Distinct extended amygdala circuits for divergent motivational states

Abstract

The co-morbidity of anxiety and dysfunctional reward processing in illnesses such as addiction and depression suggests that common neural circuitry contributes to these disparate neuropsychiatric symptoms. The extended amygdala, including the bed nucleus of the stria terminalis (BNST), modulates fear and anxiety, but also projects to the ventral tegmental area (VTA), a region implicated in reward and aversion, thus providing a candidate neural substrate for integrating diverse emotional states. However, the precise functional connectivity between distinct BNST projection neurons and their postsynaptic targets in the VTA, as well as the role of this circuit in controlling motivational states, have not been described. Here we record and manipulate the activity of genetically and neurochemically identified VTA-projecting BNST neurons in freely behaving mice. Collectively, aversive stimuli exposure produced heterogeneous firing patterns in VTA-projecting BNST neurons. By contrast, in vivo optically identified glutamatergic projection neurons displayed a net enhancement of activity to aversive stimuli, whereas the firing rate of identified GABAergic (γ-aminobutyric acid-containing) projection neurons was suppressed. Channelrhodopsin-2-assisted circuit mapping revealed that both BNST glutamatergic and GABAergic projections preferentially innervate postsynaptic non-dopaminergic VTA neurons, thus providing a mechanistic framework for in vivo circuit perturbations. In vivo photostimulation of BNST glutamatergic projections resulted in aversive and anxiogenic behavioural phenotypes. Conversely, activation of BNST GABAergic projections produced rewarding and anxiolytic phenotypes, which were also recapitulated by direct inhibition of VTA GABAergic neurons. These data demonstrate that functionally opposing BNST to VTA circuits regulate rewarding and aversive motivational states, and may serve as a crucial circuit node for bidirectionally normalizing maladaptive behaviours.

Figure 1. Optogenetic identification of BNSTv→VTA projection neurons

a. Sagittal image showing the BNSTv→VTA projection (ac, anterior commissure; D, dorsal; V, ventral; P, posterior; A, anterior; scale = 500 µm). b. Optogenetic collision test. c. Example traces from a single CaMKIIa^BNSTv→VTA unit demonstrating antidromic-orthodromic spike collision. d. Significant reduction in antidromic spike fidelity (%) at short antidromic-orthdromic photostimulation intervals (O, orthodromic photostimulation; A, antidromic photostimulation; F_5,65 = 48.63, P < 0.0001; n = 12 units). e. Antidromic spike latencies were significantly greater than orthodromic latencies (P < 0.0001; n = 12 units). f. Antidromic-initiated spikes displayed significantly greater latency stability compared to orthodromic-activated spikes (P < 0.001; n = 12 units). g. Antidromic spikes responded more reliably to 40 Hz photostimulation compared to orthodromic spikes (F_2,18 = 11.2, P = 0.003, n = 4 units). h. Optogenetic identification of BNSTv→VTA projection neurons in behaving mice. i. Representative peri-event histogram and raster of a single unit timelocked to 5 ms antidromic photostimulation. j. Mean first-spike latencies following antidromic photostimulation for all identified CaMKIIa^BNSTv→VTA projection neurons (n = 53 units, n = 7 mice). All values for all figures represent mean ± s.e.m. * P < 0.05, ** P < 0.01.

Figure 2. Excitatory and inhibitory synapses onto non-dopaminergic VTA neurons from neurochemically distinct BNSTv neurons

a – b. ChR2-eYFP in the BNSTv (top) and fibers in the VTA (bottom) in Vglut2-ires-cre (a) and Vgat-ires-cre (b) mice (BNSTld, lateral-dorsal BNST; oval, oval nucleus BNST; D, dorsal; V, ventral; L, lateral; M, medial; green = ChR2-eYFP; cyan = fluorescent Nissl stain; scale bars = 200 µm (top), 20 µm (bottom)). c. Optically-evoked EPSCs recorded in VTA neurons following Vglut2^BNSTv→VTA::ChR2 stimulation before and after application of the glutamate receptor antagonist, DNQX (bottom) (n = 4 cells, P = 0.0307). d. Optically-evoked IPSCs recorded in VTA neurons following Vgat^BNSTv→VTA::ChR2 stimulation before and after application of the GABA_A receptor antagonist, Gabazine (bottom) (n = 4 cells, P = 0.0378. e - f. Location of light-responsive and non light-responsive dopaminergic and non-dopaminergic neurons in horizontal VTA slices following photostimulation of Vglut2^BNSTv→VTA::ChR2 (e) and Vgat^BNSTv→VTA::ChR2 (f) projections. * P < 0.05.

Figure 3. Vglut2^BNSTv→VTA and Vgat^BNSTv→VTA projection neurons display distinct firing patterns in response to foot shock and shock-associated contextual cues

a. Color-coded normalized firing rates for all identified Vglut2^BNSTv→VTA neurons in response to the first foot-shock session. b. Average normalized firing rate of classified shock-excited Vglut2^BNSTv→VTA neurons is significantly altered compared to no effect classified neurons during and following the foot-shock session (F_99,2900 = 3.13, P < 0.0001, n = 34 units, n = 7 mice). Inset: percentages of classified neurons. c. Color-coded normalized firing rates for all identified Vgat^BNSTv→VTA neurons in response to the first foot-shock session. d. Average normalized firing rate of classified shock-inhibited Vgat^BNSTv→VTA neurons is significantly altered compared to no effect classified neurons during and following the foot-shock session (F_99,2600 = 2.66, P < 0.0001, n = 33 units, n = 5 mice). e. Color-coded normalized firing rates of identified Vglut2^BNSTv→VTA neurons in response to cue re-exposure. f. Average normalized firing rate of classified cue-excited Vglut2^BNSTv→VTA neurons is significantly altered compared to no effect classified neurons during and following cue re-exposure (F_99,3100 = 5.135, P < 0.0001, n = 37 units, n = 4 mice). g. Color-coded normalized firing rates of Vgat^BNSTv→VTA neurons in response to cue re-exposure. h. Average normalized firing rate of classified cue-inhibited Vgat^BNSTv→VTA neurons is significantly altered compared to no effect classified neurons during and following cue re-exposure (F_99,4,900 = 8.285, P < 0.0001, n = 56 units, n = 4 mice).

Figure 4. Photostimulation of theVglut2^BNSTv→VTA pathway promotes aversion and anxiety

a. Representative real-time place preference tracks from Vglut2^BNSTv→VTA::ChR2 (top) and control (bottom) mice. b. Intra-VTA infusions of a glutamate antagonist cocktail, followed by Vglut2^BNSTv→VTA::ChR2 stimulation during real-time place preference blocked aversion (F_3,15 = 12.811, P < 0.001, n = 6 mice). c. Representative heat maps displaying average time spent in an open field for 10 min following stimulation from Vglut2^BNSTv→VTA::ChR2 (top) and Vglut2^BNSTv→VTA::Control (bottom) mice. Vglut2^BNSTv→VTA::ChR2 mice spent significantly more time in the corners (P = 0.008) and less time in the center (P = 0.007) of an open field immediately following constant 20 Hz stimulation compared to Vglut2^BNSTv→VTA::Control mice (n = 6 mice per group).

Figure 5. Photostimulation of theVgat^BNSTv→VTA pathway and inhibition ofVgat^VTA neurons produces reward-related behaviors and attenuates anxiety

a. Real-time place preference representative tracks from Vgat^BNSTv→VTA::ChR2 (top) and control (bottom) mice. b. Intra-VTA infusions of the GABA_A antagonist, Gabazine, followed by Vgat^BNSTv→VTA::ChR2 stimulation abolished place preference (F_3,15 = 13.718, P < 0.001, n = 6 mice) cVgat^BNSTv→VTA::ChR2 mice made significantly more nose pokes to obtain photostimulation compared to controls (F_4,36 = 12.42, P < 0.001, n = 5 – 7 mice per group). d. Schematic detailing Vgat^VTA::NpHR inhibition during behavioral experiments. eVgat^VTA::NpHR mice spent significantly more time in the inhibition-paired side when compared to controls (P = 0.01, n = 6 mice per group). fVgat^VTA::NpHR mice made significantly more nose pokes to obtain photoinhibition compared to controls (P < 0.001, n = 5 mice per group). gVgat^BNSTv→VTA::ChR2 mice spent significantly more time in the elevated-plus maze (EPM) open arms compared to controls during the 5 min photostimulation epoch (F_2,24 = 14.648, P < 0.001, n = 7 mice per group). f. After concurrent photostimulation during the foot-shock session, Vgat^BNSTv→VTA::ChR2 mice (n = 6 – 7) spent significantly less time frozen compared to controls (F_1,22 = 37.992, P < 0.001). * P < 0.05, ** P < 0.01, *^#P significant compared to all manipulations.