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[Preprint]. 2024 Jun 25:2024.06.25.600616.
doi: 10.1101/2024.06.25.600616.

Presynaptic GABAA receptors control integration of nicotinic input onto dopaminergic axons in the striatum

Samuel G Brill-Weil  1   2 , Paul F Kramer  1   2 , Anthony Yanez  1   2 , Faye H Clever  1   2 , Renshu Zhang  1   2 , Zayd M Khaliq  1   2
Affiliations

Presynaptic GABAA receptors control integration of nicotinic input onto dopaminergic axons in the striatum

Samuel G Brill-Weil et al. bioRxiv. .

Abstract

Axons of dopaminergic neurons express gamma-aminobutyric acid type-A receptors (GABAARs) and nicotinic acetylcholine receptors (nAChRs) which are both independently positioned to shape striatal dopamine release. Using electrophysiology and calcium imaging, we investigated how interactions between GABAARs and nAChRs influence dopaminergic axon excitability. Direct axonal recordings showed that benzodiazepine application suppresses subthreshold axonal input from cholinergic interneurons (CINs). In imaging experiments, we used the first temporal derivative of presynaptic calcium signals to distinguish between direct- and nAChR-evoked activity in dopaminergic axons. We found that GABAAR antagonism with gabazine selectively enhanced nAChR-evoked axonal signals. Acetylcholine release was unchanged in gabazine suggesting that GABAARs located on dopaminergic axons, but not CINs, mediated this enhancement. Unexpectedly, we found that a widely used GABAAR antagonist, picrotoxin, inhibits axonal nAChRs and should be used cautiously for striatal circuit analysis. Overall, we demonstrate that GABAARs on dopaminergic axons regulate integration of nicotinic input to shape presynaptic excitability.

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Figures

Figure 1:
Figure 1:. Diazepam suppresses subthreshold nicotinic input onto DA axons.
(A) Illustration of the simplified striatal environment that DA axons are subjected to. Namely, a GABAergic tone that acts upon GABAA receptors and ACh release from CINs that acts on nAChRs. (B) Schematic of recording set-up. Low-intensity electrical stimulation was applied in the vicinity of an axon recorded in perforated patch configuration. (C) Average electrically-evoked axEPSPs from a representative axon during control conditions, after diazepam (10 μM) wash-in, and after DHβE (1 μM) wash-in. (D) Normalized amplitude of evoked axEPSPs during diazepam wash-in (n = 9). (E) Quantification of data in (C) (n = 9). (F) The effect of diazepam on the rising slope of axEPSPs. Slope was measured between 30% and 70% of the peak amplitude (n = 9). In a subset of recordings (circles in E and F; triangles represent axons recorded in normal ACSF), nAChRs were partially blocked with a sub-saturating concentration of hexamethonium chloride (200 μM) to ensure that the amplitude of axEPSPs remained subthreshold.
Figure 2:
Figure 2:. jGCaMP8s as a readout of multi-component DA axon activation.
(A) Schematic of jGCaMP8s expression strategy and experimental set-up. Horizontal slices containing the dorsomedial striatum were imaged with a photodiode while stimulated with a bipolar electrode 200 μm away and out of the field of view of the photodiode. (B) Fluorescent signals following a single electrical stimulus. Each trace represents an average of each drug condition in a representative experiment. (C) Normalized amplitude in DHβE (1 μM) and TTX (500 nM) compared to control (n = 6). (D) Schematic of CIN-DA axon circuitry and time course of APs following suprathreshold electrical stimulation. (E) First derivative of traces in (B). Grey trace is the subtraction of the DHβE trace from the control trace. (F) Top: Normalized nicotinic and direct components from (E). Bottom: Onset latencies for the two components (n = 6).
Figure 3:
Figure 3:. GABAAR antagonism potentiates the nicotinic component without affecting ACh release.
(A) Average jGCaMP8s signals for each condition in a representative experiment. Inset shows the same traces on an expanded timescale. (B) Time course of the peak amplitude during gabazine (10 μM) wash-in (n = 8). (C) Quantification of the peak amplitude of the jGCaMP8s signals in gabazine compared to control. (D) Left: First derivative of traces in (A). Right: Direct component and nicotinic components overlaid. (E) Time course of normalized direct component and nicotinic component amplitude during wash-in of gabazine. (F) Normalized amplitudes of both components in gabazine compared to control. (G) Latency of the two components in gabazine compared to control (see also Fig. S2). (H) Average evoked GRABACh signals during control conditions and following wash-in of gabazine for a representative experiment. (I) Time course of the normalized amplitude of GRABACh signals during gabazine wash-in (n = 6). (J) Normalized amplitude of GRABACh signals in gabazine compared to control.
Figure 4:
Figure 4:. Picrotoxin blocks nAChRs in a dose-dependent manner.
(A) Average jGCaMP8s signals for each condition in a representative experiment. Inset shows the same traces on an expanded timescale. (B) Time course of the peak amplitude during picrotoxin (100 μM) and DHβE (1 μM) wash-in (n = 8). (C) Quantification of the peak amplitude of the jGCaMP8s signals in picrotoxin and DHβE compared to control (gabazine, 10 μM). (D) Left: First derivative of traces in (A). Right: Direct component and nicotinic components overlaid. (E) Time course of normalized direct component and nicotinic component amplitude during wash-in of picrotoxin and DHβE. (F) Normalized amplitudes of both components in picrotoxin and DHβE compared to control. (G) Dose-response curve of the nicotinic component to increasing picrotoxin concentration (30μM: n = 3; 100 μM: n = 11; 300 μM: n = 5; 1000 μM: n = 4). (H) Average evoked GRABACh signals in gabazine and following wash-in of picrotoxin for a representative experiment. (I) Time course of the normalized amplitude of GRABACh signals during picrotoxin wash-in (n = 6). (J) Normalized amplitude of GRABACh signals in picrotoxin compared to control.
Figure 5:
Figure 5:. Picrotoxin decreases ACh-evoked depolarizations in the main axon of DA neurons.
(A) Schematic of recording configuration. ACh (300 μM) was applied from a puff pipette positioned ~50 μm away from the recording site. (B) Average ACh-evoked depolarizations in the main axon of a DA neuron. (C) Time course of depolarization amplitude during wash-in of picrotoxin (100 μM) followed by DHβE (1 μM). (D) Amplitude of ACh-evoked depolarization in each condition (n = 6).
Figure 6:
Figure 6:. Picrotoxin antagonism of axonal nAChRs is not occluded by conotoxin P1A.
(A) Average jGCaMP8s signals for each condition in a representative experiment. Inset shows the same traces on an expanded timescale. (B) Time course of the peak amplitude during conotoxin-P1A (300 nM), picrotoxin (100 μM), and DHβE (1 μM) wash-in (n = 5). (C) Quantification of the peak amplitude of the jGCaMP8s signals in P1A, picrotoxin, and DHβE compared to control (gabazine, 10 μM). (D) Left: First derivative of traces in (A). Right: Direct component and nicotinic components overlaid. (E) Time course of normalized direct component and nicotinic component amplitude during wash-in of P1A, picrotoxin, and DHβE. (F) Normalized amplitudes of both components in P1A, picrotoxin, and DHβE compared to control. (G) Time course of the time-to-peak of the nicotinic component following application of either picrotoxin (n = 8) or P1A (n = 5). All slices were bathed in ACSF containing 10 μM gabazine for the duration of the experiment. (H) Left: Quantification of data shown in (G). Right: No changes were observed in the onset latency of the direct component in the same experiment (Ptx: n = 8; P1A: n = 4; see also Fig. S3). (I) Correlation of effect on nicotinic component amplitude and nicotinic component latency for gabazine, picrotoxin, and P1A (GZ: n = 8; Ptx: n = 8; P1A: n = 5).
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