Prolonged Stimulus-Induced Acetylcholine Increases in Striatal Brain Slices Acetylcholine (ACh) has modulatory effects on several different neurons and synapses in the striatum, and also has key roles in striatal synaptic plasticity. While the roles of cholinergic neurons and receptors have been examined, we know less about the dynamics of extracellular ACh levels, mainly due to lack of ACh sensors. To address this isse we expressed the genetically encoded intensity-based acetylcholine sensing fluorescent reporter (iAChSnFR, developed by the laboratory of Loren Looger) in dorsomedial striatum (DMS) and used brain slice fluorimetry/photometry to examine responses to electrical stimulation. The iAChSnFR was introduced into striatal neurons by stereotaxic injection of an adeno-associated virus (AAV) construct. We also performed photometric recording in DMS slices from expressing a mutated (iAChSnFR-Null) version of the sensor that has >1000x lower ACh affinity. Single afferent stimuli of 0.04-2 msec duration at 0.1-2 mA intensity induced fluorescence increases that persisted for more than 40 sec when measured with the iAChSnFR, but not the iAChSnFR-Null construct. In some slices small increases in fluorescence were observed in the absence of afferent stimulation, and these transients decayed within 10 sec. These transients were not observed in recordings using the iAChSnFR-Null contruct despite the fact that substantial background fluorescence was observed in these slices indicating that the Null sensor was expressed. The peak amplitude of the stimulus-induced fluorescence increases increased with increasing stimulus duration or intensity, but the long duration responses were observed even with weak stimulus parameters that activated small peak amplitude responses. Brief (2-20 msec) pressure application of ACh produced fluorescence increases that decayed within 20 sec. When iAChSnFR expression and photometry were used to examine different cortical regions, electrical stimulation in those regions produced fluorescence increases that persisted for less than 20 sec. The stimulus-induced fluorescent increases were prevented by application of 300 nM tetrodotoxin (TTX), and when extracellular calcium was lowered from 2 mM to 10 micromolar. Furthermore application of the vesicular ACh transport inhibitor vesamicol reduced both the amplitude and duration of stimulus-induced fluorescence increases. Inhibiting the ACh degrading enzyme ACh esterase prolonged the stimulus-induced fluorescence increases. Thus, responses depend on neuronal action potentials and synaptic ACh release. Cholinergic interneurons (CINs) provide the bulk of the ACh in DMS, although afferents from the pedunculopontine nucleus can also release this neuromodulator. To determine if CINs contribute to the stimulus-induced ACh release we used a Cre recombinase- and AAV-based Caspase3 lesion approach to decrease CINs in DMS expressing iAChSnFR. The amplitude and duration of stimulus-induced ACh increases was strongly reduced in the caspase-exposed DMS. Thus, CINs contribute to the release ACh. Stimulus-induced ACh increases were not altered by blocking ionotropic glutamate receptors or with MPEP, a negative allosteric modulator of the mGlu5 receptor. Blocking ionotropic GABA receptors also did not alter stimulus-induced ACh increases. Thus, the ACh release does not appear to be secondary to activation of afferents that induce fast synaptic responses through other neurotransmitters. We are currently determining what mechanisms underlie the long-lasting increase in stimulus-induced signals in striatum that are not observed in cortex. It is likely that stimulation induces persistent feedforward activation of several cholinergic neurons. It is also possible that in striatum ACh release leads to formation of choline at concentrations high enough bind iAChSnFR (i.e. 100s micromolar). We are also examining effects of other neuromodulators on ACh release measured with this sensor. Using trains of stimuli of the type that promote synaptic plasticity in striatum, we have observed ACh increases that persist for minutes, and we are currently examining the relationship of these signals to changes in other neurotransmitters. Overall, our findings indicate that large and long-lasting increases in striatal ACh can be induced by modest afferent stimulation, indicating one factor that accounts for the prominent role of this neuromodulator in striatal synaptic modulation and plasticity. Dynamics of Endocannabinoid Signaling in Striatum Endocannabinoids (eCBs) are fatty acid metabolites of arachidonic acid-containing lipids that act on the cannabinoid type 1 and 2 (CB1 and CB2) receptors to modulate neuronal function. In the striatum, eCBs have been implicated in short- and long-term synaptic depression. However the factors that drive eCB release., timing of release and termination of the eCB signal have been inferred from indirect measures. To directly measure eCB dynamics in dorsal striatal brain slices we combined expression of the fluorescent genetically encoded GPCR Activation-based Sensor for endocannabinoids (GRABeCB, a modified CB1 receptor developed by Drs. Ao Dong and Yulong Li at Peking University) and slice fluorimetry/photometry as described in the preceding section of this report. We either expressed GRABeCB in striatal neurons or in primary motor cortex (M1) neurons by AAV injection, and then collected fluorescent signals in dorsal striatum. Two GRABeCB versions, 1.0 and 2.0, were used. Brief bursts of electrical stimuli (e.g. 5 pulses at 20 Hz) elicited increases in fluorescence whether measurements were made from striatal neurons or presynaptic M1 afferents projecting into striatum. The onset of the fluorescence increases occurred 100s of msec after stimulation onset, and increases persisted for 10-15 sec after stimulus cessation. The stimulus burst-induced fluorescence increases were blocked by TTX or in low extracellular calcium and were also prevented by application of the CB1 antagonist AM251. Thus, signals were of neuronal origin. Furthermore, increases could not evoked when the F177A mutant GRAB-eCB construct with reduced eCB affinity was expressed. The amplitude and duration of stimulus burst-induced fluorescence increases was enhanced by a blocker of monoacylglycerol lipase, the enzyme that catalyzes breakdown of the eCB 2-arachadonoylglycerol (2-AG). The mGlu5 negative allosteric modulator MPEP reduced stimulus burst-induced eCB increases, consistent with the proposed role for this receptor in eCB production. We are currently examining effects of other neurotransmitters and neuromodulators on eCB signals, as well as effects of stimuli that produce striatal synaptic plasticity. Our findings to date indicate that GRABeCB is a useful tool for real-time measurement of eCB levels in brain tissue.
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