Synaptic and behavioral profile of multiple glutamatergic inputs to the nucleus accumbens

Abstract

Excitatory afferents to the nucleus accumbens (NAc) are thought to facilitate reward seeking by encoding reward-associated cues. Selective activation of different glutamatergic inputs to the NAc can produce divergent physiological and behavioral responses, but mechanistic explanations for these pathway-specific effects are lacking. Here, we compared the innervation patterns and synaptic properties of ventral hippocampus, basolateral amygdala, and prefrontal cortex input to the NAc. Ventral hippocampal input was found to be uniquely localized to the medial NAc shell, where it was predominant and selectively potentiated after cocaine exposure. In vivo, bidirectional optogenetic manipulations of this pathway attenuated and enhanced cocaine-induced locomotion. Challenging the idea that any of these inputs encode motivationally neutral information, activation of each discrete pathway reinforced instrumental behaviors. Finally, direct optical activation of medium spiny neurons proved to be capable of supporting self-stimulation, demonstrating that behavioral reinforcement is an explicit consequence of strong excitatory drive to the NAc.

Figure 1. vHipp Input to the NAc Is Uniquely Concentrated in the Medial NAc Shell

(A) Representative coronal brain slices showing expression of EYFP (green) after virus injection into the vHipp, basolateral amygdala, or prefrontal cortex. See also Figure S1. The left panel shows images from the sites of virus injection. The right panel shows images of EYFP-expressing afferents to the NAc. To enable comparisons, the NAc images were captured and processed using identical settings. The red lines indicate where patch recordings would be obtained from, as described later in the text. (B) The average fluorescent signal in each region of the NAc, relative to the brightest signal, shows vHipp input is uniquely concentrated in the medial NAc shell (n = 6, 4, and 6 for the vHipp, amygdala, and prefrontal cortex pathways, respectively; one-way ANOVA for vHipp input, F_(2,15) = 13.0, P < 0.01; post hoc tests of medial shell versus core and lateral shell, P < 0.01 for both; one-way ANOVA for other two pathways, P > 0.05). Images are counterstained with the nuclear dye DAPI (blue). All values are mean + SEM and * indicates p < 0.05. Abbreviations: ac = anterior commissure, AcbC = nucleus accumbens core, AcbSh = nucleus accumbens shell, Amyg = amygdala, vHipp = ventral hippocampus, PFC = prefrontal cortex. Scale bars are 1 mm.

Figure 2. More Neurons Project to the Medial NAc Shell from the vHipp than from the Basolateral Amygdala or Prefrontal Cortex

(A) A representative coronal brain slice showing injection site of the retrograde tracer Fluoro-Gold (red) in the medial NAc shell. (B) Representative coronal brain slices showing immunolabeled-Fluoro-Gold (red) in NAc shell-projecting cells in the vHipp, basolateral amygdala and prefrontal cortex. (C) In slices from each region that contained dense populations of NAc-projecting cells, more medial NAc shell-projecting neurons were found the vHipp than in either the basolateral amygdala or prefrontal cortex (n = 3 for each area; one-way analysis of variance (ANOVA), F_(2,6) = 29.1, P < 0.01; post hoc tests of vHipp versus amygdala and prefrontal cortex, P < 0.01 for both). (D) Greater immunolabeled-Fluoro-Gold fluorescence was measured throughout the extent of the vHipp than throughout the basolateral amygdala or prefrontal cortex, indicating a larger population of medial NAc-shell projecting neurons in that region (n = 3 for each area; one-way ANOVA, F_(2,6) = 29.1, P < 0.01; Fisher’s least significant difference post hoc tests of vHipp versus amygdala and prefrontal cortex, P < 0.05 for both). See also Figure S2. Scale bars are 1 mm.

Figure 3. Pathway-Specific EPSCs Are Largest Following Optical Stimulation of vHipp Input to the NAc Shell

(A) Representative optically-evoked EPSCs recorded in medium spiny neurons from the NAc shell. (B) EPSCs elicited from vHipp fibers are larger than those evoked from amygdala or prefrontal cortex inputs (n = 53, 40 and 50 for the vHipp, amygdala and prefrontal cortex, respectively; one-way ANOVA, F_(2,140) = 21.4, P < 0.01; post hoc tests of vHipp versus amygdala and prefrontal cortex, P < 0.01 for both). See also Figure S3. (C) Paired pulse ratios (P₂/P₁) obtained with 50 ms inter-pulse intervals show that amygdala input exhibits paired pulse facilitation, suggesting a lower presynaptic vesicle release probability in this pathway (n = 52, 39 and 37 for the vHipp, amygdala and prefrontal cortex, respectively; one-way ANOVA, F_(2,125) = 9.6, P < 0.01; post hoc tests of amygdala versus vHipp and prefrontal cortex, P < 0.01 for both). (D) Representative asynchronous EPSCs (asEPSCs) obtained from the optical stimulation of selective afferents to the medial NAc shell. Traces are clipped during the initial release event to highlight subsequent asEPSCs. (E) Averaged asEPSCs from representative cells (left). Summary of asEPSC amplitudes in each pathway (right; n = 7, 5, 6 for vHipp, amygdala, prefrontal cortex inputs, respectively; one-way ANOVA, F_(2,15) = 1.98, P > 0.05).

Figure 4. NMDARs at vHipp to NAc Synapses Pass Proportionally More Inward Current

(A) Optically-evoked, AMPAR-mediated currents recorded at several holding potentials (+40, +25, +10, −5, −25, −45, −65 mV; top). Summary of normalized current-voltage relationships in pathway-specific AMPAR populations (bottom; n = 10, 5, 8 for vHipp, amygdala, prefrontal cortex inputs, respectively; repeated measures ANOVA, pathway effect, F_(2,120) = 0.59, P > 0.05). (B) Optically-evoked, NMDAR-mediated currents recorded at several holding potentials (+35, +15, −5, −25, −45, −65, −85 mV; top). Summary of normalized current-voltage relationships in pathway-specific NMDAR populations shows vHipp to NAc synapses pass proportionally more peak inward current than other synapses (bottom; n = 6, 6, 4 for vHipp, amygdala, prefrontal cortex inputs, respectively; repeated measures ANOVA, pathway effect, F_(2,78) = 13.08, P < 0.001; post hoc test of pathway effect between vHipp and both other inputs at −25, −45 and −65 mV, p < 0.05).

Figure 5. vHipp Afferents to the NAc Shell Are Selectively Potentiated Following Cocaine Injections

(A) Experimental timeline showing brain slice recordings were obtained 10 to 14 days after 5 daily injections of either cocaine or saline. (B) The summary of asynchronous EPSC amplitudes obtained in the medial NAc shell show selective cocaine-induced increases in the quantal amplitude of vHipp input (for the saline and cocaine groups, respectively, n = 7 and 7 for the vHipp, n = 5 and 5 for the amygdala, n = 6 and 5 for the prefrontal cortex; two-way ANOVA, cocaine main effect, F_(1,29) = 5.4, P < 0.05). (C) Representative AMPA and NMDA receptor-mediated currents recorded at +40 mV in the medial NAc shell from the optical stimulation of different inputs in saline- and cocaine-treated mice. (D) Summary of AMPA/NMDA receptor response ratios show vHipp input is selectively potentiated following repeated cocaine injections (for the saline and cocaine groups, respectively, n = 10 and 9 for the vHipp, n = 13 and 12 for the amygdala, n = 8 and 7 for the prefrontal cortex; two-way ANOVA, significant interaction, F_(2,53) = 4.6, P < 0.05; post hoc test of cocaine effect on vHipp input, p < 0.01). (E) Summary of normalized current-voltage relationships in vHipp to NAc shell synaptic AMPAR populations in naïve and cocaine-treated mice (n = 10 and 6 for naïve and cocaine-treated mice, respectively). See also Figure S4.

Figure 6. Activity of vHipp Axons in the NAc Drives Cocaine-Induced Locomotion

(A) In an unfamiliar environment, optical inhibition of vHipp axons in the NAc reduces cocaine-induced locomotion (n = 6 for both groups; repeated measures ANOVA, NpHR main effect, F_(1,89) = 70.6, P < 0.01; post hoc test of group effects on days 2–9, p < 0.05). This effect strengthens over time (days 1–5) and dissipates in the absence of optical inhibition (days 8–10). During the first session, labeled day S, mice were only given saline injections. (B) Optical inhibition does not alter locomotor activity of cocaine-naïve mice, measured daily in an open field chamber (n = 4 for both groups; repeated measures ANOVA, NpHR effect, F_(1,30) = 0.6, P > 0.05). (C) In animals’ home cages, optical activation of vHipp axons in the NAc enhances cocaine-induced locomotion (n = 6 and 7 for control and ChR2 groups, respectively; repeated measures ANOVA, ChR2 main effect, F_(1,77) = 24.9, P < 0.01). This effect does not persist in the absence of optical activation (t₁₁ = 0.9, p > 0.05). (D) Optical stimulation does not alter locomotor activity of cocaine-naïve mice, measured daily in an open field chamber (n = 4 for both groups; repeated measures ANOVA, ChR2 effect, F_(1,30) = 0.6, P > 0.05). See also Figure S5.

Figure 7. Photostimulation of vHipp Axons in the NAc Can Reinforce Instrumental Behaviors

(A) Summary of time spent in different sides of a modified place preference chamber over five consecutive days in which mice had complete freedom of movement (n = 6; repeated measures ANOVA, significant interaction, F_(4,40) = 24.6, P < 0.01; post hoc test of room effect on day one, p < 0.01). During the three test sessions, vHipp axons in the NAc were optically activated whenever mice entered and remained in the ChR2-paired side of the chamber. (B) Summary of instantaneous room exit probabilities each day in the place preference chamber (n = 6; repeated measures ANOVA, significant interaction, F_(4,40) = 51.7, P < 0.05; post hoc test of room effect on days two and three, p < 0.01). See also Figure S6. (C) Cumulative-activity graph of nose pokes made in the first behavioral session to obtain optical stimulation of vHipp axons in the NAc (n = 9). Solid lines represent the mean and dashed lines are ± SEM. (D) Summary of active and inactive nose poking behavior in ChR2 and EYFP control mice made during the first behavioral session (n = 9 and 6 for ChR2 and EYFP groups, respectively).

Figure 8. Excitatory Input from Different Sources and Direct Stimulation of Medium Spiny Neurons Can Each Reinforce Instrumental Behaviors

(A) Irrespective of the specific pathway activated, mice spend more time on the side of a chamber that is paired with ChR2-mediated activation of glutamatergic axons in the NAc shell (n = 6, 5 and 6 for the vHipp, amygdala, prefrontal cortex input, respectively; repeated measures ANOVA, significant effect of session, F_(4,56) = 19.8, P < 0.01). (B) Summary of active nose pokes made in the third behavioral session to obtain pathway-specific optical activation of ChR2-expressing axons in the NAc (n = 6, 5, and 6 for vHipp, amygdala, prefrontal cortex pathways, respectively). (C) Representative coronal brain slice showing tracts of implanted optical fibers and expression of ChR2-EYFP (green) in the NAc shell after local virus injection. Image is counterstained with the nuclear dye DAPI (blue). (D) Cumulative-activity graph of nose pokes made in the first behavioral session to obtain optical stimulation of medium spiny neurons in the NAc shell (n = 5).