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Background The fimbria/fornix fiber system is an essential part of the hippocampal-VTA loop, and therefore activities that are propagated through this fiber system control the activity of the mesolimbic dopamine system. Objectives/hypothesis We hypothesized that stimulation of the fimbria/fornix with an increasing number of electrical pulses would cause increasing activity of the mesolimbic dopamine system, which coincides with concurrent changes in neuronal activities in target regions of the mesolimbic dopaminergic system. Methods Right fimbria/fornix fibers were electrically stimulated with different pulse protocols. Stimulus-induced changes in neuronal activities were visualized with BOLD-fMRI, whereas stimulus-induced release of dopamine, as measured for the activity of the mesolimbic dopamine system, was determined in the nucleus accumbens with in vivo fast-scan cyclic voltammetry. Results Dependent on the protocol, electrical fimbria/fornix stimulation caused BOLD responses in various targets of the mesolimbic dopamine system. Stimulation in the low theta frequency range (5 Hz) triggered significant BOLD responses mainly in the hippocampal formation, infralimbic cortex, and septum. Stimulation in the beta frequency range (20 Hz) caused additional activation in the medial prefrontal cortex (mPFC), nucleus accumbens, striatum, and VTA. Stimulation in the high-gamma frequency range (100 Hz) caused further activation in the hippocampus proper and mPFC. The strong activation in the mPFC during 100 Hz stimulations depended not only on the number of pulses but also on the frequency. Thus, short bursts of 5 or 20 high-frequency pulses caused stronger activation in the mPFC than continuous 5 or 20 Hz pulses. In contrast, high-frequency burst fimbria/fornix stimulation did not further activate the mesolimbic dopamine system when compared to continuous 5 or 20 Hz pulse stimulation. Conclusions There exists a frequency-dependent dissociation between BOLD responses and activation of the dopaminergic system. Low frequencies were more efficient to activate the mesolimbic dopamine system, whereas high frequencies were more efficient to trigger BOLD responses in target regions of the mesolimbic dopamine system, particularly the mPFC.

HIGHLIGHTS: eOPN3 is a mosquito-derived rhodopsin that inhibits neurotransmission in neurons. Activation of eOPN3 activates the G(i/o) pathway and reduces Ca(2+) channel activity; eOPN3 can suppress neurotransmission in a variety of cell types in vitro and in vivo. Activation of eOPN3 in nigrostriatal dopamine axons modulates locomotor behavior. SUMMARY: Information is carried between brain regions through neurotransmitter release from axonal presynaptic terminals. Understanding the functional roles of defined neuronal projection pathways requires temporally precise manipulation of their activity. However, existing inhibitory optogenetic tools have low efficacy and off-target effects when applied to presynaptic terminals, while chemogenetic tools are difficult to control in space and time. Here, we show that a targeting-enhanced mosquito homolog of the vertebrate encephalopsin (eOPN3) can effectively suppress synaptic transmission through the G(i/o) signaling pathway. Brief illumination of presynaptic terminals expressing eOPN3 triggers a lasting suppression of synaptic output that recovers spontaneously within minutes in vitro and in vivo. In freely moving mice, eOPN3-mediated suppression of dopaminergic nigrostriatal afferents induces a reversible ipsiversive rotational bias. We conclude that eOPN3 can be used to selectively suppress neurotransmitter release at presynaptic terminals with high spatiotemporal precision, opening new avenues for functional interrogation of long-range neuronal circuits in vivo.

Mapping the activity of the human mesolimbic dopamine system by BOLD-fMRI is a tempting approach to non-invasively study the action of the brain reward system during different experimental conditions. However, the contribution of dopamine release to the BOLD signal is disputed. To assign the actual contribution of dopaminergic and non-dopaminergic VTA neurons to the formation of BOLD responses in target regions of the mesolimbic system, we used two optogenetic approaches in rats. We either activated VTA dopaminergic neurons selectively, or dopaminergic and mainly glutamatergic projecting neurons together. We further used electrical stimulation to non-selectively activate neurons in the VTA. All three stimulation conditions effectively activated the mesolimbic dopaminergic system and triggered dopamine releases into the NAcc as measured by in vivo fast-scan cyclic voltammetry. Furthermore, both optogenetic stimulation paradigms led to indistinguishable self-stimulation behavior. In contrast to these similarities, however, the BOLD response pattern differed greatly between groups. In general, BOLD responses were weaker and sparser with increasing stimulation specificity for dopaminergic neurons. In addition, repetitive stimulation of the VTA caused a progressive decoupling of dopamine release and BOLD signal strength, and dopamine receptor antagonists were unable to block the BOLD signal elicited by VTA stimulation. To exclude that the sedation during fMRI is the cause of minimal mesolimbic BOLD in response to specific dopaminergic stimulation, we repeated our experiments using CBF SPECT in awake animals. Again, we found activations only for less-specific stimulation. Based on these results we conclude that canonical BOLD responses in the reward system represent mainly the activity of non-dopaminergic neurons. Thus, the minor effects of projecting dopaminergic neurons are concealed by non-dopaminergic activity, a finding which highlights the importance of a careful interpretation of reward-related human fMRI data.

Direct lineage reprogramming through genetic-based strategies enables the conversion of differentiated somatic cells into functional neurons and distinct neuronal subtypes. Induced dopaminergic (iDA) neurons can be generated by direct conversion of skin fibroblasts; however, their in vivo phenotypic and functional properties remain incompletely understood, leaving their impact on Parkinson's disease (PD) cell therapy and modeling uncertain. Here, we determined that iDA neurons retain a transgene-independent stable phenotype in culture and in animal models. Furthermore, transplanted iDA neurons functionally integrated into host neuronal tissue, exhibiting electrically excitable membranes, synaptic currents, dopamine release, and substantial reduction of motor symptoms in a PD animal model. Neuronal cell replacement approaches will benefit from a system that allows the activity of transplanted neurons to be controlled remotely and enables modulation depending on the physiological needs of the recipient; therefore, we adapted a DREADD (designer receptor exclusively activated by designer drug) technology for remote and real-time control of grafted iDA neuronal activity in living animals. Remote DREADD-dependent iDA neuron activation markedly enhanced the beneficial effects in transplanted PD animals. These data suggest that iDA neurons have therapeutic potential as a cell replacement approach for PD and highlight the applicability of pharmacogenetics for enhancing cellular signaling in reprogrammed cell-based approaches.

Mitochondrial defects have been shown to be associated with the pathogenesis of Parkinson's disease (PD). Yet, experience in PD research linking mitochondrial dysfunction, e.g., deregulation of oxidative phosphorylation, with neuronal degeneration and behavioral changes is rather limited. Using the 6-hydroxydopamine (6-OHDA) rat model of PD, we have investigated the potential role of mitochondria in dopaminergic neuronal cell death in the substantia nigra pars compacta by high-resolution respirometry. Mitochondrial function was correlated with the time course of disease-related motor behavior asymmetry and dopaminergic neuronal cell loss, respectively. Unilateral 6-OHDA injections (>2.5 μg/2 μl) into the median forebrain bundle induced an impairment of oxidative phosphorylation due to a decrease in complex I activity. This was indicated by increased flux control coefficient. During the period of days 2-21, a progressive decrease in respiratory control ratio of up to -58 % was observed in the lesioned compared to the non-lesioned substantia nigra of the same animals. This decrease was associated with a marked uncoupling of oxidative phosphorylation. Mitochondrial dysfunction, motor behavior asymmetry, and dopaminergic neuronal cell loss correlated with dosage (1.25-5 μg/2 μl). We conclude that high-resolution respirometry may allow the detection of distinct mitochondrial dysfunction as a suitable surrogate marker for the preclinical assessment of potential neuroprotective strategies in the 6-OHDA model of PD.

Background Since the mesocortical dopaminergic system of rodents has several differences to that found in primate species, including humans, there is the need for more exhaustively studying causative relationships between activation/stimulation of the ventral tegmental area (VTA) and substantia nigra (SN) and behavior in monkeys. Objective To gain causative relationships between VTA/SN stimulation and behavior, we investigated whether monkeys perform audiovisual (AV) tasks using brain stimulation reward (BSR) as the reinforcer, and how reward intensity affects performance during self-stimulation. Methods Monkeys were required to touch a bar freely when self-stimulating or when instructed by an AV stimulus, to receive BSR. Results We were able to train monkeys to successfully perform the AV task for BSR within three days. Self-stimulation revealed an increase in the bar touch rate when using higher electrical currents, with no ceiling effects observed. During a training session the touch rate decreased, often before the monkeys had received 1000 deliveries of BSR, suggesting satiation. Conclusions When BSR is applied directly to the VTA/SN, it can motivate monkeys to perform detection tasks, exhibit operant actions, and may be used as a substitute for fluid or food rewards. Monkeys ceased self-stimulation during a training session by their own volition, in contrast to work on rodents. This may be an important safety aspect for consideration in the development of electrical stimulation procedures for patients with dysfunctions of the dopaminergic system; thus, satiation may avert additional compulsions to already existing compulsive behaviors in patients.

Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder with a prevalence of about 1% in a population older than 70 years and about 3% in individuals older than 80 years (Strickland and Bertoni 2004). Pathological hallmarks include, but are not confined to, the preferential loss of dopaminergic (DAergic) neurons within the substantia nigra pars compacta and the presence of intracytoplasmic inclusions containing α-synuclein and ubiquitin, so-called Lewy bodies (Braak et al. 2003). Next to the generation of free radicals in the course of dopamine metabolism, the increased presence of iron in the midbrain of PD patients has been assumed as a contributing pathogenic factor (Berg and Hochstrasser 2006). Iron participates in metabolic processes by undergoing oxidation–reduction reactions, a common property among transition metals, which allows this metal to undergo interconversion between the divalent cationic or ferrous (Fe2+), and trivalent cationic or ferric (Fe3+) states. These electron exchange processes can also lead to significant oxidative damage via free radical production within the brain when excess iron is present. In DAergic neurons during the dopamine catabolism, hydrogen peroxide is produced that in presence of iron favors Fenton reactions and oxidative stress [reviewed in (Papanikolaou and Pantopoulos 2005)]. Other transition metals such as Mn or Zn have been suggested to contribute to the pathogenesis of PD. For example, exposure of workers to high concentrations of manganese is an established risk factor for the development of a Parkinsonian syndrome, which clinically greatly mimics idiopathic Parkinson's disease [reviewed in (Guilarte 2010)]. Similar symptoms have been observed in psychostimulant drug abusers after repetitive intravenous injection of manganese-containing substances (Sikk 2011). Excessive levels of brain manganese have been linked to the loss of dopamine in the striatum, death of non-DAergic neurons in the globus pallidus, and damage of other neuronal pathways such as glutamatergic and GABAergic projections, all of which contribute to altered behavior, motor dysfunction, and cognition deficit (Erikson 2002). Manganese elevates intracellular H2O2 and related peroxides (HaMai et al. 2001) and reduces tyrosine hydroxylase activity and intracellular antioxidant levels (GSH, thiols, catalase) in DAergic neurons (Migheli 1999). Iron and manganese are known to pass across the blood–brain barrier [reviewed in (Yokel 2009; Moos 2007)]. Because of their chemical similarity, both metals share and compete for transport proteins in organisms ranging from bacteria to mammals. As such, during conditions of low iron, abnormal manganese accumulation occurs. Conversely, when manganese concentrations are altered, the homeostasis and deposition of iron and other transition metals are disrupted. However, the knowledge on the subcellular allocation of trace elements upon exposure to manganese or iron remains incomplete. Indirect methods, like radioactive 54Mn tracing after cellular extraction, are able to quantify manganese in cell extracts, but provide only limited spatial information (Kalia et al. 2008). The principle of X-ray fluorescence (XRF) is based on the irradiation of sample atoms with X-rays with energy sufficient to eject an electron from one of the atom's inner shells. The relaxation process is accompanied by the emission of a fluorescence photon. The energy of the emitted X-rays is characteristic of the excited element enabling the identification of the composition of the atoms constitutive of the sample. Beside element composition, X-ray spectromicroscopy performed using an X-ray nanoprobe beam is a unique method to determine the oxidation state of elements in cells and tissues (Qin 2011). X-rays can penetrate thick cells and tissues, eliminating the need of invasive preparation and sectioning of the specimen. Several studies have thus recently demonstrated the use of X-rays for trace element mapping of different cell types (Ortega 2007; Bacquart 2007; Ducic 2011). This study was designed to investigate the subcellular distribution of manganese and iron in primary midbrain neurons and to compare this distribution in the DAergic and non-DAergic subpopulations by X-ray fluorescence microscopy. Previous studies have been limited to cell lines, post-mortem tissue, or did not take into account changes in the oxidative state during sample processing (Bacquart 2007; Szczerbowska-Boruchowska 2007). In this study, we visualized the transition metal distribution upon exposure in primary midbrain neurons and used the cryo-preserving (so-called cryo-embedding) technique to preserve oxidative states of the metal ions, which, to the best of our knowledge, is the first description exploiting primary midbrain neurons culture model with X-ray imaging techniques.