Dopaminergic dynamics underlying sex-specific cocaine reward methods
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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.
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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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