DOI: 10.5061/dryad.44j0zpcv7
title: Data from: Dynamic changes in chloride homeostasis coordinate midbrain inhibitory network activity during reward learning

description: The ability to associate environmental stimuli with positive outcomes is a
 fundamental form of learning. While extensive research has focused on
 midbrain dopamine neurons during associative learning, less is known about
 learning-mediated changes in the afferents that shape dopamine neuron
 responses. We demonstrate in rats that during critical phases of learning,
 anion homeostasis in midbrain inhibitory GABA neurons – a primary source
 of input to dopamine neurons – is disrupted due to downregulation of the
 chloride transporter KCC2. This alteration in GABA neurons preferentially
 impacted lateral mesoaccumbal dopamine pathways and was not observed after
 learning was established. At the network level, learning-mediated KCC2
 downregulation was associated with enhanced synchronization between
 individual GABA neurons and increased dopamine responses to rewards and
 reward-related stimuli. Conversely, enhancing KCC2 function during
 learning reduced GABA synchronization, diminished relevant dopamine
 signaling, and prevented cue-reward associations. Thus, circuit-specific
 adaptations in midbrain GABA neurons are crucial for forming new
 reward-related behaviors.
description: # Data from: Dynamic changes in chloride homeostasis coordinate midbrain
 inhibitory network activity during reward learning Dataset DOI:
 [10.5061/dryad.44j0zpcv7](https://doi.org/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 &lt; 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 &lt; 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 &lt; 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
 &lt; 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 &lt; 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 &lt; 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 &lt; 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 &lt; 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 &gt;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
 &gt; 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 &gt; 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 &gt; 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.