Distinct Ventral Pallidal Neural Populations Mediate Separate Symptoms of Depression

Abstract

Major depressive disorder (MDD) patients display a common but often variable set of symptoms making successful, sustained treatment difficult to achieve. Separate depressive symptoms may be encoded by differential changes in distinct circuits in the brain, yet how discrete circuits underlie behavioral subsets of depression and how they adapt in response to stress has not been addressed. We identify two discrete circuits of parvalbumin-positive (PV) neurons in the ventral pallidum (VP) projecting to either the lateral habenula or ventral tegmental area contributing to depression. We find that these populations undergo different electrophysiological adaptations in response to social defeat stress, which are normalized by antidepressant treatment. Furthermore, manipulation of each population mediates either social withdrawal or behavioral despair, but not both. We propose that distinct components of the VP PV circuit can subserve related, yet separate depressive-like phenotypes in mice, which could ultimately provide a platform for symptom-specific treatments of depression.

Keywords: depression; equine infectious anemia virus; neural circuits; parvalbumin; social defeat stress; susceptibility; ventral pallidum.

Figure 1. Distinct subpopulations of VP PV neurons project to the LHb and VTA

(A) PV neuronal localization in VP. Substance P (SP)-immunostaining denotes boundaries of VP. Scale bars: 100 µm; inset, 50 µm. (B) Schematic of tracing strategy. Cell bodies and efferent fibers in red and presynaptic sites in green using AAV-DIO-mRuby2-T2A-Synaptophysin-eGFP. (C) Representative image of injection site in VP. Scale bar: 80 µm. (D and E) Fibers and synaptic puncta in LHb and VTA from VP PV neurons. Scale: 200 µm; inset, 20 µm. (F) Two possible projection patterns: VP PV neurons send collateralized axons to both the LHb and VTA (top), or independent subpopulations send axons exclusively to either LHb or VTA (bottom). (G and H) Schematic of viral strategy. EIAV is pseudotyped with a fusion protein containing rabies virus glycoprotein and VSVG transmembrane (RG-EIAV; G). Injection of RG-EIAV-DIO-Flp into target area and AAV-fDIO-eGFP into VP of PV-Cre mice. (I) Images of injection site and target areas showing cell-type and projection-specific labeling. VP PV neurons selectively project to either the LHb or VTA, respectively, but not to both. Scale: 200 µm. (J) Light-evoked synaptic responses in LHb or VTA. EPSCs measured at −70 mV, IPSCs at 0 mV. Pie charts indicate whether absolute amplitude was greater for evoked EPSC, IPSC, or no response (individual responses in Figure S4). Top row, LHb neurons; bottom row, VTA neurons distinguished as either GABAergic or dopaminergic. Scales: 50pA, 100ms. (K) Strategy using PV-Flp×Ai14 mouse for labeling VP PV neurons in projection specific manner. RG-EIAV-fDIO-Cre is injected into either LHb (top) or VTA (bottom). (L) mRNA labeling of PV^VP→LHb neurons (top panel) and PV^VP→VTA neurons (bottom panel). Green arrowheads show VGAT⁺ cells, white arrowheads VGluT2⁺ cells. Scale: 10 µm. Pie charts indicate percentage of tdTomato-mRNA⁺ neurons projecting to LHb or VTA colocalizing with probes to VGAT or VGluT2 (n = 154 tdT⁺ cells for VTA-projecting, n = 24 cells for LHb-projecting). EIAV, equine infectious anemia virus; RV G, rabies virus glycoprotein; ac, anterior commissure; SNr, substantia nigra reticulata; MHb, medial habenula; CPu caudate putamen.

Figure 2. Input-output mapping reveals anatomically and molecularly distinct inputs of PV^VP→LHb and PV^VP→VTA neurons

(A) Injection regiment to map pseudotype rabies-mediated monosynaptic inputs to either LHb-projecting or VTA-projecting VP PV neurons (top). Starter cell localization of AAV-fDIO-mRuby2-TVA, AAV-fDIO-oPBG, and EnvA-RVΔG-eGFP (bottom, yellow arrows). Scale: 200 µm. (B) Whole-brain quantitation of inputs to PV^VP→LHb and PV^VP→VTA neurons. Data presented as mean ± SEM, percentage of total cells in a given brain area relative to total number of brain-wide inputs. Unpaired t-tests used for individual brain regions, * P < .05, ** P < .01, n = 4 for each condition. (C) Representative images of inputs in select brain areas. Scale bars: 200 µm. (D) Molecular characterization of CeA inputs to PV^VP→VTA neurons via FISH assay. Quantitation (top) and representative images of mRNA labelling (bottom). n = 257 cells from 2 animals. Scale: 100 µm. (E) Images of CeA neurons sending input to PV^VP→VTA neurons with cell-type specific markers. Colored arrows represent colocalization between rabies⁺ input cells and mRNA for specified probe. Scale: 10 µm. (F) Quantitation of NAc D1R- and D2R-expressing MSN inputs to PV^VP→LHb and PV^VP→VTA neurons. Scale: 100 µm. (G) Representative images in NAc showing colocalization of D1R or D2R in neurons projecting to PV^VP→LHb and PV^VP→VTA neurons. Red arrows denote D1R colocalization, white arrows D2R, yellow arrows co-express D1R and D2R. N = 1505 cells from 2 animals each. Scale: 10 µm. mPFC, medial prefrontal cortext; BNST, bed nucleus stria terminalis; LH, lateral hypothalamus; LGP, lateral globus pallidus; AHA/VMH, anterior hypothalamic area/ventromedial hypothalamus; PAG, periaqueductal grey; RMTg, rostromedial tegmental nucleus; DRN, dorsal raphe nucleus. SST, somatostatin; PENK, preproenkephalin; CRH, corticotropin releasing hormone; PKCδ, protein kinase C delta.

Figure 3. PV^VP→LHb and PV^VP→VTA neurons from susceptible animals exhibit distinct electrophysiological adaptations to SDS and can be reversed by chronic fluoxetine

(A and B) Behavioral paradigm and experimental timeline of repeated chronic social defeat stress (SDS) experiments. (C) Chronic fluoxetine (FLX) treatment ameliorates SDS-induced social withdrawal. Red line indicates bound between susceptible (< 1) and resilient animals (> 1). (D) Schematic for labeling PV^VP→LHb neurons in acute slices. (E and F) Spikes elicited after 100 pA current injection in PV^VP→LHb neurons. One-way ANOVA F_3,34 = 9.088, P < .001. Tukey post-test; *** P < .001, ** P < .01, * P < .05; n = 9, 11, 10, 8 cells from 4 animals each in control, resilient, susceptible, and FLX groups, respectively. Example traces in (F), blue bars represent duration of current injection (500 ms). For full responses see Fig S7A. (G–I) Evoked excitatory and inhibitory inputs (E/I) onto PV^VP→LHb neurons in healthy and SDS groups. Absolute amplitudes (G), ratio of E/I inputs (E/I ratio; H), and example traces (I) of cells recorded. One-way ANOVA F_3,42 = 5.499, P < .01; Tukey post-test: ** P < .01; n = 8, 9, 13, 16 cells from 5 animals each in control, resilient, susceptible, FLX groups, respectively. (J) Schematic for labeling PV^VP→VTA neurons in acute slices. (K–L) Same as in (E and F), but PV^VP→VTA neurons. One-way ANOVA F_3,36 = 8.770, P < .001. Tukey post-test; *** P < .001, ** P < .01, * P < .05; n = 12, 10, 10, 8 cells from 7, 4, 4, and 3 animals in control, resilient, susceptible, and FLX groups, respectively. For full responses see Fig S7B. (M–O) PV^VP→VTA neurons have a significant increase in E/I ratio in susceptible animals that is normalized by FLX. Absolute amplitudes (M), E/I ratio quantitation (N), and example traces (N). One-way ANOVA F_3,50 = 9.585, P < .001; n = 11, 15, 13, 15 cells from 5 animals each in control, resilient, susceptible, FLX groups, respectively. Tukey post-test: *** P < .001. Scale for (F and L): 100 ms, 10 mV; (I and O): 200 pA, 100 ms. Data presented as average mean ± SEM.

Figure 4. Silencing VP PV activity promotes resilience to a subset of depressive-like phenotypes

(A) Experimental timeline of Kir_2.1-mediated silencing. (B–D) No changes in pre-stress measures of locomotion in open field test (OFT, B), anxiety on elevated plus maze (EPM, C), or social interaction (SI, D). n = 6, 11 for eYFP, Kir_2.1, respectively. (E) No significant difference in measures of anhedonia on sucrose preference test (SPT) after SDS. (F) Kir_2.1 animals show significantly reduced time spent immobile on measure of behavioral despair, the tail suspension test (TST) after SDS. U = 69, P < .001; n = 8, 9 for eYFP and Kir_2.1, respectively. (G) Kir_2.1 animals display increased social interaction ratios relative to controls after SDS. U = 13, P < .05; n = 8, 8 for eYFP and Kir_2.1, respectively. All data tested on Mann-Whitney U-test and presented as mean ± SEM. * P < .05, ** P < .01.

Figure 5. PV^VP→LHb and PV^VP→VTA neurons mediate discrete symptoms of depression

(A and B) Schematic of viral injections and optic fiber implantations for optogenetic manipulation in stressed animals. (C and D) Representative traces of animals expressing NpHR in VP PV neurons in social interaction (SI) test in susceptible animals during light stimulation: off (left panel) and light on (589 nm; right panel). Red rectangle indicates cage in which aggressive CD1 animal was placed, yellow dashed lines outline ‘interaction zone.’ Warmer colors indicate increased time spent. (E and F) Silencing terminals of VP PV neurons in VTA, but not in LHb, alleviates social withdrawal symptoms in SI test. SI ratio = time interaction zone light ON / light OFF. Dashed line indicates no change. Each line in (E) represents single animal, bolded line indicates average ± SEM. Mann-Whitney U-test U = 80, P < .05. (F) shows population means of data in (E). VTA SI: one-way ANOVA F_2,21 = 6.132, P < .01. Tukey post-test: * P < .05; n = 5, 9, 10 and n = 6, 7, 9 animals for eYFP, ChR2, NpHR groups in VTA and LHb conditions, respectively. (G and H) Modulation of VP PV neuronal terminals in LHb, but not in VTA induces bidirectional effects on the tail suspension test (TST). For (G), LHb-ChR2: Mann-Whitney U-test U = 56, ** P < .01; LHb-NpHR U = 15, *** P < .001. For (H), LHb TST: one-way ANOVA F_2,25 = 24.49 P < .001. Tukey post-test: * P < .05; n = 5, 8, 11 and 6, 8, 14 for eYFP, ChR2, and NpHR in VTA and LHb groups, respectively. All data reported as mean ± SEM.

Figure 6. Cell type- and projection-specific inhibitory DREADDs recapitulate optogenetic behavior

(A) Experimental setup for expression of inhibitory DREADD (hM4Di) or control virus (mCherry) in PV^VP→LHb or PV^VP→VTA neurons. (B) Representative images of injection site. hM4Di expression in red, Substance P (SP) in blue to delineate VP. (C) Inhibition of PV^VP→VTA neurons expressing hM4Di by CNO injection promotes increased social interaction in susceptible animals. Mann-Whitney U-test U=6, * P < .05. Individual lines represent individual animals. n = 8, 9 for mCherry, hM4Di, respectively. (D) Inhibition of PV^VP→LHb neurons expressing hM4Di by CNO injection has no effect on social interaction in susceptible animals. (E) Changes in SI ratio in response to i.p. CNO injection. Mann-Whitney U-test U=5, * P < .05. (F) No effect of CNO injection in animals expressing hM4Di in PV^VP→VTA neurons on behavioral helplessness as measured by change in time immobile with respect to saline injections. (G) The inhibition of PV^VP→LHb neurons expressing hM4Di by CNO injection reduces time spent immobile relative to saline injections in susceptible animals. Mann-Whitney U-test U=40, * P < .05. n = 8, 7 for mCherry, hM4Di, respectively. (H) hM4Di expression in PV^VP→LHb, but not PV^VP→VTA neurons, reduces time spent immobile in response to CNO injection. Mann-Whitney U-test U=42, * P < .05. n = 8, 7 for mCherry, hM4Di, respectively.

Figure 7. Repeated, chronic stimulation of PV^VP→VTA terminals is sufficient to induce social interaction deficits

(A) Experimental timeline for B–G. (B–D) Repeated stimulation of VP PV terminals in VTA is sufficient to induce social avoidance (B), but has no effect on SPT (C) or TST (D). Mann-Whitney test; U = 162, * p < .05; n = 15, 14 for eGFP and ChR2 groups, respectively for SI and TST test, n = 7 for SPT). (E–G) No effect of repeated stimulation of VP PV terminals in LHb in SI (E), SPT (F), or TST (G); n = 5 each condition. (H–J) No change in depressive like phenotypes after subthreshold defeat stress with VP PV terminal stimulation in LHb (n = 5 each SI, SPT, TST). All data tested on Mann-Whitney U-test and presented as mean ± SEM. Red dashed lines indicate levels separating resilient and susceptible animals (SI ratio of 1 and 50% sucrose preference for SI, SPT, respectively).