Glutamatergic signaling by mesolimbic dopamine neurons in the nucleus accumbens

Abstract

Recent evidence suggests the intriguing possibility that midbrain dopaminergic (DAergic) neurons may use fast glutamatergic transmission to communicate with their postsynaptic targets. Because of technical limitations, direct demonstration of the existence of this signaling mechanism has been limited to experiments using cell culture preparations that often alter neuronal function including neurotransmitter phenotype. Consequently, it remains uncertain whether glutamatergic signaling between DAergic neurons and their postsynaptic targets exists under physiological conditions. Here, using an optogenetic approach, we provide the first conclusive demonstration that mesolimbic DAergic neurons in mice release glutamate and elicit excitatory postsynaptic responses in projection neurons of the nucleus accumbens. In addition, we describe the properties of the postsynaptic glutamatergic responses of these neurons during experimentally evoked burst firing of DAergic axons that reproduce the reward-related phasic population activity of the mesolimbic projection. These observations indicate that, in addition to DAergic mechanisms, mesolimbic reward signaling may involve glutamatergic transmission.

Figure 1.

Functional glutamatergic transmission by DAergic axons in the nucleus accumbens. A, Left, composite confocal images of a typical SPN and surrounding DAergic axons constructed from two consecutive optical sections taken at 2 μm intervals. The SPN was intracellularly labeled with Alexa Fluor 594 (red) and illustrates the morphological characteristics used for identification. DAergic axons are visualized by YFP (yellow). The limited thickness of the displayed optical section (∼4 μm in depth) is to avoid obscuring the neuron by the extremely dense DAergic axon arborization. Right, Confocal microscopic images (single optical sections) obtained at higher resolution at the two rectangular areas delineated in the panel on the left. Arrows indicate some of the observable putative close oppositions between the SPN and DAergic axons. B, AMPA receptor-mediated EPSCs in a nucleus accumbens SPN elicited by optical activation of DAergic axons with 5 ms light pulses (blue bar). The EPSC (control, blue trace) is reversibly blocked by the AMPAR-selective antagonist DNQX (10 μm, red trace; recovery, green trace). Colored traces are averages of 10 EPSCs, individual predrug control responses are shown in gray. C, NMDAR-mediated EPSC elicited with the same stimuli as in B in an SPN in the accumbens (V _h = +50 mV). The average EPSC recorded in the control condition (green), in the presence of the selective NMDAR antagonist APV (blue), and the difference in the two traces corresponding to the NMDAR EPSC (red) are shown. The black trace is the AMPAR current recorded at −70 mV. D, Top, Optically evoked EPSPs trigger spikes (arrows) in an SPN when depolarized above −52 mV in current clamp mode. Traces represent successively larger current injections. Bottom, Corresponding EPSCs in voltage clamp (blue, average; gray, individual EPSCs; V_m = −70 mV).

Figure 2.

Optically induced synaptic release is physiological. A, Reliable and temporally precise control of axonal firing with optical stimulation. Top left, Epifluorescence image of a DAergic, YFP⁺ axon bleb (yellow structure, arrowhead) patched with a pipette filled with Alexa Fluor 594 (red). Bottom left, differential interference contrast image of the recording area illustrates pipette positions (arrows) for recordings shown on the right. Optical fiber is seen at arrowhead. Top right, Averaged spikes in a DAergic axon evoked by a 5 ms light pulse (blue bar) recorded in cell-attached patch mode (black trace). Averaged whole-cell EPSCs (green traces) recorded in a nearby SPN show that the onset of the EPSC is coincident with the axonal response. Gray traces are individual EPSCs. Bottom right, Individual action potentials (colored traces, n = 12) recorded in one axon illustrate the reliability of the response and the limited variability of the latency to spike. Bottom middle, Histogram of the distribution of spike delays normalized to the mean and fitted with a Gaussian function are shown for this example (bottom histogram and curve in red). Green curves are similar Gaussian fits to spike delay distributions measured in two other axons. B, The optically evoked EPSC (average, blue trace) is blocked by TTX (1 μm, red trace). C, The EPSC is elicited by propagating action potentials. Top traces show EPSCs in one SPN in response to optical stimuli delivered at increasing distances from the neuron as indicated on the left. Bottom graph shows latency–distance relationships for four neurons. Lines are linear fits; the fit to the combined data are shown in black. Note the consistent effect of distance on latency. D, Cyclic voltammograms obtained with FSCV in a DA standard (1 μm, black trace) and at the peak of the FSCV current response to optical stimulation (St) in the core of the accumbens (Acc) (green trace). Note the identical positions of the oxidation (Ox.) and reduction (Red.) peaks, and the similarity of the waveforms indicating that the detected substance is DA. E, Time course of the extracellular DA concentration in the accumbens shell (normalized to peak) during electrical (black trace) and optical (green trace) train stimulation (five stimuli, 10 Hz, blue ticks). The raising phase of the response (top graph) during the first 700 ms is shown at higher resolution on the bottom. Note the nearly identical rates of extracellular DA accumulation in response to electrical and optical stimulation.

Figure 3.

Glutamatergic responses are independent of DAergic signaling. A, The optically elicited EPSC (control, green trace) is not altered by the blockade of D₁ and D₂ DA receptors with SCH21390 (SCH; 10 μm) and sulpiride (SUL; 5 μm, blue). B, Pharmacological depletion eliminates DA release. DA release elicited with optical train stimulation in the core (green trace) and shell (blue trace) of the accumbens in a control animal is absent in slices from a DA-depleted animal (red and black traces). C, Normal AMPA receptor-mediated EPSCs are elicited optically (blue bar) in an SPN after complete DA depletion (averaged response, green trace; individual responses, gray traces). The response is reversibly blocked by an AMPAR antagonist (CNQX, 10 μm, red trace; wash, blue trace).

Figure 4.

Glutamatergic responses of SPNs to a behaviorally relevant pattern of activity of DAergic axons. A, Simultaneous cell-attached patch recording from a DAergic axon (black traces) and somatic whole-cell recording from a nearby SPN (green traces) during optical train stimulation demonstrate reliable axonal firing at 10 or 33.3 Hz (blue ticks, five pulses, 5 ms duration each). Evoked EPSCs are coincident with the axonal spikes. B, Optically evoked action potential bursts (blue ticks; 33.3 Hz; three pulses; 2 ms each) in DAergic axons elicit a train of AMPAR-mediated EPSCs (black trace; average EPSC, Vm = −70 mV) exhibiting pronounced short-term depression. A train of EPSCs recorded at Vm = 50 mV shows effective temporal summation and an increasing peak current during the stimulus. Inset shows higher time resolution of the first 160 ms of the response (total current in green; CNQX, 10 μm, in blue).