Optogenetic Measurement of Presynaptic Calcium Transients Using Conditional Genetically Encoded Calcium Indicator Expression in Dopaminergic Neurons

Calcium triggers dopamine release from presynaptic terminals of midbrain dopaminergic (mDA) neurons in the striatum. However, calcium transients within mDA axons and axon terminals are difficult to study and little is known about how they are regulated. Here we use a newly-developed method to measure presynaptic calcium transients (PreCaTs) in axons and terminals of mDA neurons with a genetically encoded calcium indicator (GECI) GCaMP3 expressed in transgenic mice. Using a photomultiplier tube-based system, we measured electrical stimulation-induced PreCaTs of mDA neurons in dorsolateral striatum slices from these mice. Single-pulse stimulation produced a transient increase in fluorescence that was completely blocked by a combination of N- and P/Q-type calcium channel blockers. DA and cholinergic, but not serotoninergic, signaling pathways modulated the PreCaTs in mDA fibers. These findings reveal heretofore unexplored dynamic modulation of presynaptic calcium in nigrostriatal terminals.


Introduction
The striatum has the richest DA innervation in the CNS, provided by the widely arborized axons of mDA neurons [1].Dopamine regulates neuronal function at both pre-and postsynaptic loci [2], and disruption of nigrostriatal DA signaling contributes to basal ganglia circuit dysfunction and psychomotor disorders, including Parkinson's disease [3,4].Although calcium (Ca 2+ ) is required for dopamine release [5], little is known about mechanisms and modulation of presynaptic Ca 2+ dynamics in striatal DA terminals.
The ability to measure Ca 2+ in DA terminals has been hampered by the small dimensions of DA axons and terminals, the massive distribution of DA axonal ramifications in the striatum [1], and difficulties associated with loading Ca 2+ indicators selectively into DA axons/terminals while avoiding nearby cellular elements.Genetically Encoded Calcium Indicator (GECIs) based on chimeric fluorescent proteins allow investigators to avoid many limitations of conventional small-molecule Ca 2+ dyes [6].They can easily be targeted for expression in specific cell types, avoiding indiscriminate loading.Genetic expression also avoids dye injection that can damage tissues [6].In particular, GCaMP3 has been used to examine neuronal Ca 2+ transients in brain slices and in vivo, following infection with adeno-associated virus or transgenic expression [7].However, the utility of GCaMPs for measuring presynaptic Ca 2+ has not been widely assessed [8,9].
Recently, expression of transgenic GCaMP3 and Ca 2+ signaling in mice was characterized in various neuronal subpopulations using expression under the control of the Thy1 promoter, with a focus on postsynaptic calcium transients [10].The sensor detected action potential bursts with good response linearity and photostability.Owing to these features, we selected GCaMP3 for the generation of a new line of transgenic mice, which express GCaMP3 in mDA neurons using a binary tetracycline-dependent inducible gene expression system [11,12].The expression of GCaMP3 selectively in the axons and axon terminals of mDA neurons in the striatum allowed us to systematically examine the dynamics of presynaptic Ca 2+ transients in mDA neurons under various conditions.Our findings reveal the role of specific voltagegated Ca 2+ channels in Ca 2+ entry into presynaptic elements of these neurons, and potent and selective modulation of presynaptic Ca 2+ by dopaminergic and cholinergic receptor signaling.

Generation of Transgenic Mice
To develop a conditional GCaMP3 transgenic mouse model, the cDNA fragment encoding GCaMP3 (Addgene Plasmid 26974) [13] was inserted into the mouse prion protein (pPrP)-tetO gene expression vector (a gift from Dr. David Borchelt, University of Florida, Gainesville, FL), which is controlled by the tetracyclineresponsive promoter (tetP) [14].The tetO-GCaMP3 expression construct was then purified and microinjected into fertilized oocytes derived from C57BL/6J mice.The founder mice were crossed with wild-type C57BL/6J mice to produce the F1 generation.
PITX3/IRES2-tTA mice were obtained as previously reported [12].PITX3 is predominantly expressed in mDA neurons, where it is involved in developmental processes, cell-specific gene expression and regulation of dopaminergic neurons [15].PITX3/IRES2-tTA mice with 95% C57BL/6J strain background were crossbred with tetO-GCaMP3 transgenic mice in C57BL/6J strain background.Mice were housed in a 12-h light/dark cycle and fed regular diet ad libitum.Generation of D1-CRE/GFP mice was previously described [16].

Behavior Tests
Accelerating rotarod test.As described previously [12,17], mice were placed onto a rotating rod with auto-acceleration from 0 to 40 rpm for 1 min (San Diego Instruments, San Diego, CA).The length of time the mouse stayed on the rotating rod was recorded, across 10 trials.
Open-field test.As described previously [17], the ambulatory and rearing activities of mice were measured by the Flex-Field activity system (San Diego Instruments, San Diego, CA).Flex-Field software was used to trace and quantify mouse movement in the unit as the number of beam breaks per 25 min.All behavioral results were compared using two-way ANOVA.

Photometric Recording
Hemisections were transferred to a recording chamber and constantly superfused with aCSF at 29-31uC at a rate of 1.5 ml/ min using a peristaltic pump.A bipolar concentric electrode was positioned in the corpus callosum.Calcium transients were recorded in the dorsolateral stratum using a 40x/0.8N.A. water-immersion objective on a Zeiss microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).Fluorescence in regions of interest (ROI: 180 mm 6180 mm in a 250-mm thick slice) was excited using light emitted by a mercury burner (Zeiss FluoArc Variable Intensity Lamp Control for HBO 100, Carl Zeiss Microscopy GmbH, Jena, Germany) and attenuated to 35% brightness.A shutter (model V25; Uniblitz, Vincent Associates, Rochester, NY), with exposure controlled by a driver under TTL control (model D122, Uniblitz), was used to reduce exposure time and photobleaching.Ca 2+ transients were evoked from populations of axon fibers within the ROIs considered, by rectangular, electrical pulse stimulation (120 mA, 10 ms, monophasic, unless otherwise noted) using an isolated constant current stimulator (DS3/GG2A system Digitimer Ltd, Hertfordshire, UK).Excitation exposures of 5-sec duration were recorded every 30 sec.Slices were allowed to sit in the recording chamber with afferent stimulation for 20 min before recording session began.The light emitted from the ROI was filtered at 535 nm and sent to a photomultiplier tube (PMT, model C6271; Hamamatsu Photonic Systems, Bridgewater, NJ).The PMT voltage output (time constant: 5 ms; gain: 400 6 10-1 mA/V) was fed into a computer interface (Digidata 1322A, Axon Instruments, Molecular Devices LLC, Sunnyvale, CA).Data were sampled at 100 Hz, and stored on a PC hard drive using PCLAMP 9.2 software (Axon Instruments, Molecular Devices LLC, Sunnyvale, CA).Analysis of transients was performed offline using cursor-based measurements in Clampex.Five sweeps evoked over each 5-min period were averaged, and corrected for gradual reductions in background fluorescence (most likely due to photobleaching and/or loss of signal from a small subpopulation of axons), using linear regression calculated from fluorescence measures with the stimulus-induced transient period removed (see Figure S1 in File S1).A 2-sec period was selected from the original averaged sweep and the peak of the time period containing the stimulus-induced transient was deleted and linear regression was performed on the remaining data points.A linear function was calculated and values at each point on this line were estimated.Each point in the original raw exported data was divided by the fit-generated estimated value at the same time point to obtain the normalized baseline value across the 2-sec period originally exported.Transients were measured as the ratio of the peak amplitude of the transient (DF) to the averaged baseline value (F) measured before the stimulus.
For pharmacological experiments, fluorescent transients were expressed as a percentage of the average of the first 10 min predrug control baseline, and compared to non-treated conditions, to account for the slight time-dependent decrease in the amplitude of the transient in the absence of treatment.Two-way ANOVA and MANOVA were used for statistical comparisons between different conditions.For better representation of the data, Bonferroni's Post Hoc tests showed in Figures were applied to data collected during drug treatments, with the only exception being experiments with limited-duration exposure to Ca 2+ -free aCSF and experiments involving scopolamine treatment.Pearson r was used to calculate correlations between time constants and amplitude of the fluorescent transients.

Fast-Scan Cyclic Voltammetry
Cylindrical carbon-fiber microelectrodes (50-100 mm of exposed fiber) were prepared with T650 fibers (6 mm diameter; Goodfellow, Coraopolis, PA) and inserted into a glass pipette (Cahill et al., 1996).The carbon-fiber electrode was held at 2 0.4 V, and the potential was increased to 1.2 V and back at 400 V/s every 100 ms using a triangle waveform.Dopamine release was evoked by rectangular, electrical pulse stimulation (120 mA, 10 ms, monophasic, unless otherwise noted) applied every 3 min.Data collection and analysis were performed using the Demon Voltammetry and Analysis software suite [19].Ten cyclic voltammograms of charging currents were recorded as background before stimulation, and the average of these responses was subtracted from data collected during and after stimulation.The maximum amplitudes of extracellular DA transients were obtained from input/output (I/O) curves.I/O curves were constructed by plotting stimulus current versus concentration of DA response amplitude over a range of stimulus intensities.Twoway ANOVA were used for statistical comparisons between groups.

Extracellular Field Potential Recording
Extracellular field recordings were acquired from 250 mm-thick coronal brain slices using glass recording electrodes filled with 0.9% NaCl solution (wt/vol).Population spikes ranging in amplitude from 0.6 to 1.4 mV were elicited at 0.033 Hz in the DLS by electrical stimulation (0.8-1.0 mA, 40 ms) from a concentric bipolar electrode placed just ventral to the overlaying white matter of the external capsule.Recordings were filtered at 1 kHz and digitized at 6.67 kHz using Clampex 9.2.

Simultaneous Photometric and Voltammetric or Field Potential Recordings
For FSCV/photometry and extracellular field potential/photometry simultaneous recordings, two systems were synchronized to the same electrically evoked event for recordings.In the FSCV/ photometry double recording, the intensity of the light was lower than the standard protocol (from 35% to 20%), to minimize the interference described in Figure S1 in File S1.To synchronize the two independent systems, Demon software was used to start the cyclic scanning together with a TTL signal for triggering the Clampex recording protocol.Subsequently, Clampex controlled both shutter status and electrical stimulation delivered through the isolated stimulator unit.For extracellular field potential/photometry simultaneous recordings, note that the frequencies of acquisition rate was increased from 100 Hz to 6.67 kHz, to be able to record the faster field potential events.

Immunohistochemistry
For basal localization of tyrosine hydroxylase (TH) and GFP expression, mice were perfusion fixed with 4% formaldehyde (PFA) and 40-mm coronal and sagittal sections were prepared as described previously [20].Slices were fixed overnight with 4% formaldehyde in PBS, beginning at room temperature for at least 30 min and then sections were transferred to 4-uC.After washing overnight in PBS plus 0.2% triton X-100 (PBST), slices were rinsed in deionized water (dH 2 O), then incubated twice for 10 min in freshly prepared sodium borohydride (5 mg/m: in dH 2 O).After rinsing again with PBST, the slices were incubated for at least 4 h in 5% BSA in PBST.Antibodies specific to green fluorescent protein (GFP; 1:2000; Sigma-Aldrich) and, Tyrosine Hydroxylase (TH; 1:2000, Dynal Biotech) were used for incubation for 48 h at 4uC.After three rinses in PBS, tissue was incubated with 488-or 568-conjugated secondary antibodies (1:500; Invitrogen) for 24 h.Fluorescence images were captured using a stereoscope (SteREO discovery, Zeiss, Thornwood, NJ) and a laser scanning confocal microscope (LSM 510; Zeiss, Thornwood, NJ).The stereoscopic images were collected using light emitted by a mercury burner filtered at 535 nm set at 12x magnification, using the same gain, focus from the surface and offset settings for each slice using AxioVision software (Rel.4.8., Zeiss, Thornwood, NJ).The confocal images were collected using LSM imager or Leica LCS software, as single optic layer with a 100x/1.4NA oil immersion objective.Excitation filter of 480/35 and emission filter of 535/30 were used for green fluorescence, while excitation filter of 540/25 and emission filter of 605/55 were used for red fluorescence.Postcollection processing was applied uniformly to all paired images using ImageJ (http://imagej.nih.gov).

Generation and Characterization of GCaMP3 Inducible Transgenic Mice
We generated PITX3-IRES2-tTA/tetO-GCaMP3 (PITX3/ GC) inducible transgenic mice by crossbreeding PITX3-IRES2-tTA (PITX3/2) heterozygous knock-in mice expressing the tetracycline transactivator (tTA) under the control of mouse endogenous PITX3 promoter [12], with tetO-GCaMP3 (2/GC) transgenic mice that express GCaMP3 under the regulation of the tetracycline operator (tetO) (Fig. 1A).GCaMP3 expression in mDA neurons of PITX3/GC transgenic mice was visualized by co-immunostaining with specific antibodies against GFP and tyrosine hydroxylase (TH).The GFP signals appeared in the cytosol of soma and processes, in both substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) mDA neurons, as well as in dorsal striatal DA axons and terminals (Fig. 1B).Additional GFP immunoreactivity was detected in non-mDA neurons located in the posterior cerebral cortex, cerebellum, hippocampus, and other brain regions (Fig. 1C).The same expression pattern of GCaMP3 in non-mDA neurons was also observed in the brain of 2/GC single transgenic mice, suggesting that tetO alone allows a tTA-independent ''leaky'' expression of GCaMP3 in non-mDA neurons, a phenomenon found previously using the same tetO expression vector [12].Importantly, little or no GFP immunoreactivity was detected in striatum in the 2/GC mouse slices (Fig. 1D).In contrast, strongly enhanced GCaMP3 expression was observed only in the somata and neurites of mDA neurons in PITX3/GC double transgenic mice (Fig. 1C, D).Thus, in the midbrain and striatum of this double transgenic line, the large majority of GCaMP3 expression is driven by the PITX-tTA driver in mDA neurons and axonal projections.

Serotonin Signaling Does Not Affect the PreCaTs in the mDA Axons and Terminals
Increasing endogenous serotonin (5-HT) levels using the selective serotonin reuptake inhibitor (SSRI) citalopram (10 mM), or directly activating 5-HT1B receptors using the agonist CP-93,129 (2 mM) did not affect PreCaTs in mDA inputs to striatum (Fig. 5G).Thus, our experiments did not reveal evidence of any direct serotonergic modulation of presynaptic Ca 2+ that would contribute to inhibition of DA release by this monoamine neuromodulator.

Simultaneous Paired Recordings of PreCaTs with [DA] Release and Extracellular Field Potentials
To determine if PreCaTs were evoked by stimuli that produce single presynaptic action potentials, we also performed simultaneous photometry and extracellular field potential recordings.In these experiments, we used shorter stimulation pulses (40 ms) that reliably evoked single fiber volleys (N1 field potential component, non-synaptically driven potentials in afferent fibers generated by stimulation), and synaptically driven population spikes (N2 field potential component), as previously observed [24] (Fig. 6A).The same stimulation simultaneously evoked PreCaTs with duration similar to that observed with the longer-duration stimuli (Fig. 6B,  C, D).Application of a cocktail of ionotropic glutamate receptor antagonists (NBQX, 10 mM, APV, 50 mM) and a GABA A receptor antagonist (picrotoxin, 50 mM) eliminated the N2, but not N1 field potential component within ,10 min, as expected (Fig. 6A), but produced only a marginal decrease in PreCaT amplitude (Figure 6B).We took advantage of our ability to simultaneously record the N1/fiber volley and PreCaT to compare the time course of presynaptic Ca 2+ increases in relation to single afferent action potentials generated in a population of fibers.Figure 6C shows that the PreCaT onset occurs with a very short latency after the peak of the fiber volley/N1 component of the field potential, as one would expect for a response that reflects Ca2+ entry directly driven by presynaptic afferent activation.The increase in presynaptic Ca2+ occurs in close temporal relationship to N1, and before the expected onset of synaptic responses, as observed in past photometric recordings of PreCaTs [25].

Discussion
Recent improvements in GECI kinetic properties and sensitivity have resulted in reporters that allow for ever-improving monitoring of activity-driven Ca 2+ changes [26].Investigators can now begin to use these tools to study the activation of DA terminals but also to probe pathological changes in models of neurological disorders.While GCaMP3 lacks the sensitivity of more recently developed GECIs [26], our findings indicate that it is clearly usable for detection of Ca 2+ transients from populations of densely packed presynaptic elements even under stimulation conditions where only one action potential is elicited.This approach allowed us to investigate PreCaTs using real-time measurements in DA axons/terminals within striatal slices, which would be very difficult using Ca 2+ -sensitive dye-based techniques.The characteristics of these Ca 2+ transients corresponded with known mechanisms of DA release.While the photometric method does not allow for measurement of Ca 2+ specifically within the active zones of DA terminals, the strong relationship between factors that underlie and modulate the PreCaTs and DA release suggests that we can learn a great deal about presynaptic functions related to DA dynamics using this approach.Our reported effects of ionic manipulation and toxins strongly support the conclusion that the stimulus-induced increases in fluorescence measured in striatal slices arise from presynaptic action potential-dependent Ca 2+ influx via mechanisms implicated in DA release.
While we report kinetic parameters of the PreCaTs, we are aware that such parameters do not always faithfully report the kinetics of intracellular free Ca 2+ concentration changes that occur naturally in the absence of a Ca 2+ indicator.The onset and duration of Ca 2+ transients are affected not only by the effective Ca 2+ flow in and out of terminals, but they also reflect GCaMP binding and kinetic properties.GCaMP3 in particular showed slower kinetics compared to the most recent generation GCaMP constructs [26], but still within the range of decay times of PreCaTs observed with the most common Ca 2+ indicators [27].In the absence of robust data on the true kinetics of PreCaTs in dopaminergic neurons, the time constants measured in our PITX3/GC mice might be considered as a limited estimation of rise and clearance of Ca 2+ within the terminals.It is reassuring that DA release was not altered by the presynaptic expression of GCaMP3, as this finding indicates that the GECI is not buffering presynaptic Ca 2+ at levels/location that would disrupt sensitive excitation/secretion coupling mechanisms.The observation that Ca 2+ kinetics were not greatly altered at different stimulus durations or intensities indicates that GCaMP3 is reporting a relatively uniform process of increased presynaptic Ca 2+ even when the number of afferents stimulated is greatly increased.An important feature of DA transmission in striatum is presynaptic autoinhibition, in which dopamine modulates its own release and synthesis through presynaptic D2AR located at DA axon terminals in the striatum [28,29].Activation of D2AR or other G i/o subtype G-protein-coupled receptors (GPCRs) may reduce neurotransmitter release through inhibition of presynaptic Ca 2+ entry, a common mechanism underlying presynaptic GPCR effects [30,31].Data from simultaneous dual FSCV/photometry recordings presented in this study indicate different potencies of D2AR agonists for inhibition of PreCaTs and DA release.One explanation for this finding is that inhibition of release could involve multiple signaling pathways, some of which may require lower receptor occupancy or more post-receptor amplification than inhibition of Ca 2+ channels.Alternatively, given the strong cooperativity in the relationship between presynaptic Ca 2+ levels and vesicle fusion [32], small decreases in Ca 2+ transients may have large effects on DA release.
Interactions between striatal ACh and dopamine influence many aspects of striatal function and related behaviors [33].ACh stimulates DA release through nicotinic acetylcholine receptors (nAChRs) on DA axons [34], mainly via b2-subunit-containing receptors [35,36].Activation of cholinergic striatal interneurons directly stimulates DA release [37].Meanwhile, muscarinic acetylcholine receptors (mAChRs) modulate DA release in a number of ways [38,39,40].Our work shows a direct influence of AChRs on presynaptic Ca 2+ in mDA neurons.Single-stimulusevoked presynaptic Ca 2+ rises in mDA neurons are suppressed when b2-nAChR on DA axons are directly antagonized.This finding is consistent with the known role of these receptors in DA release in striatal slices [37,38,41].Activation of mACh autoreceptors decreased PreCaTs in the dopaminergic afferents, and blockade of mACh appeared to relieve endogenous cholinergic tone that inhibits PreCaTs.These actions are good candidate mechanisms for the known AChR effects on dopamine release.
In brain areas containing dopaminergic terminals, various serotonin (5-HT) receptors have been shown to modulate dopamine release [42].In striatum, dopamine release is reduced by presynaptic 5-HT1B heteroreceptors [43].We did not observe any effect of 5-HT on PreCaTs in dopaminergic neurons.It is possible that these receptors have indirect effects that decrease DA release, perhaps involving disinhibition of GABAergic interneurons, as described in the dorsal striatum of 5-HT1B knockout mice using in vivo microdialysis [44].
Finally, we were also able to make simultaneous field potential and PreCaT recordings with an appreciable noise to signal ratio.From these experiments we can clearly see that PreCaTs are driven by, and temporally associated with, single action potentials driven in populations of afferent fibers.
In conclusion, combining GCaMP expression with photometry in brain slices allowed us to examine the molecular basis and modulation of PreCaTs in mDA neurons, and should make it possible to measure these presynaptic responses in a variety of afferent inputs made by projection neurons.Aberrant striatal dopaminergic transmission contributes to various neurological disorders.Although pathological changes and motor dysfunction that characterize diseases like Parkinson's disease are well documented, the mechanism(s) responsible for dysfunction of DA neurons and terminals have yet to be clearly established.We can use the present approach to study the involvement of Ca 2+ signaling simultaneously with other conventional striatal physiology techniques and during the progression of severe neuropathology of the DA system.Transients in double PITX3-IRES2-tTA/tetO-GCaMP3 (PITX3/GC) and single tetO-GCaMP3 (2/GC) transgenic mice were increased by the potassium channel inhibitor 4-AP and decreased by inhibition of the firing of action potentials with TTX.Note that PITX3-IRES2-tTA (PITX3/2) mouse slices expressed a small, drug-insensitive transient similar to the transients observed in slices from the other two lines after TTX application.This step-like response is most likely mainly a consequence of stimulation-induced changes in autofluorescence easily detected against a low fluorescence background (D).In fact, no such transient was detectable in slices from D1-cre/GFP transgenic mice, presumably due to their relatively high background fluorescence (as reported in Fig. 3B).(E) Comparison of different inter-stimulus interval (ISI) effects on stability of PreCaTs.Stimulation with 5-min and 3-min ISIs revealed a minor (7-15%) decrease in current amplitude that overlapped with that observed during to our standard 30-sec ISI condition (10-20% decrease).Thus, the time-dependent loss of signal is probably due to a combination of both GFP-photobleaching and loss of signal from some afferents.As such, we routinely used the 30-sec ISI to obtain more time points for averaging traces that reduced interference from noise in the PreCaT calculations.Note that we included no-treatment conditions for experiments with drug exposure and prolonged recordings (e.g.Fig. 4 and 5) to control for the time-dependent loss of signal.(PDF)

Supporting Information
All mouse work follows the guidelines approved by the Institutional Animal Care and Use Committees of the National Institute of Child Health and Human Development, US National Institutes of Health.The research was approved by the Animal Care and Use Committee of the Division of Intramural Clinical and Biological Research, National Institute on Alcohol Abuse and Alcoholism, NIH.

Figure 1 .
Figure 1.Generation of GCaMP3 conditional transgenic mice.(A) Schematic depicts the generation of PITX3-IRES2-tTA/tetO-GCaMP3 (PITX3/GC) double-transgenic mice.(B) Sample images of immunohistochemistry for tyrosine hydroxylase (TH) and the GFP moiety of GCaMP show GCaMP3 distribution in the dorsolateral striatum and SNpc of 3month-old PITX3/GC mice.Scale bars: 10 mm.(C) Sagittal and (D) coronal sections showing TH (left, merged in red on the right) and GFP

Figure 3 .
Figure 3. Ex vivo photometric measurement of Ca 2+ transients in dopaminergic axons.(A) Cartoon illustrating the photometry setup (PMT: photomultiplier tube, FITC: Fluorescein isothiocyanate filter, PC: personal computer, ROI: region of interest).(B) I/O curves showing the peak amplitude of the fluorescence transient as a function of stimulus intensity for striatum in the four mouse lines.Representative traces are shown on the right.(black arrowhead: 120 mA, 10 ms, monophasic).(C) Comparison of I/O curves showing peak fluorescence transient amplitude as a function of stimulus intensity in different areas of coronal brain slices in PITX3-IRES2-tTA/tetO-GCaMP3 (PITX3/GC) mice.Dorsolateral striatum PreCaTs exhibited higher amplitudes compared with ventral striatum (Nucleus Accumbens, shell), with no detectable transients observed in the cortex (V layer of motor cortex).(D) tetO-GCaMP3 (2/GC) single transgenic mice showed minimal fluorescence changes after electrical stimulation in the striatum, about 10% compared to double transgenic mice at the highest stimulus intensities.(E) Scatterplots showing the time constant (t) of the fluorescence transient rise and decay times in relation to peak amplitude of the PreCaT (normalized to maximum amplitude) for several individual dorsolateral striatum recordings.No significant correlations were observed.doi:10.1371/journal.pone.0111749.g003

Figure 6 .
Figure 6.Simultaneous PreCaT and extracellular field potential recordings.(A) Representative field potential recordings and simultaneously measured PreCaT traces (B) induced by electrical stimulation (white arrowhead: 900 mA, 40 ms, monophasic).Effects of application of a cocktail of antagonists of ionotropic glutamate and GABA receptors ((2R)-amino-5-phosphonovaleric acid (APV, 50 mM); 2,3-dihydroxy-6-nitro-7sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBXQ, 10 mM)) are also shown.Perfusion of the drug cocktail for 10 min eliminated the synaptically driven postsynaptic component (N2) of the field potential, leaving intact the directly electrical stimulation-driven fiber volley component (N1).A marginal decrease in the PreCaT was observed following application of the antagonist cocktail.Subsequent application of the nAChR competitive antagonist DHbE (1 mM) produced strong inhibition of the PreCaT.(C) Traces from simultaneous record of the N1/fiber volley and PreCaT.The time course of presynaptic Ca2+ increases in relation to single afferent action potentials generated in a population of fibers.(D) PreCaTs time constant of slopes calculated from two different stimulation conditions (FSCV vs field potential double recording sessions).No significant difference was observed.doi:10.1371/journal.pone.0111749.g006

File
S1 Contains the following files: Figure S1.Description of raw data and mathematical correction for sampling epochs.Related to Data Analysis Procedures (A) Raw data traces showing the total fluorescence (F) and stimulus-induced change in fluorescence (DF) during several trials in a single slice from a PITX3-IRES2-tTA/tetO-GCaMP3 (PITX3/GC) mouse.Box indicates segment of the traces used for linear regression and baseline normalization in the entire trace (left) and with expanded axes (right).Each sweep shows a 6-sec window in which the shutter was open (excitation light exposure) for 5 sec.Two sec after the opening of the shutter, electrical stimulation was delivered, with a consequent increase of the fluorescence recorded by the PMT.(B) The boxed portion of averaged sweeps (2 sec total) was exported and analyzed.For baseline normalization, the peak of the time period containing the stimulus-induced transient was deleted and linear regression was performed on the remaining data points.A linear function was calculated and values at each point on this line were estimated.Each point in the original raw exported data was divided by the fit-generated estimated value at the same time point to obtain the normalized baseline value across the entire 2 sec period.(C) The final result of this baseline normalization is shown for the sweeps in (A) and (B).(D) UV light interfered with basal current recorded by FSCV electrode, producing an increase in current proportional to the intensity of the light used.(E) Representative DA traces (with cyclic voltammograms in inset) and color plots showing responses to single-pulse stimulation (black arrowhead: 120 mA, 10 ms, monophasic) with exposure to light from the mercury burner (20% intensity).Note that the lightinduced current is strongest at electrode potentials more positive than the voltages associated with DA oxidation.In the same fashion described above for correction of photometric correction traces shown in panel B, we corrected for the light-induced change voltammetric current in the boxed portion of the DA transient.(F) shows the signals before and after linear regression calculation and subtraction, and compared with a light OFF trace sample.Note the lack of effect of light exposure or the correction procedure on the DA transient.