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Ventral striatal islands of Calleja neurons control grooming in mice

A Publisher Correction to this article was published on 06 January 2022

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Abstract

The striatum comprises multiple subdivisions and neural circuits that differentially control motor output. The islands of Calleja (IC) contain clusters of densely packed granule cells situated in the ventral striatum, predominantly in the olfactory tubercle (OT). Characterized by expression of the D3 dopamine receptor, the IC are evolutionally conserved, but have undefined functions. Here, we show that optogenetic activation of OT D3 neurons robustly initiates self-grooming in mice while suppressing other ongoing behaviors. Conversely, optogenetic inhibition of these neurons halts ongoing grooming, and genetic ablation reduces spontaneous grooming. Furthermore, OT D3 neurons show increased activity before and during grooming and influence local striatal output via synaptic connections with neighboring OT neurons (primarily spiny projection neurons), whose firing rates display grooming-related modulation. Our study uncovers a new role of the ventral striatum’s IC in regulating motor output and has important implications for the neural control of grooming.

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Fig. 1: The islands of Calleja contain densely packed granule cells expressing the D3 dopamine receptor.
Fig. 2: Activation of olfactory tubercle D3-Cre/ChR2 neurons induces grooming.
Fig. 3: Activation of olfactory tubercle D3-Cre/ChR2 neurons induces grooming while suppressing alternative ongoing behaviors.
Fig. 4: Inactivation of D3-Cre/eArchT neurons halts ongoing grooming.
Fig. 5: Ablation of olfactory tubercle D3 neurons reduces spontaneous grooming.
Fig. 6: Whole-brain mapping of presynaptic partners of olfactory tubercle D3 neurons.
Fig. 7: Olfactory tubercle D3 neurons make local synaptic contacts.
Fig. 8: Olfactory tubercle neurons show grooming-related activity.

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Data availability

The raw data generated and/or analyzed during the current study are available from the corresponding authors on reasonable request. The Allen Mouse Brain Connectivity Atlas (https://connectivity.brain-map.org/transgenic/experiment/304168043/) was used for Fig. 1d and the Allen Mouse Brain Common Coordinate Framework version 3 (https://scalablebrainatlas.incf.org/mouse/ABA_v3/) was used to outline brain structures in Figs. 2b and 4a and Extended Data Figs. 4 and 5. Source data are provided with this paper.

Code availability

All commercial software used to collect and analyze the data in this study are described. Custom code for CLARITY brain imaging has been previously published and is publicly available74,75.

Change history

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Acknowledgements

We thank Acal BFi and Andor for providing access to the Dragonfly 500 spinning-disk confocal microscope platform. This work was supported by the NIH (R01NS117061 to D.W.W., M.V.F. and M.M., R01DC006213 to M.M., R01DA049545 and R01DA049449 to M.M. and D.W.W., R01DC016519 and R01DC014443 to D.W.W., R01MH118369 to M.V.F., R21DC019193 to J.P.B., F32DC018452 to K.N.W., F31DC017054 to M. Schreck and F31MH124372 to E.J.), by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation; 368482240/GRK2416 to M. Spehr and 269953372/GRK2150 to J.M. and M. Spehr) and by the Whitehall Foundation and Foundation for OCD Research to M.V.F. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization, Y.-F.Z., D.W.W., M. Spehr, M.V.F. and M.M.; methodology, all authors; investigation, Y.-F.Z., L.V.C., K.N.W., J.P.B., J.M., D.F., E.J., S.L.C., N.G., M. Schreck, A.H.M., Y.Y., J.S. and D.W.W.; formal analysis, data curation and visualization, Y.-F.Z., L.V.C., K.N.W., J.P.B., J.M., D.F., E.J., C.J., J.S., D.W.W., M. Spehr, M.V.F. and M.M.; writing—original draft, Y.-F.Z., D.W.W., M. Spehr, M.V.F. and M.M.; writing—review and editing, all authors; resources, B.R.A., J.N.B., W.L., J.S., D.W.W., M. Spehr, M.V.F. and M.M.; supervision and funding acquisition, D.W.W., M. Spehr, M.V.F. and M.M.

Corresponding authors

Correspondence to Daniel W. Wesson, Marc Spehr, Marc V. Fuccillo or Minghong Ma.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks Eric Burguiere, Christiane Schreiweis, Xin Jin, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Quantification of D3-Cre/tdTomato neurons in the ventral striatum.

a, Left, ventral view of location(s) of D3-Cre/tdTomato neurons within a mouse brain mapped onto a sample-adjusted version of the Allen Mouse Brain Atlas. The OT and hippocampus are outlined as light blue volumes. Right, 3D projection of the OT region outlined (black rectangle). Scale bars = 400 µm (upper left), 100 µm (upper right), 150 µm (lower left), and 50 µm (lower right). b, Left, frontal view projection of the location(s) of D3-Cre/tdTomato neurons. Right, 3D projection of the ventral striatum. Scale bar = 300 µm. c, Left, frontal side view projection of the location(s) of D3-Cre/tdTomato neurons. Right, 3D surface rendering of the IC network. Scale bar = 500 µm. d-e, Quantification of D3 neurons in the ventral striatum and ventral pallidum. Absolute numbers of dense versus loose neurons in d, total numbers of neurons in e (left) and cell density in e (right) in the VP, the NAc and the OT. n = 6 hemispheres from 3 mice. All averaged data are shown as mean ± s.e.m. OT, olfactory tubercle. HF, hippocampal formation. IC, islands of Calleja. islm, major island. VP, ventral pallidum. NAc, nucleus accumbens.

Source data

Extended Data Fig. 2 Quantification of the IC between hemispheres and among individuals.

a, Left, 3D reconstruction of D3-Cre/tdTomato neurons in the OT demonstrating that the IC form a continuous branched network. Similar results were observed in 6 OTs from 3 mice. Right, map of registered neurons categorized as dense or loose neurons, respectively. Scale bars = 600 µm (left) and 500 µm (right). b, Top, 3D reconstructions of OT IC structures in individual hemispheres (left and middle panel), and the area of maximal overlap (right panel). Bottom left, IC overlap between the two hemispheres from the same mouse. Mirrored 3D objects were merged and aligned to create maximum overlap (white voxels). Scale bar = 500 µm. Bottom right, maximum volume overlap of the IC network between two hemispheres and among individuals. All averaged data are shown as mean ± s.e.m.

Source data

Extended Data Fig. 3 D3-Cre/tdTomato neurons in the piriform cortex, the hypothalamus and the hippocampus.

Location(s) of D3 neurons within the mouse brain are mapped onto a sample-adjusted version of the Allen Mouse Brain Atlas. The OT and hippocampal formation (HF) are outlined as light blue volumes. a, D3 neurons in the piriform cortex do not project to targets outside this region. a’, 3D projection of the piriform cortex region outlined in a (black rectangle). Different areas are shown at higher magnification. Note that no projection fibers are evident; in frontal view, D3 neurons appear to adhere to a layer-specific organization. Scale bars = 500 μm (left), 200 μm (middle), and 100 μm (right). b, Two areas in the hypothalamus harbor D3 neurons. Of these, neurons in the caudal aspect of the hypothalamus exhibit some projections. b’, 3D maximum projection of the hypothalamic region outlined in b (black rectangle). Different areas are shown at higher magnification. Relatively sparse, but consistent tdTomato expression is observed at both a relatively caudal and rostral region within the hypothalamus (close to midline). Note that few fibers are evident at the caudal site, whereas no fibers are found at the rostral site. Scale bars = 1000 μm (left), 300 μm (middle), and 100 μm (right). c, D3 neurons in the hippocampus. c’, 3D projection of the hippocampal region outlined in c (black rectangle). Different areas are shown at higher magnification. Scale bars = 1000 μm (left), 1000 μm (middle, upper panel), 100 μm (middle, lower panel), 200 μm (right, upper panel) and 100 μm (right, lower panel). Similar results were observed in 3 mice for a-c.

Extended Data Fig. 4 Retrograde tracing of presynaptic partners of OT D3 neurons.

Representative images showing labeled presynaptic partners of OT D3 neurons from the anterior (upper left) to posterior brain sections (lower right). Similar results were observed in 3 mice. MOB, main olfactory bulb. ORB, orbital area. AI, agranular insular area. AON, anterior olfactory nucleus. PC, piriform cortex. NAc, nucleus accumbens. VP, ventral pallidum. OT, olfactory tubercle. LS, lateral septal nucleus. NDB, diagonal band nucleus. SH, septohippocampal nucleus. LPO, lateral preoptic area. LHA, lateral hypothalamic area. AMY (AAA), anterior amygdalar area. AMY (CEA), central amygdalar nucleus. AMY (MEA), medial amygdalar nucleus. AMY (LA), lateral amygdalar nucleus. AMY (COA), cortical amygdalar area. PVH, paraventricular hypothalamic nucleus. TU, tuberal nucleus. ARH, arcuate hypothalamic nucleus. PH, posterior hypothalamic nucleus. PMv, ventral premammillary nucleus. TH, thalamus (mostly in the subparafascicular area). AMY (BLA), basolateral amygdalar nucleus. SN, substantia nigra. VTA, ventral tegmental area. MRN, midbrain reticular nucleus. PAG, periaqueductal gray. RAmb (CRN), medbrain raphe nuclei, central part. RM, nucleus raphe magnus. PRNc, pontine reticular nucleus, caudal part. RAmb (DRN), medbrain raphe nuclei, dorsal part. PCG, pontine central gray. PB, parabrachial nucleus. Brain atlas images are modified from Allen Mouse Brain Common Coordinate Framework version 3 (https://scalablebrainatlas.incf.org/mouse/ABA_v3). Scale bars = 500 µm.

Extended Data Fig. 5 Viral injection/expression sites and optical fiber placements for experiments in Figs. 28.

Coronal brain sections at the bregma levels showing viral injection sites (dots)/expression areas (gray shadow areas with red borders) and/or optical fiber tracts (vertical bars) for mice included. a-b, Optogenetic experiments (Figs. 2, 3 in a and Fig. 4 in b). c, DTA ablation experiments (Fig. 5). d-e, Retrograde (Fig. 6 in d) and anterograde (Fig. 7 in e) tracing experiments. f, Electrophysiological recordings (Fig. 7 in f), and (g) fiber photometry experiments (Fig. 8). For clarity, an optical fiber (400 µm) is shown as a thin vertical bar at the center of the tract. h, Electrode array locations from in vivo unit recording experiments (Fig. 8). i, Schematic showing viral expression and fiber covered areas for the two coordinates used in the OT. Scale bar = 500 µm. Each dot or line represents one animal except for bilateral AAV8-DTA injection in c. Brain atlas images are modified from Allen Mouse Brain Common Coordinate Framework version 3 (https://scalablebrainatlas.incf.org/mouse/ABA_v3). IC, islands of Calleja. OT, olfactory tubercle. NAc, nucleus accumbens. PVH, paraventricular hypothalamic nucleus.

Extended Data Fig. 6 Properties of postsynaptic currents (PSCs) upon optogenetic stimulation of D3-Cre/ChR2 neurons in the OT.

a and c, Latency to PSC onset in D3-Cre/tdTomato neurons (4.73 ± 0.19 ms) (a) and SPNs (4.63 ± 0.11 ms) (c). b and d, Jitter of PSCs (SD of latencies during repeated light stimuli) in D3-Cre/tdTomato neurons (1.28 ± 0.08 ms) (b) and SPNs (1.14 ± 0.10 ms) (d). Data are quantified in 16 D3-Cre/tdTomato neurons and 38 SPNs (6-10 traces/neuron) showing light-evoked PSCs. All averaged data are shown as mean ± s.e.m.

Source data

Extended Data Fig. 7 Single unit quality control metrics.

a, Upper panel, PCA plot of two putative single units recorded from the same electrode. Ellipses denote 2.5x SD of each K-means cluster. Lower panel, overlaid waveforms of the same neurons. b-c, Inter-spike intervals (ISIs, 2 ms bins) for the same two neurons as in (a) indicating significantly different distributions (two-sample Kolmogorov-Smirnov test D (2493) = 0.22, p <0.0001). Insets, ISI distributions (1 ms bins) showing limited numbers (< 2%) of ISI events < 2ms. d, Distribution of the proportion of ISI violations (< 2ms between spikes) among all single units. 100% of units had <2% of their spikes occurring within 2ms of each other. e, Distribution of mean firing rates during entire recording session of all single units (median: 1.97 Hz, typical of spiny projection neurons). f, Distribution of spike amplitude: noise floor values for all single units.

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Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and legends of Supplementary Videos 1–13.

Reporting Summary

Supplementary Video 1

3D reconstruction of the IC in the OT. Red, D3-Cre/tdTomato neurons. Green, axonal fibers from the MOB projection neurons labeled via viral injection of AAV9-EGFP. Blue, cell nuclei labeled by DRAQ5TM, a far-red fluorescent DNA dye.

Supplementary Video 2

3D reconstruction of individual D3 neurons in and between two IC in the OT showing the neuronal processes of these neurons. Colors are the same as in Supplementary Video 1.

Supplementary Video 3

Blue-light (but not green-light) activation of OT D3-Cre/ChR2 neurons induces grooming.

Supplementary Video 4

Grooming behavior induced by blue-light stimulation of OT D3-Cre/ChR2 neurons with different durations (1 to 20 s at 20 Hz).

Supplementary Video 5

Grooming behavior induced by blue-light stimulation of OT D3-Cre/ChR2 neurons with different frequencies (1–20 Hz for 10 s).

Supplementary Video 6

Genotype and stimulation site controls. Blue-light stimulation of OT D3-Cre/tdTomato neurons or blue-light stimulation of D3-Cre/ChR2 neurons in the NAc and hippocampus (dentate gyrus and CA3 region) do not induce grooming.

Supplementary Video 7

Grooming behavior induced by blue-light activation of the OT in D3-Cre/tdTomato mice injected with Cre-dependent AAV1-DIO-ChR2-EYFP virus. Additional conditions include green light or optical fiber implanted in the PVH.

Supplementary Video 8

Blue-light (but not green-light) activation of OT D3 neurons stops social investigation and initiates grooming in D3-Cre/ChR2 mice.

Supplementary Video 9

Blue-light (but not green-light) activation of OT D3 neurons stops feeding and initiates grooming in D3-Cre/ChR2 mice.

Supplementary Video 10

Blue-light (but not green-light) activation of OT D3 neurons stops itch-induced scratching and initiates grooming in D3-Cre/ChR2 mice.

Supplementary Video 11

Blue-light (but not green-light) activation of OT D3 neurons stops body licking and initiates grooming in D3-Cre/ChR2 mice.

Supplementary Video 12

Green-light (but not blue-light) inactivation of OT D3-Cre/eArchT neurons shortens spontaneous grooming.

Supplementary Video 13

Green-light (but not blue-light) inactivation of OT D3-Cre/eArchT neurons halts water-spray-induced grooming.

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Zhang, YF., Vargas Cifuentes, L., Wright, K.N. et al. Ventral striatal islands of Calleja neurons control grooming in mice. Nat Neurosci 24, 1699–1710 (2021). https://doi.org/10.1038/s41593-021-00952-z

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