Axonal mechanisms mediating GABA-A receptor inhibition of striatal dopamine release

Axons of midbrain dopaminergic neurons innervate the striatum where they contribute to movement and reinforcement learning. Past work has shown that striatal GABA tonically inhibits dopamine release, but whether GABA-A receptors directly modulate transmission or act indirectly through circuit elements is unresolved. Here, we use whole-cell and perforated-patch recordings to test for GABA-A receptors on the main dopaminergic neuron axons and branching processes within striatum. Application of GABA depolarized axons, but also decreased the amplitude of axonal spikes, limited propagation and reduced striatal dopamine release. The mechanism of inhibition involved sodium channel inactivation and shunting. Lastly, we show that the positive allosteric modulator diazepam enhanced GABA-A currents on dopaminergic neuron axons and directly inhibited release, but also likely acts by reducing excitatory drive from cholinergic interneurons. Thus, we reveal the mechanisms of GABA-A receptor modulation of dopamine release and provide new insight into the actions of benzodiazepines within the striatum.

was then applied onto the axon with a short pressure ejection (80 -300 ms in duration) 165 using a PV 820 Pneumatic PicoPump (WPI). 166 Fast-scan cyclic voltammetry (FSCV) 167 For all voltammetry experiments the methods are as follow. Cylindrical carbon-fiber 168 electrodes (CFEs) were prepared with T650 fibers (6 μm diameter, ~150 μm of exposed 169 fiber) inserted into a glass pipette and filled with KCl (3 M). Before use, the CFEs were    Prism, and an extra sum-of-squares F test was performed to determine significant 243 differences in slope between data sets on the same plot, and to determine whether a 244 line or exponential decay model fits the data better. For exponential fits, the One Phase 245 Decay analysis function in Prism was used to fit a standard curve.  previously reported values (Khaliq and Bean, 2008). A plot of the slope of the interspike 276 voltage against the axonal recording distance followed a roughly exponential 277 relationship with the interspike slope, such that it becomes more shallow with increasing 278 recording distance ( Figure 1G; single exponential fit, length constant, λ=211 µm, n=27; 279 R 2 =0.70; data were fit with a single exponential significantly better than with a line:  Main axon recorded in whole-cell mode with a connected soma (filled with neurobiotin, imaged with streptavidin-Cy5, slice cleared with CUBIC, red); GFP driven by the TH promoter (green). B. Trace of spontaneous action potentials recorded from a dopaminergic axon (left). Firing rate from somatic (n=10) and axonal recordings (n=41; p=0.298) (right). C. Overlay of an axonal and somatic spike (left). Phase plot for a somatic and axonal action potential (right). D. Half-peak width from somatic (n=10) and axonal (n=27) APs (**p=0.008) E. AP threshold between soma (n=10) and axon (n=26)(****p<0.0001) F. Example traces of interspike voltage from obtained from axonal (blue) and somatic (gray) recordings. G. Slope of interspike voltage plotted against recording distance between axonal recording site (blue) and soma (gray). H. Post-hoc reconstruction of a patched striatal axon I. Trace of subthreshold depolarization (left) and axonal AP (right) evoked by 250 pA and 275 pA current injection. J. Input resistance values for main axon (n=28) and striatal axons (n=74) APs (****p<0.0001) K. Comparison of the mean interspike voltage between soma (n=10) main axon (n=21) and striatal axon, which was measured as the average resting membrane potential (n=74) (*p=0.032; ***p=0.0007; ns p=0.87).

387
To dissect the contribution from these two processes, we compared the GABA puff 388 experiments in Figure 4A to separate experiments where depolarization was evoked 389 instead by direct current injection ( Figure 4B). We reasoned that the effects of current    upstroke denotes area of measurement for rate of rise, arrows denoting measurement of 7 change in AP peak and change in membrane potential C. Effect of the amount of baseline 8 depolarization on the decrease in peak AP amplitude, compared between GABA (green; n=14) 9 and current injection (tan; n=15) (*p=0.047) D. Effect of the amount of baseline depolarization 10 on the normalized decrease in rate of AP upstroke, compared between GABA (green; n=11) 11 and current injection (tan; n=10) (ns p=0.564). E. A plot showing the relationship between 12 decrease in rate of AP rise and decrease in AP peak, for injection of current (brown; n=8) and a 13 brief pressure ejection of GABA (green; n=11). F. Example axonal recording showing 14 spontaneously firing action potentials before the application of TTX (top, black) and after TTX 15 bath perfusion, just before the action potentials cease (bottom, red). inset: Example phase plots 16 for axonal action potentials before (black) and after (red) TTX perfusion. Right: Averaged data 17 showing the effect of TTX on action potential peak amplitude (graphs were aligned to the 18 beginning of TTX effect, n=5). The decrease in peak amplitude is plotted in G. G. Data from five 19 individual axons showing the effect of TTX wash-in on the change in rate of action potential rise, 20 and the change in the peak of the action potential. Each dot in data from an individual action 21 potential, normalized to just before the perfusion of TTX. In red is the average effect. H. Slope 22 and R 2 values from E and G compared. 23 These data suggested the peak amplitude of the action potential was susceptible 406 to both depolarizations and shunting inhibition, while the rate of rise was only affected 407 by depolarizations. In order to combine these two effects and distinguish between 408 shunting inhibition and depolarization-mediated inactivation of sodium channels, the 409 change in rate of rise was graphed against the change in peak spike amplitude. From  To experimentally test the effect of sodium channel inhibition on axonal action 415 potentials, TTX was bath perfused while recording axonal action potentials ( Figure 4F).

416
As the effect of TTX developed, the amplitude of the peak of the action potential was 417 progressively reduced, and the rate of rise was progressively slowed ( Figure 4F-H). We 418 compared the relationship of the reduction in the peak and the slowing of the rate of rise 419 across groups and found that the average of the TTX condition was similar to the direct 420 depolarization, indicating this effect was mainly through inactivation of sodium channels.    Example dopamine transient in control (black) and in GZ/CGP (red). Right: Plot of the average 5 increase in peak optically-evoked dopamine release (each dot is one slice; n=10, p = 0.015). C. 6 Example traces of imaged dopamine release from control (black) and diazepam bath perfusion 7 (green) conditions. Right: Group effect of diazepam on peak dopamine release (n=6; *p=0.044). 8 D. Example traces of imaged dopamine release from control (black) and diazepam bath 9 perfusion (green) conditions. Right: time course showing the effect of diazepam bath perfusion 10 on peak dopamine release (n=5). E. Example step depolarization (left) and muscimol pressure 11 ejection (right) recorded in the main axon in control (black) and diazepam bath application 12 (green).
Step depolarization and muscimol puff were performed within the same cell. axonal input resistance. 20 the input resistance by giving a small voltage step ( Figure 5E). We found that, in the 468 main axon, there was no effect of diazepam perfusion on the axonal input resistance 469 ( Figure 5F). This set of experiments show that diazepam directly targets axonal GABA-470 A receptors on dopamine neurons, but in the medial fiber bundle GABA-A agonists must 471 be exogenously applied to observe the effects of the drug.    The hyperpolarized membrane potential of the axon suggests that further 527 hyperpolarization by Kv1 may be limited by the potassium reversal potential and may 528 not be the main mechanism of dopamine inhibition. Consistent with the proposal by 529 Martel and colleagues (2011), shunting and/or changes in spike shape are likely to 530 underlie the D2-dependent inhibition of dopamine release.

531
In somatic recordings of pacemaking, dopaminergic neurons exhibit a gradual 532 depolarization of the interspike voltage thought to be critical for the generation of 533 spontaneous activity (Kang and Kitai, 1993;Khaliq and Bean, 2008). By contrast, our 534 data from distal recordings show that the slope of the interspike axonal membrane 535 potential was shallow. The shallower interspike depolarization in the axon suggests that 536 pacemaking in dopaminergic neurons results largely from excitability of the soma and 537 dendrites. Furthermore, hyperpolarized axonal threshold potential suggests that our 538 recording site in the axon is distal to the site of action potential initiation, which is the 539 axon initial segment (Hausser et al., 1995;Shu et al., 2007). Therefore, these 540 observations argue against the axon as a third site of oscillation generation after the 541 soma and dendrites (Pissadaki and Bolam, 2013). It is important to note that although     Furthermore, we found that these two mechanisms of inhibition differentially affect 620 action potential waveforms. While depolarization-mediated sodium channel inactivation both reduces spike height and slows the rate of action potential rise, shunting inhibition 622 only affects spike height. 2019). Our study shows that the GABA tone acts also through presynaptic GABA-A 647 receptors located on dopamine axons. Furthermore, drugs that potentiate GABA-A 648 receptors like diazepam will act on this tone to inhibit dopamine release. Yet, it is 649 important to also consider that tonic GABA activity within the striatum may not only 650 affect dopamine neuron axons. Indeed, we observed that dopamine release was more 651 inhibited by diazepam with nicotinic receptors available rather than inhibited. This finding hints at an additional circuit mechanism of action for diazepam, and perhaps for 653 GABA-A receptors more generally, within the striatum. As GABA-A receptors have been  In sum, this report shows direct evidence for GABA-A receptors on dopamine