Substantia Nigra Pars Reticulata Projections to the Pedunculopontine Nucleus Modulate Dyskinesia.

BACKGROUND: Long-term use of levodopa for Parkinson's disease (PD) treatment is often hindered by development of motor complications, including levodopa-induced dyskinesia (LID). The substantia nigra pars reticulata (SNr) and globus pallidus internal segment (GPi) are the output nuclei of the basal ganglia. Dysregulation of SNr and GPi activity contributes to PD pathophysiology and LID. OBJECTIVE: The objective of this study was to determine whether direct modulation of SNr GABAergic neurons and SNr projections to the pedunculopontine nucleus (PPN) regulates PD symptoms and LID in a mouse model. METHODS: We expressed Cre-recombinase activated channelrhodopsin-2 (ChR2) or halorhodopsin adeno-associated virus-2 (AAV2) vectors selectively in SNr GABAergic neurons of Vgat-IRES-Cre mice in a 6-hydroxydopamine model of PD to investigate whether direct optogenetic modulation of SNr neurons or their projections to the PPN regulates PD symptoms and LID expression. The forepaw stepping task, mouse LID rating scale, and open-field locomotion were used to assess akinesia and LID to test the effect of SNr modulation. RESULTS: Akinesia was improved by suppressing SNr neuron activity with halorhodopsin. LID was significantly reduced by increasing SNr neuronal activity with ChR2, which did not interfere with the antiakinetic effect of levodopa. Optical stimulation of ChR2 in SNr projections to the PPN recapitulated direct SNr stimulation. CONCLUSIONS: Modulation of SNr GABAergic neurons alters akinesia and LID expression in a manner consistent with the rate model of basal ganglia circuitry. Moreover, the projections from SNr to PPN likely mediate the antidyskinetic effect of increasing SNr neuronal activity, identifying a potential novel role for the PPN in LID. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Phase-adaptive brain stimulation of striatal D1 medium spiny neurons in dopamine-depleted mice.

Brain rhythms are strongly linked with behavior, and abnormal rhythms can signify pathophysiology. For instance, the basal ganglia exhibit a wide range of low-frequency oscillations during movement, but pathological "beta" rhythms at ~ 20 Hz have been observed in Parkinson's disease (PD) and in PD animal models. All brain rhythms have a frequency, which describes how often they oscillate, and a phase, which describes the precise time that peaks and troughs of brain rhythms occur. Although frequency has been extensively studied, the relevance of phase is unknown, in part because it is difficult to causally manipulate the instantaneous phase of ongoing brain rhythms. Here, we developed a phase-adaptive, real-time, closed-loop algorithm to deliver optogenetic stimulation at a specific phase with millisecond latency. We combined this Phase-Adaptive Brain STimulation (PABST) approach with cell-type-specific optogenetic methods to stimulate basal ganglia networks in dopamine-depleted mice that model motor aspects of human PD. We focused on striatal medium spiny neurons expressing D1-type dopamine receptors because these neurons can facilitate movement. We report three main results. First, we found that our approach delivered PABST within system latencies of 13 ms. Second, we report that closed-loop stimulation powerfully influenced the spike-field coherence of local brain rhythms within the dorsal striatum. Finally, we found that both 4 Hz PABST and 20 Hz PABST improved movement speed, but we found differences between phase only with 4 Hz PABST. These data provide causal evidence that phase is relevant for brain stimulation, which will allow for more precise, targeted, and individualized brain stimulation. Our findings are applicable to a broad range of preclinical brain stimulation approaches and could also inform circuit-specific neuromodulation treatments for human brain disease.

Linear Predictive Approaches Separate Field Potentials in Animal Model of Parkinson's Disease.

Parkinson's disease (PD) causes impaired movement and cognition. PD can involve profound changes in cortical and subcortical brain activity as measured by electroencephalography or intracranial recordings of local field potentials (LFP). Such signals can adaptively guide deep-brain stimulation (DBS) as part of PD therapy. However, adaptive DBS requires the identification of triggers of neuronal activity dependent on real time monitoring and analysis. Current methods do not always identify PD-related signals and can entail delays. We test an alternative approach based on linear predictive coding (LPC), which fits autoregressive (AR) models to time-series data. Parameters of these AR models can be calculated by fast algorithms in real time. We compare LFPs from the striatum in an animal model of PD with dopamine depletion in the absence and presence of the dopamine precursor levodopa, which is used to treat motor symptoms of PD. We show that in dopamine-depleted mice a first order AR model characterized by a single LPC parameter obtained by LFP sampling at 1 kHz for just 1 min can distinguish between levodopa-treated and saline-treated mice and outperform current methods. This suggests that LPC may be useful in online analysis of neuronal signals to guide DBS in real time and could contribute to DBS-based treatment of PD.

Age-dependent nigral dopaminergic neurodegeneration and α-synuclein accumulation in RGS6-deficient mice.

Parkinson's is primarily a non-familial, age-related disorder caused by α-synuclein accumulation and the progressive loss of dopamine neurons in the substantia nigra pars compacta (SNc). G protein-coupled receptor (GPCR)-cAMP signaling has been linked to a reduction in human Parkinson's incidence and α-synuclein expression. Neuronal cAMP levels are controlled by GPCRs coupled to Gs or Gi/o, which increase or decrease cAMP, respectively. Regulator of G protein signaling 6 (RGS6) powerfully inhibits Gi/o signaling. Therefore, we hypothesized that RGS6 suppresses D2 autoreceptor- Gi/o signaling in SNc dopamine neurons promoting neuronal survival and reducing α-synuclein expression. Here we provide novel evidence that RGS6 critically suppresses late-age-onset SNc dopamine neuron loss and α-synuclein accumulation. RGS6 is restrictively expressed in human SNc dopamine neurons and, despite their loss in Parkinson's, all surviving neurons express RGS6. RGS6-/- mice exhibit hyperactive D2 autoreceptors with reduced cAMP signaling in SNc dopamine neurons. Importantly, RGS6-/- mice recapitulate key sporadic Parkinson's hallmarks, including: SNc dopamine neuron loss, reduced nigrostriatal dopamine, motor deficits, and α-synuclein accumulation. To our knowledge, Rgs6 is the only gene whose loss phenocopies these features of human Parkinson's. Therefore, RGS6 is a key regulator of D2R-Gi/o signaling in SNc dopamine neurons, protecting against Parkinson's neurodegeneration and α-synuclein accumulation.

Dorsal Raphe Serotonin Neurons Mediate CO2-Induced Arousal from Sleep.

Arousal from sleep in response to CO2 is a critical protective phenomenon. Dysregulation of CO2-induced arousal contributes to morbidity and mortality from prevalent diseases, such as obstructive sleep apnea and sudden infant death syndrome. Despite the critical nature of this protective reflex, the precise mechanism for CO2-induced arousal is unknown. Because CO2 is a major regulator of breathing, prevailing theories suggest that activation of respiratory chemo- and mechano-sensors is required for CO2-induced arousal. However, populations of neurons that are not involved in the regulation of breathing are also chemosensitive. Among these are serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) that comprise a component of the ascending arousal system. We hypothesized that direct stimulation of these neurons with CO2 could cause arousal from sleep independently of enhancing breathing. Dialysis of CO2-rich acidified solution into DRN, but not medullary raphe responsible for modulating breathing, caused arousal from sleep. Arousal was lost in mice with a genetic absence of 5-HT neurons, and with acute pharmacological or optogenetic inactivation of DRN 5-HT neurons. Here we demonstrate that CO2 can cause arousal from sleep directly, without requiring enhancement of breathing, and that chemosensitive 5-HT neurons in the DRN critically mediate this arousal. Better understanding mechanisms underlying this protective reflex may lead to interventions to reduce disease-associated morbidity and mortality.SIGNIFICANCE STATEMENT Although CO2-induced arousal is critical to a number of diseases, the specific mechanism is not well understood. We previously demonstrated that serotonin (5-HT) neurons are important for CO2-induced arousal, as mice without 5-HT neurons do not arouse to CO2 Many have interpreted this to mean that medullary 5-HT neurons that regulate breathing are important in this arousal mechanism. Here we found that direct application of CO2-rich aCSF to the dorsal raphe nucleus, but not the medullary raphe, causes arousal from sleep, and that this arousal was lost with genetic ablation or acute inhibition of 5-HT neurons. We propose that 5-HT neurons in the dorsal raphe nucleus can be activated directly by CO2 to cause arousal independently of respiratory activation.

Optogenetic Stimulation of Frontal D1 Neurons Compensates for Impaired Temporal Control of Action in Dopamine-Depleted Mice.

Disrupted mesocortical dopamine contributes to cognitive symptoms of Parkinson's disease (PD). Past work has implicated medial frontal neurons expressing D1 dopamine receptors (D1DRs) in temporal processing. Here, we investigated whether these neurons can compensate for behavioral deficits resulting from midbrain dopamine dysfunction. We report three main results. First, both PD patients and mice with ventral tegmental area (VTA) dopamine depletion had attenuated delta activity (1-4 Hz) in the medial frontal cortex (MFC) during interval timing. Second, we found that optogenetically stimulating MFC D1DR neurons could increase ramping activity among MFC neurons. Finally, stimulating MFC D1DR neurons specifically at delta frequencies (2 Hz) compensated for deficits in temporal control of action caused by VTA dopamine depletion. Our results suggest that cortical networks can be targeted by frequency-specific brain stimulation to improve dopamine-dependent cognitive processing.

Axial levodopa-induced dyskinesias and neuronal activity in the dorsal striatum.

Levodopa-induced dyskinesias are abnormal involuntary movements that limit the effectiveness of treatments for Parkinson's disease. Although dyskinesias involve the striatum, it is unclear how striatal neurons are involved in dyskinetic movements. Here we record from striatal neurons in mice during levodopa-induced axial dyskinesias. We developed an automated 3-dimensional motion tracking system to capture the development of axial dyskinesias at ∼10ms resolution, and correlated these movements with neuronal activity of striatal medium spiny neurons and fast-spiking interneurons. The average firing rate of medium spiny neurons increased as axial dyskinesias developed, and both medium spiny neurons and fast-spiking interneurons were modulated around axial dyskinesias. We also found that delta field potential power increased in the striatum with dyskinesia, and that this increased delta power coupled with striatal neurons. Our findings provide insight into how striatal networks change as levodopa-induced dyskinesias develop, and suggest that increased medium spiny neuron firing, increased delta field potential power, and abnormal delta-coupling may be neurophysiological signatures of dyskinesias. These data could be helpful in understanding the role of the striatum in the pathogenesis of dyskinesias in Parkinson's disease.

Optogenetic approaches to evaluate striatal function in animal models of Parkinson disease.

Optogenetics refers to the ability to control cells that have been genetically modified to express light-sensitive ion channels. The introduction of optogenetic approaches has facilitated the dissection of neural circuits. Optogenetics allows for the precise stimulation and inhibition of specific sets of neurons and their projections with fine temporal specificity. These techniques are ideally suited to investigating neural circuitry underlying motor and cognitive dysfunction in animal models of human disease. Here, we focus on how optogenetics has been used over the last decade to probe striatal circuits that are involved in Parkinson disease, a neurodegenerative condition involving motor and cognitive abnormalities resulting from degeneration of midbrain dopaminergic neurons. The precise mechanisms underlying the striatal contribution to both cognitive and motor dysfunction in Parkinson disease are unknown. Although optogenetic approaches are somewhat removed from clinical use, insight from these studies can help identify novel therapeutic targets and may inspire new treatments for Parkinson disease. Elucidating how neuronal and behavioral functions are influenced and potentially rescued by optogenetic manipulation in animal models could prove to be translatable to humans. These insights can be used to guide future brain-stimulation approaches for motor and cognitive abnormalities in Parkinson disease and other neuropsychiatric diseases.

La optogenética se refiere a la capacidad de controlar células que han sido modificadas genéticamente para expresar canales iónicos sensibles a la luz. La introducción de las estrategias optogenéticas ha facilitado la disección de los circuitos neurales. La optogenética permite precisar la estimulación e inhibición de conjuntos específicos de neuronas y sus proyecciones con una alta especificidad temporal. Estas técnicas idealmente están adaptadas para investigar los circuitos neurales que subyacen a la disfunción motora y cognitiva en modelos animales de la enfermedad humana. Este artículo se enfoca en cómo se ha empleado la optogenética durante la última década para explorar los circuitos neurales que están involucrados en la Enfermedad de Parkinson, una condición neurodegenerativa que incluye alteraciones motoras y cognitivas resultantes de la degeneración de neuronas dopaminérgicas del mesencéfalo. Aunque las estrategias optogenéticas están algo alejadas del empleo clínico, el conocimiento a partir de estos estudios puede ayudar a identificar nuevos blancos terapéuticos y puede inspirar nuevos tratamientos para la Enfermedad de Parkinson. El esclarecer cómo las mediciones neurales y conductuales son influenciadas y potencialmente recuperadas por la manipulación optogenética podría llegar a ser traducible a los humanos. Estos conocimientos pueden ser empleados para guiar futuras estrategias de estimulación cerebral para anormalidades motoras y cognitivas en la Enfermedad de Parkinson y otras enfermedades neuropsiquiátricas.

L'optogénétique est une méthode permettant de contrôler des cellules qui ont été préalablement génétiquement modifiées pour exprimer des canaux ioniques sensibles à la lumière. Son utilisation a ouvert la voie à l'analyse des circuits neuronaux car elle permet la stimulation et l'inhibition précises de groupes spécifiques de neurones et de leurs projections avec une excellente spécificité temporale. Ces techniques sont parfaitement adaptées à l'examen des circuits neuronaux sous-tendant une dysfonction motrice et cognitive dans des modèles animaux de pathologies humaines. Cet article met l'accent sur la façon dont l'optogénétique a été utilisée ces 10 dernières années pour examiner les circuits striataux impliqués dans la maladie de Parkinson, une maladie neurodégénérative dont les troubles moteurs et cognitifs résultent d'une dégénérescence des neurones dopaminergiques du mésencéphale. Les mécanismes précis sous-tendant la contribution du striatum au dysfonctionnement moteur et cognitif de la maladie de Parkinson sont encore méconnus. Bien que l'optogénétique soit quelque peu éloignée de l'usage clinique, les connaissances issues de ces études peuvent aider à identifier de nouvelles cibles thérapeutiques et suggérer de nouveaux traitements pour la maladie de Parkinson. Une fois élucidés, les mécanismes par lesquels les manipulations optogénétiques peuvent influencer et potentiellement restaurer les fonctions neuronales et comportementales pourraient être transposés chez l'homme. Ces connaissances pourraient alors être utilisées pour mener de futures stratégies de stimulation cérébrale dans les anomalies motrices et cognitives de la maladie de Parkinson et d'autres maladies neuropsychiatriques.

The Vulnerable Ventral Tegmental Area in Parkinson's Disease.

INTRODUCTION: The involvement of dopaminergic neurons in the ventral tegmental area (VTA) in Parkinson's disease (PD) has not been universally recognized by neuroscientists and neurologists. Here, we conduct a review of previous research documenting dopaminergic neuronal loss in both the substantia nigra pars compacta (SNpc) and VTA and add three new post-mortem PD cases to the literature. METHODS: PD and control brains were sectioned, stained for tyrosine hydroxylase, and cells in the SNpc and VTA were counted. RESULTS: Based on the review, we report two main results: 1) the VTA does degenerate in PD, and 2) the VTA degenerates less than the SNpc. CONCLUSION: Inconsistent clinical information about these cases limits our ability to interpret how the VTA contributes to PD symptoms. However, our data in combination with prior PD neuropathological cases in the literature unequivocally establish that the VTA is involved in PD, and could be relevant for future investigation of non-motor symptoms in PD.

Organic light emitting diode improves diabetic cutaneous wound healing in rats.

A major complication for diabetic patients is chronic wounds due to impaired wound healing. It is well documented that visible red wavelengths can accelerate wound healing in diabetic animal models and patients. In vitro and in vivo diabetic models were used to investigate the effects of organic light emitting diode (OLED) irradiation on cellular function and cutaneous wound healing. Human dermal fibroblasts were cultured in hyperglycemic medium (glucose concentration 180 mM) and irradiated with an OLED (623 nm wavelength peak, range from 560 to 770 nm, power density 7 or 10 mW/cm2 at 0.2, 1, or 5 J/cm2). The OLED significantly increased total adenosine triphosphate concentration, metabolic activity, and cell proliferation compared with untreated controls in most parameters tested. For the in vivo experiment, OLED and laser (635 ± 5 nm wavelength) treatments (10 mW/cm2 , 5 J/cm2 daily for a total of seven consecutive days) for cutaneous wound healing were compared using a genetic, diabetic rat model. Both treatments had significantly higher percentage of wound closure on day 6 postinjury and higher total histological scores on day 13 postinjury compared with control. No statistical difference was found between the two treatments. OLED irradiation significantly increased fibroblast growth factor-2 expression at 36-hour postinjury and enhanced macrophage activation during initial stages of wound healing. In conclusion, the OLED and laser had comparative effects on enhancing diabetic wound healing.

New therapeutic strategies targeting D1-type dopamine receptors for neuropsychiatric disease.

The neurotransmitter dopamine acts via two major classes of receptors, D1-type and D2-type. D1 receptors are highly expressed in the striatum and can also be found in the cerebral cortex. Here we review the role of D1 dopamine signaling in two major domains: L-DOPA-induced dyskinesias in Parkinson's disease and cognition in neuropsychiatric disorders. While there are many drugs targeting D2-type receptors, there are no drugs that specifically target D1 receptors. It has been difficult to use selective D1-receptor agonists for clinical applications due to issues with bioavailability, binding affinity, pharmacological kinetics, and side effects. We propose potential therapies that selectively modulate D1 dopamine signaling by targeting second messengers downstream of D1 receptors, allosteric modulators, or by making targeted modifications to D1-receptor machinery. The development of therapies specific to D1-receptor signaling could be a new frontier in the treatment of neurological and psychiatric disorders.

In vitro and in vivo optimization of infrared laser treatment for injured peripheral nerves.

BACKGROUND AND OBJECTIVE: Repair of peripheral nerve injuries remains a major challenge in restorative medicine. Effective therapies that can be used in conjunction with surgical nerve repair to improve nerve regeneration and functional recovery are being actively investigated. It has been demonstrated by a number of peer reviewed publications that photobiomodulation (PBM) supports nerve regeneration, reinnervation of the denervated muscle, and functional recovery after peripheral nerve injury. However, a key issue in the use of PBM as a treatment for peripheral nerve injury is the lack of parameter optimization for any given wavelength. The objective of this study was to demonstrate that for a selected wavelength effective in vitro dosing parameters could be translated to effective in vivo parameters. MATERIALS AND METHODS: Comparison of infra-red (810 and 980 nm wavelengths) laser treatment parameters for injured peripheral nerves was done beginning with a series of in vitro experiments using primary human fibroblasts and primary rat cortical neurons. The primary rat cortical neurons were used for further optimization of energy density for 980 nm wavelength light using measurement of total neurite length as the bioassay. For these experiments, the parameters included a 1 W output power, power density of 10 mW/cm(2) , and energy densities of 0.01, 0.1, 0.5, 2, 10, 50, 200, 1,000, and 5,000 mJ/cm(2) . For translation of the in vitro data for use in vivo it was necessary to determine the transcutaneous penetration of 980 nm wavelength light to the level of the peroneal nerve. Two anesthetized, male White New Zealand rabbits were used for these experiments. The output power of the laser was set at 1.0 or 4.0 W. Power density measurements were taken at the surface of the skin, sub-dermally, and at the level of the nerve. Laser parameters used in the in vivo studies were calculated based on data from the in vitro studies and the light penetration measurements. For the in vivo experiments, a total of 22 White New Zealand rabbits (2.34-2.89 kg) were used. Translated dosing parameters were refined in a pilot study using a transection model of the peroneal nerve in rabbits. Output powers of 2 and 4 W were tested. For the final set of in vivo experiments, the same transection nerve injury model was used. An energy density of 10 mW/cm(2) at the level of the peroneal nerve was selected and the laser parameters were further refined. The dosing parameters used were: 1.5 W output power, 43 seconds exposure, 8 cm(2) area and a total energy of 65 J. RESULTS: In vitro, 980 nm wavelength light at 10 mW/cm(2) significantly improved neurite elongation at energy densities between 2 and 200 mJ/cm(2) . In vivo penetration of the infrared light measured in anesthetized rabbits showed that on average, 2.45% of the light applied to the skin reached the depth of the peroneal nerve. The in vivo pilot study data revealed that the 4 W parameters inhibited nerve regeneration while the 2 W parameters significantly improved axonal regrowth. For the final set of experiments, the irradiated group performed significantly better in the toe spread reflex test compared to the control group from week 7 post-injury, and the average length of motor endplates returned to uninjured levels. CONCLUSION: The results of this study demonstrate that treatment parameters can be determined initially using in vitro models and then translated to in vivo research and clinical practice. Furthermore, this study establishes that infrared light with optimized parameters promotes accelerated nerve regeneration and improved functional recovery in a surgically repaired peripheral nerve.

Pulsed light irradiation improves behavioral outcome in a rat model of chronic mild stress.

BACKGROUND AND OBJECTIVE: Transcranial laser therapy (TLT) has been used successfully for the treatment of stroke in animal models and clinical trials. These results support the hypothesis that TLT could be used to treat other central nervous system conditions, such as depression. Current therapy for depression emphasizes pharmaco-therapeutics. However, these interventions often cause unwanted side effects. Here, TLT as a treatment for depression was studied in a rat model of chronic mild stress (CMS). STUDY DESIGN/MATERIAL AND METHODS: Wistar rats were randomized into four experimental groups (n = 8): (1) No-stress; (2) stress without treatment (Stress); (3) stress treated with an antidepressant (Drug); and (4) stress treated with TLT (TLT). The rats in the stress groups were exposed sequentially to a variety of mild stressors for 8 weeks. Rats were weighed weekly. After 5 weeks of stressing, the Drug group received a daily injection of fluoxetine (10 mg/kg), and the TLT group was irradiated transcranially 3 times a week (810 nm wavelength laser, 3 mm diameter probe, 350 mW peak power, 100 Hz with 20% duty cycle, 2-minute treatment time, 120 J/cm(2) average energy density on skin surface). After 3 weeks of treatment, a forced swimming test (FST) was performed and recorded for behavioral assessment. Animals were euthanized after 8 weeks of the study. RESULTS: The No-stress group had significantly higher body weight than stress groups from week 5 (P < 0.05). No weight difference was found between the stress groups before treatment. However, the Drug group had significantly less body weight than both Stress and TLT groups after 2 weeks of treatment (P < 0.05). FST showed that the Stress group had significantly more immobility than the No-stress group (P < 0.05). Both Drug and TLT groups had significantly less immobility than the stress group (P < 0.05). There was no significant difference in immobility between both Drug and TLT groups (P = 0.62). CONCLUSIONS: TLT was comparable to fluoxetine in improving the behavioral outcome after CMS. TLT did not cause weight loss, which is consistently seen in patients treated with fluoxetine. This study demonstrates that TLT has potential as an effective treatment for depression.