My Andersson

Pharmacological validation of behavioural measures of akinesia and dyskinesia in a rat model of Parkinson's disease

In an attempt to define clinically relevant models of akinesia and dyskinesia in 6‐hydroxydopamine (6‐OHDA)‐lesioned rats, we have examined the effects of drugs with high (l‐DOPA) vs. low (bromocriptine) dyskinesiogenic potential in Parkinsons disease on three types of motor performance, namely: (i) abnormal involuntary movements (AIMs) (ii) rotational behaviour, and (iii) spontaneous forelimb use (cylinder test). Rats with unilateral 6‐OHDA lesions received single daily i.p. injections of l‐DOPA or bromocriptine at therapeutic doses. During 3 weeks of treatment, l‐DOPA but not bromocriptine induced increasingly severe AIMs affecting the limb, trunk and orofacial region. Rotational behaviour was induced to a much higher extent by bromocriptine than l‐DOPA. In the cylinder test, the two drugs initially improved the performance of the parkinsonian limb to a similar extent. However, l‐DOPA‐treated animals showed declining levels of performance in this test because the drug‐induced AIMs interfered with physiological limb use, and gradually replaced all normal motor activities. l‐DOPA‐induced axial, limb and orolingual AIM scores were significantly reduced by the acute administration of compounds that have antidyskinetic efficacy in parkinsonian patients and/or nonhuman primates (−91%, yohimbine 10 mg/kg; −19%, naloxone 4–8 mg/kg; −37%, 5‐methoxy 5‐N,N‐dimethyl‐tryptamine 2 mg/kg; −30%, clozapine 8 mg/kg; −50%, amantadine 40 mg/kg). l‐DOPA‐induced rotation was, however, not affected. The present results demonstrate that 6‐OHDA‐lesioned rats do exhibit motor deficits that share essential functional similarities with parkinsonian akinesia or dyskinesia. Such deficits can be quantified using novel and relatively simple testing procedures, whereas rotometry cannot discriminate between dyskinetic and antiakinetic effects of antiparkinsonian treatments.

Striatal fosB expression is causally linked with l-DOPA-induced abnormal involuntary movements and the associated upregulation of striatal prodynorphin mRNA in a rat model of Parkinson's disease.

Rats with unilateral dopamine-denervating lesions sustained a 3-week treatment with a daily l-DOPA dose that is in the therapeutic range for Parkinsons disease. In most of the treated animals, chronic l-DOPA administration gradually induced abnormal involuntary movements affecting cranial, trunk, and limb muscles on the side of the body contralateral to the lesion. This effect was paralleled by an induction of FosB-like immunoreactive proteins in striatal subregions somatotopically related to the types of movements that had been elicited by l-DOPA. The induced proteins showed both regional and cellular colocalization with prodynorphin mRNA. Intrastriatal infusion of fosB antisense inhibited the development of dyskinetic movements that were related to the striatal subregion targeted and produced a local specific downregulation of prodynorphin mRNA. These data provide compelling evidence of a causal role for striatal fosB induction in the development of l-DOPA-induced dyskinesia in the rat and of a positive regulation of prodynorphin gene expression by FosB-related transcription factors.

Persistent changes in striatal gene expression induced by long-term L-DOPA treatment in a rat model of Parkinson's disease

Current knowledge of the molecular changes induced by dopamine denervation and subsequent treatment with l‐DOPA is based on studies performed on relatively acute and young animal models of parkinsonism. It is highly warranted to ask how well these models simulate the state of chronic denervation and sustained l‐DOPA pharmacotherapy which are typical of advanced Parkinsons disease. This study investigates the effects of time postdenervation and l‐dopa treatment duration on the striatal expression of opioid precursor mRNAs and FosB/ΔFosB‐related proteins. Unilaterally 6‐hydroxydopamine‐lesioned rats were treated with therapeutical doses of l‐DOPA for one year (long‐term group) or a few weeks (short‐term group). Age‐matched lesioned rats received injections of vehicle or bromocriptine, an antiparkinsonian compound which does not produce dyskinesia when administered de novo. The lesion‐induced up‐regulation of preproenkephalin mRNA expression persisted at more than one year postlesion, and was unaffected by the pharmacological treatments applied. l‐DOPA, but not bromocriptine, induced high striatal levels of FosB/ΔFosB immunoreactivity and prodynorphin mRNA, and these did not differ between short‐term and long‐term l‐DOPA‐treated rats. The present data provide the first demonstration that l‐DOPA maintains high striatal levels of fosB and prodynorphin gene expression during a prolonged course of treatment, which simulates the clinical practice in Parkinsons disease more closely than the short‐treatment paradigms studied thus far.

Time course of striatal DeltaFosB-like immunoreactivity and prodynorphin mRNA levels after discontinuation of chronic dopaminomimetic treatment.

ΔFosB‐like proteins are particularly stable transcription factors that accumulate in the brain in response to chronic perturbations. In this study we have compared the time‐course of striatal FosB/ΔFosB‐like immunoreactivity and prodynorphin mRNA expression after discontinuation of chronic cocaine treatment to intact rats and chronic L‐DOPA treatment to unilaterally 6‐hydroxydopamine (6‐OHDA) lesioned rats. The animals were killed between 3 h and 16 days after the last drug injection. In both treatment paradigms, the drug‐induced FosB/ΔFosB immunoreactivity remained significantly elevated in the caudate putamen even at the longest withdrawal period examined. The concomitant upregulation of prodynorphin mRNA, a target of ΔFosB, paralleled the time‐course of ΔFosB‐like immunoreactivity in the 6‐OHDA‐lesion/L‐DOPA model, but was more transient in animals treated with cocaine. These results suggest that ΔFosB‐like proteins have exceptional in vivo stability. In the dopamine‐denervated striatum, these proteins may exert sustained effects on the expression of their target genes long after discontinuation of L‐DOPA pharmacotherapy.

Optogenetics reveal delayed afferent synaptogenesis on grafted human induced pluripotent stem cell-derived neural progenitors.

Reprogramming of somatic cells into pluripotency stem cell state has opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human‐induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here, we present a detailed characterization of the functional properties and synaptic integration of hiPSC‐derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSCs), and in adult rats in vivo. The hiPSCs were first differentiated into long‐term self‐renewing neuroepithelial stem (lt‐NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for 6 weeks, lt‐NES cell‐derived neurons displayed neuronal properties such as tetrodotoxin‐sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential, and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt‐NES cell‐derived neurons, the host neurons were transduced with Channelrhodopsin‐2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt‐NES cell‐derived neurons using whole‐cell patch‐clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the lt‐NES cell‐derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt‐NES cell‐derived neurons receive ample afferent input from the host brain. Since the lt‐NES cells used in this study show a strong propensity for GABAergic differentiation, the host‐to‐graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, for example, such as epilepsy. Stem Cells 2014;32:3088–3098

Optogenetic inhibition of chemically induced hypersynchronized bursting in mice

Synchronized activity is common during various physiological operations but can culminate in seizures and consequently in epilepsy in pathological hyperexcitable conditions in the brain. Many types of seizures are not possible to control and impose significant disability for patients with epilepsy. Such intractable epilepsy cases are often associated with degeneration of inhibitory interneurons in the cortical areas resulting in impaired inhibitory drive onto the principal neurons. Recently emerging optogenetic technique has been proposed as an alternative approach to control such seizures but whether it may be effective in situations where inhibitory processes in the brain are compromised has not been addressed. Here we used pharmacological and optogenetic techniques to block inhibitory neurotransmission and induce epileptiform activity in vitro and in vivo. We demonstrate that NpHR-based optogenetic hyperpolarization and thereby inactivation of a principal neuronal population in the hippocampus is effectively attenuating seizure activity caused by disconnected network inhibition both in vitro and in vivo. Our data suggest that epileptiform activity in the hippocampus caused by impaired inhibition may be controlled by optogenetic silencing of principal neurons and potentially can be developed as an alternative treatment for epilepsy.

Optogenetic control of human neurons in organotypic brain cultures

Optogenetics is one of the most powerful tools in neuroscience, allowing for selective control of specific neuronal populations in the brain of experimental animals, including mammals. We report, for the first time, the application of optogenetic tools to human brain tissue providing a proof-of-concept for the use of optogenetics in neuromodulation of human cortical and hippocampal neurons as a possible tool to explore network mechanisms and develop future therapeutic strategies.

Differential Effect of Neuropeptides on Excitatory Synaptic Transmission in Human Epileptic Hippocampus

Development of novel disease-modifying treatment strategies for neurological disorders, which at present have no cure, represents a major challenge for todays neurology. Translation of findings from animal models to humans represents an unresolved gap in most of the preclinical studies. Gene therapy is an evolving innovative approach that may prove useful for clinical applications. In animal models of temporal lobe epilepsy (TLE), gene therapy treatments based on viral vectors encoding NPY or galanin have been shown to effectively suppress seizures. However, how this translates to human TLE remains unknown. A unique possibility to validate these animal studies is provided by a surgical therapeutic approach, whereby resected epileptic tissue from temporal lobes of pharmacoresistant patients are available for neurophysiological studies in vitro. To test whether NPY and galanin have antiepileptic actions in human epileptic tissue as well, we applied these neuropeptides directly to human hippocampal slices in vitro. NPY strongly decreased stimulation-induced EPSPs in dentate gyrus and CA1 (up to 30 and 55%, respectively) via Y2 receptors, while galanin had no significant effect. Receptor autoradiographic binding revealed the presence of both NPY and galanin receptors, while functional receptor binding was only detected for NPY, suggesting that galanin receptor signaling may be impaired. These results underline the importance of validating findings from animal studies in human brain tissue, and advocate for NPY as a more appropriate candidate than galanin for future gene therapy trials in pharmacoresistant TLE patients.

DREADDs suppress seizure-like activity in a mouse model of pharmacoresistant epileptic brain tissue

Epilepsy is a neurological disorder with a prevalence of ≈1% of general population. Available antiepileptic drugs (AEDs) have multiple side effects and are ineffective in 30% of patients. Therefore, development of effective treatment strategies is highly needed, requiring drug-screening models that are relevant and reliable. We investigated novel chemogenetic approach, using DREADDs (designer receptors exclusively activated by designer drugs) as possible inhibitor of epileptiform activity in organotypic hippocampal slice cultures (OHSCs). The OHSCs are characterized by increased overall excitability and closely resemble features of human epileptic tissue. Studies suggest that chemically induced epileptiform activity in rat OHSCs is pharmacoresistant to most of AEDs. However, high-frequency electric stimulus train-induced bursting (STIB) in OHSCs is responsive to carbamazepine and phenytoin. We investigated whether inhibitory DREADD, hM4Di, would be effective in suppressing STIB in OHSC. hM4Di is a mutated muscarinic receptor selectively activated by otherwise inert clozapine-N-oxide, which leads to hyperpolarization in neurons. We demonstrated that this hyperpolarization effectively suppresses STIB in mouse OHSCs. As we also found that STIB in mouse OHSCs is resistant to common AED, valproic acid, collectively our findings suggest that DREADD-based strategy may be effective in suppressing epileptiform activity in a pharamcoresitant epileptic brain tissue.

Prolonged life of human acute hippocampal slices from temporal lobe epilepsy surgery

Resected hippocampal tissue from patients with drug-resistant epilepsy presents a unique possibility to test novel treatment strategies directly in target tissue. The post-resection time for testing and analysis however is normally limited. Acute tissue slices allow for electrophysiological recordings typically up to 12 hours. To enable longer time to test novel treatment strategies such as, e.g., gene-therapy, we developed a method for keeping acute human brain slices viable over a longer period. Our protocol keeps neurons viable well up to 48 hours. Using a dual-flow chamber, which allows for microscopic visualisation of individual neurons with a submerged objective for whole-cell patch-clamp recordings, we report stable electrophysiological properties, such as action potential amplitude and threshold during this time. We also demonstrate that epileptiform activity, monitored by individual dentate granule whole-cell recordings, can be consistently induced in these slices, underlying the usefulness of this methodology for testing and/or validating novel treatment strategies for epilepsy.