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

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-1(tox) MUT mice, a transgenic line in which cortical Drd1a-expressing pyramidal neurons were selectively ablated. Emx-1(tox) 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-1(tox) MUT mice overlapped with those in CamKIIα(tox) MUT transgenic mice, a line in which both striatal and cortical Drd1a-expressing cells were ablated. Although Emx-1(tox) MUT mice had normal striatal anatomy, both Emx-1(tox) MUT and CamKIIα(tox) 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.

Role of α4- and α6-containing nicotinic receptors in the acquisition and maintenance of nicotine self-administration.

Tobacco smoking is a major cause of death and disease and as such there is a critical need for the development of new therapeutic approaches to treat nicotine addiction. Here, we utilize genetic and pharmacological tools to further investigate the nicotinic acetylcholine receptor (nAChR) subtypes that support intravenous self-administration of nicotine. α4-S248F mice contain a point mutation within the α4 nAChR subunit which confers increased sensitivity to nicotine and resistance to mecamylamine. Here, we show that acute administration of mecamylamine (2 mg/kg, i.p.) reduces established nicotine self-administration (0.05 mg/kg/infusion) in wild-type (WT), but not in α4-S248F heterozygous mice, demonstrating a role for α4* nAChRs in the modulation of ongoing nicotine self-administration. Administration of N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI), a selective α6ß2* nAChR antagonist, dose dependently (5 and 10 mg/kg, i.p.) impairs the acquisition of nicotine self-administration and reduces established nicotine self-administration in WT mice when administered acutely (10 mg/kg, i.p.). This was not due to a general reduction in locomotor activity and the same dose of bPiDI did not affect operant responding for sucrose. bPiDI treatment (10 mg/kg, i.p.) also impaired both the acquisition and maintenance of nicotine self-administration in α4-S248F heterozygous mice. This provides further evidence for the involvement of α6ß2* nAChRs in the reinforcing effects of nicotine that underlies its ability to support ongoing self-administration. Taken together, selective targeting of α6ß2* or α4α6ß2* nAChRs may prove to be an effective strategy for the development of smoking cessation therapies.

Resolving pathobiological mechanisms relating to Huntington disease: gait, balance, and involuntary movements in mice with targeted ablation of striatal D1 dopamine receptor cells.

Progressive cell loss is observed in the striatum, cerebral cortex, thalamus, hypothalamus, subthalamic nucleus and hippocampus in Huntington disease. In the striatum, dopamine-responsive medium spiny neurons are preferentially lost. Clinical features include involuntary movements, gait and orofacial impairments in addition to cognitive deficits and psychosis, anxiety and mood disorders. We utilized the Cre-LoxP system to generate mutant mice with selective postnatal ablation of D1 dopamine receptor-expressing striatal neurons to determine which elements of the complex Huntington disease phenotype relate to loss of this neuronal subpopulation. Mutant mice had reduced body weight, locomotor slowing, reduced rearing, ataxia, a short stride length wide-based erratic gait, impairment in orofacial movements and displayed haloperidol-suppressible tic-like movements. The mutation was associated with an anxiolytic profile. Mutant mice had significant striatal-specific atrophy and astrogliosis. D1-expressing cell number was reduced throughout the rostrocaudal extent of the dorsal striatum consistent with partial destruction of the striatonigral pathway. Additional striatal changes included up-regulated D2 and enkephalin mRNA, and an increased density of D2 and preproenkephalin-expressing projection neurons, and striatal neuropeptide Y and cholinergic interneurons. These data suggest that striatal D1-cell-ablation alone may account for the involuntary movements and locomotor, balance and orofacial deficits seen not only in HD but also in HD phenocopy syndromes with striatal atrophy. Therapeutic strategies would therefore need to target striatal D1 cells to ameliorate deficits especially when the clinical presentation is dominated by a bradykinetic/ataxic phenotype with involuntary movements.

Phenotyping dividing cells in mouse models of neurodegenerative basal ganglia diseases.

BACKGROUND: Mice generated by a Cre/LoxP transgenic paradigm were used to model neurodegenerative basal ganglia disease of which Huntington disease (HD) is the prototypical example. In HD, death occurs in striatal projection neurons as well as cortical neurons. Cortical and striatal neurons that express the D1 dopamine receptor (Drd1a) degenerate in HD. The contribution that death of specific neuronal cell populations makes to the HD disease phenotype and the response of the brain to loss of defined cell subtypes is largely unknown. METHODS: Drd1a-expressing cells were targeted for cell death and three independent lines generated; a striatal-restricted line, a cortical-restricted line and a global line in which Drd1a cells were deleted from both the striatum and cortex. Two independent experimental approaches were used. In the first, the proliferative marker Ki-67 was used to identify proliferating cells in eighty-week-old mice belonging to a generic global line, a global in which Drd1a cells express green fluorescent protein (GFP-global) and in eighty-week-old mice of a cortical line. In the second experiment, the proliferative response of four-week-old mice belonging to GFP-global and striatal lines was assessed using the thymidine analogue BrdU. The phenotype of proliferating cells was ascertained by double staining for BrdU and Olig2 (an oligodendrocyte marker), Iba1 (a microglial cell marker), S100ß (an astroglial cell marker), or NeuN (a neuronal cell marker). RESULTS: In the first study, we found that Ki-67-expressing cells were restricted to the striatal side of the lateral ventricles. Control mice had a greater number of Ki-67+ cells than mutant mice. There was no overlap between Ki-67 and GFP staining in control or mutant mice, suggesting that cells did not undergo cell division once they acquired a Drd1a phenotype. In contrast, in the second study we found that BrdU+ cells were identified throughout the cortex, striatum and periventricular region of control and mutant mice. Mutant mice from the GFP-global line showed increased BrdU+ cells in the cortex, striatum and periventricular region relative to control. Striatal line mutant mice had an increased number of BrdU+ cells in the striatum and periventricular region, but not the cortex. The number of microglia, astrocytes, oligodendrocytes and neurons generated from dividing progenitors was increased relative to control mice in most brain regions in mutant mice from the GFP-global line. In contrast, striatal line mutant mice displayed an increase only in the number of dividing microglia in striatal and periventricular regions. CONCLUSIONS: Genetically programmed post-natal ablation of Drd1a-expressing neurons is associated with an extensive proliferative response involving multiple cell lineages. The nature of the tissue response has the potential not only to remove cellular debris but also to forge physiologically meaningful brain repair. Age related deficits in proliferation are seen in mutant lines. A blunted endogenous reparative response may underlie the cumulative deficits characteristic of age related neurodegeneration.

Proconvulsant-induced seizures in alpha(4) nicotinic acetylcholine receptor subunit knockout mice.

The genetic basis of a number of epilepsy syndromes has been identified but the precise mechanism whereby these mutations produce seizures is unknown. Three mutations of the alpha(4) subunit of the neuronal nicotinic acetylcholine receptor (nAChR) have been identified in autosomal dominant nocturnal frontal lobe epilepsy. In vitro studies of two mutations suggest an alteration of receptor function resulting in decreased ion channel current flow. We investigated the response of alpha(4) nAChR subunit knockout mice to the gamma-aminobutyric acid (GABA) receptor antagonists; pentylenetetrazole (PTZ) and bicuculline (BIC), the glutamate receptor agonist kainic acid (KA), the glycine receptor antagonist strychnine and the K(+) channel blocker 4-aminopyridine (4-AP). Mutant (Mt) mice had a greater sensitivity to PTZ and BIC, with an increase in major motor seizures and seizure-related deaths. Furthermore, Mt mice were more sensitive to KA and strychnine, but the effects were much smaller compared to those seen with the GABA receptor antagonists. Paradoxically, Mt mice appeared to be relatively protected from 4-AP-induced major motor seizures and death. The results show that a functional deletion of the alpha(4) nAChR subunit in vivo is associated with a major increase in sensitivity to GABA receptor blockers.

Essential conservation of D1 mutant phenotype at the level of individual topographies of behaviour in mice lacking both D1 and D3 dopamine receptors.

RATIONALE: In the absence of agonists and antagonists evidencing appropriate selectivities, individual and interactive properties of D(1) and D(3) dopamine receptors would be illuminated most powerfully by their co-deletion. OBJECTIVES: To define and contrast the behavioural phenotype of D(1)/D(3) double knockout mice in comparison with wild types, and with individual D(1) and D(3 )mutants. METHODS: Behavioural phenotype was characterised using an ethologically based topographical technique. RESULTS: On comparison with wild types, D(1)/D(3) double mutants were characterised topographically as follows: increases in sniffing and locomotion, which evidenced delayed habituation; reductions in rearing free, rearing seated, grooming, chewing and stillness. Though the D(1)/D(3) double mutant ethogram comprised elements of both single mutant D(1) and D(3) lines, this phenotype was largely reflective of the D(1) mutant component. CONCLUSIONS: Distinct patterns of initial exploratory behaviour and of temporal change over subsequent habituation were evident across the three genotypes, with particular conservation of the D(1) phenotype in D(1)/D(3 )double mutants. Under the present conditions, there was little systematic evidence for D(1):D(3) interactions in the regulation of these aspects of behaviour.

Neurochemical changes in dopamine D1, D3 and D1/D3 receptor knockout mice.

Neurochemical changes were examined in dopamine D1 receptor knockout (D1(-/-)), dopamine D3 receptor knockout (D3(-/-)) and dopamine D1/D3 receptor double knockout (D1(-/-)D3(-/-)) mice. The level of dopamine D1- and D2-like receptors and gamma-aminobutyric acid (GABA(A)) receptor was assessed by ligand autoradiography and dopamine D1- and D2 receptor, enkephalin, dynorphin and substance P transcripts measured by in situ hybridization. D1(-/-) mice had normal GABA(A) receptor levels, reduced dynorphin and substance P, and increased enkephalin mRNA and dopamine D2-like binding. D1(-/-)D3(-/-) mice evidenced decreased dynorphin and substance P but normal enkephalin expression, whereas dopamine D2-like and GABA(A) receptor binding were increased. Major changes occur in substance P and dynorphin expression in D1(-/-) mice and these changes are unaffected by loss of dopamine D3 receptors. Upregulated dopamine D2-like binding and enkephalin in D1(-/-) mice may be due to decreased dopamine turnover. Upregulated enkephalin in D1(-/-) mice is dependent on functional dopamine D3 receptors.