Dopaminergic dynamics underlying sex-specific cocaine reward methods
Aim. Evidence-backed execution summary for Dopaminergic dynamics underlying sex-specific cocaine reward methods from Dopaminergic dynamics underlying sex-specific cocaine reward.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Fluctuations in the VTA-NAc circuit are oestrous dependent
reagent used in the protocol.
- Use
- Cocaine's ability to alter dopaminergic transmission from the VTA to the NAc has been proposed as a central mechanism underlying its reinforcing and rewarding actions. To examine real-time activity of these neurons in vivo during associative learning for drug rewards, we injected male and female C57BL/6J mice with...
Oestrus females show enhanced dopamine function
reagent used in the protocol.
- Use
- Both drug reward and cue-reward associations (that is, associative learning) are dependent on dopaminergic transmission from the ventral tegmental area (VTA) to the nucleus accumbens (NAc). Phasic firing of VTA dopamine neurons is a critical component in associative learning between environmental cues and rewarding...
Oestrus females show enhanced dopamine function
reagent used in the protocol.
- Use
- Using anaesthetized, single-unit in vivo electrophysiology in drug-naive male mice, dioestrus female mice and oestrus female mice, we found that female mice overall exhibit increased basal putative VTA dopamine neuron activity when compared with male mice ( ). However, the increased putative VTA dopamine neuron firi...
Oestrus females show enhanced dopamine function
reagent used in the protocol.
- Use
- Together, these data suggest that the dopaminergic VTA-NAc circuit of oestrus females is more active, even before cocaine conditioning. Increases in dopamine release can be due to increases in dopamine synthesis or in alterations in feedback mechanisms at the level of the nerve terminal. Levels of tyrosine hydroxyla...
Increased cocaine potency during oestrus
reagent used in the protocol.
- Use
- To assess whether these effects could be due to circulating hormone levels, we assayed progesterone and estradiol levels in serum using enzyme-linked immunosorbent assay (ELISA) within each animal on blood collected immediately before FSCV recordings. We found that circulating estradiol levels were significantly hig...
Long-lasting VTA-NAc neural responses to drug-associated cues
reagent used in the protocol.
- Use
- The VTA-NAc pathway is a key neural substrate of cue-reward associations, as it mediates multiple aspects of this process including learning, selecting and executing goal-oriented behaviours. Over time, contextual cues in the absence of drug can elicit neural responses that resemble that of the drug itself and trig...
Long-lasting VTA-NAc neural responses to drug-associated cues
reagent used in the protocol.
- Use
- We found that increases in VTA Ca 2+ activity preceded entry into the cocaine-paired, but not saline-paired, chamber ( and ). Interestingly, the magnitude of these responses (Δ F / F ) was significantly higher in oestrus-conditioned female mice ( ). It is important to note that these females are no longer in oe...
Long-lasting VTA-NAc neural responses to drug-associated cues
reagent used in the protocol.
- Use
- Although the data presented above outline a role for the VTA-NAc pathway in this process, they do not rule out the contribution of non-dopaminergic projections originating in the VTA in encoding this information. To this end, we used male and female mice that express Cre-recombinase under the control of the TH promo...
Oestrus females show enhanced dopamine function
Using anaesthetized, single-unit in vivo electrophysiology in drug-naive male mice, dioestrus female mice and oestrus female mice, we found that female mice overall exhibit increased basal putative VTA dopamine neuron activity when compared with male mice ( ). However, the increased putative VTA dopamine neuron firi...
- Use
- Using anaesthetized, single-unit in vivo electrophysiology in drug-naive male mice, dioestrus female mice and oestrus female mice, we found that female mice overall exhibit increased basal putative VTA dopamine neuron activity when compared with male mice ( ). However, the increased putative VTA dopamine neuron firi...
Oestrus females show enhanced dopamine function
Together, these data suggest that the dopaminergic VTA-NAc circuit of oestrus females is more active, even before cocaine conditioning. Increases in dopamine release can be due to increases in dopamine synthesis or in alterations in feedback mechanisms at the level of the nerve terminal. Levels of tyrosine hydroxyla...
- Use
- Together, these data suggest that the dopaminergic VTA-NAc circuit of oestrus females is more active, even before cocaine conditioning. Increases in dopamine release can be due to increases in dopamine synthesis or in alterations in feedback mechanisms at the level of the nerve terminal. Levels of tyrosine hydroxyla...
Fluctuations in the VTA-NAc circuit are oestrous dependent
Consistent with electrophysiology data, we show enhanced basal VTA activity in awake and behaving oestrus female mice as compared with both male and dioestrus female mice ( and ). Further, we show that although cocaine suppressed VTA Ca 2+ events as measured by both frequency (events per minute) and magnitude (%...
- Use
- Consistent with electrophysiology data, we show enhanced basal VTA activity in awake and behaving oestrus female mice as compared with both male and dioestrus female mice ( and ). Further, we show that although cocaine suppressed VTA Ca 2+ events as measured by both frequency (events per minute) and magnitude (%...
Increased cocaine potency during oestrus
Next, we determined whether the effects of estradiol on cocaine potency represented a direct effect at dopamine terminals or an indirect effect via activation of VTA cell bodies. Notably, for all groups, the K m (affinity) of dopamine for DAT was not significantly different before cocaine bath application, suggestin...
- Use
- Next, we determined whether the effects of estradiol on cocaine potency represented a direct effect at dopamine terminals or an indirect effect via activation of VTA cell bodies. Notably, for all groups, the K m (affinity) of dopamine for DAT was not significantly different before cocaine bath application, suggestin...
Long-lasting VTA-NAc neural responses to drug-associated cues
We found that increases in VTA Ca 2+ activity preceded entry into the cocaine-paired, but not saline-paired, chamber ( and ). Interestingly, the magnitude of these responses (Δ F / F ) was significantly higher in oestrus-conditioned female mice ( ). It is important to note that these females are no longer in oe...
- Use
- We found that increases in VTA Ca 2+ activity preceded entry into the cocaine-paired, but not saline-paired, chamber ( and ). Interestingly, the magnitude of these responses (Δ F / F ) was significantly higher in oestrus-conditioned female mice ( ). It is important to note that these females are no longer in oe...
VTA dopamine neuron firing controls DAT function
We show that oestrous cycle-dependent changes in reward system function at the level of both the VTAs and terminals in the NAc lead to fluctuations in the ability to associate rewards with contextual cues; however, the mechanism was unclear. We hypothesized that this enhancement of VTA firing was the mechanism by wh...
- Use
- We show that oestrous cycle-dependent changes in reward system function at the level of both the VTAs and terminals in the NAc lead to fluctuations in the ability to associate rewards with contextual cues; however, the mechanism was unclear. We hypothesized that this enhancement of VTA firing was the mechanism by wh...
Voltammetric data analysis
Demon voltammetry and analysis software was used for all analysis of FSCV data. Data were modelled using Michaelis-Menten kinetics to determine dopamine release, V max and cocaine potency. The quinpirole concentration-response curves were assessed by determining the peak height of dopamine release at ea...
- Use
- Demon voltammetry and analysis software was used for all analysis of FSCV data. Data were modelled using Michaelis-Menten kinetics to determine dopamine release, V max and cocaine potency. The quinpirole concentration-response curves were assessed by determining the peak height of dopamine release at ea...
Conditioned place preference
Daily oestrous cycle staging was conducted 1.5 h before experiments. All mice were acclimated to the behaviour room for at least 1 h before testing. CPP was performed in a rectangular apparatus consisting of two side chambers measuring 28 × 24 cm each and a centre chamber measuring 11.5 ×...
- Use
- Daily oestrous cycle staging was conducted 1.5 h before experiments. All mice were acclimated to the behaviour room for at least 1 h before testing. CPP was performed in a rectangular apparatus consisting of two side chambers measuring 28 × 24 cm each and a centre chamber measuring 11.5 ×...
Statistics
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Graph Pad Prism (version 6, La Jolla, CA, USA) or R were used to statistically analyse data sets and create graphs. Normality of data was assessed and, when data were not normally distributed, non-parametric analyses were performed. Data for CPP across oestrous cycle, VTA dopamine neuron percent spikes within a burs...
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Long-lasting VTA-NAc neural responses to drug-associated cues
Although the data presented above outline a role for the VTA-NAc pathway in this process, they do not rule out the contribution of non-dopaminergic projections originating in the VTA in encoding this information. To this end, we used male and female mice that express Cre-recombinase under the control of the TH promoter ( TH-BAC-Cre ), combined with viral expression of Cre-dependent AAV5-DIO-GCaMP6f in the VTA, to record dopamine neuron-specific Ca 2+ transients through two separate optic fibres implanted in the VTA and the NAc during cocaine and saline conditioning ( ). Consistent with electrophysiology data and the global imaging data, we show enhanced basal VTA activity of dopaminergic neurons in awake, behaving oestrus female mice as compared with both male and dioestrus female mice ( and ). Further, we show that, although cocaine suppressed VTA neural activity ( ), the effect of...
Voltammetric data analysis
Demon voltammetry and analysis software was used for all analysis of FSCV data. Data were modelled using Michaelis-Menten kinetics to determine dopamine release, V max and cocaine potency. The quinpirole concentration-response curves were assessed by determining the peak height of dopamine release at each dose and expressed as a percentage of the pre-drug basal dopamine release.
Drugs
CNO (Sigma-Aldrich) was dissolved at a concentration of 2.5 mg ml -1 in 5% dimethyl sulfoxide in normal saline (Sigma-Aldrich). CNO was administered in the following dose of 1 mg kg -1, i.p. An equal (matched) volume of vehicle (saline with 5% dimethyl sulfoxide) was used as the control. Cocaine HCl was provided by the NIDA drug supply programme and administered at a 10 mg kg -1 doses i.p. For slice voltammetry experiments, Cocaine (NIDA Drug Supply), Quinpirole (Sigma-Aldrich) and Estradiol (Sigma-Aldrich) were perfused over slices in artificial cerebrospinal fluid (aCSF) solution.
Stereotaxic virus injection and optic fiber implantation
Under ketamine (100 mg kg -1 )/xylazine (10 mg kg -1 ) anaesthesia, mice were positioned in a stereotaxic frame (Kopf Instruments) and the VTA was targeted (Bregma coordinates: anterior/posterior, -3.3 mm; medial/lateral, 0.75 mm; dorsal/ventral, -4.6 mm; 7° angle). Ophthalmic ointment was applied to the eyes to prevent drying. A midline incision was made down the scalp and a craniotomy was made using a dental drill. A 10 µl Nanofil Hamilton syringe (WPI, Sarasota, FL) with a 34-gauge beveled metal needle was used to infuse 0.5 µl virus at a rate of 100 nl min -1. Following infusion, the needle was kept at the injection site for 10 min and then slowly withdrawn. Chronically implantable optic fibres constructed with 400 µm core 0.48 numerical aperture (NA) op...
CPP with DREADD-induced excitation of VTA dopamine neurons
To determine whether basal increases in VTA firing were sufficient to enhance reward processing, AAV-DIO-hM3Dq was injected into the VTA of TH-BAC-Cre mice. The virus was allowed to express for 5 weeks and then the animals went through CPP as described above. CNO (1 mg kg -1; Sigma) was administered 2 h before cocaine pairing sessions. Time spent in each area of the conditioning chamber was recorded as before and animals were considered to have developed a preference when the difference in time spent between the drug paired and chamber paired with saline was significantly greater than before the pairings.
Single-unit recordings data analysis
Putative dopamine cells in the VTA were identified according to standard electrophysiological criteria: a stark, triphasic waveform with set filters; action potential duration from start to negative trough ≥1.1 ms; slow firing rate (<10 spikes per second); and bursting behaviour with onset beginning when two spikes occurred within <80 ms and offset set when no activity occurred for ≥160 ms. Spike times were measured using Clampfit 10.2 and data were further processed using R as previously described. Activity was quantified by overlapping 60 s windows shifted every 15 s. Percentage of spikes within bursts corresponds to the percentage of spikes discharged within a burst. Further, burst length was determined after measuring the number of spikes within a burst and averaged per cell.
Fast-scan cyclic voltammetry
Ex vivo FSCV was used to characterize D2/D3 autoreceptor function, DAT activity, dopamine release and cocaine potency in the NAc. Voltammetry experiments were conducted 1-2 weeks following the completion of the CPP experiments. Recordings were made directly at the end of the fiberoptic probe, to ensure that the recordings were from the same location in the NAc as calcium-imaging experiments. A vibrating tissue slicer was used to prepare 250 µm-thick coronal brain sections containing the NAc, which were immersed in oxygenated aCSF containing (in mM): NaCl (126), KCl (2.5), NaH 2 PO 4 (1.2), CaCl 2 (2.4), MgCl 2 (1.2), NaHCO 3 (25), glucose (11), L -ascorbic acid (0.4) and pH was adjusted to 7.4. The slice was transferred to the testing chambers containing aCSF at 32 °C with a 1 ml min -1 flow rate. A carbon fibre microelectrode (100-2...
Fast-scan cyclic voltammetry
To determine the local effects of estradiol on NAc brain slices, estradiol was bath applied at 1 or 3 µM. Recordings were taken for 60 min at each dose, to ensure that estradiol had sufficient time to induce changes. Once dopamine measurements were stable, a concentration response curve for cocaine was run (0.1-30 µmol l -1 ) within each group.
Measurement outputs
What raw and processed outputs should exist?
Although phasic activity of VTA dopamine neurons is a critical mediator of reward encoding, ultimately dopamine release from nerve terminals in the NAc is the outcome that encod...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
To assess whether these effects could be due to circulating hormone levels, we assayed progesterone and estradiol levels in serum using enzyme-linked immunosorbent assay (ELISA)...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Although the data presented above outline a role for the VTA-NAc pathway in this process, they do not rule out the contribution of non-dopaminergic projections originating in th...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
We show that oestrous cycle-dependent changes in reward system function at the level of both the VTAs and terminals in the NAc lead to fluctuations in the ability to associate r...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Graph Pad Prism (version 6, La Jolla, CA, USA) or R were used to statistically analyse data sets and create graphs.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Although phasic activity of VTA dopamine neurons is a critical mediator of reward encoding, ultimately dopamine release from nerve terminals in the NAc is the outcome that encod...; To assess whether these effects could be due to circulating hormone levels, we assayed progesterone and estradiol levels in serum using enzyme-linked immunosorbent assay (ELISA)...; Although the data presented above outline a role for the VTA-NAc pathway in this process, they do not rule out the contribution of non-dopaminergic projections originating in th...; We show that oestrous cycle-dependent changes in reward system function at the level of both the VTAs and terminals in the NAc lead to fluctuations in the ability to associate r....
from paperStatistical comparison
Graph Pad Prism (version 6, La Jolla, CA, USA) or R were used to statistically analyse data sets and create graphs. Normality of data was assessed and, when data were not normal...
from paperReporting output
Report representative outputs alongside summary comparisons for Although phasic activity of VTA dopamine neurons is a critical mediator of reward encoding, ultimately dopamine release from nerve terminals in the NAc is the outcome that encod..., To assess whether these effects could be due to circulating hormone levels, we assayed progesterone and estradiol levels in serum using enzyme-linked immunosorbent assay (ELISA)..., Although the data presented above outline a role for the VTA-NAc pathway in this process, they do not rule out the contribution of non-dopaminergic projections originating in th..., We show that oestrous cycle-dependent changes in reward system function at the level of both the VTAs and terminals in the NAc lead to fluctuations in the ability to associate r....
inferred from protocolStructured statistical methods
Graph Pad Prism (version 6, La Jolla, CA, USA) or R were used to statistically analyse data sets and create graphs. Normality of data was assessed and, when data were not normal...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
Although the data presented above outline a role for the VTA-NAc pathway in this process, they do not rule out the contribution of non-dopaminergic projections originating in the VTA in encoding this information. To this end, we used male and female mice that express Cre-recombinase under the control of the TH promoter ( TH-BAC-Cre ), combined with viral expression of Cre-dependent AAV5-DIO-GCaMP6f in the VTA, to record dopamine neuron-specific Ca 2+ transients through two separate optic fibres implanted in the VTA and the NAc during cocaine and saline conditioning ( ). Consistent with electrophysiology data and the global imaging data, we show enhanced basal VTA activity of dopaminergic neurons in awake, behaving oestrus female mice as compared with both male and dioestrus female mice ( and ). Further, we show that, although cocaine suppressed VTA neural activity ( ), the effect of cocaine in the VTA was significantly higher in oestrus females when compared with male and dioestrus female mice ( ).
Demon voltammetry and analysis software was used for all analysis of FSCV data. Data were modelled using Michaelis-Menten kinetics to determine dopamine release, V max and cocaine potency. The quinpirole concentration-response curves were assessed by determining the peak height of dopamine release at each dose and expressed as a percentage of the pre-drug basal dopamine release.
CNO (Sigma-Aldrich) was dissolved at a concentration of 2.5 mg ml -1 in 5% dimethyl sulfoxide in normal saline (Sigma-Aldrich). CNO was administered in the following dose of 1 mg kg -1, i.p. An equal (matched) volume of vehicle (saline with 5% dimethyl sulfoxide) was used as the control. Cocaine HCl was provided by the NIDA drug supply programme and administered at a 10 mg kg -1 doses i.p. For slice voltammetry experiments, Cocaine (NIDA Drug Supply), Quinpirole (Sigma-Aldrich) and Estradiol (Sigma-Aldrich) were perfused over slices in artificial cerebrospinal fluid (aCSF) solution.
Under ketamine (100 mg kg -1 )/xylazine (10 mg kg -1 ) anaesthesia, mice were positioned in a stereotaxic frame (Kopf Instruments) and the VTA was targeted (Bregma coordinates: anterior/posterior, -3.3 mm; medial/lateral, 0.75 mm; dorsal/ventral, -4.6 mm; 7° angle). Ophthalmic ointment was applied to the eyes to prevent drying. A midline incision was made down the scalp and a craniotomy was made using a dental drill. A 10 µl Nanofil Hamilton syringe (WPI, Sarasota, FL) with a 34-gauge beveled metal needle was used to infuse 0.5 µl virus at a rate of 100 nl min -1. Following infusion, the needle was kept at the injection site for 10 min and then slowly withdrawn. Chronically implantable optic fibres constructed with 400 µm core 0.48 numerical aperture (NA) optic fibre and unilaterally implanted into the VTA (Bregma coordinates: anterior/posterior, -3.3 medial/lateral, +1.0; dorsal/ventral, -4.4; 7° angle) and NAc (bregma coordinates: anterior/posterior, 1.4 mm; medial/lateral, 1.5 mm; dorsal/ventral, -4.3 mm; 10&...
To determine whether basal increases in VTA firing were sufficient to enhance reward processing, AAV-DIO-hM3Dq was injected into the VTA of TH-BAC-Cre mice. The virus was allowed to express for 5 weeks and then the animals went through CPP as described above. CNO (1 mg kg -1; Sigma) was administered 2 h before cocaine pairing sessions. Time spent in each area of the conditioning chamber was recorded as before and animals were considered to have developed a preference when the difference in time spent between the drug paired and chamber paired with saline was significantly greater than before the pairings.
Putative dopamine cells in the VTA were identified according to standard electrophysiological criteria: a stark, triphasic waveform with set filters; action potential duration from start to negative trough ≥1.1 ms; slow firing rate (<10 spikes per second); and bursting behaviour with onset beginning when two spikes occurred within <80 ms and offset set when no activity occurred for ≥160 ms. Spike times were measured using Clampfit 10.2 and data were further processed using R as previously described. Activity was quantified by overlapping 60 s windows shifted every 15 s. Percentage of spikes within bursts corresponds to the percentage of spikes discharged within a burst. Further, burst length was determined after measuring the number of spikes within a burst and averaged per cell.
Ex vivo FSCV was used to characterize D2/D3 autoreceptor function, DAT activity, dopamine release and cocaine potency in the NAc. Voltammetry experiments were conducted 1-2 weeks following the completion of the CPP experiments. Recordings were made directly at the end of the fiberoptic probe, to ensure that the recordings were from the same location in the NAc as calcium-imaging experiments. A vibrating tissue slicer was used to prepare 250 µm-thick coronal brain sections containing the NAc, which were immersed in oxygenated aCSF containing (in mM): NaCl (126), KCl (2.5), NaH 2 PO 4 (1.2), CaCl 2 (2.4), MgCl 2 (1.2), NaHCO 3 (25), glucose (11), L -ascorbic acid (0.4) and pH was adjusted to 7.4. The slice was transferred to the testing chambers containing aCSF at 32 °C with a 1 ml min -1 flow rate. A carbon fibre microelectrode (100-200 µM length, 7 µM radius) and bipolar stimulating electrode were placed into the NAc where GCaMP-GFP terminal expression was high and near the optic fibre track to get the best representation of release near the recorded regions. Dopamine release was evoked by a single electri...
To determine the local effects of estradiol on NAc brain slices, estradiol was bath applied at 1 or 3 µM. Recordings were taken for 60 min at each dose, to ensure that estradiol had sufficient time to induce changes. Once dopamine measurements were stable, a concentration response curve for cocaine was run (0.1-30 µmol l -1 ) within each group.
Machine-readable layer
[
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"name": "Dopaminergic dynamics underlying sex-specific cocaine reward methods",
"description": "Evidence-backed execution summary for Dopaminergic dynamics underlying sex-specific cocaine reward methods from Dopaminergic dynamics underlying sex-specific cocaine reward.",
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"text": "Although the data presented above outline a role for the VTA-NAc pathway in this process, they do not rule out the contribution of non-dopaminergic projections originating in the VTA in encoding this information. To this end, we used male and female mice that express Cre-recombinase under the control of the TH promoter ( TH-BAC-Cre ), combined with viral expression of Cre-dependent AAV5-DIO-GCaMP6f in the VTA, to record dopamine neuron-specific Ca 2+ transients through two separate optic fibres implanted in the VTA and the NAc during cocaine and saline conditioning ( ). Consistent with electrophysiology data and the global imaging data, we show enhanced basal VTA activity of dopaminergic neurons in awake, behaving oestrus female mice as compared with both male and dioestrus female mice ( and ). Further, we show that, although cocaine suppressed VTA neural activity ( ), the effect of..."
},
{
"@type": "HowToStep",
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"name": "Voltammetric data analysis",
"text": "Demon voltammetry and analysis software was used for all analysis of FSCV data. Data were modelled using Michaelis-Menten kinetics to determine dopamine release, V max and cocaine potency. The quinpirole concentration-response curves were assessed by determining the peak height of dopamine release at each dose and expressed as a percentage of the pre-drug basal dopamine release."
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"text": "CNO (Sigma-Aldrich) was dissolved at a concentration of 2.5 mg ml -1 in 5% dimethyl sulfoxide in normal saline (Sigma-Aldrich). CNO was administered in the following dose of 1 mg kg -1, i.p. An equal (matched) volume of vehicle (saline with 5% dimethyl sulfoxide) was used as the control. Cocaine HCl was provided by the NIDA drug supply programme and administered at a 10 mg kg -1 doses i.p. For slice voltammetry experiments, Cocaine (NIDA Drug Supply), Quinpirole (Sigma-Aldrich) and Estradiol (Sigma-Aldrich) were perfused over slices in artificial cerebrospinal fluid (aCSF) solution."
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"name": "CPP with DREADD-induced excitation of VTA dopamine neurons",
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"text": "Ex vivo FSCV was used to characterize D2/D3 autoreceptor function, DAT activity, dopamine release and cocaine potency in the NAc. Voltammetry experiments were conducted 1-2 weeks following the completion of the CPP experiments. Recordings were made directly at the end of the fiberoptic probe, to ensure that the recordings were from the same location in the NAc as calcium-imaging experiments. A vibrating tissue slicer was used to prepare 250 µm-thick coronal brain sections containing the NAc, which were immersed in oxygenated aCSF containing (in mM): NaCl (126), KCl (2.5), NaH 2 PO 4 (1.2), CaCl 2 (2.4), MgCl 2 (1.2), NaHCO 3 (25), glucose (11), L -ascorbic acid (0.4) and pH was adjusted to 7.4. The slice was transferred to the testing chambers containing aCSF at 32 °C with a 1 ml min -1 flow rate. A carbon fibre microelectrode (100-2..."
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