Organization of the connections between claustrum and cortex in the mouse

Abstract

The connections between the claustrum and the cortex in mouse are systematically investigated with adeno-associated virus (AAV), an anterograde viral tracer. We first define the boundary and the three-dimensional structure of the claustrum based on a variety of molecular and anatomical data. From AAV injections into 42 neocortical and allocortical areas, we conclude that most cortical areas send bilateral projections to the claustrum, the majority being denser on the ipsilateral side. This includes prelimbic, infralimbic, medial, ventrolateral and lateral orbital, ventral retrosplenial, dorsal and posterior agranular insular, visceral, temporal association, dorsal and ventral auditory, ectorhinal, perirhinal, lateral entorhinal, and anteromedial, posteromedial, lateroposterior, laterointermediate, and postrhinal visual areas. In contrast, the cingulate and the secondary motor areas send denser projections to the contralateral claustrum than to the ipsilateral one. The gustatory, primary auditory, primary visual, rostrolateral visual, and medial entorhinal cortices send projections only to the ipsilateral claustrum. Primary motor, primary somatosensory and subicular areas barely send projections to either ipsi- or contralateral claustrum. Corticoclaustral projections are organized in a rough topographic manner, with variable projection strengths. We find that the claustrum, in turn, sends widespread projections preferentially to ipsilateral cortical areas with different projection strengths and laminar distribution patterns and to certain contralateral cortical areas. Our quantitative results show that the claustrum has strong reciprocal and bilateral connections with prefrontal and cingulate areas as well as strong reciprocal connections with the ipsilateral temporal and retrohippocampal areas, suggesting that it may play a crucial role in a variety of cognitive processes. J. Comp. Neurol. 525:1317-1346, 2017. © 2016 Wiley Periodicals, Inc.

Keywords: AAV; RRID:AB_10000344; RRID:AB_10048713; RRID:AB_306956; RRID:AB_509998; RRID:SCR_008848; claustrum; connectivity; cortex; tract tracing.

Figure 1

Double IHC for NeuN (red) and NF‐160 (green; A–F) and SMI‐32 (red) and parvalbumin (PV, green; G–L). Counterstaining with DAPI is in blue. Overlapping images from A and B, D and E, G and H, and J and K are, respectively, shown in C, F, I, and L. The boxes in A–C and G–I indicate, respectively, the enlargements in D–F and J–L. The dashed lines indicate approximate borders between cortical areas. For abbreviations see list. Scale bars = 737 μm in C (applies to A–C); 258 μm in F (applies to D–F); 737 μm in I (applies to G–I); 258 μm in L (applies to J–L).

Figure 2

Molecular markers for mouse claustrum. A–C: Expression of Ntng2 by ISH, with the boxed area in A enlarged in B and a nearby Nissl‐stained section shown in C. D–F: Similarly for Gnb4. G–I: Similar for Gng2. J–L: Similar for Lxn. For abbreviations see list. Scale bars = 839 μm in J (applies to A,D,G,J); 300 μm in L (applies to B,C,E,F,H,I,K,L).

Figure 3

Transgenic Cre driver lines crossed to Ai14 reporter showing tdTomato signal in the claustrum. A,B: A TissueCyte section image from a Pvalb‐IRES‐Cre;Ai14 mouse, with the boxed area in A enlarged in B. Similarly for C,D (Cux2‐CreERT2;Ai14) and E,F (Rbp4‐Cre_KL100;Ai14). For abbreviations see list. Scale bars = 650 μm in E (applies to A,C,E); 200 μm in F (applies to B,D,F).

Figure 4

Transgenic Cre driver lines crossed to Ai14 reporter showing tdTomato signal absent or very weak in the claustrum. A,B: A TissueCyte section image from a Ctgf‐2A‐dgCre;Ai14 mouse, with the boxed area in A enlarged in B. Similarly for C,D (Rorb‐IRES2‐Cre;Ai14.) and E,F (Ntsr1‐Cre_GN220;Ai14). For abbreviations see list. Scale bars = 650 μm in E (applies to A,C,E); 200 μm in F (applies to B,D,F).

Figure 5

3D reconstruction of the mouse claustrum, shown in medial view (A), dorsal view (B), and lateral view (C). A‐P: Anterior to posterior direction. The dark blue lines depict anterior–posterior, medial–lateral, and dorsal–ventral axes in 3D. The mouse claustrum stretches for approximately 2.9 mm along the anterior–posterior axis, with a volume of 0.275 mm³. Scale bar = 250 μm.

Figure 6

Representative images showing AAV‐GFP injection sites in 42 neocortical and allocortical areas of the right hemisphere. Injection sites are in prelimbic area (PL; A), infralimbic area (ILA; B), orbital area, medial part (ORBm; C), orbital area, ventrolateral part (ORBvl; D), orbital area, lateral part (ORBl; E), secondary motor area (MOs; F), primary motor area (MOp; G), anterior cingulate area, ventral part (ACAv; H), anterior cingulate area, dorsal part (AVAd; I), retrosplenial area, dorsal part (RSPd; J), retrosplenial area, ventral part (RSPv; K), agranular insular area, dorsal part (AId; L), agranular insular area, posterior part (AIp; M), gustatory area (GU; N), visceral area (VISC; O), piriform area (PIR; P), primary somatosensory area, barrel field (SSp‐bfd; Q), primary somatosensory area, trunk (SSp‐tr; R), primary somatosensory area, lower limb (SSp‐ll; S), primary somatosensory area, upper limb (SSp‐ul; T), primary somatosensory area, mouth (SSp‐m; U), supplemental somatosensory area (SSs; V), temporal association area (TEa; W), perirhinal area (PERI; X), ectorhinal area (ECT; Y), primary auditory area (AUDp; Z), dorsal auditory area (AUDd; AA), ventral auditory area (AUDv; BB), primary visual area (VISp; CC), posteromedial visual area (VISpm; DD), anteromedial visual area (VISam; EE), rostrolateral visual area (VISrl; FF), anterolateral visual area (VISal; GG), posterolateral visual area (VISpl; HH), laterointermediate visual area (VISli; II), postrhinal visual area (VISpor; JJ), entorhinal area, lateral part (ENTl; KK), entorhinal area, medial part (ENTm; LL), subiculum, dorsal part (SUBd; MM), postsubiculum (POST; NN), presubiculum (PRE; OO), and parasubiculum (PAR; PP). Fluorescent signal is green, and background is red. Scale bar = 800 μm.

Figure 7

A–PP: Dorsal view of the AAV‐GFP injection sites in 42 neocortical and allocortical areas of the right hemisphere and their brain‐wide projections, corresponding to the panels in Figure 6. In each panel, the red cross within the black circle indicates the injection site, the size of the black circle is proportional to the size of the injection site, and the red arrows point to the claustrum in both hemispheres. For abbreviations see list. Scale bar = 1,000 μm.

Figure 8

A–PP: Dorsal‐ventral view of the voxel‐level projection densities in ipsilateral claustrum (right) and contralateral claustrum (left) from the AAV‐GFP injections in 42 neocortical and allocortical areas of the right hemisphere, corresponding to the panels in Figures 6 and 7. D, dorsal; V, ventral; A, anterior; P, posterior. Color bar: white 0 to dark purple 1 in linear scale.

Figure 9

A–PP: Higher‐magnification coronal images of the ipsilateral (right) and contralateral (left) claustrum, showing incoming projections from the AAV‐GFP injections in 42 neocortical and allocortical areas of the right hemisphere, corresponding to the panels in Figures 6, 7, 8. Dashed ovals outline the proximate locations of the left and right claustrum. L, left claustrum; R, right claustrum. Scale bar = 100 μm.

Figure 10

AAV‐GFP injections in the claustrum of the right hemisphere and their projections to the cortex. A,B: Low‐magnification TissueCyte images of injection sites in Ntng2‐IRES2‐Cre (485846989) and Gnb4‐IRES2‐Cre (485903475) mice, respectively. Note that laser excitation power during TissueCyte scanning was adjusted to maximize detection of single axon fibers anywhere in the brain, which led to oversaturation at the injection sites, preventing clear visualization of AAV‐infected cell bodies. C,D: Low‐magnification confocal images of the same sections shown in A and B, counterstained with DAPI. Arrows point to the claustrum and endopiriform nucleus in A–D. Insets: Higher‐magnification confocal images of the injection sites. Arrows point to the claustrum (outlined by the dashed lines). Note that laser excitation power during confocal imaging of the sections was adjusted to proper levels to distinguish AAV‐infected cell bodies from those uninfected. E,F: Dorsal view of the voxel‐level claustrocortical projection densities from injections shown in A and B, respectively. Asterisks indicate injection sites in the right hemisphere. Black lines in F indicate approximate levels of coronal sections shown in Figure 11. For abbreviations see list. A, anterior; L, lateral. Color bar: white 0 to dark purple 1 in linear scale. Scale bars = 700 μm in D (applies to A–D); 1,000 μm in E (applies to E,F); 80 μm in insets.

Figure 11

A–H: Low‐magnification coronal images of projections from the AAV‐GFP injection into the claustrum of the right hemisphere in a Gnb4‐IRES2‐Cre mouse (485903475). Sections in A–G are in 1‐mm intervals from anterior to posterior, and the interval between sections in G and H is 0.7 mm. Large and small boxes indicate regions where axon terminals are shown at higher magnification in Figure 12 for the right (ipsilateral) and left (contralateral) hemispheres, respectively. Arrow indicates injection site of the claustrum. Arrowheads indicate cortical area borders. For abbreviations see list. Scale bar = 800 μm.

Figure 12

Higher‐magnification images of projections from the AAV‐GFP injection in the claustrum of the right hemisphere in a Gnb4‐IRES2‐Cre mouse (485903475) reveal laminar distribution of axon terminals in different cortical regions. Regions shown correspond to the large (ipsilateral) and small (contralateral) boxes in Figure 11: ORBm (A), AId (B), MOs (C), ACAv (D), SSp (E), RSPd (F), AUDp (G), RSPagl (H), VISp (I), ECT (J), contralateral ACAv (K), contralateral MOs (L), contralateral ACAd (M), contralateral RSP (N), and contralateral SSp (O). Images are oriented with pial surface at top and white matter at bottom. I–VI represent cortical layers. Lines indicate borders between cortical layers. Scale bars = 100 μm in J (applies to A–J); 180 μm in O (applies to K–O).

Figure 13

Histograms showing fractions of projection densities from the claustrum to different neocortical and allocortical areas in four AAV‐GFP injection experiments in three Cre mice and one wild‐type mouse. Each inset reveals the location of the injection site and projections in a dorsal view. The four injections are ordered from anterior to posterior.

Figure 14

Circle diagrams showing the normalized projection densities from different neocortical and allocortical areas to claustrum and vice versa. A: Ipsilateral connections. B: Contralateral connections. The width of the line is proportional to the normalized projection density. Corticoclaustral projections are color‐coded in red and claustrocortical projections in blue. Yellow dots indicate no injections or lack of good injections. Cortical areas are color‐coded as in the Allen mouse reference atlas.