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Dopamine D1 Receptor–Positive Neurons in the Lateral Nucleus of the Cerebellum Contribute to Cognitive Behavior

Open AccessPublished:February 03, 2018DOI:https://doi.org/10.1016/j.biopsych.2018.01.019

      Abstract

      Background

      Studies in humans and nonhuman primates have identified a region of the dentate nucleus of the cerebellum, or the lateral cerebellar nucleus (LCN) in rodents, activated during performance of cognitive tasks involving complex spatial and sequential planning. Whether such a subdivision exists in rodents is not known. Dopamine and its receptors, which are implicated in cognitive function, are present in the cerebellar nuclei, but their function is unknown.

      Methods

      Using viral and genetic strategies in mice, we examined cellular phenotypes of dopamine D1 receptor–positive (D1R+) cells in the LCN with whole-cell patch clamp recordings, messenger RNA profiling, and immunohistochemistry to examine D1R expression in mouse LCN and human dentate nucleus of the cerebellum. We used chemogenetics to inhibit D1R+ neurons and examined behaviors including spatial navigation, social recognition memory, prepulse inhibition of the acoustic startle reflex, response inhibition, and working memory to test the necessity of these neurons in these behaviors.

      Results

      We identified a population of D1R+ neurons that are localized to an anatomically distinct region of the LCN. We also observed D1R+ neurons in human dentate nucleus of the cerebellum, which suggests an evolutionarily conserved population of dopamine-receptive neurons in this region. The genetic, electrophysiological, and anatomical profile of mouse D1R neurons is consistent with a heterogeneous population of gamma-aminobutyric acidergic, and to a lesser extent glutamatergic, cell types. Selective inhibition of D1R+ LCN neurons impairs spatial navigation memory, response inhibition, working memory, and prepulse inhibition of the acoustic startle reflex.

      Conclusions

      Collectively, these data demonstrate a functional link between genetically distinct neurons in the LCN and cognitive behaviors.

      Keywords

      The cerebellum is well known for its role in coordinating motor output and adapting involuntary reflexes to support sensory prediction error–based learning (
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      A computational model of four regions of the cerebellum based on feedback-error learning.
      ,
      • Medina J.F.
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      ,
      • Raymond J.L.
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      Neural learning rules for the vestibulo-ocular reflex.
      ). A lesser appreciated, but no less important, function of the cerebellum is its role in goal-directed cognitive functions. Consistent with this role, numerous neuropsychiatric disorders and developmental syndromes are associated with alterations in cerebellar function, including schizophrenia, autism, frontotemporal dementia, and Joubert syndrome (
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      Cerebellar involvement in Pick’s disease: Affliction of mossy fibers, monodendritic brush cells, and dentate projection neurons.
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      ). In many individuals with altered cerebellar anatomy, cognitive and behavioral perturbations occur in the absence of gross motor impairments (
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      Disorders of the cerebellum: Ataxia, dysmetria of thought, and the cerebellar cognitive affective syndrome.
      ). Several of these disorders are associated with alterations in dentate nucleus of the cerebellum (DCN)/lateral cerebellar nucleus (LCN) anatomy and gene expression (
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      • Arai K.
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      Cerebellar involvement in Pick’s disease: Affliction of mossy fibers, monodendritic brush cells, and dentate projection neurons.
      ,
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      The LCN is the most lateral of the cerebellar nuclei and is the major output nucleus of the neocerebellum (
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      Evidence for topographic organization in the cerebellum of motor control versus cognitive and affective processing.
      ). In addition to classical cerebellar output pathways to thalamic nuclei and red nucleus, the LCN has direct, reciprocal connections with limbic circuitry such as the ventral tegmental area (VTA) (
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      The basilar pontine nuclei and the nucleus reticularis tegmenti pontis subserve distinct cerebrocerebellar pathways.
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      ). Consistent with this connectivity, functional imaging and anatomical mapping studies in primates demonstrate both motor and cognitive regions within the LCN (
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      ). For example, in humans undergoing a functional magnetic resonance imaging study, DCN activation increased three- to fourfold during attempts to solve a pegboard puzzle compared with simply moving these pegs (
      • Kim S.G.
      • Ugurbil K.
      • Strick P.L.
      Activation of a cerebellar output nucleus during cognitive processing.
      ). Neocerebellar lesions in humans can elicit changes in affect, spatial navigation, working memory (WM), behavioral flexibility, and interval timing (
      • Ghajar J.
      • Ivry R.B.
      The predictive brain state: Asynchrony in disorders of attention?.
      ,
      • Schmahmann J.D.
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      ,
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      ), and lesions of lateral cerebellar cortex (CCtx) and LCN in rodents impair spatial learning and memory (
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      ,
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      ), reduce reinforcement learning and motivation (
      • Bauer D.J.
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      Cerebellar dentate nuclei lesions reduce motivation in appetitive operant conditioning and open field exploration.
      ), and impair absolute timing (
      • Yamaguchi K.
      • Sakurai Y.
      Inactivation of cerebellar cortical Crus II disrupts temporal processing of absolute timing but not relative timing in voluntary movements.
      ). Several human disorders with cerebellar dysfunction are associated with deficits in social behavior (
      • Wassink T.H.
      • Andreasen N.C.
      • Nopoulos P.
      • Flaum M.
      Cerebellar morphology as a predictor of symptom and psychosocial outcome in schizophrenia.
      ,
      • Webb S.J.
      • Sparks B.F.
      • Friedman S.D.
      • Shaw D.W.
      • Giedd J.
      • Dawson G.
      • et al.
      Cerebellar vermal volumes and behavioral correlates in children with autism spectrum disorder.
      ). Aberrant cerebellar development in rodent models is also associated with altered WM and social behavior without motor impairment (
      • Kim Y.S.
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      • Wine R.N.
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      Altered cerebellar development in nuclear receptor TAK1/TR4 null mice is associated with deficits in GLAST+ glia, alterations in social behavior, motor learning, startle reactivity, and microglia.
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      Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice.
      ). Collectively, these data point to a key role of the cerebellum in the regulation of cognitive and social behaviors. Given the strong correlation between cerebellar function and mental illness, there is a strong desire to understand the relationship between cerebellar anatomy and function and the etiology of symptom domains of these disorders.
      Multiple cell types that reside within the LCN have unique morphological and projection profiles (
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      • Czubayko U.
      • Sultan F.
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      Two types of neurons in the rat cerebellar nuclei as distinguished by membrane potentials and intracellular fillings.
      ,
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      The GABAergic cerebello-olivary projection in the rat.
      ,
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      • et al.
      Excitatory cerebellar nucleocortical circuit provides internal amplification during associative conditioning.
      ,
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      Cerebellar premotor output neurons collateralize to innervate the cerebellar cortex.
      ,
      • Husson Z.
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      • Broll I.
      • Zeilhofer H.U.
      • Dieudonne S.
      Differential GABAergic and glycinergic inputs of inhibitory interneurons and Purkinje cells to principal cells of the cerebellar nuclei.
      ,
      • Najac M.
      • Raman I.M.
      Integration of Purkinje cell inhibition by cerebellar nucleo-olivary neurons.
      ,
      • Schwarz C.
      • Schmitz Y.
      Projection from the cerebellar lateral nucleus to precerebellar nuclei in the mossy fiber pathway is glutamatergic: A study combining anterograde tracing with immunogold labeling in the rat.
      ,
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      Anatomical and physiological evidence for a cerebellar nucleo-cortical projection in the cat.
      ,
      • Uusisaari M.
      • Knopfel T.
      GlyT2+ neurons in the lateral cerebellar nucleus.
      ,
      • Uusisaari M.
      • Obata K.
      • Knopfel T.
      Morphological and electrophysiological properties of GABAergic and non-GABAergic cells in the deep cerebellar nuclei.
      ). Input-mapping and gene-mapping studies have revealed distinct cerebellar nuclei subregions, including at least two subdivisions of the LCN in rodents (
      • Chung S.H.
      • Marzban H.
      • Hawkes R.
      Compartmentation of the cerebellar nuclei of the mouse.
      ,
      • Sugihara I.
      • Shinoda Y.
      Molecular, topographic, and functional organization of the cerebellar nuclei: Analysis by three-dimensional mapping of the olivonuclear projection and aldolase C labeling.
      ). However, studies interrogating the functional specificity of these region- or cell type-specific cerebellar divisions are lacking.
      Dopamine and dopamine receptors are broadly implicated in mental illness (
      • Knable M.B.
      • Weinberger D.R.
      Dopamine, the prefrontal cortex and schizophrenia.
      ) and localize to microanatomical regions of the cerebellum (
      • Barili P.
      • Bronzetti E.
      • Ricci A.
      • Zaccheo D.
      • Amenta F.
      Microanatomical localization of dopamine receptor protein immunoreactivity in the rat cerebellar cortex.
      ,
      • Delis F.
      • Mitsacos A.
      • Giompres P.
      Dopamine receptor and transporter levels are altered in the brain of Purkinje cell degeneration mutant mice.
      ,
      • Melchitzky D.S.
      • Lewis D.A.
      Tyrosine hydroxylase- and dopamine transporter-immunoreactive axons in the primate cerebellum: Evidence for a lobular- and laminar-specific dopamine innervation.
      ,
      • Nelson T.E.
      • King J.S.
      • Bishop G.A.
      Distribution of tyrosine hydroxylase-immunoreactive afferents to the cerebellum differs between species.
      ). Furthermore, in rodents, cerebellar nuclei are reported to have dopamine at concentrations greater than in hippocampus or CCtx and similar levels relative to frontal cortex (
      • Versteeg D.H.
      • Van Der Gugten J.
      • De Jong W.
      • Palkovits M.
      Regional concentrations of noradrenaline and dopamine in rat brain.
      ). Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, is found in fibers projecting to all cerebellar lobules, laminae, and nuclei in mouse (
      • Nelson T.E.
      • King J.S.
      • Bishop G.A.
      Distribution of tyrosine hydroxylase-immunoreactive afferents to the cerebellum differs between species.
      ). Dopamine D1 receptor (D1R) is implicated in cognitive functions, including WM, spatial navigation, and cognitive flexibility, but whether D1Rs in cerebellum could regulate these is unknown (
      • El-Ghundi M.
      • O’Dowd B.F.
      • George S.R.
      Insights into the role of dopamine receptor systems in learning and memory.
      ,
      • Williams G.V.
      • Castner S.A.
      Under the curve: Critical issues for elucidating D1 receptor function in working memory.
      ). Based on these observations, we hypothesized that D1Rs may identify subdivisions within the LCN that would allow for manipulation and interrogation of LCN function, with relevance to cognition in neuropsychiatric disorders. Here, we confirm the presence of D1R+ neurons in human DCN and mouse LCN. Molecular profiling, projection analysis, and electrophysiological characterization of these neurons reveal a partially heterogeneous population of neurons that is largely inhibitory. Consistent with a role in cognition, cell-specific chemogenetic silencing of these neurons alters spatial navigation memory, response inhibition, WM, and sensorimotor gating, thereby demonstrating a cellular specialization within the LCN for these functions.

      Methods and Materials

      Standard Techniques for Characterization of LCN D1R-positive Neurons and Their Role in Behavior

      We used several standard molecular and cellular techniques, including immunohistochemistry, RiboTag, quantitative polymerase chain reaction, slice electrophysiology, and in vivo electrophysiology, for characterizing cellular phenotypes as well as several cognitive, motivational, sensory, and motor behavioral tasks. These techniques are described in detail in the Supplement.

      Results

      D1R Neurons Occupy a Specific Subregion of the LCN

      We confirmed D1R expression in subsets of cells in mouse and human DCN (Figure 1A–C and Supplemental Figure S2A, B). Immunohistochemical analysis suggests that dopamine D2 receptor (D2R) is broadly expressed in mouse LCN, whereas D1R has a lower, regionally restricted expression (Figure 1D and Supplemental Figures S3A, C, E and S4A–P). D1R staining was not present in striatum or LCN of Drd1aCre/Cre knockout mice but was present in these regions in wild-type littermate control mice, validating the antibody (Supplemental Figures S3A–G and S4A–P). To determine whether D1R would isolate discrete populations of neurons in mouse LCN, we performed an anatomical analysis of virally labeled D1R neurons. Cell-specific expression of yellow fluorescent protein (YFP) was achieved by injecting a conditional adeno-associated viral (AAV) vector (AAV-FLEX-YFP) into the LCN of Drd1aCre/+ mice (Figure 1E). YFP-positive neurons were counted along the rostral-caudal extent of the LCN (Figure 1F, G), with the highest numbers of labeled neurons localized to caudal, medial, and ventral zones of the LCN (Figure 1H–J). Because this method of identifying cells could be confounded by viral spread, we performed a similar experiment using Drd1aCre/+ mice crossed with a conditional tdTomato reporter line (
      • Madisen L.
      • Zwingman T.A.
      • Sunkin S.M.
      • Oh S.W.
      • Zariwala H.A.
      • Gu H.
      • et al.
      A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.
      ). Tomato-positive neurons were counted along the rostral-caudal extent of the LCN (Supplemental Figure S1B, C), with a similar pattern of the highest number of labeled neurons localized to caudal, medial, and ventral zones of the LCN (Supplemental Figure S1D–F). Although a complete stereological assessment of their anatomical distribution in humans could not be performed, these data strongly support a highly conserved population of D1R-expressing neurons in the cerebellar nuclei.
      Figure thumbnail gr1
      Figure 1Immunohistochemistry reveals staining for dopamine D1 receptor (D1R) in human dentate nucleus of the cerebellum and mouse lateral cerebellar nucleus (LCN). Mapping reveals subregional localization of D1R neurons in the LCN. (A, B) Immunohistochemistry for the D1R protein in human dentate nucleus of the cerebellum (in a parasagittal section), square in (A) is zone at higher magnification in (B). Staining for D1R was positive in each of five cases. (C) Staining of D1R in the green color channel in lateral nucleus of the cerebellum in a coronal section. (D) Staining of dopamine D2 receptor in the red color channel in lateral nucleus of the cerebellum in a coronal section. (E) Illustration depicting the lateral (dentate) nucleus (DN/LAT) from the rostral (R) to caudal (C) extent that was analyzed for D1R neuron location. (F) Illustration of the rostral region of LCN in coronal plane (top) and representative image of D1R:Tmto expression in rostral LCN overlaid with divisions of dorsal, ventral, medial, and lateral zones (bottom). Dorsal (D) and lateral (L) orientations are denoted in the inset. Scale bar = 120 μm. (G) Illustration of the caudal region of LCN in coronal plane (top) and representative image of D1R:Tmto expression in caudal LCN overlaid with divisions of dorsal, ventral, medial, and lateral zones (bottom), In addition to the LCN, few cells were observed in the parvocellular region of the LCN and the interposed nuclei. (H) Quantification of distribution of D1R:yellow fluorescent protein (YFP)-positive cells in rostral vs. caudal zones (t29 = 7.42). (I) Quantification of distribution of D1R:green fluorescent protein–positive cells in medial vs. lateral zones (t56 = 19.20). (J) Quantification of distribution of D1R:green fluorescent protein–positive cells in dorsal vs. ventral zones (t54 = 2.83). Results were acquired from 4 mice; four sections per mouse were counted bilaterally and are represented as the average number of cells per section per side or the percentage of cells within each region per section per side. Illustrations in (A–C) are from Paxinos and Franklin
      (
      • Paxinos G.
      • Keith B.J.
      Paxinos and Franklin's The Mouse Brain in Stereotaxic Coordinates.
      )
      . **p < .01, ****p < .0001, Student’s t test, two tailed. IntA, interposed nucleus, anterior part; IntP, interposed nucleus, posterior part; PC, dentate (or lateral) nucleus, parvocellular part; Y, nucleus Y of the vestibular complex.

      Electrophysiological Properties of D1R LCN Neurons in Slice

      Different cell types within the LCN have distinct electrophysiological profiles (
      • Czubayko U.
      • Sultan F.
      • Thier P.
      • Schwarz C.
      Two types of neurons in the rat cerebellar nuclei as distinguished by membrane potentials and intracellular fillings.
      ,
      • Husson Z.
      • Rousseau C.V.
      • Broll I.
      • Zeilhofer H.U.
      • Dieudonne S.
      Differential GABAergic and glycinergic inputs of inhibitory interneurons and Purkinje cells to principal cells of the cerebellar nuclei.
      ,
      • Najac M.
      • Raman I.M.
      Integration of Purkinje cell inhibition by cerebellar nucleo-olivary neurons.
      ,
      • Uusisaari M.
      • Knopfel T.
      GlyT2+ neurons in the lateral cerebellar nucleus.
      ,
      • Uusisaari M.
      • Obata K.
      • Knopfel T.
      Morphological and electrophysiological properties of GABAergic and non-GABAergic cells in the deep cerebellar nuclei.
      ). To characterize the electrophysiological properties of D1R neurons, we performed whole-cell patch clamp recordings on fluorescently identified neurons from acute cerebellar slices. D1R cells had diverse intrinsic properties and could be sorted into two categories (type I [62% of cells] and type II [38% of cells]) based on properties of action potential (AP) waveforms, including half width and time to after hyperpolarization (AHP) (Figure 2A). AP threshold, peak amplitude, and AHP amplitude did not differ between populations (Figure 2B). Type I neurons had broad APs with a slow AHP, whereas type II neurons had narrow APs with a fast AHP (Figure 2C, D). The majority of type I neurons (15/16) were spontaneously active, whereas the majority of type II neurons (7/10) fired APs only following current injection (Figure 2E).
      Figure thumbnail gr2
      Figure 2Electrophysiological characterization of dopamine D1 receptor lateral cerebellar nucleus neurons in mice. (A) Average action potential (AP) waveforms of type I and type II neurons. Scale bar = 20 mV, 2 ms. (B) AP threshold, AP peak, and after hyperpolarization (AHP) peak in type I and type II neurons. (C) Time to AHP peak in type I and type II neurons. ***p < .001, Student’s t test, two tailed, t24 = 4.04. (D) AP half width of type I and type II neurons. ****p < .0001, Student’s t test, two tailed, t24 = 5.33. (E) Example type I (top) and type II (middle) neurons before, during, and after 50 pA current injection (bottom). Scale bar = 20 mV, 200 ms. (F) Capacitance of type I and type II neurons. Type I n = 16, Type II n = 10, ****p < .0001, Student’s t test, two tailed, t24 = 5.87. (G) Distribution of measured surface areas of dopamine D1 receptor neurons in the dentate nucleus of the cerebellum, revealing a non-normal distribution (n = 306 cells, Shapiro-Wilk, p < .05).
      Membrane capacitance of type I neurons was significantly smaller than that of type II neurons (Figure 2F). Consistent with two (or more) distinct cell sizes, histological measurements of cell surface area revealed a significantly skewed non-normal distribution, with the majority of neurons being of smaller size (Figure 2G).

      Molecular Characterization and Projections of D1R LCN Neurons

      Our electrophysiological results were remarkably similar to established properties of two neuronal populations, local glycinergic/gamma-aminobutyric acidergic (GABAergic) neurons and nucleocortical projecting glycinergic neurons (
      • Uusisaari M.
      • Knopfel T.
      GlyT2+ neurons in the lateral cerebellar nucleus.
      ), and possibly a third type, putatively glutamatergic neurons (three type II neurons differed in that they did fire spontaneous APs, similar to a described population) (
      • Uusisaari M.
      • Obata K.
      • Knopfel T.
      Morphological and electrophysiological properties of GABAergic and non-GABAergic cells in the deep cerebellar nuclei.
      ). To determine the neurotransmitter phenotype of LCN D1R neurons, we selectively isolated actively translating messenger RNA (mRNA) using a RiboTag approach (
      • Sanz E.
      • Quintana A.
      • Deem J.
      • Steiner R.A.
      • Palmiter R.
      • McKnight G.S.
      Fertility-regulating Kiss1 neurons arise from hypothalamic Pomc-expressing progenitors.
      ). Cell-specific expression of the affinity-tagged ribosomal protein Rpl22-HA was achieved by injecting a conditional AAV vector [AAV-FLEX-Rpl22-HA (
      • Sanz E.
      • Quintana A.
      • Deem J.
      • Steiner R.A.
      • Palmiter R.
      • McKnight G.S.
      Fertility-regulating Kiss1 neurons arise from hypothalamic Pomc-expressing progenitors.
      )] into the LCN of Drd1aCre/+ mice (Figure 3A).
      Figure thumbnail gr3
      Figure 3Translational profiling and immunohistochemistry reveal identities of dopamine D1 receptor (D1R) lateral cerebellar nucleus (LCN) neurons in mice. (A) Expression of Rpl22-HA in D1R neurons. Scale bar = 60 μm (inset scale bar = 10 μm) in the LCN. (B) Schematic of RiboTag methodology. Following cell lysis
      (
      • Kawato M.
      • Gomi H.
      A computational model of four regions of the cerebellum based on feedback-error learning.
      )
      , human influenza hemagglutinin antibody-coupled magnetic beads immuno-isolate tagged polysomes and associated messenger RNA
      (
      • Medina J.F.
      • Nores W.L.
      • Ohyama T.
      • Mauk M.D.
      Mechanisms of cerebellar learning suggested by eyelid conditioning.
      )
      . Messenger RNA is isolated
      (
      • Raymond J.L.
      • Lisberger S.G.
      Neural learning rules for the vestibulo-ocular reflex.
      )
      , and complementary DNA is generated from both input and immunoprecipitated messenger RNA. (C) Quantitative reverse transcription polymerase chain reaction analysis of immunoprecipitate relative to input demonstrating significant enrichment of Drd1a, Drd2, Vgat, Gad1, Glyt2, and Penk (enriched markers in black), relative to Cnp, an oligodendrocyte marker in D1R cells of the LCN. Drd3, encoding the dopamine D3 receptor, and oligodendroglial marker (Cnp) were de-enriched (white), while the marker of glutamatergic neurons (Vglut2) was neither enriched nor de-enriched (gray). ****p < .0001, **p < .01, *p < .05, one-way analysis of variance, n = 3 pooled samples of 7 mice/pool (F9,20 = 15.3). (D) Quantitative reverse transcription polymerase chain reaction analysis of immunoprecipitate relative to input demonstrating significant enrichment of Drd2, Penk, Glyt2, Gad1, and Vgat (enriched markers in black), relative to Cnp, in Vgat+ cells of the LCN. Cnp, Vglut2, and Drd3 were de-enriched (white), while Drd1a was neither enriched nor de-enriched (gray). ****p < .0001, **p < .01, *p < .05, one-way analysis of variance, n = 4 pooled samples of 4 mice/pool (F8,32 = 20.1). (E) Quantitative reverse transcription polymerase chain reaction analysis of immunoprecipitate relative to input demonstrating significant enrichment of Drd3 in Vglut2+ cells (black). Glyt2, Gad1, and Vgat were de-enriched (white), while Drd1a, Drd2, and Penk were neither enriched nor de-enriched (gray). ****p < .0001, **p < .01, *p < .05, one-way analysis of variance, n = 4 samples of 1 mouse/sample (F8,32 = 23.3). (F) Venn diagram illustrating distribution of D1R expression in neural subtypes residing in the LCN. AAA, polyadenylate tail; n.s., not significant.
      Following immunoprecipitation and isolation of polyribosomal-associated mRNA (Figure 3B), quantitative polymerase chain reaction demonstrated significant enrichment of Drd1a expression relative to total input mRNA (Figure 3C), confirming the efficacy of the enrichment in Drd1aCre/+ mice. We also observed enrichment of Drd2 expression (Figure 3C), implying some degree of specific coexpression of D1R and D2R in LCN neurons. We found no enrichment of Drd3 (Figure 3C). Neurons in the striatum coexpressing D1R and D2R express the neuropeptide enkephalin, similar to canonical D2R-expressing neurons (
      • Perreault M.L.
      • Hasbi A.
      • Alijaniaram M.
      • Fan T.
      • Varghese G.
      • Fletcher P.J.
      • et al.
      The dopamine D1–D2 receptor heteromer localizes in dynorphin/enkephalin neurons: Increased high affinity state following amphetamine and in schizophrenia.
      ); D1R LCN neurons had enrichment of the proenkephalin (Penk) mRNA (Figure 3C). In addition, we observed enrichment of several markers of inhibitory neurotransmission, that is, mRNA for the GABA synthesizing enzyme Gad67 (Gad1), the vesicular GABA and glycine transporter Vgat (Slc32a1), and the membrane glycine transporter GlyT2 (Slc6a5) (Figure 3C). Surprisingly, expression of the vesicular glutamate transporter Vglut2 (Slc17a6) was neither enriched nor deenriched, suggestive of a third minor population of cells coexpressing both Vglut2 and Drd1a. To clarify this issue, we performed a similar analysis in VgatCre/+ and Vglut2Cre/+ mice (Figure 3D, E). In VgatCre/+ mice, we observed enrichment of Drd2 but only partial enrichment of Drd1a. In Vglut2Cre/+ mice, we found enrichment only of Vglut2 and Drd3, implying that Vglut2 neurons make up only a small percentage of the D1R population. These data indicate that D1R neurons are a regionally restricted population of predominantly inhibitory neurons, with a much smaller proportion being glutamatergic (Figure 3F).
      To determine projections of D1R neurons, we coinjected Cre-dependent AAVs encoding mCherry (AAV-FLEX-mCherry) to fill cells and a green fluorescent protein (GFP)-tagged synaptophysin (AAV-FLEX-synapto-GFP) to label axon terminals (Figure 4A–O). The most prominent projections were a number of small synapto-GFP-positive puncta within the LCN (Figure 4D), concordant with local connectivity. We detected large clusters of synapto-GFP in the granular layer of CCtx, most prominently in areas of lateral CCtx Crus I, Crus II, paraflocculus, and flocculus (Figure 4G). Higher magnification imaging of these synapses revealed morphology similar to previously reported nucleocortical rosettes (
      • Gao Z.
      • Proietti-Onori M.
      • Lin Z.
      • Ten Brinke M.M.
      • Boele H.J.
      • Potters J.W.
      • et al.
      Excitatory cerebellar nucleocortical circuit provides internal amplification during associative conditioning.
      ,
      • Houck B.D.
      • Person A.L.
      Cerebellar premotor output neurons collateralize to innervate the cerebellar cortex.
      ,
      • Tolbert D.L.
      • Bantli H.
      • Bloedel J.R.
      Anatomical and physiological evidence for a cerebellar nucleo-cortical projection in the cat.
      ) (Figure 4J), consistent with glutamatergic projections to CCtx (
      • Ankri L.
      • Husson Z.
      • Pietrajtis K.
      • Proville R.
      • Lena C.
      • Yarom Y.
      • et al.
      A novel inhibitory nucleo-cortical circuit controls cerebellar Golgi cell activity.
      ,
      • Gao Z.
      • Proietti-Onori M.
      • Lin Z.
      • Ten Brinke M.M.
      • Boele H.J.
      • Potters J.W.
      • et al.
      Excitatory cerebellar nucleocortical circuit provides internal amplification during associative conditioning.
      ,
      • Houck B.D.
      • Person A.L.
      Cerebellar premotor output neurons collateralize to innervate the cerebellar cortex.
      ). We also observed a small number of beadlike synapses in the granular and Purkinje cell layers (Figure 4K), consistent with projections from the LCN, which may represent GABAergic/glycinergic projections (
      • Ankri L.
      • Husson Z.
      • Pietrajtis K.
      • Proville R.
      • Lena C.
      • Yarom Y.
      • et al.
      A novel inhibitory nucleo-cortical circuit controls cerebellar Golgi cell activity.
      ).
      Figure thumbnail gr4
      Figure 4Projection patterns of dopamine D1 receptor (D1R) lateral cerebellar nucleus neurons in D1R-Cre mice compared with Vgat-Cre and Vglut2-Cre mice. (A–C) Illustration depicting the lateral dentate nucleus location of injection for Synapto-green fluorescent protein (GFP) and mCherry (mCh) viral constructs (B, C) depicted in (D–G). (B) Illustration of Synapto-GFP viral construct. (C) Illustration of mCherry viral construct. (D–F) Histochemistry demonstrating synaptophysin-GFP (Syn-GFP) expression in lateral nucleus of the cerebellum of Drd1aCre/+ (D), VgatCre/+ (E), and Vglut2Cre/+ (F) mice. Scale bar = 60 μm. (G–I) mCherry-expressing neurons are observed in the cerebellar cortex along with a number of Syn-GFP puncta. Scale bar = 200 μm. (J–O) Nucleocortical rosette-like or beadlike synapses in cerebellar cortex in each of three strains of mice. Scale bar = 2 μm. AAV, adeno-associated viral; CAG, (C) the cytomegalovirus (CMV) early enhancer element, (A) the promoter, the first exon and the first intron of chicken beta-actin gene, (G) the splice acceptor of the rabbit beta-globin gene; ITR, inverted terminal repeat sequence loxP; loxP, locus of X-over P1 sequence polyA; polyA, polyadenylate tail sequence; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element sequence.
      Previous studies have shown that GABAergic cells in the LCN project to inferior olive (
      • Najac M.
      • Raman I.M.
      Integration of Purkinje cell inhibition by cerebellar nucleo-olivary neurons.
      ) and locally (
      • Husson Z.
      • Rousseau C.V.
      • Broll I.
      • Zeilhofer H.U.
      • Dieudonne S.
      Differential GABAergic and glycinergic inputs of inhibitory interneurons and Purkinje cells to principal cells of the cerebellar nuclei.
      ,
      • Uusisaari M.
      • Knopfel T.
      GlyT2+ neurons in the lateral cerebellar nucleus.
      ), whereas glycinergic cells project to CCtx with small beadlike synapses (
      • Ankri L.
      • Husson Z.
      • Pietrajtis K.
      • Proville R.
      • Lena C.
      • Yarom Y.
      • et al.
      A novel inhibitory nucleo-cortical circuit controls cerebellar Golgi cell activity.
      ). In contrast, glutamatergic cells project to thalamic targets and to CCtx, where they form large mossy fiberlike rosette synapses (
      • Gao Z.
      • Proietti-Onori M.
      • Lin Z.
      • Ten Brinke M.M.
      • Boele H.J.
      • Potters J.W.
      • et al.
      Excitatory cerebellar nucleocortical circuit provides internal amplification during associative conditioning.
      ,
      • Houck B.D.
      • Person A.L.
      Cerebellar premotor output neurons collateralize to innervate the cerebellar cortex.
      ). Analysis of projections from Vglut2Cre/+ and VgatCre/+ mouse lines (Figure 4E–O) confirmed nucleocortical rosettes in Vglut2Cre/+ mice. In VgatCre/+ mice, we observed local projections and beadlike synapses in CCtx (Figure 4D–O). D1R+ projections were found in extracerebellar targets such as ventromedial thalamic nucleus, VTA, and locus ceruleus. A complete list of extracerebellar projections in Drd1aCre/+, Vglut2Cre/+, and VgatCre/+ mice is presented in Table 1 (example sections with synapto-GFP-positive puncta are shown in Supplemental Figure S5).
      Table 1Brain Regions Targeted by Lateral Nucleus of the Cerebellum Cell Populations
      StructureGenotype
      D1R-CreVgat-CreVglut2-Cre
      Diencephalon
       Nucleus of the vertical limb of the diagonal band++
       Lateral preoptic area++
       Medial forebrain bundle++
       Nucleus reuniens++
       Zona incerta++++
       Ventral anterior/Ventral lateral nucleus of the thalamus++
       Ventral medial nucleus of the thalamus++++
       Prerubral field+++
      Midbrain
       Superior cerebellar peduncle (fibers)+++++++
       Prosomere 1 reticular formation+++
       Mesencephalic reticular nucleus+++
       Red nucleus++++++
       Retrorubral fields+++
       Ventral tegmental area+++
      Pons
       Reticulotegmental nucleus of the pons+++++
       Basilar pontine nuclei++++
       Pedunculopontine tegmental nucleus+++
       Medial lemniscus++++
       Gigantocellular reticular nucleus+++
       Locus ceruleus+++
      Medulla
       Superior vestibular nucleus+++
       Medial vestibular nucleus++
       Lateral vestibular nucleus++
       Inferior olive, primary nucleus++++
      Weak projection (+), moderate projection (++), and strong projection (+++) in 4/4 animals examined in each group.

      Inhibition of D1R LCN Neurons With Designer Receptors Exclusively Activated by a Designer Drug

      To assess the function of D1R LCN neurons, we inhibited these cells through conditional viral-mediated expression of the inhibitory designer receptor exclusively activated by a designer drug (DREADD) Hm4Di (
      • Armbruster B.N.
      • Li X.
      • Pausch M.H.
      • Herlitze S.
      • Roth B.L.
      Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand.
      ) fused to YFP (AAV-FLEX-Hm4Di-YFP) (Supplemental Figure S6A, B). Bath application of the selective ligand clozapine-n-oxide (CNO) (5 μM) to acute cerebellar slices reduced the firing frequency of spontaneously active YFP-positive cells by an average of 44.3 ± 11.9% (12 cells) and hyperpolarized nonspontaneous cells by an average of 4.0 ± 1.3 mV (6 cells) (Supplemental Figure S6C).
      Analysis of the in vivo effects of CNO-mediated inhibition (1 mg/kg; intraperitoneal injection of CNO) using chronic tetrode recordings revealed a number of cells (13/43) that were inhibited, while a similar number (13/43) were excited and the remainder were unaffected (17/43) (Supplemental Figure S6D–G). Based on observations of local, likely inhibitory, projections of D1R neurons within the LCN, neurons activated by CNO are likely a reflection of disinhibition caused by silencing inhibitory D1R cells.

      D1R LCN Neurons Influence Spatial Navigation

      Lesions to the LCN disrupt spatial memory (
      • Lalonde R.
      • Strazielle C.
      The effects of cerebellar damage on maze learning in animals.
      ,
      • Noblett K.L.
      • Swain R.A.
      Pretraining enhances recovery from visuospatial deficit following cerebellar dentate nucleus lesion.
      ,
      • Petrosini L.
      • Leggio M.G.
      • Molinari M.
      The cerebellum in the spatial problem solving: A co-star or a guest star?.
      ). To establish whether D1R LCN neurons influence spatial memory, we inhibited D1R LCN neurons during a Barnes maze task. Experimental D1R:Hm4Di and control D1R:GFP mice both were pretreated with CNO (1 mg/kg, intraperitoneal injection) prior to behavioral assessment during both training and memory recall. During training, both groups showed improvements in performance (decreases in total distance traveled, center crossings, and latency to goal [Supplemental Figure S7A–C]), but D1R:Hm4Di mice showed deficits in measures of learning such as time spent and nose pokes in the target quadrant during training trials relative to littermate D1R:GFP control mice (Figure 5A). We next probed mice for memory recall following removal of the escape tunnel; D1R:Hm4Di mice had reduced nose pokes in the goal and goal quadrant (Figure 5B, C). Because we observed differences between groups in learning across training, in a separate cohort of mice we injected CNO only prior to the probe trial. In this experiment, D1R:Hm4Di mice showed a difference on only one measure during training (D1R:Hm4Di mice had increased duration in the target quadrant on day 2) but not nose pokes in the target quadrant (Supplemental Figure S7D). There were no differences in performance on the probe trial when CNO was administered (Supplemental Figure S7E, F). Thus, the effect of CNO treatment on time spent in the target quadrant during training is not specific to neuronal silencing. In contrast, the effect of silencing on nose poke accuracy is specific to neuronal silencing given that differences in this measure throughout training (and in the probe trial) occurred only when CNO was given throughout training.
      Figure thumbnail gr5
      Figure 5Designer receptor exclusively activated by a designer drug (DREADD) expression in dopamine D1 receptor (D1R) lateral cerebellar nucleus neurons and clozapine-n-oxide (CNO) application results in altered performance on the Barnes maze. (A) Schema for when CNO was injected intraperitoneally prior to training and probe tests above performance of each group during training trials, as measured by average duration in the goal quadrant (left) and as measured by nose pokes in target quadrant holes and goal hole (right). n = 17 D1R:Hm4Di mice and n = 23 littermate D1R:green fluorescent protein (GFP) control mice. For goal quadrant duration, error bars are SEM. *p < .05, two-way repeated-measures analysis of variance (ANOVA). Two factors were significant: interaction between training and presence of Hm4Di (F3,114 = 3.14, *p < .05) and training (F3,114 = 5.30, **p < .01). The factor of presence of Hm4Di alone was not significant. Holm-Sidak’s post hoc multiple comparisons test did not indicate any difference in target quadrant duration for any days of training. For nose pokes in goal quadrant (two-way repeated-measures ANOVA), two factors were significant: interact ion between training and presence of Hm4Di (F3,114 = 3.92, *p < .05) and training (F3,114 = 8.49, *p < .0001). The factor of presence of Hm4Di alone was not significant. Holm-Sidak’s post hoc multiple comparisons test indicated that the difference in nose pokes between groups during training was significantly different only on day 4 of training, *p < .05, df = 152. (B) Performance of each group during memory recall in the probe trial of the Barnes maze, as measured by nose pokes in target quadrant holes. n = 17 D1R:Hm4Di mice and n = 23 littermate D1R:GFP control mice. Error bars are SEM. *p < .05, unpaired Student’s t test, two tailed, t38 = 2.52. (C) Performance of each group during memory recall in the probe trial of the Barnes maze, as measured by nose pokes in goal hole. D1R:GFP mice had significantly more nose pokes in goal hole than D1R:Hm4Di mice on probe trial. *p < .05, unpaired Student’s t test, two tailed, t38 = 2.23. (D, E) Representative path traces of D1R:GFP after intraperitoneal injection of CNO (D) and D1R:Hm4Di after intraperitoneal injection of CNO (E) on the probe trial on day 5 of the Barnes maze. (F) Social approach as measured by time spent in arena with a novel mouse or novel object. Both groups preferred social interaction (F1,72 = 48.52, p < .0001). n = 22 D1R:GFP mice and n = 17 D1R:Hm4Di mice, two-way ANOVA. No significant differences were found for presence of Hm4Di or interaction (presence of Hm4Di × zone). (G) Social preference as measured by time spent in arena with a novel mouse or familiar mouse. Factors for interaction (presence of Hm4Di × zone, F1,72 = 4.59, *p < .05) and zone (F1,72 = 4.19, p < .05) were significant (n = 22 D1R:GFP mice and n = 17 D1R:Hm4Di mice, **p < .01, two-way ANOVA, Sidak’s multiple comparisons test). (H) Prepulse inhibition of the acoustic startle reflex (n = 22 D1R:GFP mice and n = 17 D1R:Hm4Di mice) was significantly different for presence of Hm4Di (F1,37 = 6.95, *p < .05) and decibels of prepulse (F2,74 = 44.22, p < .0001), but not for interaction (two-way repeated-measures ANOVA).
      To establish whether spatial navigation deficits in mice with inhibited D1R LCN neurons is due to motor incoordination, CNO-treated D1R:Hm4Di and D1R:GFP mice were assayed on an accelerating rotarod task. D1R:Hm4Di mice showed no deficits in performance relative to D1R:GFP mice across multiple days of training (Supplemental Figure S7G). We also analyzed gait variability and found no difference between groups on measures of stance and stride (Supplemental Figure S7H).

      D1R LCN Neurons Influence Social Recognition Memory and Sensorimotor Gating Behaviors

      Mental illnesses with associated changes in cerebellar function are associated with decreased social cognition and alterations in prepulse inhibition (PPI) of the acoustic startle reflex (
      • Silverman J.L.
      • Yang M.
      • Lord C.
      • Crawley J.N.
      Behavioural phenotyping assays for mouse models of autism.
      ,
      • Swerdlow N.R.
      • Braff D.L.
      • Geyer M.A.
      Animal models of deficient sensorimotor gating: What we know, what we think we know, and what we hope to know soon.
      ). To test social approach, interaction, and preference, we used a three-chamber assay (
      • Silverman J.L.
      • Yang M.
      • Lord C.
      • Crawley J.N.
      Behavioural phenotyping assays for mouse models of autism.
      ). Both CNO-treated D1R:Hm4Di and D1R:GFP mice showed a preference for exploring a chamber and in sniffing a zone containing a mouse compared with one containing a novel object (Figure 5F and Supplementary Figure S7J). We next replaced the object with a novel mouse, establishing a familiar mouse in one chamber and an unfamiliar mouse in the second chamber. While D1R:GFP mice spent significantly more time in the chamber with the novel mouse, D1R:Hm4Di mice failed to discriminate in this task (Figure 5G and Supplemental Figure S7K). Deficits in social preference in D1R:HM4 mice were not associated with an overall reduction in exploratory behavior (Supplemental Figure S7L, M).
      Although motor coordination was not altered in D1R:Hm4Di mice, we did observe a significant deficit in PPI (Figure 5H), primarily at the lowest amplitude prepulse (70 dB). This is consistent with a deficit in sensorimotor gating and attentional processes (
      • Swerdlow N.R.
      • Braff D.L.
      • Geyer M.A.
      Animal models of deficient sensorimotor gating: What we know, what we think we know, and what we hope to know soon.
      ). The amplitude of responses to different amplitudes of startle pulses in the absence of acoustic prepulse did not differ between groups, indicating intact basic motor reflexes in D1R:Hm4Di mice (Supplementary Figure S7I). Cerebellar modulation of PPI in mice has been documented previously (
      • Takeuchi T.
      • Kiyama Y.
      • Nakamura K.
      • Tsujita M.
      • Matsuda I.
      • Mori H.
      • et al.
      Roles of the glutamate receptor epsilon2 and delta2 subunits in the potentiation and prepulse inhibition of the acoustic startle reflex.
      ).

      D1R LCN Neurons Influence Temporally Dependent Response Inhibition

      A classic function attributed to dopamine in the prefrontal cortex is temporally dependent response inhibition (
      • Sokolowski J.D.
      • Salamone J.D.
      Effects of dopamine depletions in the medial prefrontal cortex on DRL performance and motor activity in the rat.
      ). To determine whether D1R LCN neurons modulate temporally dependent response inhibition, we used a paradigm known as differential reinforcement of low-rate responses (DRL), which reinforces a subject’s ability to refrain from responding for a set time period. Optimal performance requires a subject to accurately time the interval between lever presses, and inhibit responses before a set time has elapsed (Figure 6A, B). CNO-injected D1R:Hm4Di male mice had right-shifted responses compared with control mice and increased response inhibition in the 10-second interval version of this task (DRL-10) (Figure 6C). The peak (mode) of the response distribution for each animal was averaged by group and was significantly increased for D1R:Hm4Di mice on DRL-10 trials (inset in Figure 6C). Assessment of this behavior in female mice showed similar deficits in D1R:Hm4Di mice, although the overall training time required for control mice to perform the task was greater than in male mice (Supplemental Figure S8A–F).
      Figure thumbnail gr6
      Figure 6Temporally dependent response inhibition and working memory are regulated by dopamine D1 receptor (D1R) neurons in the lateral cerebellar nucleus. (A, B) Schematics describing contingencies of differential reinforcement of low-rate responses (DRL) operant timing task. (C) Distribution of latencies of lever presses after reward in the 10-second interval version of the task for D1R:green fluorescent protein (GFP) control mice (black circles) and D1R:Hm4Di mice (red diamonds). Group mean ± SEM are presented for latencies binned in 2-second intervals. Dotted line represents minimum response latency that is rewarded for the DRL paradigm (two-way repeated-measures analysis of variance, presence of Hm4Di × time interaction: week 3, F29,261 = 5.61, p < .0001; n = 6 D1R:Hm4Di mice and n = 5 D1R:GFP mice). Inset is the mean peak of response distribution of each group for this week of training in this 10-second DRL paradigm. (D) Schematic describing the delayed alternation operant task. (E–J) Performance of D1R:GFP control mice (black lines) and D1R:Hm4Di mice (red lines) on increasingly difficult versions of the task as measured by proportion correct (E–G) and pellets rewarded (H–J). Group mean ± SEM are presented in (E–J). (E) A 2-second delay, with the only significant factor being for training: F5,140 = 101.5, p < .0001; no significant differences between groups or interaction between training and presence of Hm4Di. n = 12 D1R:Hm4Di mice and n = 18 D1R:GFP mice. (F) An 8-second delay, with significant differences for factors of training, F4,112 = 16.16, p < .0001, and between groups, F1,28 = 7.2, *p < .05, but not for interaction. n = 12 D1R:Hm4Di mice and n = 18 D1R:GFP mice. (G) A 16-second delay, with the factor of presence of Hm4Di being significant: F1,28 = 4.83, *p < .05; interaction between groups and training not significant. n = 12 D1R:Hm4Di mice and n = 18 D1R:GFP mice. (H) A 2-second delay, with the only significant factor being for training: F5,140 = 50.92, p < .0001; no significant differences between groups or interaction between presence of training and presence of Hm4Di. n = 12 D1R:Hm4Di mice and n = 18 D1R:GFP mice. (I) An 8-second delay, with no significant differences found for factors of training, Hm4Di groups, or interaction between groups and training. n = 12 D1R:Hm4Di mice and n = 18 D1R:GFP mice. (J) A 16-second delay, with the factor of presence of Hm4Di being significant: F1,28 = 4.35, *p < .05; interaction between groups and training not significant (two-way repeated-measures analysis of variance). n = 12 D1R:Hm4Di mice and n = 18 D1R:GFP mice.
      The altered performance of the D1R:Hm4Di male mice was not due to changes in motivation or to ability to lever press, given that performance in instrumental conditioning, a progressive ratio reinforcement task, and overall pellets rewarded in DRL were equivalent between groups (Supplemental Figure S9A–C). Female D1R:Hm4Di mice had significantly fewer pellets rewarded when performing DRL (Supplemental Figure S9D).

      D1R LCN Neurons Influence WM

      Lateral CCtx and LCN in species ranging from rodents to humans are also implicated in WM (
      • Kim S.G.
      • Ugurbil K.
      • Strick P.L.
      Activation of a cerebellar output nucleus during cognitive processing.
      ,
      • Kuper M.
      • Dimitrova A.
      • Thurling M.
      • Maderwald S.
      • Roths J.
      • Elles H.G.
      • et al.
      Evidence for a motor and a non-motor domain in the human dentate nucleus—An fMRI study.
      ,
      • Lalonde R.
      • Strazielle C.
      The effects of cerebellar damage on maze learning in animals.
      ). Thus, we chose the delayed alternation (DA) protocol to test WM in rodents, which also requires dopamine (
      • Brozoski T.J.
      • Brown R.M.
      • Rosvold H.E.
      • Goldman P.S.
      Cognitive deficit caused by regional depletion of dopamine in prefrontal cortex of rhesus monkey.
      ,
      • Rossi M.A.
      • Hayrapetyan V.Y.
      • Maimon B.
      • Mak K.
      • Je H.S.
      • Yin H.H.
      Prefrontal cortical mechanisms underlying delayed alternation in mice.
      ). This operant paradigm requires a subject to learn to press one of two levers, wait for a delay, and then press the other lever (Figure 6D). CNO-injected D1R:Hm4Di mice showed no differences on acquisition of the task with a 2-second delay (Figure 6E, H), but they had poorer performance on one measure (proportion correct) at an 8-second delay (Figure 6F) and on two measures (proportion correct and pellets rewarded) with a 16-second delay (Figure 6G, J), indicating that D1R cells are necessary for when WM demands are higher.

      Discussion

      We have identified D1R expression in human DCN, a population of D1R neurons in mouse ventrocaudal LCN that demonstrates restricted anatomical localization, and converging evidence that activity in this population is required for normal performance in several cognitive domains. This population of LCN D1R neurons in the mouse is similar to previous reports of neurons in ventrocaudal DCN that make up a cognitive domain in monkeys and humans (
      • Tellmann S.
      • Bludau S.
      • Eickhoff S.
      • Mohlberg H.
      • Minnerop M.
      • Amunts K.
      Cytoarchitectonic mapping of the human brain cerebellar nuclei in stereotaxic space and delineation of their co-activation patterns.
      ,
      • Dum R.P.
      • Li C.
      • Strick P.L.
      Motor and nonmotor domains in the monkey dentate.
      ,
      • Kuper M.
      • Dimitrova A.
      • Thurling M.
      • Maderwald S.
      • Roths J.
      • Elles H.G.
      • et al.
      Evidence for a motor and a non-motor domain in the human dentate nucleus—An fMRI study.
      ,
      • Ashmore R.C.
      • Sommer M.A.
      Delay activity of saccade-related neurons in the caudal dentate nucleus of the macaque cerebellum.
      ). Prefrontal D1Rs have been shown to modulate cognitive behaviors such as interval timing, risk-based decision making, WM, and behavioral flexibility (
      • Durstewitz D.
      • Seamans J.K.
      The computational role of dopamine D1 receptors in working memory.
      ,
      • Narayanan N.S.
      • Land B.B.
      • Solder J.E.
      • Deisseroth K.
      • DiLeone R.J.
      Prefrontal D1 dopamine signaling is required for temporal control.
      ,
      • Ragozzino M.E.
      The effects of dopamine D1 receptor blockade in the prelimbic-infralimbic areas on behavioral flexibility.
      ,
      • St. Onge J.R.
      • Abhari H.
      • Floresco S.B.
      Dissociable contributions by prefrontal D1 and D2 receptors to risk-based decision making.
      ,
      • Vijayraghavan S.
      • Wang M.
      • Birnbaum S.G.
      • Williams G.V.
      • Arnsten A.F.
      Inverted-U dopamine D1 receptor actions on prefrontal neurons engaged in working memory.
      ). Previous studies linking cerebellum to these functions proposed modulation of catecholamines in prefrontal cortex via connections through the thalamus or VTA (
      • Parker K.L.
      • Kim Y.C.
      • Kelley R.M.
      • Nessler A.J.
      • Chen K.H.
      • Muller-Ewald V.A.
      • et al.
      Delta-frequency stimulation of cerebellar projections can compensate for schizophrenia-related medial frontal dysfunction.
      ,
      • Rogers T.D.
      • Dickson P.E.
      • Heck D.H.
      • Goldowitz D.
      • Mittleman G.
      • Blaha C.D.
      Connecting the dots of the cerebro-cerebellar role in cognitive function: Neuronal pathways for cerebellar modulation of dopamine release in the prefrontal cortex.
      ); this is the first study to link a specific cerebellar cell population to these behaviors.
      D1R LCN neurons appear to be a heterogeneous population containing principally inhibitory neurons and a small number of excitatory neurons. In cerebellar slices, we observed electrophysiological properties consistent with putative glycinergic, GABAergic/glycinergic, and glutamatergic neurons (
      • Ankri L.
      • Husson Z.
      • Pietrajtis K.
      • Proville R.
      • Lena C.
      • Yarom Y.
      • et al.
      A novel inhibitory nucleo-cortical circuit controls cerebellar Golgi cell activity.
      ,
      • Uusisaari M.
      • Knopfel T.
      GlyT2+ neurons in the lateral cerebellar nucleus.
      ,
      • Uusisaari M.
      • Obata K.
      • Knopfel T.
      Morphological and electrophysiological properties of GABAergic and non-GABAergic cells in the deep cerebellar nuclei.
      ,
      • Chen S.
      • Hillman D.E.
      Colocalization of neurotransmitters in the deep cerebellar nuclei.
      ). D1R cells are enriched with markers of inhibitory neurotransmission, and we observed synapses both within the LCN and in CCtx, consistent with inhibitory local and nucleocortical projections. D1R cells were neither enriched nor de-enriched in the glutamatergic marker Vglut2, and we observed rosette-like synapses in CCtx similar to those seen in Vglut2Cre/+ mice. We also observed a small number of larger cells in slice that were consistent with glutamatergic neurons. These observations are consistent with a smaller proportion of D1R LCN cells being glutamatergic. The extracerebellar targets of D1R+ and Vglut2+ cell projections lend further evidence to the idea that these cells can regulate cognitive functions via regulation of brain targets associated with them. The restricted distribution of D1R LCN neurons suggests that this population of cells may represent a specific behavioral control segment involved in cognitive operations. Why encompass inhibitory and excitatory neurons? One possibility is that the glutamatergic D1R neurons represent the minimal essential component for regulation of specific cognitive functions, with the larger number of inhibitory neurons projecting locally within the LCN and back to CCtx to constrain output from other behavioral control regions. In this way, performance of specific spatial, WM, and temporally dependent tasks can proceed without competing behavioral processes running concurrently.
      When we induced functional inhibition of D1R neurons in the LCN, similar numbers of neurons were inhibited as were excited. The implication of this is that LCN D1R cells are significantly contributing to local inhibition. Previous studies have found that only a few neurons in cerebellar nuclei are sufficient to transmit temporally precise information for a given effector system and that inhibitory Purkinje cell input is suitable to modulate neurons in the cerebellar nuclei at this level of precision (
      • Gauck V.
      • Jaeger D.
      The control of rate and timing of spikes in the deep cerebellar nuclei by inhibition.
      ). Furthermore, we reported D1R+ nucleocortical synapses in the granular layer of Crus I, Crus II, paraflocculus, and flocculus. Crus I and Crus II are classically associated with prefrontal and parietal cortices (
      • Allen G.I.
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      Cerebrocerebellar communication systems.
      ,
      • Sasaki K.
      • Oka H.
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      • Mizuno N.
      Electrophysiological studies of the projections from the parietal association area to the cerebellar cortex.
      ,
      • Schmahmann J.D.
      • Pandya D.N.
      Prefrontal cortex projections to the basilar pons in rhesus monkey: Implications for the cerebellar contribution to higher function.
      ,
      • Schmahmann J.D.
      • Pandya D.N.
      Anatomic organization of the basilar pontine projections from prefrontal cortices in rhesus monkey.
      ), and paraflocculus and flocculus are associated with modulation of eye movements and adaptation of the vestibulo-ocular reflex (
      • Rambold H.
      • Churchland A.
      • Selig Y.
      • Jasmin L.
      • Lisberger S.G.
      Partial ablations of the flocculus and ventral paraflocculus in monkeys cause linked deficits in smooth pursuit eye movements and adaptive modification of the VOR.
      ,
      • Zee D.S.
      • Yamazaki A.
      • Butler P.H.
      • Gucer G.
      Effects of ablation of flocculus and paraflocculus of eye movements in primate.
      ), so D1R cells could possibly exert differential control over different behavioral control regions at the level of CCtx. Thus, it is likely that D1R cells regulate adjacent neurons via local inhibition, and more distant cerebellar cortical neurons via nucleocortical projections, suggesting more levels of cerebellar output regulation, potentially by extracerebellar catecholaminergic inputs.
      A remaining question is whether the D1R cells we describe are responding to dopamine or some other neurotransmitter in the behaviors we examined. Distinct dopamine and norepinephrine uptake mechanisms into synaptosomes isolated from cerebellum have been identified (
      • Efthimiopoulos S.
      • Giompres P.
      • Valcana T.
      Kinetics of dopamine and noradrenaline transport in synaptosomes from cerebellum, striatum and frontal cortex of normal and reeler mice.
      ). Adrenergic afferents to the cerebellar nuclei likely come from locus ceruleus (catecholamine group A6) and an adjacent nucleus subceruleus (catecholamine group A4), but not other adrenergic nuclei (
      • Grzanna R.
      • Molliver M.E.
      The locus coeruleus in the rat: An immunohistochemical delineation.
      ,
      • Schuerger R.J.
      • Balaban C.D.
      Immunohistochemical demonstration of regionally selective projections from locus coeruleus to the vestibular nuclei in rats.
      ). While it is clear that the LCN has receptors for dopamine, there is some disagreement about where it may be coming from. In primates, there is robust distinction between dopamine transporter–positive and dopamine-β-hydroxylase-positive axons in different parts of CCtx (
      • Melchitzky D.S.
      • Lewis D.A.
      Tyrosine hydroxylase- and dopamine transporter-immunoreactive axons in the primate cerebellum: Evidence for a lobular- and laminar-specific dopamine innervation.
      ). Retrograde mapping with Cav2-Cre in a tomato reporter line failed to reveal any tomato labeling in the VTA of mice (
      • Wagner M.J.
      • Kim T.H.
      • Savall J.
      • Schnitzer M.J.
      • Luo L.
      Cerebellar granule cells encode the expectation of reward.
      ). In rats, the VTA sends glutamatergic projections, but not dopaminergic projections, to the LCN (
      • Ikai Y.
      • Takada M.
      • Shinonaga Y.
      • Mizuno N.
      Dopaminergic and non-dopaminergic neurons in the ventral tegmental area of the rat project, respectively, to the cerebellar cortex and deep cerebellar nuclei.
      ). All cerebellar nuclei are reported to have dopamine transporter–like binding, which is not blocked by norepinephrine (
      • Delis F.
      • Mitsacos A.
      • Giompres P.
      Dopamine receptor and transporter levels are altered in the brain of Purkinje cell degeneration mutant mice.
      ,
      • Delis F.
      • Mitsacos A.
      • Giompres P.
      Pharmacological characterization and anatomical distribution of the dopamine transporter in the mouse cerebellum.
      ). Other possible sources of catecholamines in the LCN include a small population of Purkinje cells in the caudal cerebellum (
      • Fujii T.
      • Sakai M.
      • Nagatsu I.
      Immunohistochemical demonstration of expression of tyrosine hydroxylase in cerebellar Purkinje cells of the human and mouse.
      ); the zona incerta (catecholamine group A13), which has projections to the interposed cerebellar nuclei (
      • Mitrofanis J.
      • deFonseka R.
      Organisation of connections between the zona incerta and the interposed nucleus.
      ); and the locus ceruleus, which has been previously shown to release dopamine in the hippocampus, an area with less dopamine than cerebellar nuclei (
      • Versteeg D.H.
      • Van Der Gugten J.
      • De Jong W.
      • Palkovits M.
      Regional concentrations of noradrenaline and dopamine in rat brain.
      ,
      • Kempadoo K.A.
      • Mosharov E.V.
      • Choi S.J.
      • Sulzer D.
      • Kandel E.R.
      Dopamine release from the locus coeruleus to the dorsal hippocampus promotes spatial learning and memory.
      ,
      • Laatikainen L.M.
      • Sharp T.
      • Harrison P.J.
      • Tunbridge E.M.
      Sexually dimorphic effects of catechol-O-methyltransferase (COMT) inhibition on dopamine metabolism in multiple brain regions.
      ,
      • Smith C.C.
      • Greene R.W.
      CNS dopamine transmission mediated by noradrenergic innervation.
      ,
      • Takeuchi T.
      • Duszkiewicz A.J.
      • Sonneborn A.
      • Spooner P.A.
      • Yamasaki M.
      • Watanabe M.
      • et al.
      Locus coeruleus and dopaminergic consolidation of everyday memory.
      ).
      We found that inhibition of D1R neurons results in decreased spatial navigation, social recognition, WM performance, and alterations in response inhibition, which is in agreement with findings of animals and humans with lateral cerebellar lesions (
      • Lalonde R.
      • Strazielle C.
      The effects of cerebellar damage on maze learning in animals.
      ,
      • Noblett K.L.
      • Swain R.A.
      Pretraining enhances recovery from visuospatial deficit following cerebellar dentate nucleus lesion.
      ,
      • Petrosini L.
      • Leggio M.G.
      • Molinari M.
      The cerebellum in the spatial problem solving: A co-star or a guest star?.
      ,
      • Gooch C.M.
      • Wiener M.
      • Wencil E.B.
      • Coslett H.B.
      Interval timing disruptions in subjects with cerebellar lesions.
      ). These findings are relevant in the context of sensory prediction error functions for which the cerebellum is specialized. The cerebellum integrates predicted sensory outcomes of motor commands with sensory feedback to achieve optimal kinematic performance (
      • Kawato M.
      • Gomi H.
      A computational model of four regions of the cerebellum based on feedback-error learning.
      ,
      • Parker K.L.
      • Kim Y.C.
      • Kelley R.M.
      • Nessler A.J.
      • Chen K.H.
      • Muller-Ewald V.A.
      • et al.
      Delta-frequency stimulation of cerebellar projections can compensate for schizophrenia-related medial frontal dysfunction.
      ) by generating appropriately timed anticipatory signals for accurate feedforward predictions, particularly in adaptation of involuntary reflexes during limb movements, eyeblink conditioning, and the vestibulo-ocular reflex (
      • Raymond J.L.
      • Lisberger S.G.
      Neural learning rules for the vestibulo-ocular reflex.
      ,
      • Jimenez-Diaz L.
      • Navarro-Lopez Jde D.
      • Gruart A.
      • Delgado-Garcia J.M.
      Role of cerebellar interpositus nucleus in the genesis and control of reflex and conditioned eyelid responses.
      ,
      • Koekkoek S.K.
      • Hulscher H.C.
      • Dortland B.R.
      • Hensbroek R.A.
      • Elgersma Y.
      • Ruigrok T.J.
      • et al.
      Cerebellar LTD and learning-dependent timing of conditioned eyelid responses.
      ,
      • Manto M.U.
      • Setta F.
      • Jacquy J.
      • Godaux E.
      • Hildebrand J.
      • Roland H.
      • et al.
      Different types of cerebellar hypometria associated with a distinct topography of the lesion in cerebellum.
      ,
      • Ohmae S.
      • Medina J.F.
      Climbing fibers encode a temporal-difference prediction error during cerebellar learning in mice.
      ,
      • Perrett S.P.
      • Ruiz B.P.
      • Mauk M.D.
      Cerebellar cortex lesions disrupt learning-dependent timing of conditioned eyelid responses.
      ). Notably, aspiration lesions of the cerebellum result in attenuation of the acoustic startle reflex (
      • Davis M.
      • Gendelman D.S.
      • Tischler M.D.
      • Gendelman P.M.
      A primary acoustic startle circuit: Lesion and stimulation studies.
      ) and rotarod performance (
      • Caston J.
      • Jones N.
      • Stelz T.
      Role of preoperative and postoperative sensorimotor training on restoration of the equilibrium behavior in adult mice following cerebellectomy.
      ,
      • Caston J.
      • Vasseur F.
      • Stelz T.
      • Chianale C.
      • Delhaye-Bouchaud N.
      • Mariani J.
      Differential roles of cerebellar cortex and deep cerebellar nuclei in the learning of the equilibrium behavior: Studies in intact and cerebellectomized lurcher mutant mice.
      ), which we did not see in D1R:Hm4Di animals, lending weight to the idea that the LCN is more cognitive in function than other cerebellar regions. Posterolateral CCtx and the LCN are postulated to influence cognitive functions by generating time-based predictions of sensory information and predicting and synchronizing motor or cognitive activity with selective sensory input using a forward timing model (
      • Ghajar J.
      • Ivry R.B.
      The predictive brain state: Asynchrony in disorders of attention?.
      ). Dysfunction in these regions would result in altered processing or weighting of sensory data relative to prior learning and memory, resulting in poor cognitive performance (
      • Lawson R.P.
      • Rees G.
      • Friston K.J.
      An aberrant precision account of autism.
      ). Decreased performance of D1R:Hm4Di animals in the Barnes maze (predictive self-motion and visual spatial cues), PPI (predictive acoustic prepulses), and social tasks (predictive odorants and visual/auditory cues) would make sense in this context. For example, cerebellar cortical processing of self-motion information is required to maintain stable hippocampal place fields during successful spatial navigation (
      • Rochefort C.
      • Arabo A.
      • Andre M.
      • Poucet B.
      • Save E.
      • Rondi-Reig L.
      Cerebellum shapes hippocampal spatial code.
      ).
      It may seem inconsistent that D1R:Hm4Di animals showed alterations in the DRL-10 task but did not show disruptions on two different measures of DA until it was extended to 16 seconds. However, DA and DRL may preferentially test different aspects of attention such as cueing/alerting, orientation, action selection/retrieval, timing, execution, and error checking. For example, DA may favor associating a sensory cue (levers extending after delay) with learning a rule and action selection (press the other lever) and recalling the previous lever location. Lateral cerebellum is specifically engaged during tasks requiring precise representation of temporal information in the subseconds to seconds range (
      • Yamaguchi K.
      • Sakurai Y.
      Inactivation of cerebellar cortical Crus II disrupts temporal processing of absolute timing but not relative timing in voluntary movements.
      ,
      • Ashmore R.C.
      • Sommer M.A.
      Delay activity of saccade-related neurons in the caudal dentate nucleus of the macaque cerebellum.
      ,
      • Parker K.L.
      • Kim Y.C.
      • Kelley R.M.
      • Nessler A.J.
      • Chen K.H.
      • Muller-Ewald V.A.
      • et al.
      Delta-frequency stimulation of cerebellar projections can compensate for schizophrenia-related medial frontal dysfunction.
      ,
      • Gooch C.M.
      • Wiener M.
      • Wencil E.B.
      • Coslett H.B.
      Interval timing disruptions in subjects with cerebellar lesions.
      ,
      • Breukelaar J.W.
      • Dalrymple-Alford J.C.
      Effects of lesions to the cerebellar vermis and hemispheres on timing and counting in rats.
      ,
      • Malapani C.
      • Dubois B.
      • Rancurel G.
      • Gibbon J.
      Cerebellar dysfunctions of temporal processing in the seconds range in humans.
      ,
      • Mangels J.A.
      • Ivry R.B.
      • Shimizu N.
      Dissociable contributions of the prefrontal and neocerebellar cortex to time perception.
      ,
      • Spencer R.M.
      • Zelaznik H.N.
      • Diedrichsen J.
      • Ivry R.B.
      Disrupted timing of discontinuous but not continuous movements by cerebellar lesions.
      ) as in DRL, whereas performance on DA might not require direct attention to specific time intervals (
      • Ivry R.B.
      • Spencer R.M.
      The neural representation of time.
      ). Consistent with this, humans with cerebellar lesions show increased variability in performance on timing tasks, with overestimations at shorter intervals (
      • Gooch C.M.
      • Wiener M.
      • Wencil E.B.
      • Coslett H.B.
      Interval timing disruptions in subjects with cerebellar lesions.
      ). Some humans with highly focal damage in lateral CCtx and nuclei have timing deficits in the seconds range without effects on attention, memory, or executive function (
      • Malapani C.
      • Dubois B.
      • Rancurel G.
      • Gibbon J.
      Cerebellar dysfunctions of temporal processing in the seconds range in humans.
      ).
      We hypothesize that deficits in D1R:Hm4Di mice on the cognitive tasks we performed are a reflection of disturbances in sensory prediction errors that perturb basic predictive attentional processes, consistent with forward models of cerebellar function (
      • Ghajar J.
      • Ivry R.B.
      The predictive brain state: Asynchrony in disorders of attention?.
      ,
      • Ito M.
      Control of mental activities by internal models in the cerebellum.
      ). Thus, we propose that D1R LCN neurons are a locus of integration of predictive internal models involved in cognitive processes.

      Acknowledgments and Disclosures

      This work was supported by the National Institutes of Health (NIH) Grant No. S10 OD016240 to the University of Washington W.M. Keck Microscopy Center. Support was also received from the neuroscience training track within the University of Washington Psychiatry Residency Training Program. Autopsy materials used in this study were obtained from the University of Washington Neuropathology Core, which is supported by the Alzheimer’s Disease Research Center (Grant No. AG05136 ), the Adult Changes in Thought Study (Grant No. AG006781 ), and the Morris K. Udall Center of Excellence for Parkinson’s Disease Research (Grant No. NS062684 ). This work was funded by the NIH (Grant No. R01-MH094536 to LSZ, Grant No. R01-MH094536-02S1 to LSZ and ESC, Grant No. R21-MH098177 to LSZ, and Grant No. K08-MH104281-01 to ESC).
      We thank members of the Zweifel lab (especially Bryan Gore) as well as Abigail Person and Krystal Parker for scientific discussion, Jennifer Deem and Stanley McKnight for their assistance with RiboTag experiments, Scott Ng-Evans for his help with programming operant chambers, Samantha Rice and Allison Beller for their help with human tissue immunostaining, and Albert Quintana for generously providing AAV-Rpl22-HA. We thank Matthew Carter and Richard Palmiter for the plasmid DNA used in the generation of AAV-FLEX-Hm4Di-YFP, AAV-FLEX-Synapto-GFP, and AAV-FLEX-mCherry.
      ESC, MES, TML, and LSZ designed the experiments. ESC, TML, and LSZ wrote the manuscript with help from MES. CDK performed autopsies and human tissue immunostaining. Viral injection surgeries were performed by ESC. Behavioral experiments were performed by ESC and TML with assistance from JAL and KSD. RiboTag analysis was performed by SMM and ESC. Slice electrophysiology was performed by MES.
      The authors report no biomedical financial interests or potential conflicts of interest.

      Supplementary Material

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