FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3058758979> ?p ?o ?g. }

• W3058758979 abstract "Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The cerebellar vermis, long associated with axial motor control, has been implicated in a surprising range of neuropsychiatric disorders and cognitive and affective functions. Remarkably little is known, however, about the specific cell types and neural circuits responsible for these diverse functions. Here, using single-cell gene expression profiling and anatomical circuit analyses of vermis output neurons in the mouse fastigial (medial cerebellar) nucleus, we identify five major classes of glutamatergic projection neurons distinguished by gene expression, morphology, distribution, and input-output connectivity. Each fastigial cell type is connected with a specific set of Purkinje cells and inferior olive neurons and in turn innervates a distinct collection of downstream targets. Transsynaptic tracing indicates extensive disynaptic links with cognitive, affective, and motor forebrain circuits. These results indicate that diverse cerebellar vermis functions could be mediated by modular synaptic connections of distinct fastigial cell types with posturomotor, oromotor, positional-autonomic, orienting, and vigilance circuits. Introduction The cerebellum has been implicated in several cognitive functions and neuropsychiatric disorders, which are more typically associated with the cerebral cortex and basal ganglia (Tsai et al., 2012; Hariri, 2019; Schmahmann et al., 2019). Although it is widely assumed that such nonmotor functions are mediated by the cerebellar hemispheres and their connections with the thalamus (Strick et al., 2009; Buckner, 2013; Wang et al., 2014; De Schutter, 2019), increasing functional and anatomical evidence points to roles for the vermis (Schmahmann and Sherman, 1998; Watson et al., 2009; Halko et al., 2014; Watson et al., 2014; Zhang et al., 2016; Wagner et al., 2017; Badura et al., 2018; Xiao et al., 2018; Albazron et al., 2019; Brady et al., 2019; Watson et al., 2019; Kelly et al., 2020) an evolutionarily old portion of the cerebellum best known for its influence on brainstem circuits for posture and eye movements (Chambers and Sprague, 1955; Ohtsuka and Noda, 1991). Structural and functional abnormalities of the vermis have been associated with various psychiatric disorders and conditions, including autism, schizophrenia, mood disorders, chronic pain, and addiction (Weinberger et al., 1980; Courchesne et al., 1988; Sweeney et al., 1998; Strakowski et al., 2005; Andreasen and Pierson, 2008; Moulton et al., 2010; Fatemi et al., 2012; Moulton et al., 2014). Remarkably little is known, however, about the specific cell types and circuits responsible for diverse nonmotor functions of the vermis. The cerebellum is thought to be organized in parallel circuit modules that comprise specific sets of inferior olive (IO) neurons, Purkinje cells (PCs), and vestibular or cerebellar nucleus neurons (Oscarsson, 1979; Ito, 1984; Apps and Hawkes, 2009; Voogd, 2011; Apps et al., 2018). The cerebellar vermis, caudal medial accessory olive, and fastigial nucleus (FN; medial cerebellar nucleus) collectively constitute the broad ‘A’ module (Groenewegen and Voogd, 1977), which is likely to include several submodules (Apps, 1990; Sugihara and Shinoda, 2004; Voogd and Ruigrok, 2004; Sugihara and Shinoda, 2007). Although the fastigial nucleus has been implicated in diverse motor and nonmotor functions in human imaging studies (Schmahmann, 1991; Schmahmann, 1998; Albazron et al., 2019), its small size (Diedrichsen et al., 2011; Tellmann et al., 2015) has precluded precise functional localization. Mono- and multi-synaptic connections with the midbrain, hippocampus, basal ganglia, and cerebral cortex are likely to mediate vermis and fastigial influences on affective and cognitive functions (Steriade, 1995; Teune et al., 2000; Watson et al., 2014; Badura et al., 2018; Xiao et al., 2018; Watson et al., 2019; Vaaga et al., 2020) but the specific fastigial cell types responsible for these influences are not known. In this study, we hypothesized that the diverse motor and nonmotor functions of the cerebellar vermis are mediated by multiple types of fastigial output neurons with distinct circuit connections. By combining single-cell gene expression, immunohistochemical, and circuit connectivity analyses, we identified several molecularly and anatomically distinct types of excitatory projection neurons in the mouse fastigial nucleus. Anterograde, transsynaptic, and retrograde tracing analyses indicate segregated, modular circuit connectivity; each fastigial cell type is linked with a specific set of PCs and IO neurons and makes divergent projections to functionally-related downstream targets. Disynaptic connections with the basal ganglia, basal forebrain, and cerebral cortex revealed that specific fastigial cell types are linked with circuits responsible for distinct aspects of cerebellar motor and nonmotor functions. Results Anatomically distinct cell types in the fastigial nucleus The fastigial nucleus (FN) is the medial cluster of cerebellar nucleus neurons (Dow, 1942), delineated in mammals by surrounding white matter (Larsell, 1970). Despite its small size, the FN comprises at least three subdivisions (Korneliussen, 1968; Beitz and Chan-Palay, 1979): a rostral part, a caudal part, and a dorsolateral protuberance (DLP), which is well developed in rodents (Fujita et al., 2010). Each of these subdivisions have been linked with distinct sets of Purkinje cells, inferior olive subnuclei, and downstream brain regions (Batton et al., 1977; Teune et al., 2000; Voogd and Ruigrok, 2004; Sugihara and Shinoda, 2007; Sugihara et al., 2009). To identify markers that could distinguish cell types in the FN, we searched in situ hybridization data in the Allen Brain Atlas (http://mouse.brain-map.org/) (Lein et al., 2007) for genes expressed differentially across FN subregions. We then evaluated candidate marker genes (Figure 1—figure supplement 1) with immunostaining and confocal microscopic analyses of serial cerebellar sections. Antibodies for SPP1 (osteopontin), SNCA (alpha-synuclein), and CALB2 (calretinin) reliably labeled specific subsets of neurons that were located predominantly in distinct subregions of the FN (Figure 1; Figure 1—figure supplement 2). In caudal regions of the FN (Figure 1B), SPP1-immunopositive (SPP1+) neurons were prominent in the caudal portion of the DLP (cDLP) and were sparsely distributed ventrally. In contrast SNCA+ neurons were prominent ventrally in the caudal fastigial nucleus (cFN), but were sparse in the cDLP. In rostral regions of the FN (Figure 1C), the majority of neurons were SPP1+. Neurons strongly immunopositive for CALB2 (CALB2+) were prominent in rostrally in ventral and lateral parts of the fastigial nucleus (vlFN). Figure 1 with 3 supplements see all Download asset Open asset Anatomically distinct cell types in the fastigial nucleus. (A) The fastigial nucleus (FN) in a sagittal section of the mouse cerebellar vermis identified with Nissl staining. Vertical lines indicate the location of sections in panels B and C. (B) Double immunostaining for alpha synuclein (SNCA) and osteopontin (SPP1) in a coronal section of the FN (n = 3 males). SNCA immunopositive neurons (green) are prominent in the caudal FN (cFN). SPP1 immunopositive neurons (magenta) are distributed throughout the caudal portion of the dorsolateral protuberance (cDLP). (C) Double immunostaining for calretinin (CALB2) and SPP1 in a more rostral section of the FN (n = 3 males). Intensely CALB2+ neurons (cyan) are densely clustered in the ventrolateral FN (vlFN). SPP1+ neurons (red) are distributed throughout the rostral FN (rFN) and the rostral portion of the DLP (rDLP). (D) Five subregions of the FN can be delineated by the distribution of neurons expressing SNCA, CALB2, and/or SPP1: (1) the cFN (purple) comprises SNCA+ neurons; (2) the cDLP (blue) comprises neurons which co-express SNCA and SPP1; (3) the vlFN (green) comprises neurons that express CALB2 and SNCA; (4, 5) the rDLP (pink) and rFN (yellow) each comprise exclusively SPP1+ neurons. (E–H) High magnification images of immunohistochemically revealed neurons located in the rFN (E, SPP1), cFN (F, SNCA), vlFN (G, CALB2) and cDLP (H, SPP1 and SNCA). Note considerable difference in the sizes of these neurons. Scale bar in C applies to B and C. Scale bar in H applies to E-H. Double-immunostaining revealed five populations of neurons that were distinguished by their marker gene expression and anatomical distribution. Neurons that exclusively expressed SPP1 were found in two distinct regions: the rostral FN (rFN) (Figure 1E) and the rostral DLP (rDLP). In contrast, neurons that coexpressed SPP1 and SNCA (Figure 1H) were found in the cDLP (Figure 1—figure supplement 2). They were also scattered through the cFN, where they intermingled with a distinct population of neurons which exclusively expressed SNCA (Figure 1F). CALB2+ neurons (Figure 1G), which were distributed in the vlFN, coexpressed SNCA but not SPP1. Although the majority of neurons in the cerebellar nuclei are glutamatergic, distinct populations of glycinergic and GABAergic neurons have been reported (Fredette and Mugnaini, 1991; Bagnall et al., 2009). To identify neurotransmitter associated with each cell type, we performed immunostaining for SNCA, SPP1, and CALB2 on cerebellar nucleus sections from mouse lines that express fluorescent reporters for glutamatergic neurons (VgluT2-Cre;Ai14), GABAergic neurons (Gad2-nls-mCherry) and glycinergic neurons (GlyT2-EGFP) (Figure 1—figure supplement 3). Notably, most (93%; n = 954) of the identified glutamatergic neurons, but none of the GABAergic neurons, were immunostained by either SPP1 or SNCA (Figure 1—figure supplement 3A–C,E). Glycinergic neurons in the FN comprise two distinct populations Bagnall et al., 2009; we found that large glycinergic projection neurons, located ventrally in the rFN, were robustly immunostained by SPP1 (but not SNCA) (Figure 1—figure supplement 3D). In contrast, small glycinergic neurons were not labeled by either SPP1 or SNCA (Figure 1—figure supplement 3D). Collectively, these results indicate that the majority of glutamatergic fastigial neurons can be classified into five major types by localization and expression of SPP1, SNCA, and CALB2. Single cell gene expression confirms anatomically distinct cell types To verify the anatomical analyses of cell types, we performed single-cell qPCR analyses on acutely isolated fastigial cells using a strategy previously developed for distinguishing cell classes among vestibular nucleus neurons (Figure 2—source data 1; Kodama et al., 2012). From an initial pool of 130 randomly harvested fastigial cells, we identified 50 cells which expressed ion channel genes associated with action potentials (Scn8a and Kcna1) and lacked non-neuronal markers (Mobp and Cd68), and were thus considered as neurons. Most of these neurons (84%: 42/50) expressed Slc17a6 (vesicular glutamate transporter 2, VgluT2, Figure 2A), indicating that they were glutamatergic. The remainder expressed inhibitory neuronal markers Gad1, Gad2, and/or Slc6a5. We focused subsequent expression analyses on the glutamatergic excitatory neurons. Figure 2 with 1 supplement see all Download asset Open asset Single cell gene expression analyses confirm molecularly distinct cell types. (A) Heatmap representation of quantitative gene expression profiles obtained via single-cell qPCR for individual excitatory fastigial neurons that express Slc17a6 (=VgluT2). Expression levels in Ct (cycle threshold in qPCR) are color coded, where insignificant expression (<5 copies of transcript, corresponding to Ct of 23.45) is shown in grey scale. Columns and rows correspond to individual neurons (n = 42) and genes examined, respectively. Clustering analysis for expression of Spp1, Snca, and Calb2 confirms four major types of fastigial neurons immunohistochemically identified, which are termed F1-F4 as shown in the dendrogram. Neurons included are from 7 (6 wildtype and 1 YFP-16) male mice. (B) Positive correlation of Nefl expression in the molecularly defined cell types with cell body area measured from the corresponding neurons immunohistochemically identified (n = 210). Plots are color-coded for the cell-types as indicated in A (F1, orange; F2, blue; F3, green; F4, purple). Note that smaller Ct values indicate greater expression levels. Population averaged data for each cell type are plotted. Error bars represent SEM. (C) NEFH immunostaining of glutamatergic fastigial neurons identified by nuclear-localized GFP (blue) in VgluT2-Cre;SUN1-sfGFP line. Size of the somata is identified with Nissl staining (green). Immunoreactivity for NEFH (red) is higher in the large glutamatergic neurons (top) than in the small glutamatergic neurons (bottom). (D) Linear correlation between expression levels of Scn8a vs Kcnc1. Color-code of the cell-type is the same as B. Plotted are population averaged data for each cell type. Error bars represent SEM. Figure 2—source data 1 Raw data of single-cell qPCR with excitatory fastigial neurons on selected genes Gene expression levels (in qPCR Ct) in the individual neurons are organized in columns. Ct values were determined with a common threshold for all the qPCR reactions (see Methods). Left columns of the table show the genes tested and primer/probe sequences used for the qPCR reaction. The qPCR probes which include minor groove binders (MGB, Applied Biosystems) are also indicated. Note that Lys, Trp, Phe, and Thr are spike-in control RNA (1000, 100, 20, and five copies, respectively). https://cdn.elifesciences.org/articles/58613/elife-58613-fig2-data1-v2.xlsx Download elife-58613-fig2-data1-v2.xlsx Nearly all excitatory neurons (97.6%: 41/42) significantly expressed at least one of the cell-type markers (Spp1, Snca, and Calb2), as confirmed with immunostaining in VgluT2-Cre;Ai14 line (Figure 1—figure supplement 3C). Hierarchical clustering based on these markers revealed four cell types in the fastigial glutamatergic population, which we termed F1-F4 (Figure 2A). Two cell types expressed Spp1 and were distinguished by the absence (F1) or presence (F2) of Snca. The other two cell types both expressed Snca and were separated by strong Calb2 expression in F3 but not F4. The combinatorial marker expression patterns corresponded with those observed in immunostaining (Figure 1): F1 with Spp1+ neurons in the rFN (F1R) and the rDLP (F1rDLP), F2 with Spp1+/Snca+ neurons in the cDLP, F3 with Calb2+/Snca+ neurons in the vlFN, and F4 with Snca+ neurons in the cFN. To further assess distinctions across cell types, we examined expression levels of neurofilament genes. Nefl, Nefm, and Nefh were highest in F1, intermediate in F2, and lowest in F3 and F4 neurons (Figure 2A), suggesting that the axon diameter of these cell types are in the order of F1 >F2>F3/F4 (Friede and Samorajski, 1970; Hoffman et al., 1987; Lee and Cleveland, 1996). Differences across cell types in axonal caliber were confirmed with local injections of adeno-associated virus (AAV.hSyn.TurboRFP) into the rFN (predominantly F1) and cFN (predominantly F4) (Figure 2—figure supplement 1A–C). As predicted, axons from the rFN were thicker than those from the cFN (1.9 ± 0.6 s.d. µm vs 1.0 ± 0.3 s.d. µm, n = 30 vs. 25, respectively). Neurofilament expression levels were also well correlated with cell body area assessed in immunohistochemically identified fastigial cell types (Nefl in Figure 2B; r2 = 0.996, p=0.002; Nefm and Nefh in Figure 2—figure supplement 1D–F). Excitatory projection neurons in the cerebellar nuclei can be distinguished from local interneurons and inhibitory nucleo-olivary neurons by their ability to fire at high rates (Uusisaari et al., 2007; Bagnall et al., 2009; Najac and Raman, 2015). Each of the excitatory FN neurons expressed Scn8a (Nav1.6), Scn4b (Navb4), Kcnc1 (Kv3.1), and Syt2 (synaptotagmin 2), as predicted for fast-firing neurons (Lien and Jonas, 2003; Mercer et al., 2007; Kodama et al., 2012; Kodama et al., 2020). Previous studies in vestibular nucleus neurons, which share multiple properties with cerebellar nucleus neurons, including high spontaneous firing rates (Uusisaari et al., 2007; Bagnall et al., 2009; Najac and Raman, 2015) and direct innervation by Purkinje cells (Sekirnjak et al., 2003; Shin et al., 2011), demonstrated that variations in neuronal firing capacities can be predicted by the absolute expression levels of these and other ‘fast-spiking’ genes (Kodama et al., 2020). Fastigial cell types F1-F4 exhibited different expression levels of action potential genes Scn8a and Kcnc1, but with an almost constant ratio (r2 = 0.98, Figure 2D). Nefl, Nefm, Nefh, Scn4b, and Syt2 followed the same trend (Figure 2—figure supplement 1G–I); absolute expression levels of these genes were consistently in the order of F1 >F2>F3/F4, suggesting that F1, F2, and F3 and F4 would have the fastest, intermediate, and the slowest firing capacities. Downstream targets of excitatory fastigial cell types are distinct Although projections from rostral vs caudal fastigial nucleus are known to differ (Angaut and Bowsher, 1970; Bentivoglio and Kuypers, 1982), little is known about the downstream targets of specific fastigial cell types. We performed a series of anterograde, transsynaptic, and retrograde anatomical tracing experiments to identify global and cell-type specific connectivity patterns. Pan-fastigial injections of anterograde tracer AAV9.hSyn.TurboRFP (Figure 3A) revealed divergent fastigial projections to over 60 distinct brain regions (Figure 3 and Figure 3—figure supplement 1, Figure 3—source data 1), which were confirmed with complementary injections of anterograde transsynaptic tracer AAV1.hSyn.Cre in Ai14 reporter mice (Zingg et al., 2017; Figure 3A). Projection patterns identified in these pan-fastigial injections were quite consistent across individuals (n = 6 males for axonal labeling; n = 4 male and n = 3 female for transsynaptic labeling). Fastigial axonal terminals and postsynaptic neurons contralateral to injection sites were prominent in cervical spinal cord (Figure 3C), cerebellar cortex (Figure 3—figure supplement 1F), and several regions of the medulla (Figure 3D), pons (Figure 3—figure supplement 1C), midbrain (Figure 3E, Figure 3—figure supplement 1D), and diencephalon (Figure 3F,G, Figure 3—figure supplement 1E). Notably, fastigial projections to the thalamus were not limited to the 'motor thalamus' (ventrolateral (VL) and ventromedial (VM) nuclei), but also robustly to intralaminar thalamus (centrolateral (CL) and parafascicular (PF)) and the mediodorsal (MD) nucleus (Figure 3F,G and Figure 3—figure supplement 1E). These projections were considered to derive from excitatory neurons because inhibitory neurons target different brain regions; glycinergic FN neurons project ipsilaterally, to hindbrain and spinal cord (Bagnall et al., 2009), and GABAergic FN projections exclusively target the IO, as confirmed with selective anterograde tracing by injecting Cre-dependent AAV into the FN of Gad2Cre mice (Figure 3—source data 1). Figure 3 with 2 supplements see all Download asset Open asset Pan-fastigial AAV injections reveal widespread fastigial output. (A) Schematics of AAV injection experiments to label fastigial axons and terminals (left) and to transsynaptically label postsynaptic neuronal somata (right). (B) Schematic of a sagittal view of the mouse brain to show approximate levels of the coronal sections in C-G. Tracer injection to the FN is illustrated in red. (C–G) Representative low magnification confocal images obtained from pan-fastigial injections of AAVs (AAV, AAV9.RFP; tsAAV, AAV1.hSyn.Cre). Contralateral side to the injection site is shown. Blue rectangles in the drawings of the sections indicate imaged areas. In these paired images, the left image shows labeled fastigial axons and terminals in red and counterstained with Nissl in blue; the right image shows transsynaptically labeled neurons in black. Asterisks indicate axonal bundles. Results from these axonal labeling and transsynaptic tracing experiments were consistent across mice (n = 6 males for axonal labeling; n = 4 male and n = 3 female for transsynaptic labeling. (C) Shows fastigial projections to the cervical spinal cord, dense at the lamina VII-VIII, at the level of C1-C2. Dotted lines circumscribe the gray matter. (D) Shows fastigial projections to the medulla. Labeled fastigial axons crossed the midline within the cerebellum, exit via the cerebellar peduncle and provide dense projections to the vestibular and reticular nuclei, including the lateral vestibular nucleus (LVN), nucleus reticularis gigantocellularis (NRG), intermediate reticular nucleus (IRt), and parvocellular reticular nucleus (PCRt), with additional collateralization to the nucleus prepositus hypoglossi (NPH) and facial nucleus (7N). (E) Shows fastigial projections to the midbrain. Labeled fastigial axons innervate perioculomotor nuclei including the interstitial nucleus of Cajal (INC), lateral periaqueductal gray (lPAG), and nucleus of Darkschewitsch (Dk), and more laterally located nuclei including the superior colliculus (SC), mesencephalic reticular nucleus (mRt), and substantia nigra pars compacta (SNc). Innervation of the ventral tegmental area (VTA) is very sparse. Note that the dense labeling in the red nucleus (+) derived from axons from the anterior interpositus that were labeled by AAV that leaked from the injection center in the FN (Figure 3—figure supplement 1B, bottom). Injections specifically localized to FN subregions did not significantly label the red nucleus (Figure 5—figure supplement 1). (F and G) Fastigial projections to the thalamus. Labeled fastigial terminals and transsynaptically labeled somata are distributed at the PF, CL, MD, VM, and VL thalamic nuclei and the zona incerta (ZI). Fastigial axons projecting to the contralateral thalamus traverse the midline at the level of G, and innervate the ipsilateral thalamus. Scale bar in G applies to all confocal images in C-G. Abbreviations, 7N, facial nucleus; CL, centrolateral thalamic nucleus; Dk, nucleus of Darkschewitsch; INC, interstitial nucleus of Cajal; IRt, intermediate reticular nucleus; lPAG, lateral periaqueductal gray; LVN, lateral vestibular nucleus; MD, mediodorsal thalamic nucleus; mRt, mesencephalic reticular nucleus; NRG, nucleus reticularis gigantocellularis; NPH, nucleus prepositus hypoglossi; PCRt, parvocellular reticular nucleus; PF, parafascicular thalamic nucleus; SC, superior colliculus; SNc, substantia nigra pars compacta; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VTA, ventral tegmental area; ZI, zona incerta. Figure 3—source data 1 Fastigial projection targets identified by localized anterograde tracer injections. Rows list the brain regions and specific nuclei that were anterogradely labeled in pan-fastigial tracer injections. Color-coded columns show distinct projections from each of the fastigial subregions identified by localized anterograde injections of AAV and/or BDA to the rFN (n = 2 males and n = 2 females), rDLP (n = 2 males and n = 1 female), cDLP (n = 2 males), vlFN (n = 3 males), and cFN (n = 3 males). Significant vs sparse terminal innervations are indicated with + vs ±. Absence of labeled terminals is indicated with –. Sparse projections are listed separately at the bottom. https://cdn.elifesciences.org/articles/58613/elife-58613-fig3-data1-v2.xlsx Download elife-58613-fig3-data1-v2.xlsx Figure 3—source data 2 List of tracers, coordinates, injection volume, and mice used for tracing experiments. Experimental parameters used in the tracing experiments, including tracers, coordinates, injection volumes, injection side (left or right), and mouse sex. These parameters were associated with 'Injection Site'. AP, ML, DV coordinates are given in mm. For the tracer injections with angled approach, AP and ML correspond with the location of craniotomy before tilting the manipulator or stage, and DV corresponds with the distance from the surface of the brain to the target in the angled setting. https://cdn.elifesciences.org/articles/58613/elife-58613-fig3-data2-v2.xlsx Download elife-58613-fig3-data2-v2.xlsx To identify linkages between specific fastigial cell types and downstream target nuclei, we performed localized anterograde tracing experiments via stereotaxic injections of AAVs (AAV9.hSyn.eGFP, AAV9.hSyn.TurboRFP, and AAV1.hSyn.Cre) into anatomically defined subregions of the FN (Figure 1; Figure 3—source data 2). Subsequent retrograde tracing combined with immunostaining for the fastigial cell-type markers was performed to confirm FN cell types of origin. Rostral fastigial projection targets To distinguish projections from F1R, F3, and F1rDLP, we made localized injections of AAVs into the rFN, vlFN, and rDLP, respectively, which resulted in differential terminal labeling in several regions of the hindbrain, including nucleus reticularis gigantocellularis (NRG), ventral medullary (MdV) and intermediate (IRt) portions of the reticular formation, and inferior vestibular nucleus (IVN) (Figure 4A,B, Figure 3—source data 1). Distinct projections from each cell type were confirmed with complementary retrograde tracing experiments in which FastBlue or retrobeads were injected into NRG/MdV, IVN, and IRt, which are the major projection targets of F1R, F3, and F1rDLP, respectively. Those injections resulted in labeling of SPP1+ large neurons in the rFN (Figure 4C), CALB2+ small neurons in the vlFN (Figure 4D), and SPP1+ large neurons in the rDLP (Figure 4E). Figure 4 with 1 supplement see all Download asset Open asset Segregated output channels from rostral parts of the FN. (A and B) AAV-mediated anterograde tracing with injections localized to subregions of the rostral fastigial nucleus that distinguish projections from F1R, F3, and F1rDLP neurons. (A) Shows a dual AAV injection in the rFN (F1R region, AAV9.RFP, magenta in confocal image) and vlFN (F3 region, AAV9.GFP, green in confocal image) and the resultant labeling in the inferior vestibular nucleus (IVN) and medulla. Results were consistent across four mice (n = 2 male and n = 2 female) for rFN projections, and across three male mice for vlFN projections. (B) Shows an AAV9.RFP injection in the rDLP (F1rDLP region, inset) and the resultant labeling (black in confocal image) in the IRt and PCRt. Results were consistent across three mice (n = 2 male and n = 1 female). (C–E) Retrograde tracing experiments to confirm segregation among rostral fastigial output projections. Injections were made into NRG/MdV (C: n = 3 males), IVN (D: n = 3 males), and IRt (D: n = 2 males). (C) Shows retrograde tracer (FastBlue, cyan in the right image) injection to the NRG/MdV. Retrogradely labeled neurons are mapped onto the rFN (middle) and overlapped with SPP1+ (red in the right image) neurons of large cell bodies, indicating that F1R neurons project to the NRG/MdV. Similarly, (D and E) Show results of retrograde labeling from the IVN (FastBlue) and IRt (retrobead). The localization of labeled cells and immunoreactivity for CALB2 and SPP1 indicate that F3 and F1rDLP neurons project to the IVN and IRt, respectively. Dotted lines in the middle panels indicate approximate borders for the fastigial subregions. (F) Key projection targets of F1R neurons. AAV and tsAAV indicate labeling by AAV9.RFP and AAV1.hSyn.Cre, respectively. Panels show dense fastigial axonal innervation (cyan) of large neurons in the NRG and LVN, which are fluorescently labeled in YFP16 mouse line (red). Also shown are transsynaptically labeled SPP1+ PMn neuron and a TH+ SubC neuron (white arrowheads) (axonal tracing, n = 2 males and n = 2 females; transsynaptic tracing, n = 3 females). (G) F3 neurons project to the Kölliker-Fuse (KF) nucleus, as demonstrated by anterograde transsynaptic labeling from the vlFN. Inset shows location of the labeled KF neurons (axonal tracing, n = 2 males; transsynaptic tracing, n = 2 males). (H) F1R and F3 differentially innervate large and small neurons in the IVN, respectively, as demonstrated by anterograde transsynaptic labeling from the rFN and vlFN. (I) Projections of F1rDLP neurons to IRt and CHAT+ facial nucleus neurons, demonstrated by anterograde transsynaptic tracing from the rDLP (axonal tracing, n = 2 males and n = 1 female; transsynaptic tracing, n = 3 females). (J–L) Summary of the major output targets of F1R, F3, and F1rDLP, respectively. Dotted vertical line indicates midline. Targets are arranged rostro-caudally from top to bottom. Scale bars in the middle and right panels in E applies to similar panels in C and D. Scale bar in H applies to both left and right images in H. Figure 3—source data 1 contains a complete list of projection targets. Abbreviations, 7N, facial nucleus; AAV, adeno associated virus; cord, spinal cord; IRt, intermediate reticular nucleus; IVN, inferior vestibular nucleus; KF, Kölliker-Fuse nucleus; LPGi, lateral paragigantocellular nucleus; LVN, lateral vestibular nucleus; MdV, medullary reticular nucleus, ventral; NRG, nucleus reticularis gigantocellularis; PCRt, parvocellular reticular nucleus; PMn, paramedian reticular nucleus; SubC, subcoeruleus nucleus; tsAAV, anterograde transsynaptic labeling with AAV; Y, dorsal group Y. Anterograde axonal tracing paired with transsynaptic tracing identified the projections of F1R, F3, and F1rDLP as follows. Consistent with the known functions of the rostral fastigial nucleus in posture and locomotion (Chambers and Sprague," @default.
• W3058758979 created "2020-08-24" @default.
• W3058758979 creator A5083296072 @default.
• W3058758979 creator A5085271296 @default.
• W3058758979 creator A5087353199 @default.
• W3058758979 date "2020-07-03" @default.
• W3058758979 modified "2023-10-14" @default.
• W3058758979 title "Author response: Modular output circuits of the fastigial nucleus for diverse motor and nonmotor functions of the cerebellar vermis" @default.
• W3058758979 doi "https://doi.org/10.7554/elife.58613.sa2" @default.
• W3058758979 hasPublicationYear "2020" @default.
• W3058758979 type Work @default.
• W3058758979 sameAs 3058758979 @default.
• W3058758979 citedByCount "5" @default.
• W3058758979 countsByYear W30587589792021 @default.
• W3058758979 countsByYear W30587589792023 @default.
• W3058758979 crossrefType "peer-review" @default.
• W3058758979 hasAuthorship W3058758979A5083296072 @default.
• W3058758979 hasAuthorship W3058758979A5085271296 @default.
• W3058758979 hasAuthorship W3058758979A5087353199 @default.
• W3058758979 hasBestOaLocation W30587589791 @default.
• W3058758979 hasConcept C101468663 @default.
• W3058758979 hasConcept C111919701 @default.
• W3058758979 hasConcept C169760540 @default.
• W3058758979 hasConcept C2776139471 @default.
• W3058758979 hasConcept C2779652256 @default.
• W3058758979 hasConcept C2780168142 @default.
• W3058758979 hasConcept C41008148 @default.
• W3058758979 hasConcept C86803240 @default.
• W3058758979 hasConceptScore W3058758979C101468663 @default.
• W3058758979 hasConceptScore W3058758979C111919701 @default.
• W3058758979 hasConceptScore W3058758979C169760540 @default.
• W3058758979 hasConceptScore W3058758979C2776139471 @default.
• W3058758979 hasConceptScore W3058758979C2779652256 @default.
• W3058758979 hasConceptScore W3058758979C2780168142 @default.
• W3058758979 hasConceptScore W3058758979C41008148 @default.
• W3058758979 hasConceptScore W3058758979C86803240 @default.
• W3058758979 hasLocation W30587589791 @default.
• W3058758979 hasOpenAccess W3058758979 @default.
• W3058758979 hasPrimaryLocation W30587589791 @default.
• W3058758979 hasRelatedWork W1741988749 @default.
• W3058758979 hasRelatedWork W182660780 @default.
• W3058758979 hasRelatedWork W1998726361 @default.
• W3058758979 hasRelatedWork W2014929484 @default.
• W3058758979 hasRelatedWork W2027378262 @default.
• W3058758979 hasRelatedWork W2038790559 @default.
• W3058758979 hasRelatedWork W2060421877 @default.
• W3058758979 hasRelatedWork W2123989256 @default.
• W3058758979 hasRelatedWork W2309171844 @default.
• W3058758979 hasRelatedWork W2334445845 @default.
• W3058758979 isParatext "false" @default.
• W3058758979 isRetracted "false" @default.
• W3058758979 magId "3058758979" @default.
• W3058758979 workType "peer-review" @default.