Dataset DOI: 10.5061/dryad.44j0zpcv7

Description of the data and file structure

We utilized multidisciplinary approach in transgenic rats to demonstrate that midbrain GABA neurons exhibit altered ionic homeostasis through functional downregulation of the potassium chloride cotransporter, KCC2, within fine temporal windows during reward learning. Specifically using rat Pavlovian learning tests, slice patch clamp recordings, and immunostaining, we show that during the acquisition phase of learning, KCC2 undergoes transient downregulation, manifesting as altered inhibitory synaptic transmission in VTA GABA neurons (Figures 1 and 2). Using a variety of physiological methods, we show that KCC2 alterations were associated with increased firing synchrony within midbrain GABA neuronal networks and enhanced phasic bursting in midbrain dopamine neurons (Figures 4-6). Most importantly, pharmacological or genetic manipulation of KCC2 function in the midbrain during acquisition of learning altered the formation of cue-reward associations (Figure 3).

Files and variables

File: 'Figure_1C.csv'

Description: Behavioral data of animals undergoing paired and unpaired pavlovian protocols. Paired protocols entail a 5 second cue followed by reward delivery. Unpaired protocols have randomized cue and reward deliveries, but never contiguous presentations. Rate difference values are the number of port entries during cues minus the number of port entries in the 5 seconds preceding cue start over time.

Variables:

• Rows list the rate difference at a given day.
• Each column has the animal ID, and corresponding condition (paired vs. unpaired).

File: 'Figure_1G_1H.csv'

Description: Gramicidin perforated patch clamp technique was used to examine the reversal potential of GABA. The reversal value was calculated from an I-V curve where the voltage of the eIPSCs amplitude was zero.

Variables:

• Group: Experimental Condition (Paired or Unpaired)
• Day: Learning phase (1, 7, or 13)
• Animal ID: Identifier of each animal
• Cell ID: identifier for each recorded cell within animal
• Reversal: Reversal value of EGABA (Vm)
• Membrane potential (Vm)
• Animal Count: Count of animals per group

File: 'Figure_1J.csv'

Description: Western blot quantification of phosphorylated and total KCC2 protein expression (normalized to GAPDH) across timepoints (Day 1 and Day 7).

Variables: Each row corresponds to a single animal, with ratio values for phosphorylated (pKCC2/ GAPDH) over total KCC2 (Total KCC2/ GAPDH), and the percent change of Day 7 values over Day 1 values.

• Column A: Animal ID: identifier for each animal
• Column B: (pKCC2/GAPDH) / (Total/GAPDH) *100 of Dimer per animal
• Column C: (pKCC2/GAPDH) / (Total/GAPDH) *100 of Monomer per animal
• Column E&G: Ratio of Day 7/ Day 1 animals of Column B.
• Column F&H: Ratio of Day 7/ Day 1 animals of Column C.

File: 'Figure_1L.csv'

Description: Western blot quantification of phosphorylated and total KCC2 protein expression (normalized to GAPDH) across timepoints (Day 1 and Day 13).

Variables:

• Column A: Animal ID: identifier for each animal
• Column B: (pKCC2/GAPDH) / (Total/GAPDH) *100 of Dimer per animal
• Column C: (pKCC2/GAPDH) / (Total/GAPDH) *100 of Monomer per animal
• Column E&G: Ratio of Day 13/ Day 1 animals of Column B.
• Column F&H: Ratio of Day 13/ Day 1 animals of Column C.

File: 'Figure_2C.jpg'

Description: Full uncropped immunostaining of lateral shell projecting DA neurons (TH, red; eGFP, green).

File: 'Figure_2E.csv'

Description: This dataset summarizes inter event interval (IEI) frequencies < 10 ms across learning phases (Day 1, 7, 13). Each cell is a recording from a specific animal, quantified for short-latency (synchronous) events.

Variables:

• Cell ID: Identifier for each recorded neuron (including animal ID and cell number)
• Heading (ie. Day 1): Learning phase or treatment
• Numerical variables: % of events that had IEI that were < 10 ms.
• Left, Middle, Right: corresponds to panel placement in the figure.

File: 'Figure_2F.csv'

Description: This dataset looks at the sIPSC event frequency measurements following diazepam across different learning phases (Day 1, 7, 13) in lateral shell projecting DA neurons. Each entry represents an individual recorded cell.

Variables:

• Cell ID: Identifier for each recorded neuron (including animal ID and cell number)
• Heading (ie. Day 1): Learning phase or treatment
• Numerical variables: % change in sIPSC frequency after diazepam application.
• Left, Right: corresponds to panel placement in the figure.

File: 'Figure_2G.jpg'

Description: Full uncropped immunostaining of Medial Shell projecting DA neurons (TH, red; eGFP, green).

File: 'Figure_2H.csv'

Description: This dataset summarizes inter event interval (IEI) frequencies < 10 ms across learning phases (Days 1, 7, 13). Each cell is a recording from a specific animal, quantified for short-latency (synchronous) events.

Variables:

• Cell ID: Identifier for each recorded neuron (including animal ID and cell number)
• Heading (ie. Day 1): Learning phase
• Numerical variables: % of events that had IEI that were < 10 ms.

File: 'Figure_2I.csv'

Description: This dataset looks at the sIPSC event frequency measurements following diazepam across different learning phases (Days 1, 7, 13) in medial shell projecting DA neurons. Each entry represents an individual recorded cell.

Variables:

• Cell ID: Identifier for each recorded neuron (including animal ID and cell number)
• Heading (ie. Day 1): Learning phase
• Numerical variables: % change in sIPSC frequency after diazepam application.

File: 'Figure_2J.jpg'

• Description: Full uncropped immunostaining of Core projecting DA neurons (TH, red; eGFP, green).

File: 'Figure_2K.csv'

Description: This dataset summarizes inter event interval (IEI) frequencies < 10 ms across learning phases (Day 1, 7, 13). Each cell is a recording from a specific animal, quantified for short-latency (synchronous) events.

Variables:

• Cell ID: Identifier for each recorded neuron (including animal ID and cell number)
• Heading (ie. “Day 1”): Learning phase
• Numerical variables: % of events that had IEI that were < 10 ms.

File: 'Figure_2L.csv'

Description: This dataset looks at the sIPSC event frequency measurements following diazepam across different learning phases (Day 1, 7, 13) in core projecting DA neurons. Each entry represents an individual recorded cell.

Variables:

• Cell ID: Identifier for each recorded neuron (including animal ID and cell number)
• Heading (ie. “Day 1”): Learning phase
• Numerical variables: % change in sIPSC frequency after diazepam application.

File: 'Figure_3B.csv'

Description: Behavioral data of animals undergoing pavlovian protocols with and without optogenetic stimulation of VTA GABA neurons. Animals were administered green light throughout CS and US presentations.

Rate difference values are the number of port entries during cues minus the number of port entries in the 5 seconds preceding cue start over time.

Variables:

• Rows lists the rate difference at a given day.
• Each column has the animal ID, and corresponding condition (Arch vs. Control). “Arch” corresponds to Archaerhodopsin injected animals.

File: 'Figure_3C.csv'

Description: The number days each animal spent below the acquisition midpoint. The rate difference at the acquisition midpoint (Criterion, dashed line from Figure 3B) was half that at plateau.

Variables:

• Animal IDs
• Number of days with rate differences below the value calculated for the middle of acquisition (Criterion).
• Condition (Control vs. Arch).

File: 'Figure_3E.csv'

Description: This dataset summarizes behavioral performance (rate difference) across Arch-expressing and GFP control animals. Light was administered during CS and US presentations the day after mid-acquisition was reached.

Variables:

• Day: Days from mid-acquisition
• Animal IDs
• Condition: Arch vs. GFP (Controls)
• Rate Differences

File: 'Figure_3G.csv'

Description: Rate differences were measured across experimental days under two treatment conditions: Vehicle and CLP 290 (60 micromolar) administered every other day. Each entry is an individual’s rate difference.

Variables:

• Condition: CLP290 vs. Vehicle (Controls)
• Animal IDs

File: 'Figure_3H.csv'

Description: The number days each animal was below the acquisition midpoint. The rate difference at the acquisition midpoint (Criterion, dashed line from Figure 3B) was half that at plateau.

Variables:

• Animal IDs
• Number of days with rate differences below the value calculated for the middle of acquisition (Criterion).
• Condition (CLP290 vs. Vehicle).

File: 'Figure_3J.csv'

Description: Animals were injected with CLP290 or vehicle every other day after they reached the mid-acquisition point (Criterion, in dashed lines). This dataset summarizes rate differences in normalized to the rate difference values at mid-acquisition.

Variables:

• Animal IDs
• Condition (CLP290 vs. Vehicle)
• Day (0-4 days from acquisition mid-point)

File: 'Figure_3M.csv'

Description: Rate differences were measured across experimental days under two treatment conditions: Scrambled shRNA and KCC2 shRNA. Each entry is an individual’s rate difference.

Variables:

• Condition: Scrambled shRNA (Controls) and KCC2 shRNA
• Animal IDs

File: 'Figure_4D.csv'

Description: This dataset summarizes pairwise synchrony from in vivo electrophysiological recordings during Pavlovian conditioning. Each row represents one recording session (identified by animal ID, genotype, and recording number), showing the number of neuron pairs with significant synchrony before, during, and after the conditioned stimulus (CS).

Variables: Columns quantify the number and percentage of significantly synchronized neuron pairs across different task epochs (Pre-CS, CS, and Post-CS).

• Path: identifier for each recording session
• epoch significant: Number of neuron pairs with significant synchrony during each epoch
• Tot Pairs: Total number of GABA identified neurons in that session
• epoch % Synch (columns F-H): percentage of significantly synchronized pairs relative to total pairs

File: 'Figure_4F.csv'

Description: Pairwise synchrony amongst VTA GABA neurons following CLP290 or Vehicle treatment during Pavlovian conditioning sessions. Animals were administered with CLP290 or vehicle treatment after they reach the mid-point of acquisition. Each row corresponds to one in vivo recording session, showing the number and percentage of synchronized neuron pairs before, during, and after the conditioned stimulus (CS).

• Path: identifier for each recording session. These variables also indicate whether animals were CLP290 or Vehicle treated.
• epoch synch pairs: Number of neuron pairs with significant synchrony during each epoch
• Total Pairs: Total number of GABA identified neurons in that session
• epoch % Synch (columns F-H): percentage of significantly synchronized pairs relative to total pairs
• Row below % synch (ie. 4F-H): % Change of % significant pairs before vs. after CLP290 or Vehicle injections.

File: 'Figure_4H_I.csv'

Description: Pairwise synchrony amongst VTA GABA neurons before and after VU0463271 injections.

Variables:

• Groups: Before and after VU04632731 injections
• Column A: Animal IDs
• Column B: Averages of values in columns C-L.
• Columns C-L: % of neuron pairs significantly synchronized across increasing increments of 5 seconds.
• Mean: Mean % synchronized values per time bin per group
• SEM: Standard error of the mean per time bin per group
• Number: number of animals.

File: 'Figure_5B.csv'

Description: Burst firing activity of VTA dopamine neurons recorded in vivo during different phases of Pavlovian learning. It separately captures burst characteristics during cue-evoked (CS) and reward-evoked (US) periods across Baseline, Acquisition, and Plateau stages of training.

Variables:

• Columns A-C: Average cue-evoked spikes per burst per neuron at each stage of learning.
• Columns E-G: Average reward-evoked spikes per burst per neuron at each stage of learning.

File: 'Figure_5D.csv'

Description: Spikes per burst of VTA dopamine neurons recorded in vivo after vehicle or CLP290 injections during cue-evoked (CS) and reward-evoked (US) periods normalized to % change of averaged spikes per burst after mid-point of acquisition.

Variables:

• Columns A-B: Average % change in cue-evoked spikes per burst per neuron after vehicle or CLP290 injection.
• Columns E-G: Average % change in reward-evoked spikes per burst per neuron after vehicle or CLP290 injection.
• Extra note: “Nan” variable comes from a unit that was responsive to cues, but not to rewards.

File: 'Figure_5F.csv'

Description: Spikes per burst of VTA dopamine neurons recorded in vivo after optogenetic inhibition of VTA GABA neurons during cue-evoked (CS) and reward-evoked (US) periods normalized to averaged spikes per burst after mid-point of acquisition.

Variables:

• Columns A-B: Average % change in cue-evoked spikes per burst per neuron after light stimulation in GFP or Arch expressing animals.
• Columns D-E: Average % change in reward-evoked spikes per burst per neuron after light stimulation in GFP or Arch expressing animals.

File: 'Figure_5K.csv'

Description: Area under the curve (AUC) of z-scored cue and reward photometry responses after CLP290 or Vehicle injections, normalized to AUC values after mid-acquisition. AUC was defined as the integral between the first peak since stimulus presentation and its eventual decay back to 0.

Variables:

• Columns: Treatment (Vehicle or CLP290, after cue (CS) or reward (US))
• Row: % Change in AUC values

File: 'Figure_6D.csv'

Description: Burst analysis of DA neurons recorded ex vivo, comparing burst firing without optogenetic stimulation of VTA GABA neurons, and burst firing after 10 hz light stimulation of VTA GABA neurons.

Variables:

• Column A: Average number of spikes per burst in glutamate-evoked bursts in VTA DA neurons without optogenetic stimulation (10 hz) per cell (row).
• Column B: Average number of spikes per burst in glutamate-evoked bursts in VTA DA neurons with optogenetic stimulation (10 hz) per cell (row).

File: 'Figure_6H.csv'

Description: Burst analysis of DA neurons recorded in vivo, comparing burst firing without optogenetic stimulation of VTA GABA neurons, and burst firing after 10 hz light stimulation of VTA GABA neurons.

Variables:

• Column A: Average number of spikes per burst in glutamate-evoked bursts in VTA DA neurons without optogenetic stimulation (10 hz) per cell (row).
• Column B: Average number of spikes per burst in glutamate-evoked bursts in VTA DA neurons with optogenetic stimulation (10 hz) per cell (row).

File: 'Figure_S1A.csv'

Description: This dataset quantifies behavioral performance in male and female rats in the paired learning group.

Variables:

• Column: Males and Females
• Day: Day of pavlovian conditioning
• Rate difference: averaged rate difference values across all male or female animals across time.
• SD: standard deviation of rate difference values across all male or female animals across time.
• N: Number of animals per day

File: 'Figure_S1B.csv'

Description: This dataset summarizes variability in behavioral measures across learning phases, specifically comparing early learning (Days 5–7) vs. late learning (Days 11–13).

Variables:

• Header: early learning (Days 5–7) vs. late learning (Days 11–13).
• Rows: Average % change in rate difference per animal.

File: ‘Figure_S1C.jpg’

Description: Full, uncropped immunostaining for CaMPARI2 (green), BFP-labeled VTA GABA neurons (blue), and photoconverted CaMPARI2 (red) across learning. Each entry represents the number of cells showing red fluorescence (photoconverted) out of total BFP-positive cells (GABAergic) in a defined ROI. The proportion of converted cells

File: 'Figure_S1D.csv'

Description: This dataset quantifies CaMPARI2 photoconversion in VTA GABA neurons across learning days. Each entry represents the proportion of converted cells per animal and averaged across learning days. It also examines the number of CaMPARI expressing cells (green) across days.

Variables:

• Columns A: image ID
• Day: Day of learning for the sample
• Positive: number of cells that co-expressed red and blue fluorescence.
• Negative: number of cells that show blue fluorescence, but no red.
• Area of ROI: area of defined ROI
• Cell count: number of cells that express CaMPARI2 (green)
• “% of BFP converted to Red”: summarizes % of BFP expressing neurons converted to red across time
• “No. BFP”: summarizes the number of BFP expressing cells
• “CaMPARI green expressing cell count” summarizes the cell count of neurons expressing CaMPARI over time.

File: 'Figure_S1E.csv'

Description: This dataset compares the reversal potentials (EGABA) in VTA GABA neurons between male and female animals across learning

Variables:

• Header: Phase (Day 1, Day 7, Day 13)
• Rows: Sex
• Individual variables: reversal potential (Vm)

File: 'Figure_S1F.csv'

Description: Resting membrane potentials in VTA GABA neurons across learning

Variables:

• Header: phase of learning
• Rows: resting membrane potentials (Vm)

File: 'Figure_S1G.csv'

Description: Gramicidin perforated patch clamp technique was used to examine the reversal potential of non-CaMPARI2 activated GABA neurons. The reversal value was calculated from an I-V curve where the voltage of the eIPSCs amplitude was zero.

Variables:

• Header: “Reversal values” or “membrane potentials” on Day 7
• Rows: each entry is an individual reversal value or membrane potential of each non-CaMPARI converted GABA neuron

File: ‘Figure_S1J.jpg’

Description: Full, uncropped immunostaining for CaMPARI2 (green), BFP-labeled VTA GABA neurons (blue), and photoconverted CaMPARI2 (red) in paired or unpaired groups at acquisition.

File: 'Figure_S1I.csv'

Description: Gramicidin perforated patch clamp technique was used to examine the reversal potential of CaMPARI activated DA neurons. The reversal value was calculated from an I-V curve where the voltage of the eIPSCs amplitude was zero.

Variables:

• Group: Day 1 or Day 7
• Reversal: Reversal value of EGABA (Vm)
• Membrane potential (Vm)

File: 'Figure_S1K.csv'

Description: This dataset quantifies CaMPARI2 photoconversion in VTA GABA neurons on day 7 in paired and unpaired animals. Each entry represents the proportion of converted cells per animal and averaged per group. It also examines the number of CaMPARI expressing cells (green) across groups.

Variables:

• Columns A: image ID
• Condition Paired or unpaired
• Positive: number of cells that co-expressed red and blue fluorescence.
• Negative: number of cells that show blue fluorescence, but no red.
• Area of ROI: area of defined ROI

File: 'Figure_S1L.csv'

Description: This dataset reports densitometric quantification of phosphorylated KCC2 (pS940) expression for dimeric and monomeric bands, separated by sex. Values represent normalized band intensities from Figure S1L.

Variables:

• Header: males/females, dimer or monomer
• Numerical variables: entries represent normalized band intensities: (pS940 KCC2/ GAPDH) / (total KCC2/ GAPDH) * 100

File: 'Figure_S1M.csv'

Description: This dataset contains western blot densitometry data quantifying total KCC2 values/ GAPDH across day 1 vs. days 7 or 13. Each row represents a single biological sample (animal or lane), with raw intensity values extracted from individual blot images. The “Day 1 vs Day 7” or “Day 1 vs 13” ratio column provides normalized expression levels used for group-level statistical comparisons.

Variables:

• Header: Day of learning, Dimer or monomer
• Rows: each entry in columns A and B show ratios of total KCC2/ GAPDH per animal per day
• Column D: % of Day 7 or 13 to Day 1 values.
• Columns G-H: summary of ratios (Dimers and monomers comparing Day 7/Day 1) from column D for statistical analysis.
• Columns J-K: summary of ratios (Dimers and monomers comparing Day 13/Day 1) from column D for statistical analysis.

File: 'Figure_S1N.csv'

Description: This dataset contains western blot densitometry data quantifying NKCC1 expression normalized to GAPDH across VTA tissue samples. Each row represents a single biological sample (animal or lane), with raw intensity values extracted from individual blot images. The NKCC1/GAPDH ratio column provides normalized expression levels used for group-level statistical comparisons.

Variables:

• Animal ID: Unique identifier for each animal
• Header: Raw data source file (B-C are GAPDH, D-E are NKCC1)
• Rows: Entries are raw densitometry data
• Columns I-J: NKCC1/GAPDH ratios
• Under Headers “Day 1” and “Day 7”, summary of ratios for statistical analysis.

File: 'Figure_S2A.csv'

Description: This dataset looks at basal sIPSC frequency in lateral shell projecting DA neurons in slices across learning.

Variables:

• Heading (ie. “Baseline”): Learning phase
• Numerical variables: sIPSC event frequency (hz)

File: 'Figure_S2B.csv'

Description: This dataset examines rate differences (left) and corresponding proportion of inter-event-interval (IEI) frequencies < 10 ms recorded in slice preparations (right) across learning (Day 1, 3, 5).

Variables:

• Heading (ie. “Day 1”): Learning Day
• Numerical variables (left): Rate differences at a given day
• Numerical variables (right): % of events with IEI < 10 ms.

File: 'Figure_S2C.csv'

Description: This dataset looks at basal sIPSC frequency in lateral shell projecting DA neurons in slices during acquisition with and without 1 hour incubation of CLP290.

Variables:

• Heading (ie. “acquisition + CLP290”): treatment
• Numerical variables: sIPSC event frequency (hz)

File: 'Figure_S2D.csv'

Description: This dataset looks at sIPSC frequency in lateral shell projecting DA neurons in naïve slices before and after VU0463271 application.

Variables:

• Heading (ie. “basal”): treatment
• Numerical variables: sIPSC event frequency (hz)

File: 'Figure_S2E.csv'

Description: This dataset looks at percent change in sIPSC amplitudes in lateral shell projecting DA neurons after diazepam application across learning.

Variables:

• Heading (ie. “Day 1”): Learning phase
• Numerical variables: % change in amplitude after diazepam application

File: 'Figure_S2F.csv'

Description: This dataset looks at percent change in sIPSC amplitudes in lateral shell projecting DA neurons after diazepam application in slices from baseline or acquisition incubated for 1 hour with CLP290.

Variables:

• Heading (ie. “Baseline”): Learning phase
• Numerical variables: % change in amplitude after diazepam application

File: 'Figure_S2G.csv'

Description: This dataset looks at the sIPSC event frequency measurements across different learning phases (Day 1, 7, 13) in medial shell projecting DA neurons. Each entry represents an individual recorded cell.

Variables:

• Heading (ie. “Day 1”): Learning phase
• Numerical variables: sIPSC event frequency (hz)

File: 'Figure_S2H.csv'

Description: This dataset looks at the sIPSC event amplitude measurements following diazepam across different learning phases (Day 1, 7, 13) in medial shell projecting DA neurons. Each entry represents an individual recorded cell.

Variables:

• Heading (ie. “Day 1”): Learning phase
• Numerical variables: % change in sIPSC amplitude after diazepam application.

File: 'Figure_S2I.csv'

Description: This dataset looks at the sIPSC event frequency measurements across different learning phases (Day 1, 7, 13) in core projecting DA neurons. Each entry represents an individual recorded cell.

Variables:

• Heading (ie. “Day 1”): Learning phase
• Numerical variables: sIPSC event frequency (hz)

File: 'Figure_S2J.csv'

Description: This dataset looks at the sIPSC event amplitude measurements following diazepam across different learning phases (Day 1, 7, 13) in core projecting DA neurons. Each entry represents an individual recorded cell.

Variables:

• Heading (ie. “Day 1”): Learning phase
• Numerical variables: % change in sIPSC amplitude after diazepam application.

File: 'Figure_S3B.csv'

Description: Comparison of rate differences of two control conditions: eGFP + light or Arch without light.

Variables:

• Columns: animals corresponding to eGFP+ light stimulation or Arch-expressing + no light stimulation group
• Rows: individual rate differences at a given day

File: 'Figure_S3C.csv'

Description: This dataset examines sex differences in rate differences in control or opto-inhibition groups.

Variables:

• Columns: individual males or female animals corresponding to (male/female x inhibited/control)
• Rows: individual rate differences at a given day

File: 'Figure_S3H.csv'

Description: Animals were injected with CLP290 or vehicle every other day after they reached the plateau. This dataset summarizes rate differences in normalized to the rate difference values at plateau (averaged rate differences at days 11-13).

Variables:

• Animal IDs
• Condition (CLP290 vs. Vehicle)
• Day of pavlovian learning

File: 'Figure_S3I.csv'

Description: This dataset shows immunostaining results (Mean fluorescence/Area) of KCC2 antibody against shRNA Scrambled vs. shRNA KCC2. Images were taken on a confocal microscope (Leica Microsystems) and analyzed on Fiji.

Variables:

• Column: Condition (Scrambled or shRNA KCC2)
• Row (Mean fluorescence normalized to area per animal)

File: 'Figure_S3J.csv'

Description: Gramicidin perforated patch clamp technique was used to examine the reversal potential of GABA neurons expressing shRNA KCC2 or shRNA scrambled. The reversal value was calculated from an I-V curve where the voltage of the eIPSCs amplitude was zero.

Variables:

• Group: Experimental Condition (shRNA KCC2 or Scrambled)
• Animal ID: Identifier of each animal and each recorded cell
• Reversal: Reversal value of EGABA (Vm)
• Membrane potential (Vm)

File: 'Figure_S4F.csv'

Description: This is the performance summary of a cell-type classifier that predicts whether VTA neurons are DA or GABA based on key electrophysiological features on a test dataset. These results has two parallel sections: Left (VTA DA neurons, TH-Cre, ChR2 tagged) and Right (VTA GABA neurons (GAD-Cre, ArchT tagged).

Variables:

• Recording session name/path
• Optotagged neuron IDs
• Number of units correctly identified by the classifier
• Total number of opto-tagged neurons tested in that session

File: 'Figure_S4G.csv'

Description: Waveform metrics between DA or GABA neurons.

Variables:

• Firing frequency: mean or instantaneous firing rate of identified DA or GABA neurons (hz)
• Peak to trough ratio: Spike waveform ratio (peak amplitude to trough duration) for DA or GABA neurons. Used to classify broad vs. narrow spikes.

File: 'Figure_S5A.csv'

Description: This dataset shows the rate differences across learning in animals implanted with electrophysiological probes. The plateau and criterion values were calculated separately than from Figures 1 and 3.

Variables:

• Column: Day
• Animal ID: identifier for each animal
• Rate differences values across time.
• Row 14: rate differences averaged across time
• Row 15: Standard error of the mean
• Row 16: Ns per group
• Row 17: % change from previous day (rate difference(day) – rate difference (day-1) / 100)
• Row 23-24: Calculation of the plateau and half-max or mid-acquisition point.

File: 'Figure_S5C.csv'

Description: Distribution of VTA DA neurons that exhibit “type 1” or “type 2” phenotypes.

Variables:

• Number of units that fall under type 1 or two
• Condition: phases of learning

File: 'Figure_S5D.csv'

Description: Distribution of VTA GABA neurons that exhibit “type 1” or “type 2” phenotypes.

Variables:

• Number of units that fall under type 1 or two
• Condition: phases of learning

File: 'Figure_S6.csv'

Description: Deeplabcut output of movement metrics during pavlovian conditioning.

Variables:

• Animal ID: identifier for each animal
• Distance moving: total movement distance for vehicle or CLP290 treated animals (pixels)
• Speed: average speed for vehicle or CLP290 treated animals (units/sec)
• Time spent moving: seconds moving per vehicle or CLP290 treated animals

File: 'Figure_S7A.csv'

Description: Number of bursts post cue and reward onset (5s each) after CLP290 or vehicle injections, normalized to number of bursts after mid-acquisition. Bursts were defined as a series of >2 spikes with an initial interspike interval of ≤ 80 ms and terminated when the ISI exceeded 160 ms.

Variables:

• Columns: Condition (phase, after cue (CS) or reward US))
• Row: Number of bursts after cue and reward

File: 'Figure_S7B.csv'

Description: Burst frequency post cue and reward onset (5s each) across learning. Bursts were defined as a series of > 2 spikes with an initial interspike interval of ≤ 80 ms and terminated when the ISI exceeded 160 ms.

Variables:

• Columns: Condition (phase, after cue (CS) or reward (US))
• Row: Burst frequency (hz) after cue and reward

File: 'Figure_S7C.csv'

Description: Percent change in number of bursts post cue and reward onset (5s each) after CLP290 or vehicle injections, normalized to number of bursts after mid-acquisition. Bursts were defined as a series of > 2 spikes with an initial interspike interval of ≤ 80 ms and terminated when the ISI exceeded 160 ms.

Variables:

• Columns: Condition (CLP290 or Vehicle, after cue (CS) or reward US)
• Row: % Change in number of bursts after cue and reward

File: 'Figure_S7D.csv'

Description: Percent change in burst frequency post cue and reward onset (5s each) after CLP290 or vehicle injections, normalized to burst frequency values after mid-acquisition. Bursts were defined as a series of > 2 spikes with an initial interspike interval of ≤ 80 ms and terminated when the ISI exceeded 160 ms.

Variables:

• Columns: Condition (CLP290 or Vehicle, after cue (CS) or reward (US))
• Row: % Change in burst frequency after cue and reward

File: 'Figure_S7F.csv'

Description: Area under the curve (AUC) of z-scored photometry outputs after cue and reward onset. AUC was defined as the integral between the first peak since stimulus presentation and its eventual decay back to 0.

Variables:

• Columns: Phase (Acquisition or Baseline, after cue (CS) or reward US)
• Row: AUC values per animal

File: 'Figure_S8C.csv'

Description: Inter-Synch intervals (Hz) that were generated from joint peristimulus time histograms that enabled the quantification of time intervals between two consecutive instances of synchrony in spike trains from pairs of VTA GABA neurons.

Variables: Columns indicate frequency values per neuron during acquisition.

File: 'Figure_S9C.csv'

Description: Analysis of DA neurons recorded ex vivo, comparing spontaneous firing without optogenetic stimulation of VTA GABA neurons, and spontaneous firing after 10 hz light stimulation of VTA GABA neurons.

Variables:

• Column A: Average spontaneous firing in VTA DA neurons without optogenetic stimulation (10 hz) per cell (row).
• Column B: Average spontaneous firing in VTA DA neurons with optogenetic stimulation (10 hz) per cell (row).

File: 'Figure_S9E.csv'

Description: Burst analysis of DA neurons recorded ex vivo, comparing intra-burst frequency without optogenetic stimulation of VTA GABA neurons, and burst firing after 10 hz light stimulation of VTA GABA neurons.

Variables:

• Column A: Average intra-burst frequency in glutamate-evoked bursts in VTA DA neurons without optogenetic stimulation (10 hz) per cell (row).
• Column B: Average intra-burst frequency in glutamate-evoked bursts in VTA DA neurons with optogenetic stimulation (10 hz) per cell (row).

File: 'Figure_S9G.csv'

Description: Analysis of DA neurons recorded in vivo, comparing spontaneous firing without optogenetic stimulation of VTA GABA neurons, and spontaneous firing after 10 hz light stimulation of VTA GABA neurons.

Variables:

• Column A: Average spontaneous firing in VTA DA neurons without optogenetic stimulation (10 hz) per cell (row).
• Column B: Average spontaneous firing in VTA DA neurons with optogenetic stimulation (10 hz) per cell (row).

File: 'Figure_S9H.csv'

Description: Burst analysis of DA neurons recorded in vivo, comparing intra-burst frequency without optogenetic stimulation of VTA GABA neurons, and burst firing after 10 hz light stimulation of VTA GABA neurons.

Variables:

• Column A: Average intra-burst frequency in glutamate-evoked bursts in VTA DA neurons without optogenetic stimulation (10 hz) per cell (row).
• Column B: Average intra-burst frequency in glutamate-evoked bursts in VTA DA neurons with optogenetic stimulation (10 hz) per cell (row).

File: 'NCOMMS_Source_Data_102825.xlsx'

Description: An excel file that combines all figures above.

Code/Software

File: 'Pavlovian_ArchT.MPC'

Med-associates code used to provide optogenetic light stimulation at cue and reward onset, with a 5 second tone preceding sugar pellet presentations. 

File: 'spike_raster.m' 

Script used to generate spike raster in figure 4B. 

File: 'Pavlovian_Ephys.MPC'

Med-associates code used for pavlovian conditioning, with a 5 second tone preceding sugar pellet presentations for in vivo ephys recordings. 

File: 'Jitter_simulation.m'

Jitter method of spike resampling adapted from Amarasingham et al., 2012 (PMID: 22031767)

File: 'CS_Conditioning_Tone_Opto_CS.MPC'

Med-associates code used for pavlovian conditioning, with a 5 second tone preceding sugar pellet presentations.