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Preclinical models of Parkinson’s disease are imperative to gain insight into the neural circuits that contribute to gait dysfunction in advanced stages of the disease. The PTEN-induced putative kinase 1 (P1) knockout (KO) early onset model of Parkinson’s disease may be a useful rodent model to study the effects of neurotransmitter degeneration caused by loss of P1 function on brain activity during volitional gait. The goal of this study was to measure changes in neural activity at the cerebellar vermis (CBLv) at 8 months of age. Gait deficits, except run speed, were not significantly different from age-matched wild-type (WT) controls as previously reported. P1KO (n=4) and WT (n=4) rats were implanted with a micro-electrocorticographic array placed over CBLv lobules VI (a, b, and c) and VII. Local field potential recordings were obtained during volitional gait across a runway. Power spectral analysis and coherence analysis were used to quantify network oscillatory activity in frequency bands of interest. CBLv power was hypoactive in the beta (VIb, VIc, and VII) and alpha (VII) bands at CBLv lobules VIb, VIc, and VII in P1KO rats compared to WT controls during gait (p

Parkinson’s disease is a neurodegenerative disease characterized by gait dysfunction in the advanced stages of the disease. The unilateral 6-OHDA toxin-induced model is the most studied animal model of Parkinson’s disease, which reproduces gait dysfunction after greater than 68% dopamine (DA) loss in the substantia nigra pars compacta (SNc). The extent to which the neural activity in hemi-parkinsonian rats correlates to gait dysfunction and DAergic cell loss is not clear. In this paper we report the effects of unilateral DA depletion on cerebellar vermis activity using micro-electrocorticography (μECoG) during walking and freezing on a runway. Gait and neural activity were measured in 6-OHDA lesioned and sham lesioned rats at 14d, 21d, and 28d after infusion of 6-OHDA or control vehicle into the medial forebrain bundle (MFB) (n=20). Gait deficits in 6-OHDA rats were different from sham rats at 14d (pp=0.018). No differences in gait deficits were observed in sham lesioned rats at any time points. Hemiparkinsonian rats showed hyperactivity in the cerebellar vermis at 21d (pp

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by gait dysfunction in later stages of the disease. PD hallmarks include a decrease in stride length, run speed, and swing time; an increase in stride time, stance time, and base of support; dopaminergic degeneration in the basal ganglia; and cholinergic degeneration in the pedunculopontine nucleus (PPN). A progressive animal model of PD is needed to identify treatments for gait dysfunction. The goal of this study was to quantify progressive gait degeneration in PTEN-induced putative kinase 1 knockout (P1KO) rats and investigate neurodegeneration as potential underlying mechanisms. Gait analysis was performed in male P1KO and wild-type rats at 5 and 8 months of age and immunohistochemical analysis at 8 months. Multiple parameters of volitional gait were measured using a runway system. P1KO rats exhibited significant gait deficits at 5 months, but not 8 months. Gait abnormalities improved over time suggesting compensation during behavioral testing. At 8 months a 15% loss of tyrosine hydroxylase (TH) in the striatum, a 27% loss of TH-positive cells in the substantia nigra pars compacta, and no significant loss of choline acetyltransferase-positive cells in the PPN was found. Dopaminergic cell loss may contribute to gait deficits in the P1KO model, but not cholinergic cell loss. The P1KO rat with the greatest dopamine loss exhibited the most pronounced PD-like gait deficits, highlighting variability within the model. Further analysis is required to determine the suitability of the P1KO rat as a progressive model of gait abnormalities in PD.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by distal (i.e., tremor, bradykinesia, and rigidity) and axial motor symptoms (i.e., gait and postural disturbances). Deep brain stimulation (DBS) is a neurosurgical approach that uses electrical current delivered by an implantable pulse generator to modulate neural activity. Although DBS at the subthalamic nucleus (STN) and the internal globus pallidus (GPi) are well established for the treatment of the distal symptoms in PD, long-term studies of axial symptoms show a decline in efficacy with progression of the disease. Currently, there is no pharmacological or neurosurgical treatment available for the axial symptoms of advanced PD. Thus, the design of novel stimulation strategies to treat gait disturbances and postural instability has been investigated, including targets such as the pedunculopontine nucleus (PPN) and the substantia nigra pars reticulata (SNr). Here, we reviewed the current state of understanding regarding the effects of STN/GPi DBS, PPN DBS, and SNr DBS on gait and postural disturbances in PD and the proposed underlying mechanisms of action. The stimulation parameters (i.e., location, frequency, amplitude, and pulse width) and localization criteria for accurate placement of DBS electrodes within each target are discussed. As DBS at spatially distinct subregions of a target impacts the effectiveness of the therapy, electrode misplacement may directly contribute to the mixed results of DBS on the gait and postural disturbances of PD. We highlight the need for future studies to provide details on the specific subregion of the stimulation target to further advance the field.

Methamphetamine-induced circling is used to quantify the behavioral effects of subthalamic nucleus (STN) deep brain stimulation (DBS) in hemiparkinsonian rats. We observed a frequency-dependent transient effect of DBS on circling, and quantified this effect to determine its neuronal basis. High frequency STN DBS (75 – 260 Hz) resulted in transient circling contralateral to the lesion at the onset of stimulation, which was not sustained after the first several seconds of stimulation. Following the transient behavioral change, DBS resulted in a frequency-dependent steady-state reduction in pathological ipsilateral circling, but no change in overall movement. Recordings from single neurons in globus pallidus externa (GPe) and substantia nigra pars reticulata (SNr) revealed that high frequency, but not low frequency, STN DBS elicited transient changes in both firing rate and neuronal oscillatory power at the stimulation frequency in a subpopulation of GPe and SNr neurons. These transient changes were not sustained, and most neurons exhibited a different response during the steady-state phase of DBS. During the steady-state, DBS produced elevated neuronal oscillatory power at the stimulus frequency in a majority of GPe and SNr neurons, and the increase was more pronounced during high frequency DBS than during low frequency DBS. Changes in oscillatory power during both transient and steady-state DBS were highly correlated with changes in firing rates. These results suggest that distinct neural mechanisms were responsible for transient and sustained behavioral responses to STN DBS. The transient contralateral turning behavior following the onset of high frequency DBS was paralleled by transient changes in firing rate and oscillatory power in the GPe and SNr, while steady-state suppression of ipsilateral turning was paralleled by sustained increased synchronization of basal ganglia neurons to the stimulus pulses. Our analysis of distinct frequency-dependent transient and steady-state responses to DBS lays the foundation for future mechanistic studies of the immediate and persistent effects of DBS.

The unilateral 6-hydroxydopamine (6-OHDA) lesioned rat model is frequently used to study the effects of subthalamic nucleus (STN) deep brain stimulation (DBS) for the treatment of Parkinson's disease. However, systematic knowledge of the effects of DBS parameters on behavior in this animal model is lacking. The goal of this study was to characterize the effects of DBS on methamphetamine-induced circling in the unilateral 6-OHDA lesioned rat. DBS parameters tested include stimulation amplitude, stimulation frequency, methamphetamine dose, stimulation polarity, and anatomical location of the electrode. When an appropriate stimulation amplitude and dose of methamphetamine were applied, high-frequency stimulation (>; 130 Hz), but not low frequency stimulation (

in microelectrode technology. The clinical benefits of brain implants are far-reaching and include improvements in quality of life for patients suffering from neurological impairments and diseases, such as Parkinson’s disease, epilepsy, blindness, and paralysis. However, a number of major bottlenecks currently hinder the realization of the therapeutic potential of brain implants, one of which being the ability of the implant to function reliably over the remaining lifetime of the patient. It is generally believed that many complications in long-term implant functionality are due to an adverse brain tissue response elicited by the implant. This inflammatory response, featured by the formation of the astroglial scar, can result in isolation of the implant from target neurons both electrically and mechanically, and is arguably the biggest stumbling block in realizing chronic recordings from neural electrodes. A common strategy to dampen the tissue response is local delivery of anti-inflammatory drugs directly at the implant-tissue interface. [3–5] Although such approach has been shown to successfully moderate host response during the acute phase, [6] alone it may be inadequate to have long-term functional consequences, due to limitations in the duration of drug release. Since the sustained chronic tissue response to the implant is considered to be a result of both foreign body reaction [7] and mechanical mismatch induced micromotion, [8,9] we propose to modulate the sustained tissue response by endowing the implant with an intrinsic anti-inflammatory surface. Here, we demonstrate through both in vitro cell culture studies and in vivo rodent studies, that immobilized alpha-MSH creates an inherently anti-inflammatory neural electrode surface such that it significantly attenuates glial response for at least 4 weeks post-implantation. Alpha-MSH is an endogenous tridecapeptide that is secreted by pituitary cells, astrocytes, monocytes, keratinocytes, etc., and is found in the skin, brain, and other tissues. [10,11] Among its broad, potent anti-inflammatory functions, studies have shown alpha-MSH inhibits pro-inflammatory cytokines and neurotoxic nitric oxide (NO) production by microglia stimulated with beta-amyloid and interferon-gamma, which simulated inflammation associated with Alzheimer’s disease. [12] Since microglia, the resident macrophages in the brain, are the frontline defense cells against invasive implants, the neuroimmunomodulatory peptide alpha-MSH was selected in our study to directly mitigate microglial response to foreign brain implants. Coupling of the active molecule to the neural implant surface was accomplished through silane chemistry and the use of a hetero-bifunctional crosslinker with both thiol- and amino-reactive moieties, [13] as demonstrated in Figure 1. The surface coverage of alpha-MSH peptide was 0.0212 nmol cm –2 , quantified by application of sulfo-SDTB that reacts with primary amine, specifically in this case, those presented from the lysine residue and the N-terminus of the alpha-MSH peptide. In order to first investigate whether the alpha-MSH peptide remains biologically active when covalently immobilized to the Si surface, we cultured primary rat microglial cells on the peptide modified surface and subjected the culture to lipopolysaccharide (LPS) bacterial endotoxin, which elicits an inflammatory response in vitro. As shown in Figure 2A, the production of nitric oxide (NO), an indicator of inflammation, was reduced to nearly 50% of that observed in the control culture, suggesting that the anti-inflammatory property of alphaMSH was preserved during the tethering process. Moreover, we examined the gene expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 1 (IL-1) using real time RT-PCR. Both of these cytokines play a key role in mediating the inflammation process in vivo, and microglia are the known major cellular source for production of these cytokines. Modulating the level of TNFalpha and IL-1 is critical as excessive production of these factors could not only amplify the inflammation process by stimulating glial cell proliferation and activation, but also cause neuronal death. LPS is commonly used to induce microglial activation and cause production of neurotoxic pro-inflammatory cytokines. This is consistent with our finding where the mRNA expression level of TNF-alpha had an over three-fold increase in the control Si group after subjecting to LPS stimulation (Fig. 2B). In contrast, TNF-alpha expression

Subthalamic nucleus (STN) deep brain stimulation (DBS) is an established treatment for the motor symptoms of Parkinson's disease (PD). However, the mechanisms of action of DBS are unknown. Random temporal patterns of DBS are less effective than regular DBS, but the neuronal basis for this dependence on temporal pattern of stimulation is unclear. Using a rat model of PD, we quantified the changes in behavior and single-unit activity in globus pallidus externa and substantia nigra pars reticulata during high-frequency STN DBS with different degrees of irregularity. Although all stimulus trains had the same average rate, 130-Hz regular DBS more effectively reversed motor symptoms, including circling and akinesia, than 130-Hz irregular DBS. A mixture of excitatory and inhibitory neuronal responses was present during all stimulation patterns, and mean firing rate did not change during DBS. Low-frequency (7–10 Hz) oscillations of single-unit firing times present in hemiparkinsonian rats were suppressed by regular DBS, and neuronal firing patterns were entrained to 130 Hz. Irregular patterns of DBS less effectively suppressed 7- to 10-Hz oscillations and did not regularize firing patterns. Random DBS resulted in a larger proportion of neuron pairs with increased coherence at 7–10 Hz compared with regular 130-Hz DBS, which suggested that long pauses (interpulse interval >50 ms) during random DBS facilitated abnormal low-frequency oscillations in the basal ganglia. These results suggest that the efficacy of high-frequency DBS stems from its ability to regularize patterns of neuronal firing and thereby suppress abnormal oscillatory neural activity within the basal ganglia.

Deep brain stimulation (DBS) improves the distal motor symptoms of Parkinson's disease, but long-term improvements in gait and postural disturbances are less pronounced. The effects of stimulation location, within the large nuclear region of the substantia nigra pars reticulata (SNr), and stimulation parameters on improvement in gait are unclear, and this lack of foundational knowledge hinders the application and optimization of SNr DBS. We quantified the effects of medial vs. lateral SNr DBS on methamphetamine-induced circling in hemiparkinsonian rats to test the hypothesis that stimulation location differentially modulates axial symptoms. The frequency tuning curves showed opposite trends with stimulation frequency; during high frequency stimulation, medial SNr DBS decreased ipsilateral circling, while lateral SNr DBS had no effect on circling. As well, we quantified the effects of 130 Hz SNr DBS on gait to test the hypothesis that SNr DBS location differentially modulates gait. High frequency DBS of the medial SNr, but not lateral SNr, improved the rat's ability to maintain walking speed. The therapeutic effects of medial SNr DBS appeared to improve with time on the same order as clinical studies (>10 min). These results suggest that improvement in gait depends on the location of the electrodes (medial vs. lateral SNr) with a time course for improvement reminiscent of human data and provide a rational basis for the appropriate selection of implant site and stimulation parameters for SNr DBS.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) is an effective treatment for the motor symptoms of Parkinson's disease (PD). The effects of DBS depend strongly on stimulation frequency: high frequencies (>90 Hz) improve motor symptoms, while low frequencies (

Deep brain stimulation (DBS) is an effective treatment of Parkinson's disease, but its mechanisms are still unclear. To test the hypothesis that DBS alleviates motor symptoms by regularizing neuronal firing, we applied regular frequency stimulation between 5-260 Hz as well as irregular high frequency stimulation with an average rate of 130Hz to rats with unilateral 6-hydroxydopamine (6-OHDA) lesions. We found that high frequency regular stimulation above 130Hz was more effective than both low frequency stimulation and high frequency irregular stimulation at normalizing pathological circling behavior. Our results support the hypothesis that DBS is effective because it is able to mask pathological firing patterns within the basal ganglia, and highlight the importance of the temporal pattern in addition to the rate of stimulation.

Neuronal density and proximity to recording electrodes is perhaps the best indicator of recording longevity from chronically implanted microelectrodes. However, little is known about this important parameter in the hours following implantation. In this study, 1 hour, 24 hours, and 2 weeks post implantation with Si "Michigan" arrays, rats were sacrificed and cortical sections were obtained and immunostained with the neuron-specific marker NeuN. Images were quantified and compared between time points within a zone of interest (0-100 mum from the insertion sites) and examined as a function of depth below the cortical surface. Differences in neuronal density between these three time points were dependent on cortical depth. These data strongly suggest that neuronal density in the cortex respond differently to injury based on their location within the cortical column. The need to account for depth information in evaluating tissue reaction to microelectrode implantation is highlighted.

Neural electrodes could significantly enhance the quality of life for patients with sensory and/or motor deficits as well as improve our understanding of brain functions. However, long-term electrical connectivity between neural tissue and recording sites is compromised by the development of astroglial scar around the recording probes. In this study we investigate the effect of a nanoscale laminin (LN) coating on Si-based neural probes on chronic cortical tissue reaction in a rat model. Tissue reaction was evaluated after 1 day, 1 week, and 4 weeks post-implant for coated and uncoated probes using immunohistochemical techniques to evaluate activated microglia/macrophages (ED-1), astrocytes (GFAP) and neurons (NeuN). The coating did not have an observable effect on neuronal density or proximity to the electrode surface. However, the response of microglia/macrophages and astrocytes was altered by the coating. One day post-implant, we observed an approximately 60% increase in ED-1 expression near LN-coated probe sites compared with control uncoated probe sites. Four weeks post-implant, we observed an approximately 20% reduction in ED-1 expression along with an approximately 50% reduction in GFAP expression at coated relative to uncoated probe sites. These results suggest that LN has a stimulatory effect on early microglia activation, accelerating the phagocytic function of these cells. This hypothesis is further supported by the increased mRNA expression of several pro-inflammatory cytokines (TNF-alpha, IL-1 and IL-6) in cultured microglia on LN-bound Si substrates. LN immunostaining of coated probes immediately after insertion and retrieval demonstrates that the coating integrity is not compromised by the shear force during insertion. We speculate, based on these encouraging results, that LN coating of Si neural probes could potentially improve chronic neural recordings through dispersion of the astroglial scar.

We used a musculoskeletal model of the cat hindlimb to compare the patterns of endpoint forces generated by all possible combination of 12 hindlimb muscles under three different muscle activation rules: homogeneous activation of muscles based on uniform activation levels, homogeneous activation of muscles based on uniform (normalized) force production, and activation based on the topography of spinal motoneuron pools. Force patterns were compared with the patterns obtained experimentally by microstimulation of the lumbar spinal cord in spinal intact cats. Magnitude and orientation of the force patterns were compared, as well as the proportion of the types found, and the proportions of patterns exhibiting points of zero force (equilibrium points). The force patterns obtained with the homogenous activation and motoneuron topography models were quite similar to those measured experimentally, with the differences being larger for the patterns from the normalized endpoint forces model. Differences in the proportions of types of force patterns between the three models and the experimental results were significant for each model. Both homogeneous activation and normalized endpoint force models produced similar proportions of equilibrium points as found experimentally. The results suggest that muscle biomechanics play an important role in limiting the number of endpoint force pattern types, and that muscle combinations activated at similar levels reproduced best the experimental results obtained with intraspinal microstimulation.

The long-term stability of implanted micromachined neural probes is compromised due to the glial scar formation at the insertion site. In this study, we developed a novel nitrocellulose-based coating for the sustained local delivery of the anti-inflammatory drug dexamethasone, a synthetic glucocorticoid that effectively reduces inflammation in the CNS. In vitro dexamethasone release was observed over 16 days, with a relatively high release in the first three days and a slow, stable release thereafter. When Michigan neural recording probes coated with and without dexamethasone-loaded nitrocellulose coatings were implanted into the adult rat brains, immunohistochemical evidence shows a marked reduction of reactive astrocytes (GFAP), reactive microglia (EDl), and chondroitin sulfate proteoglycans (CS56) expression around the insertion site compared to uncoated probes. Impedance spectroscopy showed that the dexamethasone-loaded nitrocellulose coatings slightly reduce the magnitude of electrode impedance at the biologically relevant frequency of 1 kHz through an increase of capacitance. In vivo acute recordings demonstrate that extracellular recordings with coated probes are akin to non-coated probes and it is anticipated that with time, the coated probes will exhibit superior performance. In conclusion, we developed a novel nitrocellulose-based, drug releasing coating for neural electrodes that can effectively reduce scar tissue formation without adversely affecting the electrical performance of the electrodes

Using a biomechanical model of the cat hindlimb, we studied patterns of endpoint forces created by all muscle combinations of fourteen selected muscles, and compared them to the force patterns produced by intraspinal microstimulation of the lumbar spinal gray matter. We ran the model with two different activation schemes for the muscles. The first run used combinations of the fourteen selected muscles stimulated at the same level of activation. The second run used combinations where muscle forces were normalized to produce the same maximum end-point force. These results were compared to force field patterns obtained experimentally during intraspinal microstimulation. Although there were slight variations in the force patterns produced, both methods converged to four dominant patterns. When muscles in the model were normalized, some force patterns were found that were not observed experimentally. These results show the significance of specific levels of muscle activation to the production of the experimental patterns.

We studied the structure of endpoint forces produced by microstimulation of the cat spinal cord, and from combinations of muscles using a biomechanical model of the cat hindlimb. The forces evoked by microstimulation were of four types. At some stimulation sites, the force patterns exhibited a point of convergence where the active endpoint force was zero. The endpoint forces produced by activating combinations of muscles in the biomechanical model were of types similar to the experimental ones, although they demonstrated points of convergence in locations not observed experimentally. These results suggest that the spinal circuitry uses a subset of the possible muscular combinations.

The widespread adoption of neural prosthetic devices is currently hindered by our inability to reliably record neural signals from chronically implanted electrodes. The extent to which the local tissue response to implanted electrodes influences recording failure is not well understood. To investigate this phenomenon, impedance spectroscopy has shown promise for use as a non-invasive tool to estimate the local tissue response to microelectrodes. Here, we model impedance spectra from chronically implanted rats using the well-established Cole model, and perform a correlation analysis of modeled parameters with histological markers of astroglial scar, including glial fibrillary acid protein (GFAP) and 4',6-diamidino-2- phenylindole (DAPI). Correlations between modeled parameters and GFAP were significant for three parameters studied: Py value, R(o) and |Z|(1 kHz), and in all cases were confined to the first 100 microm from the interface. Py value was the only parameter also correlated with DAPI in the first 100 microm. Our experimental results, along with computer simulations, suggest that astrocytes are a predominant cellular player affecting electrical impedance spectra. The results also suggest that the largest contribution from reactive astrocytes on impedance spectra occurs in the first 100 microm from the interface, where electrodes are most likely to record electrical signals. These results form the basis for future approaches where impedance spectroscopy can be used to evaluate neural implants, evaluate strategies to minimize scar and potentially develop closed-loop prosthetic devices.

Prosthetic devices that are controlled by intracortical electrodes recording one's 'thoughts' are a reality today, and no longer merely in the realm of science fiction. However, widespread clinical use of implanted electrodes is hampered by a lack of reliability in chronic recordings, independent of the type of electrodes used. One major hypothesis has been that astroglial scar electrically impedes the electrodes. However, there is a temporal discrepancy between stabilization of scar's electrical properties and recording failure with recording failure lagging by 1 month. In this study, we test a possible explanation for this discrepancy: the hypothesis that chronic inflammation, due to the persistent presence of the electrode, causes a local neurodegenerative state in the immediate vicinity of the electrode. Through modulation of chronic inflammation via stab wound, electrode geometry and age-matched control, we found that after 16 weeks, animals with an increased level of chronic inflammation were associated with increased neuronal and dendritic, but not axonal, loss. We observed increased neuronal and dendritic loss 16 weeks after implantation compared to 8 weeks after implantation, suggesting that the local neurodegenerative state is progressive. After 16 weeks, we observed axonal pathology in the form of hyperphosphorylation of the protein tau in the immediate vicinity of the microelectrodes (as observed in Alzheimer's disease and other tauopathies). The results of this study suggest that a local, late onset neurodegenerative disease-like state surrounds the chronic electrodes and is a potential cause for chronic recording failure. These results also inform strategies to enhance our capability to attain reliable long-term recordings from implantable electrodes in the CNS.