Elsevier

Neurobiology of Disease

Volume 76, April 2015, Pages 137-158
Neurobiology of Disease

Motor and behavioral phenotype in conditional mutants with targeted ablation of cortical D1 dopamine receptor-expressing cells

https://doi.org/10.1016/j.nbd.2015.02.006Get rights and content

Highlights

  • D1-cortical cell ablation produces dystonia, ataxia, hyperactivity and anxiety.

  • D1-cortical cell ablation impairs social interaction but not working memory.

  • D1 cortical cell ablation models many aspects of the Huntington disease phenotype.

  • D1 cortical cell loss may be involved in the pathobiology of Huntington disease.

Abstract

D1-dopamine receptors (Drd1a) are highly expressed in the deep layers of the cerebral cortex and the striatum. A number of human diseases such as Huntington disease and schizophrenia are known to have cortical pathology involving dopamine receptor expressing neurons. To illuminate their functional role, we exploited a Cre/Lox molecular paradigm to generate Emx-1tox MUT mice, a transgenic line in which cortical Drd1a-expressing pyramidal neurons were selectively ablated.

Emx-1tox MUT mice displayed prominent forelimb dystonia, hyperkinesia, ataxia on rotarod testing, heightened anxiety-like behavior, and age-dependent abnormalities in a test of social interaction. The latter occurred in the context of normal working memory on testing in the Y-maze and for novel object recognition. Some motor and behavioral abnormalities in Emx-1tox MUT mice overlapped with those in CamKIIαtox MUT transgenic mice, a line in which both striatal and cortical Drd1a-expressing cells were ablated. Although Emx-1tox MUT mice had normal striatal anatomy, both Emx-1tox MUT and CamKIIαtox MUT mice displayed selective neuronal loss in cortical layers V and VI.

This study shows that loss of cortical Drd1a-expressing cells is sufficient to produce deficits in multiple motor and behavioral domains, independent of striatal mechanisms. Primary cortical changes in the D1 dopamine receptor compartment are therefore likely to model a number of core clinical features in disorders such as Huntington disease and schizophrenia.

Introduction

D1-dopamine (Drd1a) and D2-dopamine (Drd2) receptors are highly expressed on striatal medium spiny neurons (MSN), which account for 95% of the total striatal neuron population (Gerfen, 1992, Wilson and Groves, 1980). Substantia nigra pars compacta (SNpc) dopaminergic neurons project predominantly to the dorsal striatum and release dopamine that exerts fundamental regulation of motor activity. Current models of basal ganglia connectivity divide striatal output into direct and indirect pathways (Alexander et al., 1986). In the direct pathway, Drd1a are expressed on substance P (SP)- and dynorphin (DYN)-containing GABAergic MSN that in rodents project to the substantia nigra pars reticulata (SNpr)/entopeduncular complex; this corresponds anatomically to the internal segment of globus pallidus (GPi) in primates. In the indirect pathway, Drd2 are expressed on enkephalin (ENK)-positive GABAergic MSN that project to the external segment of the globus pallidus (GPe); the GPe ultimately projects to the SNpr/entopeduncular complex via the subthalamic nucleus. According to this model, excessive, unopposed signaling within the direct pathway excites, while excessive, unopposed signaling within the indirect pathway inhibits, motor cortex activity and bodily movement. Balance between opposing inputs from the direct and indirect pathways is considered crucial for normal motor control.

The cerebral cortex provides the majority of excitatory inputs to the basal ganglia. Under resting conditions, striatal neurons are generally quiescent electrically, with activity strongly regulated by the corticostriatal pathway (Kitai et al., 1976). In neurodegenerative conditions such as Huntington disease (HD), striking neuropathological changes occur in the striatum (Reiner et al., 1988) and are accompanied by material cell loss in other regions of the brain, particularly the cerebral cortex (Braak and Braak, 1992, Halliday et al., 1998, Hedreen et al., 1991, Heinsen et al., 1994). In addition, prefrontal cortex D1 dopamine receptor binding using the ligand [11C] SCH 23390 was reduced in schizophrenia patients, in a manner associated with extent of negative symptoms and cognitive dysfunction (Okubo et al., 1997), in contrast, another study using [11C] NNC 112 ligand binding reported an increase in dorsolateral prefrontal cortex but no change in the medial prefrontal cortex or the orbital frontal cortex (Abi-Dargham et al., 2002). Interpretation of these studies is difficult for a number of reasons; first, the ligands behave differently under situations of acute and chronic dopamine deprivation, second, subjects assessed differed and third, exact regions sampled between studies may have varied (Guillin et al., 2007).

A number of studies have quantified cortical degeneration in movement disorders (Hedreen et al., 1991, Heinsen et al., 1994, Jackson et al., 1995). Neurons of the cerebral cortex are grouped into six distinctive layers (except for the frontal ‘agranular’ cortex, which lacks a well-defined granular layer 4), with each layer containing a characteristic distribution of neuronal cell types and connections with other cortical and subcortical regions (Gilbert and Sigman, 2007, Shepherd, 2009, Weiler et al., 2008). Dopaminergic input to cortical regions originates from ventral midbrain regions such as the ventral tegmental area (VTA), which lies medial to SNpc. This mesotelencephalic dopamine system targets various sites, including the frontal cortex, septum, cingulate cortex, amygdala and nucleus accumbens as well as the striatum. While dopaminergic projections from the ventral midbrain reach nearly all regions of the neocortex, both rodent and primate studies have reported a rostrocaudal gradient of decreasing dopamine concentration, with highest dopamine levels found in the prefrontal and temporal regions and lowest concentrations found in the occipital cortex (Brown et al., 1979, Kehr et al., 1976). This apparent rostrocaudal gradient of D1-like dopamine receptor activity within the cortex also exists with respect to the striatum (Babovic et al., 2010).

Dopaminergic innervation of the cortex varies according to cortical laminar pattern, with deeper cortical layers (V, VI) receiving denser innervation relative to more superficial layers (Berger et al., 1976, Ciliax et al., 1995). This profile of activation coincides with dopamine receptor expression profile, which is similarly concentrated in deeper cortical layers (Boyson et al., 1986, Dawson et al., 1986, Gaspar et al., 1995). Unlike the striatum, where dopamine receptors are predominately expressed on GABAergic MSN, cortical dopamine receptors are found on both excitatory (glutamatergic) and inhibitory (GABAergic) cell types. As a result, dopaminergic activity in the cerebral cortex has been shown to elicit both excitatory and inhibitory responses (Ellenbroek et al., 1996, Pirot et al., 1992, Yang and Seamans, 1996). Drd1 and Drd2 receptor subtypes both appear to be present on excitatory cortico-striatal and cortico-cortical projection neurons, while only Drd1a expression is found in cortico-thalamic pyramidal projection neurons (Gaspar et al., 1995). On the other hand, inhibitory cortical circuits, comprised of GABAergic interneurons, also express dopamine receptors (LeMoine and Gaspar, 1998). These dopamine receptor-expressing interneurons can be further subdivided into three subgroups based on dendritic morphology and differential immunoreactivity for the calcium-binding proteins, including calbindin D28k, parvalbumin and calretinin (Kubota et al., 1994, LeMoine and Gaspar, 1998).

In recent years, significant research has focused on dopaminergic projections to the prefrontal cortex, defining a role of this part of the brain in learning, memory formation, emotion and reward (Del Arco and Mora, 2008, Floresco and Magyar, 2006, Tzschentke, 2001). For example, modulation of dopaminergic and GABAergic activities within the prefrontal cortex has been implicated in schizophrenia. Studies have demonstrated a critical involvement of D1-dopamine expressing cells in memory, such that either excessive or impaired D1-dopamine receptor activation can precipitate functional deficits (Vijayraghavan et al., 2007). Besides working memory, animal models of attention-deficit/hyperactivity disorder have reported that the prefrontal cortex exerts an inhibitory effect on subcortical dopamine systems and thus on locomotion (Sullivan and Brake, 2003).

A number of neurodegenerative conditions are characterized by progressive loss of dopamine receptor-expressing cells in cortical and subcortical sites. In order to address the limitations inherent in existing animal models, targeted approaches, particularly conditional mutants that enable spatiotemporal resolution, are required. The Cre/Lox approach affords such flexibility, as it enables a specific population of cells to be eliminated at predetermined time points. A breeder line was generated bearing a single copy of the attenuated diphtheria toxin (tox-176) gene inserted into the Drd1a gene locus downstream of a LoxP-flanked cassette consisting of a neomycin phosphotransferase gene (NEO) for cell line selection and a DNA sequence known to inhibit downstream translation (STOP). Mice heterozygous for this “knock-in” mutation therefore display a cellular phenotype equivalent to that of wild type mice as tox-176 expression is inhibited by the upstream LoxP flanked ‘NEOSTOP’ cassette. A number of regional and temporal specific Cre recombinase-expressing transgenic lines were subsequently deployed to activate toxin-mediated cell ablation in cells that normally express the Drd1a gene.

In our first study, a CamKIIα promoter was used to drive Cre delivery in the postnatal brain (Casanova et al., 2001). Cell death follows Cre expression and Drd1a promoter-driven tox-176 production at 1–2 weeks (Drago et al., 1998) in mice that are double-transgenic for CamKIIα promoter-driven Cre and the transcriptionally silenced attenuated diphtheria toxin (tox-176) genes (Drago et al., 1998) (CamKIIαtox MUT). Expression of the Cre transgene in the Cre parental line was observed across a range of structures, including the hippocampus, cortex, amygdala, striatum, thalamus and hypothalamus. In the context of their cortical and striatal Drd1a-expressing cell ablation and a preserved Drd2-expressing compartment, CamKIIαtox MUT mice displayed a broad phenotype that included impaired rotarod performance, hind limb dystonia, locomotor hyperactivity, orofacial impairments and handling-induced, electroencephalographically verified spontaneous seizures (Gantois et al., 2007). Although this study confirmed postnatal brain Drd1a-expressing cell loss as being pivotal in a number of clinically relevant abnormalities, the question of whether the observed abnormalities were related to loss of striatal direct pathway Drd1a-expressing neurons or the loss of cortical Drd1a-expressing neurons remained unanswered.

In our second study (Kim et al., 2014), double transgenic mice with specific loss of Drd1a-expressing striatal neurons were generated by crossing dopamine and adenosine 3′,5′-cyclic monophosphate-regulated phosphoprotein, 32 kDa (DARPP-32) promoter-driven Cre recombinase-expressing transgenic mice (Bogush et al., 2005) with a transcriptionally silenced attenuated diphtheria toxin (tox-176) transgenic line (Drago et al., 1998) (DARPP-32tox MUT). In this DARPP-32/Cre line, Cre activity was not detectable until 4 weeks of age; however, evidence of Cre recombinase activity in the parental line was observed in almost all DARPP-32-expressing medium spiny projection neurons in the striatum but not in DARPP-32-expressing cells in extra-striatal regions (Bogush et al., 2005). DARPP-32tox MUT mice had reduced bodyweight, locomotor slowing, reduced rearing, ataxia, a short stride length, wide-based chaotic gait and impairment in orofacial movements. Furthermore, DARPP-32tox MUT mice displayed haloperidol-suppressible tic-like movements and an anxiolytic behavioral profile.

In the present study, Emx-1tox MUT animals were generated. Emx-1 is a homeobox-containing gene expressed in developing telencephalic, glutamatergic cortical neurons from embryonic days 10 to 12.5 (Chan et al., 2001, Gorski et al., 2002), with ongoing expression in pyramidal cells of the mature cerebral cortex. This line was designed to target only cortical Drd1a-expressing cells, so as to distinguish their functional roles vis-à-vis those of striatal Drd1a-expressing cells.

Section snippets

Animals

Mice were group housed with food and water available ad libitum. Mice were maintained in a temperature-controlled environment, on a 12-hour light/dark cycle. The transcriptionally silenced attenuated diphtheria toxin (tox-176) transgenic line (Drago et al., 1998) was backcrossed 10 times with CD1 mice. The CamKIIαCre (Casanova et al., 2001, Gantois et al., 2007), DARPP-32Cre (Bogush et al., 2005, Kim et al., 2014) and Emx-1Cre (Chan et al., 2001, Gorski et al., 2002) activator transgenic lines

Sociability and social novelty preference

The apparatus for this test consisted of a rectangular three-chambered box (left and right chambers 13.5 × 20 × 20 cm; centre chamber 9 × 20 × 20 cm; total size 36 × 20 × 20 cm); dividing walls were of clear Plexiglas with small openings (4 × 4 cm) at the bottom to allow free access into all three chambers. A small wire cage (10 × 10 × 12 cm) was placed in each of the two outermost chambers, which allowed for visual and olfactory contact. Each chamber was cleaned using ethanol wipes and fresh bedding added between

Neuroanatomical techniques

In-situ hybridization for Drd1a and Drd2 (WT, n = 8, 4 males and 4 females; Emx-1tox MUT, n = 7, 3 females and 4 males) and GFAP immunohistochemistry were undertaken at 30 weeks as described previously (Kim et al., 2014). Additional neuroanatomical methods used in this study are detailed in Supplementary information.

Statistical analysis

Data were expressed as mean ± SEM, unless stated otherwise. All statistical analyses were conducted in consultation with the Department of Mathematics and Statistics at the University of Melbourne. Analyses were performed using SigmaStat v3.5 (Systat Software Inc., CA, USA) for two-way ANOVA analyses and Student's T-tests; Tukey's Post-hoc tests were used where necessary. PASW Statistics v18.0 (SPSS Inc. IL, USA) was used for three-way repeated measures ANOVA with main factors of genotype, age

Weight, locomotor hyperactivity and orofacial movements

As identified in earlier studies on CamKIIαtox MUT (Gantois et al., 2007), Emx-1tox MUT displayed significant weight differences compared to sex matched control mice. By day 30, Emx-1tox MUT mice weighed ~ 11% less than that of sex-matched controls [effects of genotype: male weight, F1,17 = 11.09, P = 0.004; female weight, F1,26 = 7.50, P = 0.011]. Female mice gained weight at a rate comparable to that of sex-matched controls (genotype × time interaction, F = 1.043, P = 0.4076), although their weight was

Discussion

Basal ganglia neuropathology is known to occur in a number of neurodegenerative conditions, including Parkinson-plus syndromes and HD. CamKIIαtox MUT mice with ablation of Drd1a expressing cells in multiple cortical and subcortical brain regions exhibited behavioral abnormalities consistent with established animal models of HD, thereby confirming the major contribution that Drd1a-expressing cell loss makes to the HD phenotype (Gantois et al., 2007). In the current study, we generated Emx-1tox

Acknowledgments

This work was supported by project grants from the National Health & Medical Research Council (NHMRC) of Australia [509072] and [628680], the Victorian Government's Operational Infrastructure Support Program and by Science Foundation Ireland principal Investigator grant 07/IN.1/B960. JD and AJL are Fellows of the NHMRC, Australia. CLP is a Viertel Senior Medical Research Fellow, Australia.

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    1

    Current address: Faculty of Medical and Human Sciences, University of Manchester, Stopford 3.721, Oxford Road, Manchester M13 9PT, UK.

    2

    Current address: Department of Pharmacology, Monash University, Clayton, Victoria, Australia.

    3

    Current address: Department of Biochemistry & Goodman Cancer Research Centre, McGill University, Montreal, Quebec H3A 1A3, Canada.

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