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. 2021 Sep 15;41(37):7831-7847.
doi: 10.1523/JNEUROSCI.1037-20.2021. Epub 2021 Aug 4.

The Effect of Serotonin Receptor 5-HT1B on Lateral Inhibition between Spiny Projection Neurons in the Mouse Striatum

Stefan Pommer  1 , Yumiko Akamine  1 , Serge N Schiffmann  2 , Alban de Kerchove d'Exaerde  2 , Jeffery R Wickens  3
Affiliations

The Effect of Serotonin Receptor 5-HT1B on Lateral Inhibition between Spiny Projection Neurons in the Mouse Striatum

Stefan Pommer et al. J Neurosci. .

Abstract

The principal neurons of the striatum, the spiny projection neurons (SPNs), make inhibitory synaptic connections with each other via collaterals of their main axon, forming a local lateral inhibition network. Serotonin, acting via the 5-HT1B receptor, modulates neurotransmitter release from SPN terminals in striatal output nuclei, but the role of 5-HT1B receptors in lateral inhibition among SPNs in the striatum is unknown. Here, we report the effects of 5-HT1B receptor activation on lateral inhibition in the mouse striatum. Whole-cell recordings were made from SPNs in acute brain slices of either sex, while optogenetically activating presynaptic SPNs or fast-spiking interneurons (FSIs). Activation of 5-HT1B receptors significantly reduced the amplitude of IPSCs evoked by optical stimulation of both direct and indirect pathway SPNs. This reduction was blocked by application of a 5-HT1B receptor antagonist. Activation of 5-HT1B receptors did not reduce the amplitude of IPSCs evoked from FSIs. These results suggest a new role for serotonin as a modulator of lateral inhibition among striatal SPNs. The 5-HT1B receptor may, therefore, be a suitable target for future behavioral experiments investigating the currently unknown role of lateral inhibition in the function of the striatum.SIGNIFICANCE STATEMENT We show that stimulation of serotonin receptors reduces the efficacy of lateral inhibition between spiny projection neurons (SPNs), one of the biggest GABAergic sources in the striatum, by activation of the serotonin 5-HT1B receptor. The striatum receives serotonergic input from the dorsal raphe nuclei and is important in behavioral brain functions like learning and action selection. Our findings suggest a new role for serotonin in modulating the dynamics of neural interactions in the striatum, which extends current knowledge of the mechanisms of the behavioral effects of serotonin.

Keywords: GABA; MSN; SPN; lateral inhibition; serotonin; synapse.

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Figures

Figure 1.
Figure 1.
Properties of postsynaptic ChR2- neurons in ChR2 injected mice. A, Specific expression of ChR2 in a D1-Cre mouse. Position: bregma ∼0.26 mm. B, Superimposed voltage traces of an example SPN in response to a series of current steps shows inward rectification. Small depolarizations in the membrane potential are spontaneous events. Only the first AP-eliciting step is shown in black for clarity. The cell was held at −90 mV. C, Average IV-curve of subthreshold membrane potential from D1 and D2-SPNs. The voltage response curve is close to linear for hyperpolarizing steps and increases exponentially above −90 mV. V**alues are mean ± SEM. Dotted lines represent SEM were applicable. N = 14/10, D2/D1. D, Average firing frequency versus current intensity for D1 and D2-SPNs. Values are mean ± SEM. Dotted lines represent SEM were applicable. N = 14/9, D2/D1. E, Average input resistance (RI) of postsynaptic SPNs at the beginning (first minute) and end (last minute) of recording. The input resistance is slightly lower for D1-SPNs. F, Whole-cell patch clamp of SPNs recording IPSCs evoked by optogenetic stimulation of lateral ChR2+ SPNs. G, Recording protocols for ChR2-evoked IPSCs in voltage clamp configuration. Membrane current is recorded in repeated 5-s sweeps. A single 2-ms light pulse (blue bar) is given one second (double arrowheads) after the start of each sweep. H, Example IPSC response (middle graph) to presynaptic optogenetic stimulation (upper graph). APs were triggered with a 2-ms light pulse. Gray traces are an overlay of repeated stimulation. Lower graph, Example trace of an IPSC after a 2-ms light pulse in control ACSF (black trace) and in the presence of bicuculline (gray trace). All recordings were done in the presence of KA. ns, p > 0.05; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 2.
Figure 2.
AAV5-mediated ChR2 is expressed in SPNs of D1/A2a-cre mice. A–D, Images of striatal neuropil to show localization of ChR2. A, Cre-dependent expression of ChR2 in cell membranes (arrowheads) in the striatum of A2a-cre mice. B, Spiny neurons labeled with DARPP-32. C, Cell nuclei stained with DAPI. D, Overlay showing DARP-32-positive neurons were also positive for ChR2 (arrowheads). Scale bar: 10 µm.
Figure 3.
Figure 3.
AAV5-mediated ChR2 is absent in CINs of D1/A2a-cre mice. A–E, Different section showing absence of Cre-dependent ChR2 expression from CINs. A, ChR2 expression of D1-SPNs in D1-Cre mice (arrowheads). B, Example CIN. C, Serotonin 5-HT1B receptor clustered on somata of CIN and other neurons. D, Cell nuclei stained with DAPI. Scale bar: 20 µm. E, Overlay of E–H. F–J, Magnification of rectangle from A–E. F, ChR2 expressed in the cell membrane (arrowheads) and spines (arrows) of D1-Cre SPNs. G, CINs. H, Serotonin 5-HT1B receptor. I, Cell nuclei stained with DAPI. J, Overlay showing ChAT-positive cells negative for ChR2 (arrowheads). Serotonin receptor 5-HT1B is clustered on somata of CINs and SPNs. Scale bar: 10 µm.
Figure 4.
Figure 4.
Drug application does not affect ChR2-evoked APs. A, Example AP triggered in a D2-SPNs (D1-Cre mouse) by a 2-ms light pulse before (left), during (middle), and after drug treatment (black traces). Each trace is an average of 180 individual traces. Control APs (gray traces) did not receive CP-93129. B, Effect of repeated AP triggering over time. Triangles, half width; square, occurrence of AP. N = 3: D1-Cre (2), A2a-Cre (1). Values are MEAN ± SEM. C, Effect of bath-applied CP-93129. N = 3: D1-Cre (3).
Figure 5.
Figure 5.
Serotonin receptor 5-HT1B agonist CP-93129 lowers the average IPSC amplitude and synaptic success rate. A, Example IPSC amplitude recording and drug treatment for D1→D2-SPN connections (D1-Cre mouse). Bath application of serotonin receptor 5-HT1B agonist CP-93129 results in silencing of IPSCs. Recording of IPSCs resumes after washout. B, Example IPSC amplitude recording and drug treatment for D2→D1-SPN connections (A2a-Cre mouse). Bath application of serotonin receptor 5-HT1B agonist CP-93129 results in reduction of IPSCs amplitude and increased failures. IPSC recording recovers after washout. C, E, G, Average IPSC amplitude change during drug treatment for D2 SPNs (N = 12, D1-Cre mice; C), D1-SPNs (N = 6, A2a-Cre mice; E) and control conditions [N = 6: D1-Cre (3), A2a-Cre (3); G)]. Each point is an average of six IPSC events. Dotted lines represent SEM IPSC amplitude change was normalized against last 5 min of baseline. The traces on the right are examples recorded during baseline (black), drug treatment/equal period for control conditions (light gray) and washout (dark gray). The black bar represents the 2-ms light pulse. D, F, H, Average group synaptic success rate of D1→D2-SPNs (D), D2→D1-SPNs (F), and control conditions (H). Individual synaptic success rate was calculated as number of recorded IPSCs per minute divided by the number of maximal IPSCs per minute. D, F, H, same cells as C, E, G. Dotted lines represent SEM. All recordings were done in the presence of KA.
Figure 6.
Figure 6.
CP-93129 changes average IPSC amplitude and synaptic release probability in local axon collaterals. A, Statistical analysis of average IPSC amplitude change in D2-SPNs (D1-Cre mice) during baseline, drug treatment, and washout (N = 12). B, Comparison of average IPSC amplitude of drug treatment in D2-SPNs (D1-Cre mice, N = 12) with control group (N = 6). Mean ± SD. C, Analysis of average group synaptic success rate for D1→D2-SPN connections (D1-Cre mice, N = 12). D, Statistical analysis of average IPSC amplitude change in D1-SPNs (A2a-Cre mice) during baseline, drug treatment, and washout (N = 6). E, Comparison of average IPSC amplitude of drug treatment in D1-SPNs (A2a-Cre mice, N = 6) with control group (N = 6). Mean ± SD. F, Analysis of average group synaptic success rate for D2→D1-SPN connections (A2a-Cre mice, N = 6). ns, p > 0.05; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 7.
Figure 7.
CP-93129 affects ChR2-evoked IPSCs in D2-SPNs up to 30 ms after optical stimulation. A, Average distribution and amplitude of IPSCs recorded in D2-SPNs (D1-Cre mice) within the first 10 ms after the light stimulus. The yellow area indicates application of CP-93129. Each episode equals 5 s. B, Average IPSC amplitude of all recorded IPSCs in D2-SPNs (D1-Cre mice) with a latency of ≤10 ms over 60 min. The two dotted lines mark the application of CP-93129. C, Statistical analysis of average IPSC amplitude within 10 ms after the light pulse in D2-SPNs (D1-Cre mice) during baseline, drug treatment, and washout. Values are mean ± SD. D, Average distribution and amplitude of IPSCs recorded in D2-SPNs (D1-Cre mice) between 10 and 30 ms after the light stimulus. The yellow area indicates application of CP-93129. Each episode equals 5 s. E, Average IPSC amplitude of all recorded IPSCs in D2-SPNs (D1-Cre mice) with a latency between 10 and 30 ms over 60 min. The two dotted lines mark the application of CP-93129. F, Statistical analysis of average IPSC amplitude between 10 and 30 ms after the light pulse in D2-SPNs (D1-Cre mice) during baseline, drug treatment, and washout. Values are mean ± SD. G, Average distribution and amplitude of IPSCs recorded in D2-SPNs (D1-Cre mice) between 30 and 100 ms after the light stimulus. The yellow area indicates application of CP-93129. Each episode equals 5 s. H, Average IPSC amplitude of all recorded IPSCs in D2-SPNs (D1-Cre mice) with a latency between 30 and 100 ms over 60 min. I, Statistical analysis of average IPSC amplitude between 30 and 100 ms after the light pulse in D2-SPNs (D1-Cre mice) during baseline, drug treatment, and washout. Values are mean ± SD; N = 12. ns, p > 0.05; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 8.
Figure 8.
CP-93129 affects ChR2-evoked IPSCs in D1-SPNs up to 30 ms after optical stimulation. A, Average distribution and amplitude of IPSCs recorded in D1-SPNs (A2a-Cre mice) within the first 10 ms after the light stimulus. Note the higher average amplitude compared with D2-SPNs. The yellow area indicates application of CP-93129. Each episode equals 5 s. B, Average IPSC amplitude of all recorded IPSCs in D1-SPNs (A2a-Cre mice) with a latency of ≤10 ms over 60 min. The two dotted lines mark the application of CP-93129. C, Statistical analysis of average IPSC amplitude within 10 ms after the light pulse in D1-SPNs (A2a-Cre mice) during baseline, drug treatment, and washout. IPSC recovery did not reach baseline levels. Values are mean ± SD. D, Average distribution and amplitude of IPSCs recorded in D1-SPNs (A2a-Cre mice) between 10 and 30 ms after the light stimulus. The yellow area indicates application of CP-93129. Each episode equals 5 s. E, Average IPSC amplitude of all recorded IPSCs in D1-SPNs (A2a-Cre mice) with a latency between 10 and 30 ms over 60 min. The two dotted lines mark the application of CP-93129. F, Statistical analysis of average IPSC amplitude between 10 and 30 ms after the light pulse in D1-SPNs (A2a-Cre mice) during baseline, drug treatment, and washout. Values are mean ± SD. G, Average distribution and amplitude of IPSCs recorded in D1-SPNs (A2a-Cre mice) between 30 and 100 ms after the light stimulus. Each episode equals 5 s. H, Average IPSC amplitude of all recorded IPSCs in D1-SPNs (A2a-Cre mice) with a latency between 30 and 100 ms over 60 min. I, Statistical analysis of average IPSC amplitude between 30 and 100 ms after the light pulse in D1-SPNs (A2a-Cre mice) during baseline, drug treatment, and washout. Values are mean ± SD; N = 6. ns, p > 0.05; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 9.
Figure 9.
CP-93129 does not affect ChR2-IPSCs from FSIs. A, Example AP triggered in a FSI (PV-Cre mouse) by a 2-ms light pulse. Optical stimulation resulted in one (black) or two APs (gray). Blue light bar represents LED stimulus. B, Example IPSC amplitude recording and drug treatment for FSI→SPN connections (PV-Cre mouse). Bath application of serotonin receptor 5-HT1B agonist CP-93129 has no effect on IPSCs. C, Average IPSC amplitude change during CP-93129 treatment in PV-Cre mice (N = 4). Horizontal line at 100% was added as visual guideline. Dotted lines represent SEM IPSC amplitude change was normalized against minutes 10–15 (baseline). Each point is an average of six IPSC events. D, Average group synaptic success rate of SPNs with CP-93129. Dotted lines represent SEM. All recordings were done in the presence of KA.
Figure 10.
Figure 10.
Optical-evoked APs are somatic. A, Example recording of IPSC amplitudes in a D1-SPN (A2a-Cre mouse) in the presence of sodium channel blocker TTX (0.5 µm). B, Average IPSC amplitude change during TTX treatment in D1-Cre and A2a-Cre mice [N = 4: D1-Cre (1), A2a-Cre (3)]. Horizontal line at 100% was added as visual guideline. Dotted lines represent SEM IPSC amplitude change was normalized against minutes 10–15 (baseline). Each point is an average of six IPSC events. The traces on the right are examples recorded during baseline (black), drug treatment (light gray), and CP-93129 washout (dark gray). The black bar represents the 2-ms light pulse. C, Average group synaptic success rate of SPNs with TTX. Dotted lines represent SEM. D, Before and after plot of average IPSC amplitude change of SPNs with TTX. Bars represent the mean. All recordings were done in the presence of KA.
Figure 11.
Figure 11.
CP-93129 acts via 5-HT1B receptors located on presynaptic SPN terminals. A, Example recording of IPSC amplitudes in a D2-SPN (D1-Cre mouse) with serotonin receptor antagonist SB-216641 and agonist CP-93129. Recording started in the presence of SB-216641 and CP-93129 was added to the bath after 15 min. B, Average IPSC amplitude change in D2-SPNs (D1-Cre mice) during drug treatment (N = 5). Horizontal line at 100% was added as visual guideline. Each point is an average of six IPSC events. Dotted lines represent SEM IPSC amplitude change was normalized against minutes 10–15 (baseline). The traces on the right are examples recorded during baseline (black), drug treatment (light gray), and washout (dark gray). The black bar represents the 2-ms light pulse. C, Average group synaptic success rate of D2-SPNs (D1-Cre mice) with SB-216641 and CP-93129. Dotted lines represent SEM. D, Before and after plot of average IPSC amplitude change of D2-SPNs (D1-Cre mice) during drug treatment. Bars represent the mean. All recordings were done in the presence of KA. ns, p > 0.05; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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