Impaired dopamine release and uptake in R6/1 Huntington's disease model mice

Huntington's disease (HD) is a progressive, neurodegenerative movement disorder. Here, we used fast-scan cyclic voltammetry to measure dopamine release and uptake in striatal brain slices from R6/1 HD model mice. Peak dopamine release ([DA]_max) was significantly diminished in R6/1 mice (52% of wild-type at 24 weeks of age). Similarly, dopamine released per locally applied electrical stimulus pulse ([DA]_p), which is [DA]_max corrected for uptake and electrode performance, was also diminished in R6/1 mice (43% of wild-type by 24 weeks of age). Moreover, V_max, the maximum rate of dopamine uptake, obtained by modeling the stimulated release plots, was decreased at 16 and 24 weeks of age in R6/1 mice (51 and 48% of wild-type, respectively). Thus, impairments in both dopamine release and uptake appear to progress in an age-dependent manner in R6/1 mice.     

Real-time voltammetric detection of cocaine-induced dopamine changes in the striatum of freely moving mice

In the present voltammetric study, we have characterized cocaine-induced changes in evoked dopamine release and uptake in the striatum of freely moving mice in real time. Cocaine induced marked dopamine uptake inhibition measured as apparent K_m changes, producing a maximal effect 20 min following a single injection (15 mg/kg, i.p.). Changes in uptake were paralleled by increases in evoked dopamine release per stimulus pulse, revealing a high correlation between these two parameters following cocaine administration. This initial characterization of cocaine effects on striatal dopamine transmission in the commonly used C57BL/6 mouse strain provides a basis for future voltammetric studies using genetic mouse models.     

IGF-1 exacerbates the neurotoxicity of the mitochondrial inhibitor 3NP in rats

Insulin-like Growth Factor 1 (IGF-1) has broad-range neuroprotective effects and is a therapeutic candidate for Huntington's disease (HD). IGF-1 protects striatal neurons from the toxicity of mutated huntingtin in vitro and improves neuronal survival in vivo in a phenotypic model of HD involving excitotoxic cell death. Because HD is a multifactorial disease, it is important to evaluate the neuroprotective role of IGF-1 in other pathological situations involved in HD progression. We have evaluated the neuroprotective effects of IGF-1 in vivo, using the 3-nitropropionic acid (3NP) rat model which replicates the mitochondrial dysfunction observed in HD. Continuous intracerebroventricular infusion of recombinant IGF-1 at a low dose (0.025 microg/h for 5 days) did not alleviate motor impairment and weight loss induced by 3NP treatment. In addition, histological evaluation and quantification of DNA fragmentation evidenced no improvement in neuronal survival. Of interest, we found that a higher concentration of IGF-1 (0.25 microg/h) resulted in an exacerbation of 3NP toxicity on striatal neurons. These results suggest that intracerebral delivery of IGF-1 may not provide a fully effective therapeutic strategy for HD or other disorders involving mitochondrial impairment.     

APP23 mice display working memory impairment in the plus-shaped water maze

Altered glutamate transmission in the striatum has been proposed to play a critical role in the pathophysiology of Huntington's disease (HD), a genetic disorder associated with impaired activity of the mitochondrial complex II (succinate dehydrogenase, SD). In the present study, we recorded spontaneous (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs) from striatal neurons of both toxic (systemic administration of 3-nitropropionic acid in rats) and genetic models of HD (R6/2 transgenic mice). In both models, we found a significant down-regulation of glutamate transmission, suggesting that reduced synaptic excitation of the input structure of the basal ganglia represents a physiological correlate of HD.     

Single administration of 1-benzyl-1,2,3,4-tetrahydroisoquinoline increases the extracellular concentration of dopamine in rat striatum

We performed a combined neurochemical and behavioral study to determine the effects of 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-BnTIQ) on the extracellular dopamine concentrations in the striatum. Single dose administration of 1-BnTIQ (20, 40, and 80 mg/kg i.p.) increased striatal dopamine extracellular levels in a dose-dependent manner when an in vivo microdialysis technique was used to assess dopamine levels in the striatum of rats. Enhancement of striatal dopamine levels by systemic administration of a single dose of 1-BnTIQ was suppressed by perfusion of tetrodotoxin and a calcium ion-free solution into the striatum. This 1-BnTIQ-induced increase in extracellular dopamine concentration was also inhibited by pre-treatment with a dopamine uptake inhibitor, GBR12909 (1-(2-[bis(4-Fluorophenyl)-4-(3-phenylpropyl)piperazine dihydrochloride). Local application of 1-BnTIQ into the striatum via a dialysis probe failed to enhance the extracellular concentration of dopamine. However, microinjection of 1-BnTIQ into the substantia nigra pars compacta increased the extracellular dopamine levels in the striatum. Locomotor activity was increased by systemic administration of a single dose of 1-BnTIQ in a dose-dependent manner. This 1-BnTIQ-induced locomotor activity was attenuated by pre-treatment with SCH23390 (R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochlodride) and raclopride, D₁ and D₂ dopaminergic receptor antagonists, respectively. Moreover, 1-BnTIQ induced ipsilateral rotational behavior in 6-hydroxydopamine–lesioned rats. These results suggest that systemic administration of a single dose of 1-BnTIQ increases striatal extracellular dopamine concentration through activation of dopaminergic nigra striatal neurons via the dopamine transporter.     

Dopamine release and uptake are greater in female than male rat striatum as measured by fast cyclic voltammetry

The present studies investigated sexual dimorphisms in dopamine release and uptake using fast-scan cyclic voltammetry in anesthetized rats and in brain slices. Electrical stimulation of the medial forebrain bundle of anesthetized rats at high frequency (60 Hz) elicited significantly more extracellular dopamine in the caudate nucleus of females than males. This sex difference was apparent over a range of current intensities applied to the stimulating electrode. Local electrical stimulation of brain slices in vitro verified in vivo results as more extracellular dopamine was elicited by single and 10 pulse stimulations in the caudate nucleus of females. Kinetic analysis of in vivo and in vitro dopamine overflow data indicated that dopamine release (the concentration of dopamine released per stimulus pulse) and the maximal velocity of dopamine uptake are greater in female rats, but the affinity of the transporter for dopamine was the same in males and females. None of these three parameters varied across the female estrous cycle. Linear regression analysis of dopamine release versus maximal uptake velocity data indicated a significant association of release and uptake sites in each sex and regression lines for males and females virtually overlapped.One explanation for these results is greater dopamine neuron terminal density in female caudate nucleus. These sexual dimorphisms in dopaminergic neurotransmission provide a novel, plausible mechanism to explain robust sex differences in behavioral responses of rats to pyschostimulant drugs and may have implications for human neurological disorders and drug abuse.     

Autoradiographic study of striatal dopamine re-uptake sites and dopamine D1 and D2 receptors in a 6-hydroxydopamine and quinolinic acid double-lesion rat model of striatonigral degeneration (multiple system atrophy) and effects of embryonic ventral mesencephalic,striatal or co-grafts

The influence of embryonic mesencephalic, striatal and mesencephalic/striatal co-grafts on amphetamine- and apomorphine-induced rotation behaviour was assessed in a rat model of multiple system atrophy/striatonigral degeneration type using dopamine D₁ ([³H]SCH23390) and D₂ ([³H]spiperone) receptor and dopamine re-uptake ([³H]mazindol) autoradiography. Male Wistar rats subjected to a sequential unilateral 6-hydroxydopamine lesion of the medial forebrain bundle followed by a quinolinic acid lesion of the ipsilateral striatum were divided into four treatment groups, receiving either mesencephalic, striatal, mesencephalic/striatal co-grafts or sham grafts. Amphetamine- and apomorphine-induced rotation behaviour was recorded prior to and up to 10 weeks following transplantation.6-Hydroxydopamine-lesioned animals showed ipsiversive amphetamine-induced and contraversive apomorphine-induced rotation behaviour. Amphetamine-induced rotation rates persisted after the subsequent quinolinic acid lesion, whereas rotation induced by apomorphine was decreased. In 11 of 14 animals receiving mesencephalic or mesencephalic/striatal co-grafts, amphetamine-induced rotation scores were decreased by >50% at the 10-week post-grafting time-point. In contrast, only one of 12 animals receiving non-mesencephalic (striatal or sham) grafts exhibited diminished rotation rates at this time-point. Apomorphine-induced rotation rates were significantly increased following transplantation of mesencephalic, striatal or sham grafts. The largest increase of apomorphine-induced rotation rates approaching post-6-hydroxydopamine levels were observed in animals with striatal grafts. In contrast, in the co-graft group, there was no significant increase of apomorphine-induced rotation compared to the post-quinolinic acid time-point. Morphometric analysis revealed a 63–74% reduction of striatal surface areas across the treatment groups. Striatal [³H]mazindol binding on the lesioned side (excluding the demarcated graft area) revealed a marked loss of dopamine re-uptake sites across all treatment groups, indicating missing graft-induced dopaminergic re-innervation of the host. In eight (73%) of the 11 animals with mesencephalic grafts and reduced amphetamine-induced circling, discrete areas of [³H]mazindol binding (“hot spots”) were observed, indicating graft survival. Dopamine D₁ and D₂ receptor binding was preserved in the remaining lesioned striatum irrespective of treatment assignment, except for a significant reduction of D₂ receptor binding in animals receiving mesencephalic grafts. “Hot spots” of dopamine D₁ and D₂ receptor binding were observed in 10 (83%) and nine (75%) of 12 animals receiving striatal grafts or co-grafts, consistent with survival of embryonic primordial striatum grafted into a severely denervated and lesioned striatum.Our study confirms that functional improvement may be obtained from embryonic neuronal grafts in a double-lesion rat model of multiple system atrophy/striatonigral degeneration type. Co-grafts appear to be required for reversal of both amphetamine- and apomorphine-induced rotation behaviour in this model. We propose that the partial reversal of amphetamine-induced rotation asymmetry in double-lesioned rats receiving mesencephalic or mesencephalic/striatal co-grafts reflects non-synaptic graft-derived dopamine release. The changes of apomorphine-induced rotation following transplantation are likely to reflect a complex interaction of graft- and host-derived striatal projection pathways and basal ganglia output nuclei. Further studies in a larger number of animals are required to determine whether morphological parameters and behavioural improvement in the neurotransplantation multiple system atrophy rat model correlate.     

Distribution and morphological characteristics of striatal interneurons expressing calretinin in mice: A comparison with human and nonhuman primates

Striatal interneurons display a morphological and chemical heterogeneity that has been particularly well characterized in rats, monkeys and humans. By comparison much less is known of striatal interneurons in mice, although these animals are now widely used as transgenic models of various neurodegenerative diseases. The present immunohistochemical study aimed at characterizing striatal interneurons expressing calretinin (CR) in mice compared to those in squirrel monkeys and humans. The mouse striatum contains both small (9–12 μm) and medium-sized (15–20 μm) CR+ cells. The small cells are intensely stained with a single, slightly varicose and moderately arborized process. They occur throughout the striatum (77 ± 9 cells/mm³), but prevail in the area of the subventricular zone and subcallosal streak, with statistically significant anteroposterior and dorsoventral decreasing gradients. The medium-sized cells are less intensely immunoreactive and possess 2–3 long, slightly varicose and poorly branched dendrites. They are rather uniformly scattered throughout the striatum and three times more numerous (224 ± 31 cells/mm³) than the smaller CR+ cells. Double immunostaining experiments with choline acetyltransferase (ChAT) as a cholinergic marker in normal and Drd1a-tdTomato/Drd2-EGFP double transgenic mice reveal that none of the small or medium-sized CR+ cells express ChAT or D₁ and D₂ dopamine receptors. In contrast, the striatum in human and nonhuman primates harbors small and medium-sized CR+/ChAT− cells, as well as large CR+/ChAT+ interneurons that are absent in mice. Such a difference between rodents and primates must be taken into consideration if one hopes to better understand the striatal function in normal and pathological conditions.     

Striatal dopamine and glutamate release: effects of intranigral injections of substance P

The levels of extracellular striatal dopamine and glutamate were measured simultaneously in halothane-anaesthetized rats using microdialysis. Unilateral injections of substance P (0.07 nmol) into the substantia nigra, pars reticulata enhanced the levels of dopamine and glutamate in the ipsilateral striatum. Intranigral injections of neurokinin A (0.09 nmol) enhanced the levels of striatal dopamine, and intranigral injections of γ-aminobutyric acid (300 nmol) or dynorphin A (0.5 nmol) decreased the levels of striatal dopamine, but none of these had any effect on the levels of striatal glutamate. Local perfusion with the dopamine agonists apomorphine (D1/D2), SKF 38393 (DI) or pergolide (D2) (each at 10^-5 M) decreased the levels of striatal dopamine and enhanced the levels of striatal glutamate. In unilateral 6-hydroxydopamine-lesioned rats, basal striatal glutamate levels were decreased bilaterally. Furthermore, on the denervated side intranigral substance P stimulation of striatal glutamate levels was enhanced, while on the intact side intranigral substance P stimulation of striatal dopamine and glutamate levels was similar to that seen in normal rats. These findings suggest that striatonigral substance P provides a stimulatory regulation of ipsilateral striatal glutamate release. Furthermore, it is indicated that striatal glutamate release can also be regulated by dopamine terminals.     

Effect of sex and age on brain monoamines and spatial learning in rats

The concentrations of noradrenaline (NA), dopamine (DA), serotonin (5-HT), and their metabolites were measured in the prefrontal cortex, caudate-putamen, and hippocampus in young (3 months) and aged (27–31 months) Wistar rats of both sexes. Age-related changes were found in prefrontal NA and HVA/DA ratio, striatal DA and DOPAC/DA ratio, and striatal and hippocampal 5-HT and 5-HIAA/5-HT ratio. Age and sex dependent changes were found in striatal DA and DOPAC/DA ratio, and hippocampal MHPG-SO₄/NA ratio. The aged rats were tested in spatial discrimination and reversal tasks in a T maze. The effects of α₂-agonist medetomidine (3 μg/kg) on the task performance were assessed in relation to individual variation in monoamine metabolism. Medetomidine impaired spatial discrimination learning of the aged rats by interacting with the hippocampal 5-HT turnover. Medetomidine improved reversal learning through an interaction with the striatal DA turnover and reduced the number of perseverative errors after reversal, mainly due to its interaction with the prefrontal NA turnover. It is concluded that the memory enhancing effect of drugs acting through the brain monoamine systems is highly dependent on the stage of degeneration of these systems that show considerable individual variation in aged animals.     

Chronic intoxication with 3-nitropropionic acid in rats induces the loss of striatal dopamine terminals without affecting nigral cell viability

3-Nitropropionic acid (3NP) is a succinate dehydrogenase inhibitor allowing the generation of animal models of Huntington's disease. In the present study, we found that a 5-day continuous chronic infusion of 3NP produces loss of [3H]mazindol binding and tyrosine hydroxylase (TH) immunoreactivity in the striatal area of degeneration. This loss of dopamine terminals was not due to a loss of nigral neurons since the expression of TH as well as the number of TH-expressing neurons remained unaltered in the substantia nigra of rats treated by 3NP. This suggests that the 3NP-induced dopamine terminal loss is secondarily related to the striatal degeneration andlor to a direct effect of 3NP on striatal terminals and not to a primary effect on nigral cells.     

Stimulus-secretion coupling processes in brain: dependence upon extracellular calcium concentration.

The relationship between [Ca²⁺]₀ and secretion was investigated in crude synaptosomal fractions from the cerebral cortex and the corpus striatum of the rat. The sigmoid nature of the relationship was analyzed with a linearizing modification of the Michaelis-Menten equation. Higher [K⁺]₀ increased calcium-dependent release of [³H]dopamine from striatal synaptosomes. Log releaselog [Ca²⁺]⁰ curves were shifted upward and to the left. Higher [K⁺]₀ also increased the slope, or sigmoidicity, of the relationship. In the presence of 56 mM [K⁺]⁰, release of [³H]dopamine was greater than that of [¹⁴C]γ-aminobutyrate from striatal synaptosomes at all the [Ca²⁺]₀ tested. The differences in release resulted simply from an upward shift of the curves, as no differences were observed in either the apparent K_m for calcium or the sigmoidicity of the relationship.Release of [¹⁴C]γ-aminobutyrate from cortical and striatal synaptosomes was also investigated. KCl-facilitated, calcium-dependent release of γ-aminobutyrate from cortical synaptosomes was greater than release from striatal synaptosomes. In addition to an upward shift of the log release-log [Ca²⁺]₀ plot, the curve for cortical synaptosomes exhibited less sigmoidicity. No differences in the K_m for calcium were observed.The similarity of K_m values for calcium of the secretion systems for different transmitters and for different regions suggests that a similar rate-limiting process(es) may exist in the different secretion systems. However, the differences in sigmoidicity and maximal rates of secretion suggest that the systems do differ in at least some aspects.     

N-Methyl-d-aspartate (NMDA) receptor function and excitotoxicity in Huntington's disease

Many lines of evidence support a role for neuronal damage arising as a result of excessive activation of glutamate receptors by excitatory amino acids in the pathogenesis of Huntington disease. The N-methyl-d-aspartate subclass of ionotropic glutamate receptors (NMDARs) is more selective and effective than the other subclasses in mediating this damage. As well, neurons expressing high levels of NMDARs are lost early from the striatum of individuals affected with Huntington's disease (HD), and injection of NMDAR agonists into the striatum of rodents or non-human primates recapitulates the pattern of neuronal damage observed in HD. Altered NMDAR function has been reported in corticostriatal synapses in one mouse model of HD, and NMDAR-mediated current and/or toxicity have been found to be potentiated in striatal neurons from several HD mouse models as well as heterologous cells expressing the mutant huntingtin protein. Changes in NMDAR activity have been correlated with altered calcium homeostasis, mitochondrial membrane depolarization and caspase activation. NMDAR stimulation is also closely linked to mitochondrial activity, as treatment with mitochondrial toxins has been demonstrated to produce striatal damage that can be reversed by the addition of NMDAR antagonists. Recent efforts have focused on the elucidation of molecular pathways linking huntingtin to NMDARs, as well as the mechanisms which underlie the enhancement of NMDAR activity by mutant huntingtin. Here, we review the literature to date and recent findings concerning the role of NMDARs in HD pathogenesis.     

Dopamine D1 receptor-dependent modifications in the dopamine and cAMP-regulated phosphoprotein of Mr 32 kDa phosphorylation pattern in striatal areas of morphine-sensitized rats

Morphine sensitization is a model of latent, functionally inducible increase in dopamine D₁ receptor–mediated transmission, which may be unmasked by an external stimulus. Morphine-sensitized rats present dopamine D₁ receptor–dependent stereotypies upon morphine challenge and resilience to unavoidable stress-induced behavioral deficits. This tonic increase in dopamine D₁ dopaminergic transmission is counter-adaptive to an enhanced μ-opioid receptor-dependent signaling in striatal areas. Control and sensitized rats show a similar dopamine and cAMP-regulated phosphoprotein of M_r 32 kDa (DARPP-32) phosphorylation pattern in striatal areas. Acute morphine administration induced an early increase and delayed decrease in phospho-threonine (Thr)34 DARPP-32 levels accompanied by a delayed increase in phospho-Thr75 DARPP-32 levels in the nucleus accumbens and caudate-putamen of sensitized rats, while it had no effects in control animals. The administration of a selective dopamine D₁ receptor antagonist (SCH 23390) before morphine challenge prevented the behavioral and neurochemical modifications in sensitized rats. 6-Methyl-2-(phenylethynyl)-pyridine, a selective metabotropic glutamate receptor 5 (mGluR₅) antagonist, administered 1 h after morphine challenge, prevented the delayed phosphorylation changes, but it had no effect when administered before challenge. Moreover, the DARPP-32 phosphorylation pattern in the caudate-putamen of sensitized rats after unavoidable stress exposure was studied. The stress-induced neurochemical modifications and their sensitivity to receptor antagonists were similar to those observed after acute morphine administration. In conclusion, these results suggest that in the experimental conditions used an increase in dopamine output in striatal areas is followed by a complex neurochemical pattern, in which the initial stimulation of dopamine D₁ receptors triggers a sequence of signaling events that lead to an mGluR₅-mediated increase in phospho-Thr75 DARPP-32 levels. Since DARPP-32 phosphorylated in Thr75 inhibits cAMP-dependent protein kinase (PKA) activity, the final result is a decrease in the dopamine D₁ receptor–dependent phosphorylation events.     

Magnetic resonance imaging and spectroscopy in assessing 3-nitropropionic acid-induced brain lesions: an animal model of Huntington's disease

Huntington's disease (HD) is an inherited neurodegenerative disease, in which there is progressive motor and cognitive deterioration, and for which the pathogenesis of neuronal death remains controversial. Mitochondrial toxins like 3-nitropropionic acid (3-NP) and malonate, functioning as the inhibitors of the complex II of mitochondrial respiratory chain, have been found to effectively induce specific behavioral changes and selective striatal lesions in rats and non-human primates mimicking those in HD. Furthermore, several kinds of transgenic mouse models of HD have been recently developed, and used in the development and assessment of novel treatments for HD. In the past, most studies evaluating the animal models for HD were based on histological changes or in vitro neuronal cultures. With the emergence of advanced magnetic resonance technologies, non-invasive magnetic resonance imaging (MRI) and spectroscopy provide more detail of cerebral alterations, including the changes of cerebral structure, function and metabolites. These studies support the hypothesis that mitochondrial dysfunction with increased excitation of N-methyl-D-aspartate (NMDA) receptors can replicate the neurobehavioral changes, selective brain injury and neurochemical alterations in HD. The present review focuses on our work as well as that of others regarding 3-NP-induced neurotoxicity and other animal models of HD. Using both conventional and advanced MRI and spectroscopy, we summarize the pathogenesis and possible therapeutic strategies in chemical and transgenic models of HD. The results show magnetic resonance techniques to be powerful techniques in the evaluation of pathogenesis and therapeutic intervention for both chemical and transgenic models of HD.     

Striatal cells from mutant huntingtin knock-in mice are selectively vulnerable to mitochondrial complex II inhibitor-induced cell death through a non-apoptotic pathway

Extensive striatal neuronal loss occurs in Huntington's disease (HD), which is caused by an expanded polyglutamine tract in huntingtin (htt). Evidence suggests that mutant htt directly or indirectly compromises mitochondrial function, contributing to the neuronal loss. To determine the role of compromised mitochondrial function in the neuronal cell death in HD, immortalized striatal cells established from Hdh(Q7) (wild-type) and Hdh(Q111) (mutant) mouse knock-in embryos were treated with 3-nitropropionic acid (3-NP), a mitochondrial complex II toxin. 3-NP treatment caused significantly greater cell death in mutant striatal cells compared with wild-type cells. In contrast, the extent of cell death induced by rotenone, a complex I inhibitor, was similar in both cell lines. Although evidence of apoptosis was present in 3-NP-treated wild-type striatal cells, it was absent in 3-NP-treated mutant cells. 3-NP treatment caused a greater loss of mitochondrial membrane potential (deltapsim) in mutant striatal cells compared with wild-type cells. Cyclosporine A, an inhibitor of mitochondrial permeability transition pore (PTP), and ruthenium red, an inhibitor of the mitochondrial calcium uniporter, both rescued mutant striatal cells from 3-NP-induced cell death and prevented the loss of deltapsim. These data show that mutant htt specifically increases cell vulnerability to mitochondrial complex II inhibition and further switched the type of cell death induced by complex II inhibition from apoptosis to a non-apoptotic form, caused by mitochondrial membrane depolarization, probably initiated by mitochondrial calcium overload and subsequent PTP opening. These findings suggest that impaired mitochondrial complex II function in HD may contribute to non-apoptotic neuronal cell death.