Micropopulation mapping of the mouse parafascicular nucleus connections reveals diverse input-output motifs

Abstract

Introduction: In primates, including humans, the centromedian/parafascicular (CM-Pf) complex is a key thalamic node of the basal ganglia system. Deep brain stimulation in CM-Pf has been applied for the treatment of motor disorders such as Parkinson's disease or Tourette syndrome. Rodents have become widely used models for the study of the cellular and genetic mechanisms of these and other motor disorders. However, the equivalence between the primate CM-Pf and the nucleus regarded as analogous in rodents (Parafascicular, Pf) remains unclear.

Methods: Here, we analyzed the neurochemical architecture and carried out a brain-wide mapping of the input-output motifs in the mouse Pf at micropopulation level using anterograde and retrograde labeling methods. Specifically, we mapped and quantified the sources of cortical and subcortical input to different Pf subregions, and mapped and compared the distribution and terminal structure of their axons.

Results: We found that projections to Pf arise predominantly (>75%) from the cerebral cortex, with an unusually strong (>45%) Layer 5b component, which is, in part, contralateral. The intermediate layers of the superior colliculus are the main subcortical input source to Pf. On its output side, Pf neuron axons predominantly innervate the striatum. In a sparser fashion, they innervate other basal ganglia nuclei, including the subthalamic nucleus (STN), and the cerebral cortex. Differences are evident between the lateral and medial portions of Pf, both in chemoarchitecture and in connectivity. Lateral Pf axons innervate territories of the striatum, STN and cortex involved in the sensorimotor control of different parts of the contralateral hemibody. In contrast, the mediodorsal portion of Pf innervates oculomotor-limbic territories in the above three structures.

Discussion: Our data thus indicate that the mouse Pf consists of several neurochemically and connectively distinct domains whose global organization bears a marked similarity to that described in the primate CM-Pf complex.

Keywords: basal ganglia; cortical layer 5; corticothalamic; motor cortex; thalamocortical; thalamostriatal.

Figure 1

Cyto-and chemoarchitectonic heterogeneity of the mouse Pf in coronal sections. Parallel series of coronal sections stained with cresyl violet [Nissl; panels (A–D)], or immunolabeled for Calbindin 28 K [CB; panels (E–H)], μ-opioid receptor [MOR; panels (I–L)] or glycine transporter type 2 [GlyT2; (M–P)]. Coronal anterior–posterior (AP, in mm) levels for each row are indicated in panels (E–H). In panel (K), a yellow asterisk indicates some BDA-labeled pretectal cells in this experiment. Scale bar: 250 microns; CeM, central medial thalamic nucleus; CL, central lateral thalamic nucleus; Eth, ethmoid thalamic nucleus; fr, fasciculus retroflexus; Hb, habenula; MD, mediodorsal thalamic nucleus; ml, medial lemniscus; OPC, oval paracentral thalamic nucleus; p1RF, prosomere 1 reticular formation; PAG, periaqueductal gray; PC, paracentral thalamic nucleus; Pf, parafascicular thalamic nucleus; Po, posterior thalamic nucleus; PVP, paraventricular thalamic nucleus, posterior part; SPF, subparafascicular thalamic nucleus; VPPC, ventral posterior thalamic nucleus, parvocellular division.

Figure 2

Cyto-and chemoarchitectonic heterogeneity of the mouse Pf in horizontal sections. Horizontal section images at various rostral (top) to caudal (bottom) levels, stained for Nissl (A–C), CB (D–F), and cytochrome C-oxidase histochemistry [CyO; (G–I)]. Red lines are used in (A–C) to indicate the coronal levels of the images shown in Figures 1A,D. Scale bar: 250 microns; PVA, paraventricular thalamic nucleus, anterior part; VL, ventral lateral thalamic nucleus. Other abbreviations as in Figure 1.

Figure 3

Divergent axonal projections to the striatum, subthalamic nucleus and cerebral cortex labeled by an injection in the ventral and medial domain of Pf (Case 1). (A) Center of the BDA deposit in experiment #1. (B) High-power image of thalamostriatal axonal arborizations. MOR immunohistochemistry counterstain. Striosomes (S) are visible as patches of slightly darker neuropil staining that are mostly avoided by the labeled axon terminals. (C) Labeled thalamocortical axon branches in the middle layers of area FrA. (D) Diagram illustrating the extent of the BDA deposit. (E) Coronal CPu sections profiles showing Neurolucida-drawn labeled thalamostriatal arborizations (in red). For reference, striosomes made visible by MOR immunolabeling in each section are also delineated (pale gray patches). For reference, functional territories of the mouse striatum where the axons labeled in this experiment are situated are indicated: Mo, mouth sensorimotor territory; ViS, visceral sensory territory. (F) Diagram of labeled Pf axon branches in STN. (G) Coronal section diagrams of the frontal cortex showing the labeled thalamocortical arborizations. (H) Serial plotting of the position and relative density of thalamocortical axons on an “unfolded” map of the anterior half of the cerebral cortex. In this diagram, axons are schematically represented by small line segments aligned along each section contour. For reference, the blue arrows indicate the levels corresponding to sections in panel (G). Scale bars: 250 μm (A,F); 100 μm (B,C); 500 μm (E,G). A32, cingulate cortex, area 32; A24a, cingulate cortex, area 24a; A24b, cingulate cortex, area 24b; AI, agranular insular cortex; AID, agranular insular cortex, dorsal part; APTD, anterior pretectal nucleus, dorsal part; DI, disgranular insular cortex; DLG, dorsal lateral geniculate nucleus; DLO, dorsolateral orbital cortex; FL, forelimb-related; FrA, frontal association cortex; GI, granular insular cortex; LP, lateral posterior thalamic nucleus; M1, primary motor cortex; M2, secondary motor cortex; Mo, mouth-related; PV, paraventricular thalamic nucleus; S, striosome; Tr, trunk-related; VP, ventral posterior thalamic nucleus. Other abbreviations as in Figure 1.

Figure 4

Labeling of axonal projections originated from either a ventral (Case 18, top) or an intermediate (Case 10, bottom) portion of the lateral Pf. Graphic conventions as in Figure 3. (A,F) Location of the BDA deposits. (B,G) Labeling in the striatum. (C,H) Labeling in the subthalamic nucleus. (D,I) Coronal section diagrams showing labeled thalamocortical arborizations. (E,J) Serial section plots of the cortical labeling. Scale bars: 250 μm (C,H); 500 μm (B,D,G,I). bf, barrel-field; HL, hindlimb-related; MWh, whisker-related motor cortex; S1, primary somatosensory cortex; SM, somatomotor region. Other abbreviations as in Figures 1, 3.

Figure 5

Labeling of axonal projections originated from either a dorsolateral (Case 6, top) or a dorsomedial (Case 4, bottom) portion of the Pf. Graphic conventions as in Figure 3. (A,F) Location of the BDA deposits. (B,G) Labeling in the striatum. (C,H) Labeling in the subthalamic nucleus. (D,I) Coronal section diagrams showing labeled thalamocortical arborizations. (E,J) Serial section plots of the cortical labeling. Scale bars: 250 μm (C,H); 500 μm (B,D,G,I). a25, cingulate cortex, area 25; AIV, agranular insular cortex, ventral part; Cg, cingulate cortex-related; LO, lateral orbital cortex; MO, medial orbital cortex; Orb, orbital cortex-related; Tr, trunk-related; VO, ventral orbital cortex. Other abbreviations, as in Figures 1, 3, 4.

Figure 6

Labeling of axonal projections originated from either a dorsal (Case 9, top) or a ventral (Case 38, bottom) portion of the rostral Pf tip. Graphic conventions as in Figure 3. (A,F) Location of the BDA deposits. (B,G) Labeling in the striatum. (H) Microphotograph showing type II thalamostriatal axons labeled from the ventral part of the rostralmost Pf (“OPC”) arborizing in the striatum. (C,I) Labeling in the subthalamic nucleus. (D,J) Coronal section diagrams showing labeled thalamocortical arborizations. (E,K) Serial section plots of the cortical labeling. Scale bars: 250 μm (C,I); 500 μm (B,D,G,J); 100 μm (H). Abbreviations as in Figures 1, 3–5.

Figure 7

Labeling of axonal projections originated from the caudalmost cluster of Pf cells (“Eth nucleus”; case 35). Graphic conventions as in Figure 3. (A) Location of the BDA deposit. (B) Labeling in the striatum. (C) Labeling in the subthalamic nucleus. (D) Coronal section diagrams showing labeled thalamocortical arborizations. (E) Serial section plots of the cortical labeling. Scale bars: 250 μm (C); 500 μm (B,D). Ect: ectorhinal cortex. Other abbreviations, as in Figures 1, 3–6.

Figure 8

Pf projections to the subthalamic (STN) and thalamic reticular (TRN) nuclei. (A,B) Neurolucida^® drawings of the STN projection from the five BDA injection cases depicted in Figures 3-5 (Panel “A”) are overlaid in panel “B” to illustrate the fine spatial organization of this projection. (C) Microphotograph of a labeled thalamosubthalamic axon arborization (Case 6; see also Figure 5). (D) Summary diagram of the Pf-STN projection. (E–H) Microphotographs taken from two different BDA experiments at 10x (E,G) and 40x (F,H) magnification show that axons from Pf neurons leave a few varicose branches upon crossing through the TRN on their way to the striatum and cortex. Scale bars: 250 μm (E,G) or 50 μm (C,F,H). AV, anteroventral thalamic nucleus; CP, cerebral peduncle; EPN, entopeduncular nucleus; GP, globus pallidus; ic, internal capsule; ns, nigrostriatal bundle; STN, subthalamic nucleus; TRN, thalamic reticular nucleus; VM, ventral medial thalamic nucleus; ZI, zona incerta. Other abbreviations, as in previous figures.

Figure 9

Pf axon varicosities show region-specific size differences. (A) Comparison of median sizes (maximal projection area, in μm²) of axon varicosities labeled in the cerebral cortex (Cx), striatum (CPu), or STN by Pf injections. Results from six cases are averaged in this chart. (B–D) Comparison of the sizes of the thalamostriatal axon varicosities labeled in different geometrical quadrants (“Q”) and anterior–posterior levels of the striatum by a BDA injection in the ventral/lateral portion of Pf (Case 18). Panels B and C show high-magnification images of the morphology of thalamostriatal axon arborizations containing large and small (B) and only small (C) varicosities. Scale bars: 10 μm. Panel (D) shows the quantification of size (maximal projection area) range distributions of the axon varicosities in different quadrants. Kolmogorov-Smirnoff (K-S) paired comparisons. Levels of significance are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001. (E–J) Axon varicosity size distributions and paired comparisons across different quadrants of the striatum in five Pf injection experiments (E). In each plot, a continuous line represents the median, while dashed lines represent interquartile ranges. Mann–Whitney U test (MW) paired comparisons. Levels of significance are indicated by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001. The case in (G) is the one illustrated also in panels (B–D). (K) Cartoon diagram summarizing the statistical analyzes in panels (F–J). Three coronal section levels of the striatum are shown, and crossed dashed lines are used to separate the “quadrants” among which varicosities were compared. Large or small circles represent larger or smaller varicosity populations. Colors as in panel (E).

Figure 10

Retrograde CTb labeling of corticothalamic and superior colliculus neurons projecting to Pf. (A) Diagram showing the extent of four different BDA + CTb injection cases (Supplementary Figure S1). (B) The injection site in Case 19 (B) is shown as a representative example. The CTb deposit is visible as a brown DAB precipitate, whereas the BDA deposit appears as a black DAB-nickel core. Thionin counterstain. (C) High-magnification detail of the neuronal somata labeled by CTb transport (in brown) in L6 of the motor cortex [area M1, inset in panel (D)]. An isolated thalamocortical axon branch (arrowheads) labeled by the anterograde transport of BDA (blue-black) can be seen among the corticothalamic cell somata. (D–F) Coronal sections samples illustrating the labeling in the cerebral cortex ipsilateral to the injection. Large numbers of CTb-labeled corticothalamic cell bodies are visible in the lower part of L5 (putative L5b), and in L6b. The scant, isolated thalamocortical axons labeled by BDA are barely visible at this magnification [compare with panel (C)]. (G) Corticothalamic cell bodies labeled in the hemisphere contralateral to the injection. White arrows indicate corticothalamic L5 cells. Labeled somata are also visible in L6b. (H,I) High-magnification detail of labeled L5 pyramidal cells in area M1 of the injected (H) or contralateral (I) hemisphere. (J) Retrogradely labeled neurons in the intermediate gray (IG) and white (IW) layers of the superior colliculus. Inset: High magnification detail of labeled cells in the intermediate layers. Scale bars: 250 μm (B,D,E,F,G); 25 μm (C,H,I); 50 μm [inset in (J)].

Figure 11

Quantitative analysis of neurons projecting to Pf from cortical and subcortical areas of the brain and brainstem. (A) Average percent of retrogradely labeled neurons in cortical or subcortical structures over the total. (B) Average percent of retrogradely labeled L5 or L6 cells for four sensorimotor cortical areas (M1, M2, S1, S2). Ipsilateral (gray) and contralateral (black) labeling data are represented separately. (C) Percentage of retrogradely labeled cell bodies in different cortical or subcortical structures is indicated. Ipsilateral or contralateral data are separately indicated. To normalize between experiments (n = 4), the percent of cells labeled in each structure over the total of cells counted in the experiment is averaged. For simplicity, we grouped under “Cg” the various areas in the medial frontal cortex (A24, A25, A32). Individual case data are available as Supplementary Figure S4.

Figure 12

Distribution of labeled corticothalamic layer 5 (red) or layer 6 (turquouise) neurons following CTb injections in Pf. (A,B) Cells labeled by a CTb injection in ventral/medial Pf (Case 1, Figure 10); (C,D) Cells labeled by an injection in dorsal Pf (Case 19, Figure 10). Labeling is displayed on unfolded cortex maps. Due to their high density, labeled cells are represented as continuous bars. Higher saturation indicates zones of higher cell density. In the same experiments, BDA was co-injected with CTb, thus producing anterograde labeling of Pf axons; for comparison, the position of these axons is shown as black dots. PRh: perirhinal cortex. Other abbreviations, as in previous figures.

Figure 13

Distribution within Pf of corticothalamic axon terminals from different cortical areas. Two-photon tomography image samples from experiments in which AAV vectors able to drive the expression of high levels of fluorescent protein were injected in different areas. (A,B): Corticothalamic projection labeled by an AAV injection in M2 and M1 (putative Wh zone). Three sections throughout the entire rostrocaudal extent of the Pf are shown, as a representative example of our analysis methodology (C,D): Projection from M1 (putative mouth zone) (E,F): Projection from M1 (putative forelimb zone). (G,H): Projection from M1/S1 (putative hindlimb zone). (I,J): Projection from M2 (putative Mo zone). (K,L) Projection from FrA. (M,N) Projection from DLO. (O,P) Projection from AI. (Q,R) Projection from area 24b of the cingulate cortex. (S,T) Projection from VO and LO. (U,V) Projection from A32/MO areas. Images from the Allen Institute Mouse Connectivity Projection datasets https://connectivity.brain-map.org/. Experiment IDs: 183617432 (A,B); 584,903,636 (C,D); 159,651,060 (E,F); 100,141,273 (G,H); 552,757,477 (I,J); 293,433,996 (K,L); 180,709,230 (M,N); 262,536,037 (O,P); 496,576,666 (Q,R); 183,618,845 (S,T); 478,376,197 (U,V). AP: bregma level in mm. Scale bars: 1000 μm (A,C,E,G,I,K,M,O,Q,S,U); 250 μm (B,D,F,H,J,L,N,P,R,T,V). Abbreviations, as in previous figures.

Figure 14

Projections to Pf from different subcortical regions. Two-photon tomography image samples from experiments in which AAV vectors able to drive the expression of high levels of fluorescent protein were injected in some of the subcortical structures that target Pf. (A,B) Projection from the entopeduncular nucleus, anterolateral part. (C,D) Projection from the globus pallidus. (E–H) Projection from the substantia nigra pars reticulata. Injections in either a dorsolateral (E,F) or a ventromedial (G,H) region are shown. (I–L) Projection from the superior colliculus. Injections in either a lateral (I,J) or a central-lateral (K,L) region are shown Images from the Allen Institute Mouse Connectivity Projection datasets https://connectivity.brain-map.org/; experiment IDs: 539498984 (A,B), 511,942,270 (C,D), 478,096,249 (E,F), 158,914,182 (G,H), 175,158,132 (I,J), 128,001,349 (K,L). AP: bregma level in mm. Scale bars: 1000 μm (A,C,E,G,I); 250 μm (B,D,F,H,J). DG, deep gray layer; opt, optic tract layer; SC, superior colliculus; SC-all, superior colliculus, all layers; SC-int, superior colliculus, intermediate layers; SG, superficial gray layer; SPFPC, subparafascicular thalamic nucleus, parvicelular part. Rest as in previous figures.

Figure 15

Cartoon comparison between the anatomical organization patterns of the mouse and the anthropoid primate CM-Pf. (A) Schematic representation of the mouse CM-Pf complex and its afferent (bottom) and efferent (top) connections as revealed by our analysis. Connections whose neurotransmission is known to be fundamentally inhibitory are shown in orange letters. For scale comparison, the somatodendritic morphology of a typical Pf neuron (taken from the Janelia Research Campus Mouselight database; #AA1439; Winnubst et al., 2019) is illustrated. Note that the long dendrites from an individual cell extend across several input/output compartments. (B) For comparison, the afferent/efferent relationships of the CM-Pf complex in the thalamus of an anthropoid primate (squirrel monkey, Saimiri sciureus) are shown following the same graphic conventions. This representation is based on Sadikot and Rymar (2009), with additional squirrel and macaque monkey data from Mufson and Mesulam (1984), Miyata and Sasaki (1984), Goldman-Rakic and Porrino (1985), Yeterian and Pandya (1988), Nakano et al. (1993), Stepniewska et al. (1994, , Rouiller et al. (1998), Cavada et al. (2000), François et al. (2002), Sidibé et al. (2002), Kultas-Ilinsky et al. (2003), and Hsu and Price (2007). We labeled the rodent entopeduncular nucleus as “GPi” to make similarities/differences between rodents and primates more readily perceptible. Scale bars: 500 μm. A24, A25, A32, anterior cingulate cortices; bf, barrel field; Cd, caudate; CPu, caudate-putamen; DCNs, deep cerebellar nuclei; FEF, frontal eye field; FrA, frontal association cortex; GPe, external globus pallidus; GPi, internal globus pallidus; Ins, insular cortex; LC, locus coeruleus; NAc, nucleus accumbens; Orb, orbital cortex; PAG, periaqueductal gray matter; PBNs, parabrachial nuclei; PFC, prefrontal cortex; PFR, pontine reticular formation; PPN, pedunculopontine nucleus; Pu, putamen; SNr-l, substantia nigra reticular part, lateral; SNr-m, substantia nigra reticular part, medial; STN, subthalamic nucleus; TRN, thalamic reticular nucleus; VTA, ventral tegmental area; ZI, zona incerta.