective Recovery values were not significant and are omitted in the graphs, for clarity.</p

Dynamics of unitary EPSCs during single-unit HFS.

<p>A) Scheme of experiment. Note that in these experiments conditioning HFS and test pulse were applied to the same Microstim site. All recordings were performed in the presence of 15 µM gabazine. B) Estimation of threshold intensity for three single afferents to SNr neurons. C) Specimen records of responses to Microstim of a single STN fiber in the SNr. Note presence of monosynaptic (m) and disynaptic (di) uEPSCs. D) Single trial with traces with HFS and superposition of Control and HFS-conditioned traces from 20 trials. E) Plot of unitary test responses during Control (without HFS, 20 trials), HFS (50 trials) and Recovery (20 trials). Average and SE from 7 cells. F–I) Quantification of results. Arrow head denotes data from experiment illustrated in D.</p

Block of GABA(B) receptors prevents the depressant effect of HFS-STN conditioning on unitary test responses induced by microstimulation of single STN axons in the SNr.

<p>A) Example traces to illustrate partial reversal of the HFS-STN effect on unitary EPSCs elicited with Microstim. Experiment in the presence of gabazine (15 µM). B) The plot of normalized uEPSC amplitudes against time of recording. The average value before the HFS trials is 100%. More than 10 min were allowed for the wash-in of the GABA(B) antagonist CGP55845 (1 µM). Note the alleviation of amplitude depression (B,C) and the increase in the success rate (D) in the presence of the GABA(B) blocker CGP55845. E,F) Quantification of the presynaptic parameters PPR and CV. The bars above the empty and shaded columns refer to the results of paired t-test. The asterisks above each column indicate significance levels from comparison with the Control, as tested with ANOVA. The differences between Control and respective Recovery values were not significant and are omitted in the graphs, for clarity.</p