Anne Panhelainen

GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function

Degeneration of nigrostriatal dopaminergic system is the principal lesion in Parkinson’s disease. Because glial cell line-derived neurotrophic factor (GDNF) promotes survival of dopamine neurons in vitro and in vivo, intracranial delivery of GDNF has been attempted for Parkinson’s disease treatment but with variable success. For improving GDNF-based therapies, knowledge on physiological role of endogenous GDNF at the sites of its expression is important. However, due to limitations of existing genetic model systems, such knowledge is scarce. Here, we report that prevention of transcription of Gdnf 3’UTR in Gdnf endogenous locus yields GDNF hypermorphic mice with increased, but spatially unchanged GDNF expression, enabling analysis of postnatal GDNF function. We found that increased level of GDNF in the central nervous system increases the number of adult dopamine neurons in the substantia nigra pars compacta and the number of dopaminergic terminals in the dorsal striatum. At the functional level, GDNF levels increased striatal tissue dopamine levels and augmented striatal dopamine release and re-uptake. In a proteasome inhibitor lactacystin-induced model of Parkinson’s disease GDNF hypermorphic mice were protected from the reduction in striatal dopamine and failure of dopaminergic system function. Importantly, adverse phenotypic effects associated with spatially unregulated GDNF applications were not observed. Enhanced GDNF levels up-regulated striatal dopamine transporter activity by at least five fold resulting in enhanced susceptibility to 6-OHDA, a toxin transported into dopamine neurons by DAT. Further, we report how GDNF levels regulate kidney development and identify microRNAs miR-9, miR-96, miR-133, and miR-146a as negative regulators of GDNF expression via interaction with Gdnf 3’UTR in vitro. Our results reveal the role of GDNF in nigrostriatal dopamine system postnatal development and adult function, and highlight the importance of correct spatial expression of GDNF. Furthermore, our results suggest that 3’UTR targeting may constitute a useful tool in analyzing gene function.

GABA Site Agonist Gaboxadol Induces Addiction-Predicting Persistent Changes in Ventral Tegmental Area Dopamine Neurons But Is Not Rewarding in Mice or Baboons

Dopamine neurons of the ventral tegmental area (VTA) are involved at early phases of drug addiction. Even the first in vivo dose of various abused drugs induces glutamate receptor plasticity at the excitatory synapses of these neurons. Benzodiazepines that suppress the inhibitory GABAergic interneurons in the VTA via facilitation of synaptic GABAA receptors have induced neuroplasticity in dopamine neurons due to this disinhibitory mechanism. Here, we have tested a non-benzodiazepine direct GABA site agonist 4,5,6,7-tetrahydroisoxazolol[4,5-c]pyridine-3-ol (THIP) (also known as gaboxadol) that acts preferentially via high-affinity extrasynaptic GABAA receptors. A single sedative dose of THIP (6 mg/kg) to mice induced glutamate receptor plasticity for at least 6 d after administration. Increased AMPA/NMDA receptor current ratio and increased frequency, amplitude, and rectification of AMPA receptor responses suggested persistent targeting of GluA2-lacking AMPA receptors in excitatory synapses of VTA dopamine neurons ex vivo after THIP administration. This effect was abolished in GABAA receptor δ−/− mice, which have a loss of extrasynaptic GABAA receptors. In behavioral experiments, we found neither acute reinforcement in intravenous self-administration sessions with THIP at relevant doses using a yoked control paradigm in mice nor in baboons using a standard paradigm for assessing drug abuse liability; nor was any place preference found after conditioning sessions with various doses of THIP but rather a persistent aversion in 6 mg/kg THIP-conditioned mice. In summary, we found that activation of extrasynaptic δ-subunit-containing GABAA receptors leads to glutamate receptor plasticity of VTA dopamine neurons, but is not rewarding, and, instead, induces aversion.

Evidence for a role of inhibition of orexinergic neurons in the anxiolytic and sedative effects of diazepam: A c-Fos study

The classical benzodiazepine diazepam (DZ) induces anxiolysis at low doses and sedation and hypnosis at higher doses. Different brain areas and neuronal populations most likely mediate these different behavioral effects. We used c-Fos immunohistochemistry as an indirect way to study neuronal activation or inhibition induced by DZ at anxiolytic and sedative doses (0.5 and 5mg/kg, respectively) in various brain areas involved in anxiety, arousal, sedation and addiction in C57BL/6J mice. We also focused on the two neuronal populations, orexinergic and dopaminergic neuronal populations, with the help of double-immunohistochemistry using c-Fos and orexin-A antibodies and c-Fos and tyrosine hydroxylase antibodies. We found that different brain areas of unhabituated mice reacted differently to the mild stress induced by vehicle injection. Also the response to anxiolytic or sedative doses of DZ differed between the areas, suggesting that distinct brain areas mediate the behavioral effects of low and high DZ doses. Our findings propose a role for inhibition of orexin neurons in the anxiolytic and sleep-promoting effects of DZ. In addition, the activation of central amygdala neurons by DZ treatment was associated with anxiolytic and sedative effects. On the other hand, the ventral hippocampus, basolateral amygdala, ventral tegmental area and prefrontal cortex were sensitive even to the mild injection stress, but not to the anxiolytic dose of DZ.

Excessive novelty-induced c-Fos expression and altered neurogenesis in the hippocampus of GluA1 knockout mice.

α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor GluA1 subunit‐deficient (GluA1−/−) mice display novelty‐induced hyperactivity, cognitive and social defects and may model psychiatric disorders, such as schizophrenia and depression/mania. We used c‐Fos expression in GluA1−/− mice to identify brain regions responsible for novelty‐induced hyperlocomotion. Exposure to a novel cage for 2 h significantly increased c‐Fos expression in many brain regions in both wild‐type and knockout mice. Interestingly, the clearest genotype effect was observed in the hippocampus and its main input region, the entorhinal cortex, where the novelty‐induced c‐Fos expression was more strongly enhanced in GluA1−/− mice. Their novelty‐induced hyperlocomotion partly depended on the activity of AMPA receptors, as it was diminished by the AMPA receptor antagonist 2,3‐dioxo‐6‐nitro‐1,2,3,4‐tetrahydrobenzo[f]quinoxaline‐7‐sulphonamide (NBQX) and unaffected by the AMPA receptor potentiator 2,3‐dihydro‐1,4‐benzodioxin‐6‐yl‐1‐piperidinylmethanone (CX546). The hyperlocomotion of GluA1−/− mice was normalised to the level of wild‐type mice within 5–6 h, after which their locomotion followed normal circadian rhythm and was not affected by acute or chronic treatments with the selective serotonin reuptake inhibitor escitalopram. We propose that hippocampal dysfunction, as evidenced by the excessive c‐Fos response to novelty, is the major contributor to novelty‐induced hyperlocomotion in GluA1−/− mice. Hippocampal dysfunction was also indicated by changes in proliferation and survival of adult‐born dentate gyrus cells in the knockout mice. These results suggest focusing on the functions of hippocampal formation, such as novelty detection, when using the GluA1−/− mouse line as a model for neuropsychiatric and cognitive disorders.

Changes in Neuronal Dopamine Homeostasis following 1-Methyl-4-phenylpyridinium (MPP+) Exposure

Background: The neurotoxin 1-methyl-4-phenylpyridinium (MPP+) kills dopaminergic neurons by a variety of mechanisms. Results: MPP+ affects dopamine (DA) vesicular storage, plasma membrane transport, and catabolic breakdown, leading to accumulation of cytosolic DA and neurotoxicity. Conclusion: Alterations in DA homeostasis account for ∼30% of MPP+-mediated toxicity. Significance: Comprehensive analysis of the effects of MPP+ helps to understand the mechanisms underlying the development of Parkinson disease. 1-Methyl-4-phenylpyridinium (MPP+), the active metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, selectively kills dopaminergic neurons in vivo and in vitro via a variety of toxic mechanisms, including mitochondrial dysfunction, generation of peroxynitrite, induction of apoptosis, and oxidative stress due to disruption of vesicular dopamine (DA) storage. To investigate the effects of acute MPP+ exposure on neuronal DA homeostasis, we measured stimulation-dependent DA release and non-exocytotic DA efflux from mouse striatal slices and extracellular, intracellular, and cytosolic DA (DAcyt) levels in cultured mouse ventral midbrain neurons. In acute striatal slices, MPP+ exposure gradually decreased stimulation-dependent DA release, followed by massive DA efflux that was dependent on MPP+ concentration, temperature, and DA uptake transporter activity. Similarly, in mouse midbrain neuronal cultures, MPP+ depleted vesicular DA storage accompanied by an elevation of cytosolic and extracellular DA levels. In neuronal cell bodies, increased DAcyt was not due to transmitter leakage from synaptic vesicles but rather to competitive MPP+-dependent inhibition of monoamine oxidase activity. Accordingly, monoamine oxidase blockers pargyline and l-deprenyl had no effect on DAcyt levels in MPP+-treated cells and produced only a moderate effect on the survival of dopaminergic neurons treated with the toxin. In contrast, depletion of intracellular DA by blocking neurotransmitter synthesis resulted in ∼30% reduction of MPP+-mediated toxicity, whereas overexpression of VMAT2 completely rescued dopaminergic neurons. These results demonstrate the utility of comprehensive analysis of DA metabolism using various electrochemical methods and reveal the complexity of the effects of MPP+ on neuronal DA homeostasis and neurotoxicity.

Importance of GluA1 Subunit-Containing AMPA Glutamate Receptors for Morphine State-Dependency

In state-dependency, information retrieval is most efficient when the animal is in the same state as it was during the information acquisition. State-dependency has been implicated in a variety of learning and memory processes, but its mechanisms remain to be resolved. Here, mice deficient in AMPA-type glutamate receptor GluA1 subunits were first conditioned to morphine (10 or 20 mg/kg s.c. during eight sessions over four days) using an unbiased procedure, followed by testing for conditioned place preference at morphine states that were the same as or different from the one the mice were conditioned to. In GluA1 wildtype littermate mice the same-state morphine dose produced the greatest expression of place preference, while in the knockout mice no place preference was then detected. Both wildtype and knockout mice expressed moderate morphine-induced place preference when not at the morphine state (saline treatment at the test); in this case, place preference was weaker than that in the same-state test in wildtype mice. No correlation between place preference scores and locomotor activity during testing was found. Additionally, as compared to the controls, the knockout mice showed unchanged sensitization to morphine, morphine drug discrimination and brain regional μ-opioid receptor signal transduction at the G-protein level. However, the knockout mice failed to show increased AMPA/NMDA receptor current ratios in the ventral tegmental area dopamine neurons of midbrain slices after a single injection of morphine (10 mg/kg, s.c., sliced prepared 24 h afterwards), in contrast to the wildtype mice. The results indicate impaired drug-induced state-dependency in GluA1 knockout mice, correlating with impaired opioid-induced glutamate receptor neuroplasticity.

Diazepam-induced neuronal plasticity attenuates locomotor responses to morphine and amphetamine challenges in mice

A single administration of benzodiazepine-site ligands of the inhibitory GABA(A) receptors has been shown to lead to persistently potentiated AMPA receptor-mediated responses in dopaminergic neurons of the ventral tegmental area (VTA). This plasticity has been suggested to be a common property of different kinds of addictive drugs. We now wanted to test if the plasticity induced by diazepam would also affect behaviors elicited by other drugs of abuse. Activity and plasticity of the VTA dopaminergic neurons are known to be essential for the initiation and/or sensitization of the psychomotor responses to morphine and amphetamine. The effect of diazepam pre-treatment (a single dose of 5 mg/kg) was studied 24-72 h later in behaving C57BL/6J mice on locomotor activity induced by acute and repeated administration of morphine (5 mg/kg) and amphetamine (2.5 mg/kg). The pre-treatment attenuated the locomotor-activating effect of morphine. On the other hand, it reduced the amphetamine-induced locomotor sensitization in male mice in N-methyl-d-aspartate (NMDA) receptor-dependent manner. The acute amphetamine effect was not affected. The results indicate that benzodiazepine-induced neural plasticity transiently reduces the sensitivity to psychomotor stimulation by opioids and stimulants.

GABAA receptor drugs and neuronal plasticity in reward and aversion: focus on the ventral tegmental area

GABAA receptors are the main fast inhibitory neurotransmitter receptors in the mammalian brain, and targets for many clinically important drugs widely used in the treatment of anxiety disorders, insomnia and in anesthesia. Nonetheless, there are significant risks associated with the long-term use of these drugs particularly related to development of tolerance and addiction. Addictive mechanisms of GABAA receptor drugs are poorly known, but recent findings suggest that those drugs may induce aberrant neuroadaptations in the brain reward circuitry. Recently, benzodiazepines, acting on synaptic GABAA receptors, and modulators of extrasynaptic GABAA receptors (THIP and neurosteroids) have been found to induce plasticity in the ventral tegmental area (VTA) dopamine neurons and their main target projections. Furthermore, depending whether synaptic or extrasynaptic GABAA receptor populations are activated, the behavioral outcome of repeated administration seems to correlate with rewarding or aversive behavioral responses, respectively. The VTA dopamine neurons project to forebrain centers such as the nucleus accumbens and medial prefrontal cortex, and receive afferent projections from these brain regions and especially from the extended amygdala and lateral habenula, forming the major part of the reward and aversion circuitry. Both synaptic and extrasynaptic GABAA drugs inhibit the VTA GABAergic interneurons, thus activating the VTA DA neurons by disinhibition and this way inducing glutamatergic synaptic plasticity. However, the GABAA drugs failed to alter synaptic spine numbers as studied from Golgi-Cox-stained VTA dendrites. Since the GABAergic drugs are known to depress the brain metabolism and gene expression, their likely way of inducing neuroplasticity in mature neurons is by disinhibiting the principal neurons, which remains to be rigorously tested for a number of clinically important anxiolytics, sedatives and anesthetics in different parts of the circuitry.

Dampened amphetamine-stimulated behavior and altered dopamine transporter function in the absence of brain GDNF

Midbrain dopamine neuron dysfunction contributes to various psychiatric and neurological diseases, including drug addiction and Parkinsons disease. Because of its well established dopaminotrophic effects, the therapeutic potential of glial cell line-derived neurotrophic factor (GDNF) has been studied extensively in various disorders with disturbed dopamine homeostasis. However, the outcomes from preclinical and clinical studies vary, highlighting a need for a better understanding of the physiological role of GDNF on striatal dopaminergic function. Nevertheless, the current lack of appropriate animal models has limited this understanding. Therefore, we have generated novel mouse models to study conditional Gdnf deletion in the CNS during embryonic development and reduction of striatal GDNF levels in adult mice via AAV-Cre delivery. We found that both of these mice have reduced amphetamine-induced locomotor response and striatal dopamine efflux. Embryonic GDNF deletion in the CNS did not affect striatal dopamine levels or dopamine release, but dopamine reuptake was increased due to increased levels of both total and synaptic membrane-associated dopamine transporters. Collectively, these results suggest that endogenous GDNF plays an important role in regulating the function of dopamine transporters in the striatum. SIGNIFICANCE STATEMENT Delivery of ectopic glial cell line-derived neurotrophic factor (GDNF) promotes the function, plasticity, and survival of midbrain dopaminergic neurons, the dysfunction of which contributes to various neurological and psychiatric diseases. However, how the deletion or reduction of GDNF in the CNS affects the function of dopaminergic neurons has remained unknown. Using conditional Gdnf knock-out mice, we found that endogenous GDNF affects striatal dopamine homeostasis and regulates amphetamine-induced behaviors by regulating the level and function of dopamine transporters. These data regarding the physiological role of GDNF are relevant in the context of neurological and neurodegenerative diseases that involve changes in dopamine transporter function.

Prolyl Oligopeptidase Regulates Dopamine Transporter Phosphorylation in the Nigrostriatal Pathway of Mouse

Alpha-synuclein is the main component of Lewy bodies, a histopathological finding of Parkinson’s disease. Prolyl oligopeptidase (PREP) is a serine protease that binds to α-synuclein and accelerates its aggregation in vitro. PREP enzyme inhibitors have been shown to block the α-synuclein aggregation process in vitro and in cellular models, and also to enhance the clearance of α-synuclein aggregates in transgenic mouse models. Moreover, PREP inhibitors have induced alterations in dopamine and metabolite levels, and dopamine transporter immunoreactivity in the nigrostriatal tissue. In this study, we characterized the role of PREP in the nigrostriatal dopaminergic and GABAergic systems of wild-type C57Bl/6 and PREP knockout mice, and the effects of PREP overexpression on these systems. Extracellular concentrations of dopamine and protein levels of phosphorylated dopamine transporter were increased and dopamine reuptake was decreased in the striatum of PREP knockout mice, suggesting increased internalization of dopamine transporter from the presynaptic membrane. Furthermore, PREP overexpression increased the level of dopamine transporters in the nigrostriatal tissue but decreased phosphorylated dopamine transporters in the striatum in wild-type mice. Our results suggest that PREP regulates the function of dopamine transporter, possibly by controlling the phosphorylation and transport of dopamine transporter into the striatum or synaptic membrane.