Fig 1.
Experimental design for cell type-specific trans-synaptic tracing using PRV-Introvert-GFP, a modified conditional alpha-Herpes Bartha virus, in SST-Cre mice.
A) Cre-dependent Introvert sequence used for cell type-specific tracing. B) Experimental timeline for determination of monosynaptic time point. We collected tissue from 2 mice every 12 hr and observed GFP expression in known NAc afferents. By collecting sections at serial time points, we determined time points at which viral labeling is first present in known NAc afferents. PFC–Prefrontal cortex, AMY–amygdala, HYP–hypothalamus, VTA–ventral tegmental area. These four regions shown as examples of a larger number of known input regions (see Fig 2).
Fig 2.
Time course for PRV-Introvert-GFP tracing.
Representative sections from each 24 hr time point following infection with PRV-Introvert-GFP in SST-Cre mice. Scale bar = 250 μm. The first GFP(+) cells were identified in NAc at 24 hr post infection (P.I.). Representative sections shown for the following regions: PFC–prefrontal cortex, INS–insular cortex, PIR–piriform cortex, NAC–nucleus accumbens, ENT–entorhinal cortex, VTA–ventral tegmental area.
Fig 3.
First order synaptic afferents to nucleus accumbens somatostatin interneurons.
A) Experimental design for cell type-specific retrograde trans-synaptic tracing using PRV-Introvert-GFP in SST-Cre mice. The tracing data shown are from three mice, but we have replicated the tracing in three separate experiments with consistent findings obtained. B) Trans-synaptic tracing of afferents to NAc somatostatin interneurons. GFP is shown in Green. PFC–prefrontal cortex (IL+PL), AON/OLF–anterior olfactory nucleus, PIR–piriform cortex, AI/GU–anterior insular/gustatory cortex, BLA–basolateral amygdala, AMY–amygdala (non-BLA), ENTl–entorhinal cortex, HYP–hypothalamus, PVT–periventricular nucleus of the thalamus, RE–reuniens nucleus, SUB–ventral subiculum, VTA–ventral tegmental area, NAc–nucleus accumbens. TH–tyrosine hydroxylase (Red). Scale bar = 100 μm. C) Synaptic connectivity of NAc somatostatin interneurons. Baseline connectivity of mouse NAc was adapted from the Allen Brain Atlas (http://connectivity.brain-map.org/). Baseline connectivity strength for overall NAc was set at a maximum of 3.5 and used as a cutoff for counting a region as part of the global NAc connectome. The width of the line connecting each node/region to the NAc corresponds to the reported baseline connectivity strength for overall NAc. We observed GFP+ cells in green nodes shown in (C), while we did not detect GFP in regions shown in black nodes. The size of the green node represents the number of GFP+ cells that were labeled in each region after tracing in SST-Cre mice. For every region shown, the width of the line connecting the node to the NAc represents the reported/expected strength of each connection. (D) Quantification of results shown in network diagram.
Fig 4.
NAc somatostatin interneurons are innervated by first order afferents, a subset of which are activated during exposure to cocaine.
Sections of prefrontal cortex (PFC), basolateral amygdala (BLA), ventral subiculum (vSUB), and VTA at the 48 hr monosynaptic time point are shown with c-Fos staining in red. Only a subset of GFP(+) cells in each region express c-Fos 60 min after exposure to cocaine. Scale bars = 100 μm.
Fig 5.
NAc somatostatin interneurons are innervated by VTA dopamine neurons.
A section of VTA at the 48 hr monosynaptic time point is shown with TH staining in red. All GFP(+) cells identified express TH. Scale = 100 μm.