The medial prefrontal cortex (mPFC) contains populations of GABAergic interneurons that play different roles in cognition and emotion. Their local and long-range inputs are incompletely understood. We used monosynaptic rabies viral tracers in combination with fluorescence micro-optical sectioning tomography to generate a whole-brain atlas of direct long-range inputs to GABAergic interneurons in the mPFC of male mice. We discovered that three subtypes of GABAergic interneurons in two areas of the mPFC are innervated by same upstream areas. Input from subcortical upstream areas includes cholinergic neurons from the basal forebrain and serotonergic neurons (which co-release glutamate) from the raphe nuclei. Reconstruction of single-neuron morphology revealed novel substantia innominata–anteromedial thalamic nucleus–mPFC and striatum–anteromedial thalamic nucleus–mPFC circuits. Based on the projection logic of individual neurons, we classified cortical and hippocampal input neurons into several types. This atlas provides the anatomical foundation for understanding the functional organization of the mPFC.
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The data that support the findings of this study are available in the Supplementary Tables and from the corresponding author upon reasonable request. Data including the whole-brain distribution of the input neurons and the reconstructions of the neural morphology in the neocortex and hippocampus can be accessed at http://atlas.brainsmatics.org/a/sun1903.
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We thank C. Zhou, T. Luo, X. Peng, C. Tan and Z. Duan for help with experiments and data analysis. We thank the Optical Bioimaging Core Facility of HUST for support with data acquisition, as well as the Analytical and Testing Center of HUST for spectral measurements. This work was financially supported by NSFC projects (nos. 61721092, 91632302, 91749209 and 31871088) and the Director Fund of WNLO.
The authors declare no competing interests.
Peer review information: Nature Neuroscience thanks Ian Wickersham and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Integrated supplementary information
a, b. immunostaining against parvalbumin and somatostatin at the injection sites. Parvalbumin is expressed in the TVA-mCherry and EGFP positive neurons in PV-Cre mice and somatostatin is expressed in the TVA-mCherry and EGFP positive neurons in SST-Cre mice (PV+, n=6 mice, SST+, n=6 mice). c-n. Control experiments of RV monosynaptic tracing. c-e. No neuron was labeled when RV-EnvA-GFP was injected into mouse brain alone (n=3 mice). f-j. Only the specific neurons at the injection site were labeled when AAV-DIO-RG was absent (n=4 mice). k. Quantification of AAV/RV labeled neurons that are PV+ (n=4 mice, data shown as mean±s.e.m). l-n. A few neurons were labeled at the injection site when AAV helper virus and RV were injected into the wild type mouse brain but no neuron was labeled outside the injection stite (n=3 mice). Scale bar a,b 100 µm, c-e, 200 µm, f.1mm, g-j. 50 µm, l-n. 1 mm
a. Representatitve coronal sections showing distribution of PV, SST and VIP positive neurons. The PV, SST and VIP neurons were labeled by crossing PV, SST and VIP-Cre driver lines with Ai14 reporter line. b. Quantification of the layer distribution of PV, SST and VIP positive neurons in mPFC. (SST+, n=3 mice; PV+, n=3 mice; VIP+, n=3 mice, data shown as mean±s.e.m) Scale bar a. 1 mm.
a. Representatitve coronal sections showing EGFP labeled cells input to SST, PV and VIP positive neurons in different brain regions with PL targeting. The projections were 50µm. b. A sample of 210-serial section raw data set of RV labeled mouse brain imaged by fMOST system(SST+, n=18 mice; PV+, n=14 mice; VIP+, n=10 mice). The projections were 50µm. Scale bar, a.1 mm.
Supplementary Figure 4 Direct comparison of the long range inputs to SST positive neurons in two subregions of mPFC within individual brain sample.
a, Representatitve coronal sections showing EGFP labeled cells and DsRed labeled cells in different brain areas. The sections were registered to the Allen CCF3.0. b, The distribution of EGFP labeled cells and DsRed labeled cells in different brain regions (For dual-color RV labeling, SST+, n=2 mice, PV+, n=2 mice, VIP+, n=2 mice). Scale bars are 1 mm in a and b.
a, b. Conjugated CTb with different fluorescence was injected into PL and ILA, respectively. c-j. The CTb labeled neurons in major brain areas that innervate PL and ILA were shown in c-j. The number of PL projecting, ILA projecting and double projecting neurons were also quantified (one-way ANOVA followed by Tukey’s post hoc tests, **p < 0.01, ***p<0.001, ****p<0.0001, for detailed p value, see supplementary table 3, n=3 mice, data shown as mean±s.e.m). Scale bars in a and b are 1mm, those in c-j are 100µm.
Supplementary Figure 6 Quantitative analysis of whole-brain monosynaptic inputs to different GABAergic neurons in the medial prefrontal cortex at grouped brain area level, related to Fig 2.
(one-way ANOVA followed by Tukey’s post hoc tests, *p < 0.05, ***p < 0.001, ****p<0.0001,for detailed p value, see main article text, SST+, n=18 mice; PV+, n=14 mice; VIP+, n=10 mice, data shown as mean±s.e.m).
Supplementary Figure 7 Representative images showing the brain areas that differently innervated SST+, PV+ and VIP+ neurons in mPFC, related to Fig. 2.
PV+ and VIP+ neurons receive more inputs from some cortical areas (a, c, f) while SST+ neurons receive more inputs from some subcortical nuclei (b, d, e, g, h)(SST+, n=18 mice; PV+, n=14 mice; VIP+, n=10 mice). Scale bar 100 µm.
Supplementary Figure 8 Quantitative analysis of the differences between the inputs of prelimbic area and infralimbic area.
a. Brain areas that differently innervated SST+ neurons in PL and IL. b. Brain areas that differently innervated VIP+ neurons in PL and IL.(one-way ANOVA followed by Tukey’s post hoc tests, *p < 0.05, **p < 0.01, for detailed p value, see main article text, SST+, n=18 mice; PV+, n=14 mice; VIP+, n=10 mice, data shown as mean±s.e.m).
Supplementary Figure 9 Analysis of the relationship between spatial distribution of starter cells and input patterns.
a. A 3-dimensional view of the spatial distribution of starter cells in PL and ILA. The starter cells of PL targeting sample were shown in red and the starter cells of ILA targeting sample were shown in green. b. The mean vertical distance to midline of the starter cells in SST, PV and VIP-Cre mice (SST, n=12 mice, PV, n=8 mice, VIP, n=8 mice; one-way ANOVA followed by Tukey’s post hoc tests, SST+ versus VIP+, p=0.045, PV+ vs VIP+, p=0.009, data shown as mean±s.e.m). c. The mean vertical distance to brain surface of the starter cells in PL targeting samples and ILA targeting samples (SST, n=12 mice, PV, n=8 mice, VIP, n=8 mice, two-tailed unpaired t test, p<0.0001, data shown as mean±s.e.m). d. The mean vertical distance to the coronal plane under bregma point of the starter cells in PL targeting samples and ILA targeting samples (SST, n=12 mice, PV, n=8 mice, VIP, n=8 mice, two-tailed unpaired t test, p<0.0001, data shown as mean±s.e.m) (Bregma point is defined as the point on the top of the skull where the coronal and sagittal sutures meet). e. Linear regression analysis with mean vertical distance to midline, brain surface and the coronal plane under bregma point. f. Linear regression analysis with COM, Cre line and COM Cre line combination. (one-way ANOVA followed by Tukey’s post hoc tests, *p < 0.05, **p < 0.01, ****p<0.0001, SST, n=12 mice, PV, n=8 mice, VIP, n=8 mice) COM, starter cell center of mass.
Supplementary Figure 10 Characterization of the neurochemical properties of input neurons by double immunochemical staining, related to Fig. 3.
a. Input neurons in HDB express choline acetyltransferase but not EAAC1 (SST-Cre, n=6 mice; PV-Cre, n=6 mice; VIP-Cre, n=8 mice). b. Input neurons in HDB express EAAC1 but not choline acetyltransferase (SST-Cre, n=6 mice; PV-Cre, n=6 mice; VIP-Cre, n=8 mice). c. Input neurons in dorsal raphe nucleus express TPH2 but not EAAC1 (SST-Cre, n=6 mice; PV-Cre, n=6 mice; VIP-Cre, n=8 mice).d. Input neurons in dorsal raphe nucleus express EAAC1 but not TPH2 (SST-Cre, n=6 mice; PV-Cre, n=6 mice; VIP-Cre, n=8 mice). e, f. Input neurons in VTA express EAAC1 and TH, respectively (SST-Cre, n=6 mice; PV-Cre, n=6 mice; VIP-Cre, n=8 mice). g. Quantification of EGFP labelled neurons in VTA that are Dopaminergic and EAAC1 positive (SST-Cre, n=6 mice; PV-Cre, n=6 mice; VIP-Cre, n=8 mice, data shown as mean±s.e.m). h. Colocalization rate of chat and EAAC1 expression of neurons in basal forebrain that project to mPFC (SST-Cre, n=6 mice; PV-Cre, n=6 mice; VIP-Cre, n=8 mice, data shown as mean±s.e.m). i. Colocalization rate of TPH2 and EAAC1 expression of neurons in raphe nuclei that project to mPFC. (SST-Cre, n=6 mice; PV-Cre, n=6 mice; VIP-Cre, n=8 mice, data shown as mean±s.e.m) Scale bar=50 µm.
Supplementary Figure 11 Neuron in anteromedial thalamic nucleus that project to mPFC receive inputs from multiple brain areas, related to Fig. 5.
a.Brain areas that innervated AM and mPFC. b. Brain areas that innervated AM alone. n=6 mice. Scale bar=100 µm.
Supplementary Figure 12 Characterization and quantification of the neurons that directly project to mPFC and indirectly connect with mPFC via mPFC projecting neurons in AM.
a, b. Both cholinergic and non-cholinergic neurons in SI can project to mPFC and form synaptic connections with neurons in AM that project to mPFC (n=4 mice). c. Quantification of mPFC projecting neurons, AM projecting neurons and double projecting neurons in SI. d. Quantification of double projecting neurons in SI that are cholinergic. e-h. Quantification of mPFC projecting neurons, AM projecting neurons and double projecting neurons in MOs, LHA, HDB and CA1. (For quantification of neurons in SI, MOs, HDB, n=4 mice; for quantification of neurons in LHA, CA1, n=3 mice, data shown as mean±s.e.m)Scale bars in a and b are 50 µm.
Seven different types of cortical neurons innervated GABAergic neurons in mPFC.
Supplementary Figure 14 Quantitative comparison of the fine morphology of four types of different pyramidal neurons in layer V that target GABAergic neurons in mPFC, related to fig. 7.
Histograms of the dendrite length and the apical dendrite length are shown in a, b, respectively.(a, one-way ANOVA followed by Tukey’s post hoc tests, *p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001, callosal neurons, n=19, corticofugal neurons, n=17, corticospinal neurons, n=5, associative neurons, n=16, callosal neurons vs corticofugal neurons, p=0.0084, callosal neurons vs corticospinal neurons, p<0.0001, corticofugal neurons vs corticospinal neurons, p=0.033, corticospinal neurons vs associative neurons, p<0.0001, data shown as mean±s.e.m; b, two-tailed unpaired t test, p=0.0006, data shown as mean±s.e.m).
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Frontiers in Neuroscience (2019)