Cholinergic interneurons (CINs) are critical regulators of striatal network activity and output. Changes in CIN activity are thought to encode salient changes in the environment and stimulus-response-outcome associations. Here we report that the stress-associated neuropeptide corticotropin releasing factor (CRF) produces a profound and reliable increase in the spontaneous firing of CINs in both dorsal striatum and nucleus accumbens (NAc) through activation of CRF type 1 receptors, production of cAMP and reduction in spike accommodation in male mice. The increase of CIN firing by CRF results in the activation muscarinic acetylcholine receptors type 5, which mediate potentiation of dopamine transmission in the striatum. This study provides critical mechanistic insight into how CRF modulates striatal activity and dopamine transmission in the NAc to likely account for CRF facilitation of appetitive behaviors.Although the presence of CRF receptors in the dorsal and ventral striatum has been acknowledged, the cellular identity and the functional consequences of receptor activation is unknown. Here we report that striatal cholinergic interneurons express CRF-R1 receptors and are acutely activated by the neuropeptide CRF that is released in response to salient environmental stimuli. Cholinergic interneurons make Ͻ1% of the cells in the striatum but are critical regulators of the striatal circuitry and its output. CRF's fast and potent activation of cholinergic interneurons could have far reaching behavioral implications across motivated behaviors controlled by the striatum.

Axons of 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 the striatum of adult mice. 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 the positive allosteric modulator diazepam enhanced GABA-A currents on dopaminergic axons and directly inhibited release, but also likely acts by reducing excitation from cholinergic interneurons. Thus, we reveal the mechanisms of GABA-A receptor modulation of dopamine release and provide new insights into the actions of benzodiazepines within the striatum.

Dopamine (DA) signals in the striatum are critical for a variety of vital processes, including motivation, motor learning, and reinforcement learning. Striatal DA signals can be evoked by direct activation of inputs from midbrain DA neurons (DANs) as well as cortical and thalamic inputs to the striatum. In this study, we show that in vivo optogenetic stimulation of prelimbic (PrL) and infralimbic (IL) cortical afferents to the striatum triggers an increase in extracellular DA concentration, which coincides with elevation of striatal acetylcholine (ACh) levels. This increase is blocked by a nicotinic ACh receptor (nAChR) antagonist. Using single or dual optogenetic stimulation in brain slices from male and female mice, we compared the properties of these PrL/IL-evoked DA signals with those evoked by stimulation from midbrain DAN axonal projections. PrL/IL-evoked DA signals are undistinguishable from DAN evoked DA signals in their amplitudes and electrochemical properties. However, PrL/IL-evoked DA signals are spatially restricted and preferentially recorded in the dorsomedial striatum. PrL/IL-evoked DA signals also differ in their pharmacological properties, requiring activation of glutamate and nicotinic ACh receptors. Thus, both in vivo and in vitro results indicate that cortical evoked DA signals rely on recruitment of cholinergic interneurons, which renders DA signals less able to summate during trains of stimulation and more sensitive to both cholinergic drugs and temperature. In conclusion, cortical and midbrain inputs to the striatum evoke DA signals with unique spatial and pharmacological properties that likely shape their functional roles and behavioral relevance.

27Axons of midbrain dopaminergic neurons innervate the striatum where they 28 contribute to movement and reinforcement learning. Past work has shown that striatal 29 GABA tonically inhibits dopamine release, but whether GABA-A receptors directly 30 modulate transmission or act indirectly through circuit elements is unresolved. Here, we 31 use whole-cell and perforated-patch recordings to test for GABA-A receptors on the 32 main dopaminergic neuron axons and branching processes within striatum. Application 33 of GABA depolarized axons, but also decreased the amplitude of axonal spikes, limited 34 propagation and reduced striatal dopamine release. The mechanism of inhibition 35 involved sodium channel inactivation and shunting. Lastly, we show that the positive 36 allosteric modulator diazepam enhanced GABA-A currents on dopaminergic neuron 37 axons and directly inhibited release, but also likely acts by reducing excitatory drive 38 from cholinergic interneurons. Thus, we reveal the mechanisms of GABA-A receptor 39 modulation of dopamine release and provide new insight into the actions of 40 benzodiazepines within the striatum.41 42 43 101the study. Mice that underwent viral injections were injected at postnatal day 18 or older 102 and were used for ex vivo electrophysiology and imaging 3-12 weeks after injection. 103The following strains were used: DAT-Cre (SJL-Slc6a3(tm1.1(cre)Bkmn/J, The Jackson 104 Laboratory Cat#006660); Ai95-RCL-GCaMP6f-D (Cg-Gt(ROSA)26Sor(tm95.1(CAG-105 GCaMP6f)Hze)/MwarJ, The Jackson Laboratory Cat#028865); Ai9 106 (Gt(ROSA)26Sor(tm9(CAG-tdTomato)Hze), The Jackson Laboratory Cat#007909); TH-107 GFP (Tg(TH-EGFP)1Gsat) NIH MMRRC; C57/Bl6J Wild Type, The Jackson Laboratory 108 Cat#000664; Ai32 (B6.Cg -Gt(ROSA)26Sor(tm32(CAG-COP4*H143R/EYFP)Hze), The 109 Jackson Laboratory, Cat#024109). 110 Method Details 111 Viral Injections 112 All stereotaxic injections were conducted on a Stoelting QSI (Cat#53311). Mice 113 were maintained under anesthesia for the duration of the injection and allowed to 114 recover from anesthesia on a warmed pad. The AAV9-CAG-FLEX-TdTomato (Penn 115 Vector Core), AAV-Syn-FLEX-jGCaMP7f (Dana et al., 2019), and AAV9-hSyn-dLight1.2 116 (Patriarchi et al., 2018) viruses (0.5-1 µl) were injected bilaterally into either the medial 117 dorsal striatum (X: ± 1.7 Y: +0.8 Z: -3.3) or the SNc (X: ± 1.9 Y: -0.5 Z: -3.9) via a 118 Hamilton syringe. At the end of the injection, the needle was raised at a rate of 0.1 to 119 0.2 mm per minute for 10 minutes before the needle was removed. 120 Slicing and electrophysiology 121 Brain slice experiments were performed on male and female adult mice of at 122 least 6 weeks in age. Mice were anesthetized with isoflurane, decapitated, and brains 123 rapidly extracted. Horizontal sections (electrophysiology, dLight, calcium imaging) or 124 coronal sections (voltammetry) were cut at 330-400 µm thickness on a vibratome while 125 immersed in warmed, modified, slicing ACSF containing (in mM) 198 glycerol, 2.5 KCl, 126 1.2 NaH2PO4, 20 HEPES, 25 NaHC...