Ventral tegmental area-basolateral amygdala-nucleus accumbens shell neurocircuitry controls the expression of heroin-conditioned immunomodulation

Abstract

The present investigations sought to determine whether the ventral tegmental area (VTA), basolateral amygdala (BLA), and nucleus accumbens shell (NAC) comprise a circuitry that mediates heroin-induced conditioned immunomodulation. Rats were given conditioning trials in which they received an injection of heroin upon placement into a distinctive environment. Prior to testing, rats received unilateral intra-BLA microinfusion of a D(1) antagonist concomitantly with unilateral intra-NAC shell microinfusion of an NMDA antagonist. Disconnection of the VTA-BLA-NAC circuit impaired the ability of the heroin-paired environment to suppress lipopolysaccharide-induced immune responses, defining for the first time a specific neural circuit involved in conditioned neural-immune interactions.

Figure 1

Schematic representation of injection cannula placements. The symbols on the schematics (Paxinos and Watson, 1997) represent the most ventral point of the infusion cannula tracts for rats that received bilateral microinfusions into the NAC for experiment 1 (saline/home cage: closed squares; saline/heroin-paired context: open squares; AP-5/home cage, closed triangles; AP-5/heroin-paired context, open triangles; CNQX/home cage, closed circles; CNQX/heroin-paired context, open circles). For experiment 2, symbols represent the most ventral point of the infusion tract for rats receiving unilateral microinfusions into the BLA plus the contralateral NAC (home cage, closed circles; heroin-paired context, open circles) or the ipsilateral NAC (home cage, closed triangles; heroin-paired context, open triangles), or a control microinfusion of saline into the BLA and NAC (home cage, closed squares; heroin-paired context: open squares). Numbers indicate the distance from bregma in millimeters.

Figure 2

Effect of antagonist treatment and context on LPS-induced expression of iNOS mRNA in the spleen as determined by real-time RT-PCR (top panel) and nitrite/nitrate production in the serum as determined by the Greiss reagent assay (bottom panel). Exposure to the previously heroin-paired context (Saline, white bar) resulted in conditioned suppression of iNOS mRNA levels in the spleen and a conditioned suppression of the byproducts of nitric oxide production in the serum as compared to the home cage control group (Saline, black bar). Intra-NAC microinfusion of NMDA or AMPA/kainate receptor antagonist blocked the effect of the conditioned stimulus (AP5/CNQX, white bar). Real-time RT-PCR data are expressed as iNOS copy number per 10 ng cDNA based on a standard curve using Roche LightCycler software. Nitrite/nitrate data are expressed as the micromolar concentration of nitrite/nitrate.

Figure 3

Effect of antagonist treatment and context on LPS-induced expression of TNF-α mRNA in the spleen as determined by real-time RT-PCR (top panel) and TNF-α protein levels as determined by ELISA (bottom panel). Exposure to the previously heroin-paired context (Saline, white bar) resulted in a conditioned suppression of TNF-α mRNA and protein levels in the spleen as compared to the home cage control group (Saline, black bar). Intra-NAC microinfusion of NMDA or AMPA/kainate receptor antagonist blocked the effect of the conditioned stimulus (AP5/CNQX, white bar). Real-time RT-PCR data are expressed as TNF-α copy number per 10 ng cDNA based on a standard curve using Roche LightCycler software. ELISA data are expressed as pg of TNF-α protein per mg of total protein.

Figure 4

Effect of antagonist treatment and context on LPS-induced expression of IL-6 mRNA in the spleen as determined by real-time RT-PCR (top panel) and IL-6 protein in the spleen as determined by ELISA (bottom panel). Exposure to the previously heroin-paired context (Saline, white bar) resulted in a conditioned suppression of IL-6 mRNA levels in the spleen as compared to the home cage control group (Saline, black bar). Intra-NAC microinfusion of NMDA or AMPA/kainate receptor antagonist blocked the effect of the conditioned stimulus (AP5/CNQX, white bar). Real-time RT-PCR data are expressed as iNOS copy number per 10 ng cDNA based on a standard curve using Roche LightCycler software. ELISA data are expressed as pg of IL-6 protein per mg of total protein.

Figure 5

Effect of antagonist treatment and context on LPS-induced expression of iNOS mRNA in the spleen as determined by real-time RT-PCR (top panel) and nitrite/nitrate production in the serum as determined by the Greiss reagent assay (bottom panel). Exposure to the previously heroin-paired context (Saline, white bar) resulted in a conditioned suppression of iNOS mRNA levels in the spleen and a conditioned suppression of the byproducts of nitric oxide production in the serum as compared to the home cage control group (Saline, black bar). Disconnection of the VTA-BLA-NAC circuit blocked the effect of the conditioned stimulus (Contralateral, white bar). Ipsilateral administration of intra-NAC AP5 and intra-BLA SCH23390 did not alter the conditioned suppression (Ipsilateral, white bar). Real-time RT-PCR data are expressed as iNOS copy number per 10 ng cDNA based on a standard curve using Roche LightCycler software. Nitrite/nitrate data are expressed as the micromolar concentration of nitrite/nitrate.

Figure 6

Effect of antagonist treatment and context on LPS-induced expression of TNF-α mRNA in the spleen as determined by real-time RT-PCR (top panel) and TNF-α protein levels as determined by ELISA (bottom panel). Exposure to the previously heroin-paired context (Saline, white bar) resulted in a conditioned suppression of TNF-α mRNA and protein levels in the spleen as compared to the home cage control group (Saline, black bar). Disconnection of the VTA-BLA-NAC circuit blocked the effect of the conditioned stimulus (Contralateral, white bar). Ipsilateral administration of intra-NAC AP5 and intra-BLA SCH23390 did not alter the conditioned suppression of TNF-α (Ipsilateral, white bar). Real-time RT-PCR data are expressed as TNF-α copy number per 10 ng cDNA based on a standard curve using Roche LightCycler software. ELISA data are expressed as pg of TNF-α protein per mg of total protein.

Figure 7

Effect of antagonist treatment and context on LPS-induced expression of IL-6 mRNA in the spleen as determined by real-time RT-PCR (top panel) and IL-6 protein in the spleen as determined by ELISA (bottom panel). Exposure to the previously heroin-paired context (Saline, white bar) resulted in a conditioned suppression of IL-6 mRNA and protein levels in the spleen as compared to the home cage control group (Saline, black bar). Disconnection of the VTA-BLA-NAC circuit blocked the effect of the conditioned stimulus (Contralateral, white bar). Ipsilateral administration of intra-NAC AP5 and intra-BLA SCH23390 did not alter the conditioned suppression of IL-6 (Ipsilateral, white bar). Real-time RT-PCR data are expressed as IL-6 copy number per 10 ng cDNA based on a standard curve using Roche LightCycler software. ELISA data are expressed as pg of IL-6 protein per mg of total protein.