Dopamine neuron glutamate cotransmission: frequency-dependent modulation in the mesoventromedial projection - PubMed (original) (raw)

Dopamine neuron glutamate cotransmission: frequency-dependent modulation in the mesoventromedial projection

N Chuhma et al. Neuroscience. 2009.

Abstract

Mesoventromedial dopamine neurons projecting from the medial ventral tegmental area to the ventromedial shell of the nucleus accumbens play a role in attributing incentive salience to environmental stimuli that predict important events, and appear to be particularly sensitive to the effects of psychostimulant drugs. Despite the observation that these dopamine neurons make up almost the entire complement of neurons in the projection, stimulating their cell bodies evokes a fast glutamatergic response in accumbens neurons. This is apparently due to dopamine neuron glutamate cotransmission, suggested by the extensive coexpression of vesicular glutamate transporter 2 (VGLUT2) in the neurons. To examine the interplay between the dopamine and glutamate signals, we used acute quasi-horizontal brain slices made from DAT-YFP mice in which the intact mesoventromedial projection can be visualized. Under current clamp, when dopamine neurons were stimulated repeatedly, dopamine neuron glutamate transmission showed dopamine-mediated facilitation, solely at higher, burst-firing frequencies. Facilitation was diminished under voltage clamp and flipped to inhibition by intracellular Cs(+) or GDPbetaS, indicating that it was mediated postsynaptically. Postsynaptic facilitation was D1 mediated, required activation of NMDA receptors and closure of voltage gated K(+)-channels. When postsynaptic facilitation was blocked, D2-mediated presynaptic inhibition became apparent. These counterbalanced pre- and postsynaptic actions determine the frequency dependence of dopamine modulation; at lower firing frequencies dopamine modulation is not apparent, while at burst firing frequency postsynaptic facilitation dominates and dopamine becomes facilitatory. Dopamine neuron glutamate cotransmission may play an important role in encoding the incentive salience value of conditioned stimuli that activate goal-directed behaviors, and may be an important subtract for enduring drug-seeking behaviors.

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Figures

Figure 1. VGLUT2 immunoreactivity in VTA DA neurons

To assess the transmitter status of VTA DA neurons, VTA sections were double immunostained for the DA neuron marker TH (green) and for VGLUT2 (red). Confocal z-series of 5–10 sections were acquired; z-projections are shown, except for the confocal z-series in B2. (A) In a low magnification (10x) view from a horizontal section (rostral is towards the top and the midline to the right) from a juvenile mouse, many DA neurons are visualized by TH immunoreactivity (left panel). Most also immunostain for VGLUT2 (middle panel), as is evident in the merged image (right panel; colocalization appears yellow). (B1) At higher magnification (63x, same section), most TH immunoreactive neurons are also immunopositive for VGLUT2. (B2) Expanding the region of interest (ROI) outlined in B1, and showing the individual confocal sections (as used for cell counts) reveals two patterns of VGLUT2 immunoreactivity, cytoplasmic and punctate; punctate immunoreactivity is most likely in afferent terminals. In this ROI, there were 5 neurons, 3 TH+/VGLUT2+ and 2 TH−/VGLUT2+ (arrows). (C) In a higher power (63x) coronal VTA section from an adult mouse, most neurons are TH+/VGLUT2+; the arrowhead denotes a TH+/VGLUT2− neuron, and the arrow denotes a TH−/VGLUT2+ neuron. Abbreviations: medial terminal nucleus of accessory optic tract (MT), interpeduncular nucleus rostral subnucleus (IPR), medial lemniscus (ml).

Figure 2. Fast DA neuron actions in the nAcc are glutamatergic

(A) VTA stimulation. Activating a small number of DA neurons with focal stimulation in the VTA produced a fast excitatory response in the recorded MSN (grey traces), recorded either under current (A1) or voltage clamp (A2). To increase sensitivity for the detection of small DA neuron-evoked responses, DA effects were enhanced with the reuptake inhibitor nomifensine (10 μM; thick black traces). Despite the enhanced DA effect, the AMPA antagonist CNQX (40 μM; thin black traces) completely blocked the fast synaptic response, revealing no subsecond DA actions. All control records, in this and subsequent figures were obtained under continuous GABAA receptor blockade (SR95531, 10μM); records shown are the averages of 5 traces. (B) Local stimulation in the nAcc, recorded from a different neuron. DA neuron axons were activated by focal stimulation, delivered about 300 μm caudal to the recorded MSN at the border of the nAcc. Stimulus intensity was minimized to avoid direct activation of postsynaptic ion channels by current spread. Stimulus intensity for local stimulation ranged from 0.1–0.2 mA (more intense stimulation produced an artifact that distorted the evoked response); VTA stimulation ranged from 1–2 mA (which evoked a maximal response). Application of nomifensine (10 μM, black traces) again produced an enhancement of the response, confirming that the stimulation activated DA neuron axons. Then DAergic actions were isolated by blocking all other likely neurotransmitter actions with a cocktail (thin black traces) of: CNQX (40 μM), MK801 (NMDA antagonist, 10 μM), CGP55845 (GABAB antagonist, 3 μM), and scopolamine (muscarinic antagonist, 1μM). Under either current (B1) or voltage clamp (B2), the response to the local stimulation was completely blocked, revealing no subsecond DAergic action.

Figure 3. Frequency dependence of DA modulation of the VDNGR

(A) The VTA DA neuron glutamate response (VDNGR) was evoked at 0.2, 1, 4, 10 and 20 Hz in MSNs by VTA stimulation, under current clamp. Traces for 4 Hz (left) and 20 Hz (right) stimulation are shown (stimulus timing is indicated beneath the traces); control traces (gabazine only) are shown in grey. (A1) Blocking DA action with SCH23390 (D1 antagonist, 10 μM) and sulpiride (D2 antagonist, 10 μM, black traces) inhibited the VDNGR at 20 Hz selectively. Sample traces recorded from two different cells in different slices are shown. (A2) Inhibition of DA reuptake with nomifensine (10 μM, black traces) had variable effects at 4 Hz and enhanced the glutamatergic response at 20 Hz. (B) Summary of frequency dependence of DA modulation of the VDNGR. We compared the response to the first stimulation pulse (left), and the average response to the second to fifth stimulation pulses (right). Since the protocol for 0.2 Hz stimulation (10 consecutive stimulation at 0.2 Hz) was different, the values for first pulse and second to fifth pulses for 0.2 Hz stimulation were the same. The changes after DA receptor blockade (open symbols) or DAT blockade (closed symbols) are expressed as a percent of the preceding control responses. Gray dashed lines indicate no change from control (100% of control). Numbers of recorded cells are in parentheses. Points were connected by polynomial curve fitting; when error bars overlapped, only one side of the bar is shown. DA modulation was only significant (** p < 0.01, *** p < 0.001, one-sample t-test) with 20 Hz stimulation. DA antagonists and DAT blockade had opposite effects. (C) Frequency dependency of the VDNGR depends on DA. Under conditions of full DA receptor blockade (open circles, dashed line), the VDNGR, measured as the average amplitudes of the second to fifth EPSCs, showed no frequency dependence. In contrast, with physiological DA (control condition, gray circles and line) or with enhanced DA effects (DAT blockade, closed circles, black solid line) the frequency dependence became significant (one-way factorial ANOVA). ** indicates 20 Hz stimulation is significantly different from 0.2, 1 or 4 Hz stimulation, and * indicates 20Hz is significantly different from 0.2 or 4 Hz stimulation by Scheffe’s post-hoc test.

Figure 4. DA modulation of the VDNGR under voltage clamp

(A) The VDNGR was evoked at 0.2, 4 and 20 Hz, under voltage clamp (holding potential −85 mV), with a K+-based (A1) or Cs+-based (A2) intracellular solution. Control traces (gabazine only) are shown in gray and DAT blockade (nomifensine, 10 μM) traces in black. Under voltage clamp, DAT blockade had no effect on the VDNGR. When K+ currents were blocked with intracellular Cs+, DAT blockade produced significant inhibition of the VDNGR. (B) Comparison of frequency dependence of DA modulation of the VDNGR in juvenile animals (P20–34) under current clamp with K+-based intracellular solution (K+-current clamp; data shown were re-plotted from previous figure), voltage clamp with K+-based intracellular solution (K+-voltage clamp), and voltage clamp with Cs+-based pipette solution (Cs+-voltage clamp). Under voltage clamp, with intracellular Cs+, DA action significantly inhibited the VDNGR (* p < 0.05, *** p < 0.001, one sample t-test). (C) Frequency dependency of DA modulation in adult animals (P42–60). DAT blockade (nomifensine 10 μM) effect on the VTA glutamatergic response was examined at 0.2, 4 and 20 Hz under current clamp (closed circles), voltage clamp with K+-based pipette solution (open circles) and voltage clamp with Cs+-based pipette solution (open diamonds). The control level (i.e. no effect) is indicated by a dashed gray line. Numbers of recorded cells are shown in parentheses. * indicates p < 0.05, one-sample t-test. Both juvenile (B) and adult (C) groups show the same frequency dependence.

Figure 5. D2-mediated presynaptic inhibition of the VDNGR

(A) Postsynaptic DA actions were blocked with intracellular GDPβS (1–2 mM) in order to examine presynaptic DA modulation. Under current clamp (A1), DAT blockade (thick black lines) revealed DA inhibition. This was reversed by application of a D2 antagonist (sulpiride, 10 μM, left, thin black line), but not a D1 antagonist (SCH23390, 10 μM, right, thin black line). Control traces are in grey; 10 traces were averaged. Summary data (A2) show DAT blockade (left) produced significant inhibition (* indicates p < 0.05, one-sample t-test), which was reversed by D2 (middle), but not D1 antagonist (right) application; * between the gray and white bars indicates p < 0.05, unpaired-t test). Data from D1 and D2 antagonists were obtained in separate experiments. (B) Agonist pharmacology under voltage clamp and with intracellular Cs+, to block postsynaptic DA actions completely. The VDNGR was evoked with 0.2 Hz stimulation, to avoid residual DA effects. (B1) The VDNGR was inhibited by D2 agonist (quinpirole, 5 μM, left), but not by D1 agonist (SKF81297, 4 μM, right panel); 20 traces were averaged. (B2) Summary data show D2 agonist effects were significant (open bar, * p < 0.05, one-sample t-test), while D1 agonist effects were not (closed bar). (B3) A coefficient of variation analysis of the VDNGR with D2 agonist (left, open circles) and D1 agonist (right, closed circles) confirmed that the D2 modulation was presynaptic. Lines connect data points obtained under control and after agonist application for the same cells; the analysis was done on 50 consecutive traces.

Figure 6. D1-mediated postsynaptic facilitation of the VDNGR

To isolate postsynaptic DA effects, a current simulating the DA neuron glutamate synaptic current with 20 Hz stimulation (bottom left, in A) was injected every 20 sec under current clamp. The injected current was obtained by averaging 30 voltage clamp traces from 3 cells. (A) Traces show that application of DA (10 μM, top right) or D1 agonist (SKF81297, 4 μM, top left, thick black trace) produced facilitation. Addition of D2 agonist (quinpirole, 5 μM, top left, thin black trace) had no further effect. Similarly, application of D2 agonist alone (bottom right) had no effect. Control traces are in grey; 10 traces were averaged. (B) Summary of DA agonist effects. Numbers of recorded cells are in parentheses; ** indicates p < 0.01, one-sample t-test.

Figure 7. Postsynaptic facilitation was partly NMDA receptor mediated

(A) Postsynaptic depolarization enhanced DA-mediated facilitation. (A1) Sample traces of DAT blockade (nomifensine, 10 μM, upper panels) or DA receptor blockade (10 μM SCH23390 + 10 μM sulpiride, lower panels) at a holding potential of −85 mV (left panels) and −65 mV (right panels). EPSCs were evoked with 20 Hz stimulation. Control traces are shown in gray and drug traces in black. Five traces were averaged. (A2) Summary of DAT blockade effect (closed circles) and DA receptor blockade (open circles) at holding potentials of −85, −75 and −65 mV. Numbers of recorded cells are in parentheses; * and ** indicate p < 0.05 and p < 0.01 respectively (one-sample t-test). (B) Postsynaptic facilitation was partly, but not entirely, due to NMDA receptor activation. (B1) Sample traces of NMDA receptor blockade (APV, 100 μM, black trace) and DA receptor blockade followed by NMDA receptor blockade (100 μM APV + 10 μM SCH23390 + 10 μM sulpiride, thin black trace). All traces were recorded under 10 μM nomifensine to enhance DA facilitation; averages of 5 traces are shown. (B2) Summary of NMDA receptor blockade effects (left) and NMDA and DA receptor blockade (right). Data were obtained from the same sets of cells for both drug applications; * and ** indicate p < 0.05 and p < 0.01 respectively (one sample t-test); † indicates p < 0.05 (paired t-test).

Figure 8. Postsynaptic DA facilitatory effects were triggered by voltage gated K+ channel closure

Whole cell K+ currents were recorded under voltage clamp with K+-gluconate (A) or Cs+-gluconate based internal solution (B) at a holding potential of −80 mV, and a series of 10 mV step pulses (300 msec) were delivered. Amplitudes of whole cell K+ currents were measured from the average amplitude of the last 20 msec of the step pulses. Current-voltage relationships of K+ currents under control conditions (open circles) and after application of 10 μM DA (closed diamonds) are shown. Inset shows sample traces recorded at −140 to + 40 mV, with 20 mV steps under control conditions (upper traces) and after DA application (lower traces). (C) DA effects on plateau K+ current amplitude with K+-gluconate based pipette solution (open bars) and Cs+-gluconate based pipette solution (closed bars) at −120 mV (left two bars) and +10 mV (right two bars). Numbers of recorded cells are in parentheses; ** indicates p < 0.01 (one-sample t-test).

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Dopamine neuron glutamate cotransmission: frequency-dependent modulation in the mesoventromedial projection - PubMed (original) (raw)