Coordinated postnatal maturation of striatal cholinergic interneurons and dopamine release dynamics in mice

Dynamic changes in motor abilities and motivated behaviors occur during the juvenile and adolescent periods. The striatum is a subcortical nucleus critical for action selection, motor learning and reward processing. Its tonically active cholinergic interneuron (ChI) is an integral regulator of the synaptic activity of other striatal neurons, as well as afferent axonal projections of midbrain dopamine neurons. Thalamic and dopaminergic inputs initiate pauses in ChI firing following salient sensory cues that are extended for several hundred milliseconds by intrinsic regenerative currents. Here, we characterize the electrophysiological and morphological features of ChIs during mouse postnatal development. We demonstrate that ChI spontaneous activity increases with age while the duration of the pause in firing induced by depolarizing inputs decreases during postnatal development. Maturation of ChI activity is driven by two distinct physiological changes: decreased amplitude of the afterhypolarization between P14 and P18 and and increased Ih conductance between the late postnatal period and adulthood. Finally, we uncover postnatal changes in dopamine release properties that are mediated by cholinergic signalling. At P10, striatal dopamine release is diminished compared to the adult, but our data show efficient summation of dopamine relase evoked by multiple grouped stimuli that subsides by P28. Blockade of nictonic acetylcholine receptors enhances release summation in mice older than P28 but has little effect at P10. These data demonstrate a physiological maturation of ChI activity and indicate a reciprocal interaction between the postnatal maturation of striatal ChI and dopamine neurotransmission. Significance Statement Motor skills and motivated behavior regimes develop rapidly during the postnatal period. The functional development of the striatal cholinergic interneuron (ChI), which contributes to these behaviors in adulthood, remains unexplored. In this study, we tracked the ontogeny of spontaneous ChI activity and cellular morphology, as well as the developmental trajectory of ion conductances characteristic to this population. We further report a developmental link between ChI activity and dopamine release, revealing a change in the frequency-dependence of dopamine release during the early postnatal period that is mediated by cholinergic signaling. This study provides evidence that striatal microcircuits are dynamic during the postnatal period and that they undergo coordinated maturation.

In the adult, spontaneous ChI activity is driven by intrinsic ion conductances that occur in 82 the absence of synaptic activity (Bennett et al., 2000). Ih, the current mediated by 83 hyperpolarization-actived cyclic nucleotide-gated (HCN) channels, depolarizes ChIs to -60 mV,

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where HCN channels inactivate. A persistent sodium current then drives the cell to its action 85 potential threshold where CaV2 calcium channels open (Bennett et al., 2000). After the cell fires, 86 calcium-activated potassium channels, SK and BK repolarize the cell and control the magnitude 87 of a change in voltage known as the "medium afterhyperpolarization" (mAHP) (Goldberg and 88 Wilson, 2005).

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In the adult, a pause in ChI activity following salient cues is initiated by excitatory

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To address the postnatal maturation of ChI activity, we performed cell-attached and 98 whole-cell recordings of ChIs in the dorsal striatum in acute brain slices from mice over a range 99 of ages. We found that the spontaneous activity of ChIs increases linearly from postnatal day 10 100 (P10) into adulthood. Two distinct transitions in ChI physiology drive the changes in firing rate: 101 the mAHP decreases dramatically between P14 and P18, followed by an increase in the 102 putative HCN current between P28 and adulthood. In addition to the maturation of spontaneous 103 activity, the sAHP decreases in length from P10 to adulthood. Finally, using fast-scan cyclic

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Fibers were cut to an exposed length of ~100 μm, and silver leads (0.015"; A-M Systems) were 155 permanently affixed inside the pipette by coating with colloidal silver paint before insertion.

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Striatal slices were prepared as for electrophysiology experiments (see above). During   Two-photon images were acquired on a Prairie (Middleton, WI) Ultima microscope system using 181 PrairieView 4.3 software. Acute brain slices from DAT-ires-Cre x GCAMP3 mice were collected 182 as described above, transferred into a chamber, and perfused with ACSF at room temperature. tuned to 920 nm, and images were collected through a photomultiplier tube channel with a 490-185 560 nm emission filter. The objective used was a 60X, 0.9 NA water immersion lens (Olympus).

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Time series were acquired from a 100 x 100 pixel ROI located within in the same field of view

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We analyzed properties of ChI action potentials that could underlie changes in firing 250 frequency. Sample pairs of action potentials are shown in Figure 3B. In these examples, the 251 action potential threshold did not significantly differ with age ( Figure 3C). To emphasize the 252 salient features of these traces, the action potential amplitude is truncated. Neither action 253 potential width nor amplitude was significantly affected by age (data not shown).

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One possible explanation for an increased spontaneous firing frequency of ChIs during 255 postnatal development is that the action potential threshold becomes more hyperpolarized, 256 allowing the threshold to be reached more readily. Alternatively, the resting membrane potential 257 (reported as the midpoint between two action potentials) could become more depolarized. We 258 found, however, that age did not significantly affect either parameter ( Figure 3A,B).

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In contrast, the magnitude of the mAHP significantly decreased between P10 and 260 adulthood ( Figure 3C). Further, a robust negative correlation is observed between mAHP and firing rate in ChIs ( Figure 3D) Figure 4A). Thus the magnitude of the inactivated current, or difference between 272 maximal and steady-state inward current following hyperpolarizing voltage step, is a proxy for Ih 273 (Robinson and Siegelbaum, 2003). This inactivated inward current was stable between P10 and 274 P28 but was significantly increased in adulthood ( Figure 4B). These data suggest that increased

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Ih may contribute to elevated spontaneous firing frequencies in adulthood.

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The sAHP is extended during the early juvenile period.

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A unique feature of ChI physiology is a pause in firing in response to salient sensory  Figure 5A). The duration of the sAHP was longer at P10 and P14 than in adulthood ( Figure 5A,B). We conclude that the conductances that drive the sAHP are exaggerated during 288 the early juvenile period.

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Summation of DA release in response to multiple grouped stimuli.

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The pause in ChI firing following salient sensory cues is widely considered to affect local

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To address this, DA release was measured using fast-scan cyclic voltammetry (FSCV) in 303 acute brain slices from mice at P10, P28, and in adulthood, following intrastriatal electrical 304 stimulation. Consistent with our recent report, DA release following a single pulse increased 305 significantly with age ( Figure 6A,B). We found, however, that a train of stimuli significantly 306 increased evoked DA release as compared to a single stimulus at P10 but not at P28 or in 307 adulthood ( Figure 6B,C). These results are reported as the ratio of evoked DA following 100 Hz 308 stimuli to a single pulse (100Hz/1p; Figure 6C), and the ratio is significantly lower at P28 and in 309 adulthood compared to P10. We thus conclude that DA release properties mature between P10   ., 2017)). To test whether calcium dynamics were distinct at P10 318 compared to adulthood, we generated acute brain slices and imaged GCaMP3 fluorescence 319 using two-photon microscopy. A single electrical stimulation in the striatum yielded a smaller 320 change in fluorescence at P10 compared to DA axons in the adult striatum ( Figure 6D-E).

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Remarkably, stimulation with electrical pulses at 100 Hz yielded an equivalent change in 322 fluorescence at P10 and in adults ( Figure 6D-E). There was a significant interaction between 323 age and stimulation, with DA axons at P10 having a significantly increased summation of 324 depolarizing stimuli compared to adults ( Figure 6F-G). These data suggest that the postnatal 325 maturation of DA release dynamics may arise from changes in calcium dynamics.

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Evoked DA release was first measured in response to a single pulse and a train of five pulses 336 (100 Hz), after which DHbE was superfused onto the slice and evoked DA was measured in 337 response to the same stimulus paradigms. DHbE significantly increased the 100Hz/1p ratio at P28 and in the adult but had no effect at P10 ( Figure 6H). These data suggest that the 339 difference in DA release properties between P10, P28, or adults arises from increasing 340 cholinergic activity in the striatum during early postnatal maturation.

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The development and maturation of the striatal cholinergic system.

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The striatum contains the highest concentration of ACh in the brain (Fibiger, 1982). In 353 contrast to most other brain regions, however, striatal cholinergic innervation largely arises from 354 a population of interneurons rather than from afferent innervation, for example from the basal

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Although striatal ChIs appear in tandem with basal forebrain cholinergic neurons, their functional 368 development lags behind the basal forebrain system (Phelps et al., 1989), suggesting that cues 369 specific to the striatum may contribute to ChI maturation.

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At P10 and P14, the amplitude of the mAHP is significantly increased relative to ages 383 above P18 (Figure 3). In the adult, the mAHP is determined by the activity of SK and BK 384 channels (Goldberg and Wilson, 2005). SK and BK currents are mediated not only by channel 385 levels and function, but also in response to changes in calcium entry during action potential 386 firing that initiates SK and BK channel opening. Future efforts will determine how the activities of 387 these channels are altered during postnatal maturation.

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A second mechanism must drive further increases in spontaneous ChI firing beyond 389 P18, when the mAHP amplitude reaches adult levels (Figures 1 and 3). We found that the magnitude of Ih, which depolarizes ChIs from the mAHP toward the action potential threshold 391 (Kawaguchi, 1993;Bennett et al., 2000), increased from P28 to adulthood (Figure 4). Thus, the 392 maturational increase in ChI tonic firing appears to be due to the sequential decrease in mAHP 393 followed by an increase in Ih.

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We also report a maturational increase in the ChI pause with age. Although the ChI Here, we find that in contrast to these previous reports from adult mice, DA release is 427 efficiently summed across multiple grouped stimuli at P10 even without pharmacological 428 intervention. We confirmed that, in addition to DA release itself, electrically-evoked increases in 429 calcium within DA axons also underwent postnatal maturation as DA axons at P10 had 430 significantly more summation compared to the adult. Moreover, a nAChR antagonist did not 431 enhance the summation of DA release at P10, but did so at P28 and in adults. We conclude that 432 altered DA release properties in the juvenile striatum, including decreased release to a single 433 stimulus and increased summation of DA release across grouped stimuli, are mediated by 434 deficient signaling through the nAChR that then matures over the early postnatal period.

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A limitation of this study is that we did not correlate changes in ChI firing patterns with