WO2010083208A2

WO2010083208A2 - Treating neuropsychiatric diseases

Info

Publication number: WO2010083208A2
Application number: PCT/US2010/020890
Authority: WO
Inventors: Kendall H. Lee; Charles D. Blaha
Original assignee: Mayo Foundation For Medical Education And Research
Priority date: 2009-01-13
Filing date: 2010-01-13
Publication date: 2010-07-22
Also published as: WO2010083208A9

Abstract

This document relates to methods and materials involved in treating neuropsychiatric disease. For example, methods and materials for treating Parkinson's disease (PD) using deep brain stimulation are provided.

Description

TREATING NEUROPSYCHIATRIC DISEASES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is claims priority to U.S. Provisional Application Serial No. 61/144,324, filed on January 13, 2009.

BACKGROUND

1. Technical Field

This document relates to methods and materials involved in treating neuropsychiatric diseases. For example, methods and materials for treating Parkinson's disease using deep brain stimulation are provided.

2. Background Information

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder and affects approximately 1 million people in the U.S., with 50,000 diagnosed each year. Symptoms of this disorder include resting tremors in the appendages, loss of balance and slowness of gait (bradykinesia) and are due to a massive degeneration of dopaminergic neurons in the substantia nigra. Dopaminergic neurons in this midbrain nucleus form the nigrostriatal dopaminergic system that projects to the basal ganglia (striatum in the rat) in the forebrain. Although the exact cause of the disorder is unknown, i.e., idiopathic, in most

PD patients there are cellular proteins that are overexpressed in the striatum. One is the Lewy body, which is a fibrous protein that accumulates in striatal neuronal cell cytoplasm in PD patients (Shastry, "Parkinson disease: etiology, pathogenesis and future of gene therapy," Neurosci. Res., 41 : 5-12 (2001)). In some forms of PD, certain genes (α-synuclein, ubiquitin carboxy terminal hydrolase Ll (UCHLl) and parkin, a ubiquitin protein ligase) and their expressed protein products are associated with the disorder. In addition, mutations in mitochondria and expression of gene-tau, a microtubule-associated protein, have been found in families with PD. These proteins and organelles are structural components involved in neurotransmission (exocytotic mechanisms) and metabolism (oxidative-reductive mechanisms) important for the normal functioning of neurons. Research is on-going to determine the relevance of these genes and proteins in the etiology of the disorder, as well as environmental factors, including smoking and caffeine intake (Shastry (2001); Bergman et ah, "Pathophysiology of Parkinson's disease: from clinical neurology to basic neuroscience and back," Mov. Disord., 17: S28-S40 (2002); Hurley et al, "What has been learnt from study of dopamine receptors in Parkinson's disease?" Pharmacol Ther., I l l : 715-728 (2002)).

SUMMARY

This document relates to methods and materials involved in treating neuropsychiatric diseases. For example, methods and materials for treating Parkinson's disease (PD) using deep brain stimulation are provided. The methods and materials provided herein can relieve motor symptoms associated with PD and extend battery life of a neurostimulator.

In general, this document features a method for increasing dopamine release from the basal ganglia of a mammal. The method comprises applying an electrical stimulus to the medial forebrain bundle of the brain of the mammal. The electrical stimulus can comprise an intermittent stimulation. The stimulus can be delivered by an electrode connected to an implanted neurostimulator. The frequency of the electrical stimulus can be from about 100 to about 250 Hz. The current intensity of the electrical stimulus can be about 800 μA. The intermittent stimulus can comprise a rest period of between about 4 seconds and about 7 seconds subsequent to the electrical stimulus. The intermittent stimulus can cycle continuously. The electrical stimulus can comprise a burst-like stimulation. The burst-like stimulation can comprise an alternating cycle of a 0.2 second current pulse followed by a 0.2 second inter-stimulus rest. The intermittent stimulus can comprise about twenty alternating cycles. In another aspect, this document features a method for treating a human having Parkinson's disease. The method comprises applying an intermittent electrical stimulus to the medial forebrain bundle of the brain of the human. The stimulus can evoke dopamine release in the striatum of the brain, thereby treating the human. The stimulus can be delivered by an electrode connected to an implanted neurostimulator. The frequency of the electrical stimulus can be from about 100 to about 250 Hz. The current intensity of the electrical stimulus can be about 800 μA. The intermittent stimulus can comprise a rest period of between about 4 seconds and about 7 seconds subsequent to the electrical stimulus. The intermittent stimulus can cycle continuously. The electrical stimulus can comprise a burst-like stimulation. The burst-like stimulation can comprise an alternating cycle of a 0.2 second current pulse followed by a 0.2 second inter-stimulus rest. The intermittent stimulus can comprise about twenty alternating cycles.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

Figure 1. Schematic of an experimental set-up to amperometrically record dopamine release in the striatum evoked by electrical stimulation of dopamine axons contained within the medial forebrain bundle (MFB). SN refers to the substantia nigra, the site of origin of dopamine neurons in the midbrain.

Figure 2. Electrical stimulation parameters applied to the medial forebrain bundle for each of the four groups of mice. Each of the test stimulation groups consisted of 300 pulses, with Groups 1 and 3 (Gl and G3) having equal stimulation off periods and Groups 2 and 3 (G2 and G3) having equal stimulation on periods. Group 4 (G4) consisted of a continuous train of electrical stimulation applied at 100 pulses per second. For Group 1 the 300 pulses were applied in a burst fashion with 20 pulses at a time applied over a 0.2-second period, which is equivalent to 100 pulses per second for each burst.

Figure 3. Representative coronal sections illustrating the site of implantation for medial forebrain bundle (MFB) stimulating electrodes (black circles) and striatal carbon fiber recording electrodes (black vertical lines) for all mice tested in groups Gl to G4 (n=4 per site and group). Gray shaded areas correspond to the striatum (left) and MFB (right). NAc and NAs, nucleus accumbens core and shell, respectively; ac, anterior commissure; mt, mammillothalamic tract; cp, cerebral peduncle. Numbers correspond to mm from bregma in accordance with the mouse atlas of Franklin and Paxinos. (The Mouse Brain in Stereotaxic Coordinates., Academic Press, San Diego (1997)).

Figure 4. Changes in striatal dopamine oxidation current in picoAmps (pAmps; 10^~12 Amps) during the first 100 seconds of electrical stimulation of the medial forebrain bundle for representative mice taken from Groups 1-4 (Gl -4; black line in each panel). Figure 5. Steady-state levels of dopamine oxidation current (pAmps; 10^"12

Amps) recorded in the striatum during a period of 250 seconds to 300 seconds after medial forebrain stimulation began with one of the three test parameters (G 1-3) and the control stimulation parameter (G4) indicated in the box. Black lines represent the means (n=4 per group), and gray lines represent the SEMs for each group. Figure 6. Histogram showing the average steady-state levels of striatal dopamine release (as measured by summed dopamine oxidation current 250-200 seconds post-stimulation) for each of the three test stimulation parameters (Groups 1- 3, black bars) and the control stimulation parameter (Group 4, black bar). The SEM for each Group (n=4) is indicated as a single line above each bar on the graph. Figure 7. Tabulation of summed dopamine oxidation current over the period of 250-300 seconds after electrical stimulation. (* all data values are x 10^"8).

Figure 8. Tabulation of the average overall levels of striatal dopamine release evoked by stimulation of the medial forebrain bundle for each of the test stimulation parameters, and control parameter. Figure 9. Multiple t-test analysis between Groups 1 and 2 average value of summed dopamine oxidation current (p<0.0001).

Figure 10. Multiple t-test analysis between Groups 1 and 3 average value of summed dopamine oxidation current (p<0.0001).

Figure 11. Multiple t-test analysis between Groups 1 and 4 average value of summed dopamine oxidation current (p<0.0001).

Figure 12. Multiple t-test analysis between Groups 2 and 3 average value of summed dopamine oxidation current (p<0.0001).

Figure 13. Multiple t-test analysis between Groups 2 and 4 average value of summed dopamine oxidation current (p<0.0001). Figure 14. Multiple t-test analysis between Groups 3 and 4 average value of summed dopamine oxidation current (p<0.013).

Figure 15. (A.) A representative coronal MRI and corresponding fiducials (white circles) on the MRI-compatible head frame for pre -operative planning and calculation of the trajectory coordinates (dark line) for implantation of a DBS electrode in the STN of the pig. (B.) Expanded view of the pre-operatively planned trajectory for implantation of a DBS electrode in the STN. (C.) A representative coronal MRI for pre-operative planning and calculation of the trajectory coordinates (dark line) for implantation of a carbon-fiber recording electrode in the caudate of the pig. (D.) A coronal MRI showing the post-operative confirmation of the DBS electrode placement in the STN of the pig shown in (A).

Figure 16. (A.) In vivo characteristics of dopamine release recorded in pig "A" striatum during 2-sec (dark bar) STN stimulation with 5 V at 120 Hz. (Inset) A cyclic voltammogram recorded at the peak of the evoked increase in dopamine release characteristic of dopamine oxidation (ox.) and reduction (rd.) of the electro formed dopamine ortho-quinone (DOQ) back to dopamine. (B.) Pseudo-color plot obtained during (dark bar) and after 2-sec STN stimulation with 5 V at 120Hz.

Figure 17. (A.) In vivo characteristics of dopamine release recorded in pig "A" striatum during 2-sec (black bar) STN stimulation with 3 to 7 V at 120 Hz. Right panel shows representative pseudo-color plots obtain immediately prior to, during (black bars), and after 2-sec STN stimulation with 3, 4, 5, and 7 V at 120Hz. (B.) STN stimulation intensity-dependent increases in dopamine release from 3 pigs ("A", "B", and "C"). Each curve describes the best non-linear fit (square, r2 = 0. 99). (C.) STN stimulation frequency-dependent (60 to 240 Hz at 3 V) increases in dopamine release from pig "A" and pig "D".

DETAILED DESCRIPTION

This document relates to methods and materials involved in treating neuropsychiatric diseases. For example, methods and materials for treating Parkinson's disease (PD) using deep brain stimulation are provided. For example, burst- like stimulation can be delivered to the medial forebrain bundle (MFB) of the brain of a Parkinson's patient, to treat tremor and other motor symptoms.

The methods described herein can be used with a medical device capable of delivering deep brain electrical stimulation {e.g., a neurostimulator such as a Medtronic™ Soletra or Kinetra). For example, a lead (or electrode) of a neurostimulator can be positioned in the subthalamic nuclei (STN) of the brain, to provide deep brain stimulation. In some cases, a battery powered neurostimulator can be implanted (e.g. in the patient's chest) and have an extension connecting the neurostimulator and a lead in the patient's brain.

A neurostimulator lead can be positioned in a suitable part of the brain for stimulation-evoked dopamine release. For example, the lead can be positioned to stimulate the medial forebrain bundle directly rather than the STN. Stimulation of the glutamatergic innervations originating from the subthalamic nucleus and terminating in the substantia nigra can activate nigrostriatal dopaminergic neurons directly (e.g., Meltzer et al., "Modulation of dopamine neuronal activity by glutamate receptor subtypes," Neurosci Biobehav Rev. , 21 : 511-518 (1997)). Stimulation of glutamatergic neurons in the subthalamic nucleus that project to the pedunculopontine nucleus in the hindbrain can activate nigrostriatal dopaminergic neurons both by the reciprocal excitatory innervation and subsequent activation of the substantia nigra by subthalamic nucleus glutamatergic efferents, and indirectly by activating pedunculopontine cholinergic and glutamatergic inputs to the substantia nigra that act on glutamate ionotropic and metabotropic receptors located on substantia nigra dopaminergic cells (Blaha and Winn, "Modulation of dopamine efflux in the striatum following cholinergic stimulation of the substantia nigra in intact and pedunculopontine tegmental nucleus-lesioned rats," J. Neurosci., 13: 1035-1044 (1993); Blaha et al., "Modulation of dopamine efflux in the nucleus acumens after cholinergic stimulation of the ventral tegmental area in intact, pedunculopontine tegmental nucleus-lesioned, and laterodorsal tegmental nucleus-lesioned rats," J. Neurosci., 16: 714-722 (1996); Forster and Blaha, "Pedunculopontine tegmental stimulation evokes striatal dopamine efflux by activation of acetylcholine and glutamate receptors in the midbrain and pons of the rat," Eur. J. Neurosci., 17: 751- 762 (2003)). In some cases, the stimulating electrode can be placed within white matter dorsal to the subthalamic nucleus (Saint-Cyr et al, "Localization of clinically effective stimulating electrodes in the human subthalamic nucleus on magnetic resonance imaging," J. Neurosurg., 97: 1152-1166 (2002); Voges et al, "Bilateral high-frequency stimulation in the subthalamic nucleus for the treatment of Parkinson disease: correlation of therapeutic effect with anatomical electrode position," J. Neurosurg., 96: 269-279 (2002)), including the dorsolateral border of the subthalamic nucleus (Herzog et al., "Most effective stimulation site in the subthalamic deep brain stimulation of Parkinson's disease," Mov. Disord., 19: 1050-1054 (2004)), all of which contain the ascending dopaminergic axons. In some cases, a lead can be positioned unilaterally or bilaterally. An appropriate electrical signal can have a frequency of between 90 Hz and

250 Hz. In some cases, an appropriate electrical signal can have a frequency of 100 Hz. In some cases, the stimulation can be a series of applied electrical current {e.g. a train). In some cases, stimulation can include a train with intertrain rest periods. For example, stimulation parameters can include rest periods in which no current is applied. In some cases, the rest period can be from about 4 seconds to about 7 seconds. In some cases, an appropriate parameter for an electrical signal can be a burst- like stimulus. For example, a burst-like stimulus can be a stimulus that mimics the natural pattern of dopamine neuronal activity {e.g., Hyland et al., "Firing modes of midbrain dopamine cells in the freely moving rat," Neuroscience 114:475-492 (2002)). For example, burst-like stimulus can include an alternating cycle of a 0.2 second current pulse and a 0.2 second inter-stimulus rest. The stimulation cycle can last for about 5-6 seconds, or approximately twenty 0.2 second stimuli. The cumulative rest period from the inter-stimulus rests can be between about 2.5 seconds and 3 seconds. Each burst-like stimulus can be followed by a contiguous rest period of about 4 seconds. In some cases, electrical stimulation, including a period of burst- like stimuli and a subsequent contiguous rest period, can cycle continuously.

The methods described herein can be used to treat Parkinson's disease and other diseases involving motor disorders. For example, Parkinsonian tremor, essential tremor, or epilepsy can be treated using the methods described herein. In some cases, the methods described herein can be combined with pharmacological therapies {e.g., combined with Levodopa for the treatment of Parkinson's disease).

Referring to Figure 1 , methods described herein can be used to enhance dopamine release. Dopamine release in the striatum evoked by electrical stimulation of dopamine axons contained with the medial forebrain bundle can be recorded amperometrically. In some cases, dopamine levels can be assessed by placing a carbon fiber "dopamine" recording electrode into the striatum. An electrometer can apply an electrical potential (+0.8 V) to the recording electrode via the auxiliary electrode, forcing the oxidation of dopamine at the recording electrode surface. For example, two electrons can be transferred to the recording electrode for each molecule of dopamine oxidized at the surface of the recording electrode. An analog current, constituting dopamine oxidation, is subsequently converted (A/D converter) to a digital signal that can be monitored in real time on a computer screen. A dopamine oxidation current can correspond to a proportional increase in dopamine release in the striatum as a result of MFB stimulation.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES Example 1 - Determining dopamine release in response to alternative parameters of

Deep Brain Stimulation (DBS) Subjects

Wildtype C57 male mice from Jackson Laboratory (Maine, USA) were group housed with free access to food and water and placed in a temperature controlled room (21.0=1=1.0 ⁰C) on a reverse dark cycle (14:10) with the light turned on at 0800 hour. At the time of surgery, mice weighed between 35 to 45 g and were 4 to 5 months old. Experiments were approved by a local Institutional Animal Care and Use Committee and were conducted in compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Surgery

Mice were anesthetized with urethane (1.5 g/kg, i.p.; Sigma-Aldrich, MO, USA) and placed in a stereotaxic (David Kopf, CA, USA) with a mouse head-holder (Stoelting, IL, USA) within a Faraday cage. Body temperature was maintained at 38±0.5 ⁰C with a temperature-regulated heating pad (TC-1000, CWE Inc., NY, USA). Three holes were drilled through the animal's skull for an Ag/ AgCl reference/auxiliary electrode (positioned at the surface of the cortex), a concentric bipolar stimulating electrode (SNE-100; Rhodes Medical Co., CA, USA), and a dopamine recording electrode (carbon fiber 10 μm o.d., 250 μm length, Thornel Type P, Union Carbide, PA, USA) (as described in Forster and Blaha, Eur. J. Neurosci., 17:751-762 (2003)). The stimulating electrode tip was positioned within the medial forebrain bundle containing nigrostriatal dopamine axons (coordinates, in mm from bregma and dura: AP -2.1, ML +1.1, DV -4.25 to -4.6) ipsilateral to the dopamine recording electrode positioned in the dorsomedial striatum (coordinates in mm from bregma and dura: AP +1.4, ML +1.0, DV -2.5) (as described in Franklin and Paxinos (1997)).

Fixed potential amperometry and electrical stimulation Following implantation of all electrodes, a fixed positive potential (+0.8 V) was applied to the recording electrode and oxidation current monitored and recorded continuously (1OK samples/second) with an electrometer (e-corder/picostat system, e- DAQ, CO, USA), filtered at 50 Hz (see figure 1) (Forster and Blaha, 2003). Electrical stimulation of the medial forebrain bundle (counterbalanced with respect to left and right cerebrum for individual mice) consisted of 800 μA amplitude cathodic monophasic pulses (all 0.5 msecond pulse duration at 100 Hz with an interpulse duration of 10 msecond) applied via an optical isolator and programmable pulse generator (Iso-Flex/Master-8; AMPI, Jerusalem, Israel). Amperometric recordings in combination with bare carbon fiber electrodes exhibit poor chemical resolution, but can be used successfully to monitor dopamine efflux and reuptake evoked by dopaminergic axonal stimulations (as described in Dugast et al. , Neuroscience, 62:647-654 (1994)). The depths of both the stimulating and recording electrodes were adjusted for each experiment to maximize stimulation-evoked dopamine efflux tested with 15 pulses at 100 Hz applied every 30 seconds. A delay of 10-20 minutes followed to allow the recorded amperometric signal to stabilize before the start of each experiment.

Experimental Design

There were four groups with 4 mice in each group. In each group, the following electrical stimulation parameters were fixed: interpulse interval of 10 mseconds, pulse duration of 0.5 mseconds, current intensity of 800 μA, and frequency of 100 Hz. There were three sets of stimulation test parameters and a control stimulation parameter, one for each group, followed by a control experiment for each. Continuous electrical stimulation was performed on each mouse after application of a single test parameter.

Experimental Groups

The three sets of test parameters all consisted of the application of trains of 300 pulses applied intermittently over a 10-20 minute period, but differed in terms of pulse train duration, intertrain interval, and interburst interval (off then on period). The three groups (figure 2) and corresponding test parameters, including group 4 (continuous train stimulation) were as follows:

Group 1 : Pulse train on duration of 0.2 seconds with an intertrain off interval of 0.2 seconds for a total duration of 5.8 seconds followed by an interburst off interval of 4.2 seconds. Group 2: Pulse train on for a total duration of 3 seconds (no intertrain off interval) followed by an interburst off interval of 7 seconds. Group 3 : Pulse train on for a total duration of 3 seconds (no intertrain off interval) followed by an interburst off interval of 4.2 seconds.

Group 4: Continuous train stimulation.

Thirty minutes following the application of a specific test parameter, a continuous train of 100 Hz stimulation was applied for a period of 10-20 minutes. These responses were found to not differ significantly from those recorded in group 4 mice (those that received only continuous train stimulation) and were therefore not included in the analysis of the data.

Histological Analysis Immediately following each experiment, a direct current (100 μA for 10 seconds; +5 V for 5 seconds) was passed through the stimulating electrode in the medial forebrain bundle to leave iron deposits and through the recording electrode in the striatum to lesion tissue, respectively. Animals were euthanized with an intracardial injection of urethane, the brains removed, and placed in a solution consisting of reagent grade 0.1% potassium ferricyanide (III) in 10% formalin. The brains were fixed and 60 μm coronal sections were cut on a cryostat at -30 degrees C⁰. A Prussian blue spot indicative of the redox reaction of ferricyanide and iron deposits labeled the stimulating electrode tip in the medial forebrain bundle, while the placement of the recording electrodes in the striatum were determined by the position of the electrolytic lesion under light microscopy. Placements were then recorded on representative coronal diagrams (Franklin and Paxinos, (1997)).

Data analysis Data from each experiment was analyzed using multiple Dunnett's t-tests (figures 9-14). Analysis of the maximal change in amplitude of evoked dopamine release for each set of stimulation parameters tested was determined by integrating the evoked dopamine oxidation current over 250 to 300 seconds post- stimulation, a period of time that was determined empirically to represent a point at which the evoked responses attained consistent and stable responding to the electrical stimulation.

Results Stereotaxic Placements of Recording and Stimulating Electrodes.

As shown in figure 3 (Medial Forebrain Bundle), histological analysis revealed that the tips of the stimulating electrodes (n=16) were confined within the anatomical boundaries of the medial forebrain bundle for all groups of mice, ranging from 1.94 to 2.18 mm posterior to bregma, 0.8 to 1.2 mm lateral to midline, and 4.0 to 4.6 mm ventral to the cortical surface of the brain. The carbon fibers of the electrochemical recording electrodes were confined within the dorsomedial striatum for all strains of mice, ranging from 1.34 to 1.54 mm anterior to bregma, 1.0 to 1.75 mm lateral to midline, and (shank to tip) 1.75 to 2.5 mm ventral to the cortical surface of the brain (figure 3, Striatum).

Striatal Dopamine Release during the First 100 Seconds of Burst, Continuous Intermittent, and Continuous Stimulation

As shown in figure 4, the pattern and level of change in striatal dopamine oxidation current (dopamine release) recorded during the first 100 seconds of electrical stimulation of the medial forebrain bundle was markedly different for each group. Compared to all other groups of mice, the first bout of burst- like stimulation elicited a relatively lower level of striatal dopamine release (figure 4, Group 1 dark line). However, by 20 seconds of stimulation (3^rd bout) evoked dopamine release had increased to levels significantly greater than all the other groups of mice. The highest level of dopamine release evoked by medial forebrain bundle stimulation in Group 1 was attained within 60 seconds of stimulation (7^th bout) and thereafter declined to reach steady-state evoked levels within 200 seconds of stimulation.

By comparison, Group 2 (3 seconds on, 7 seconds off) achieved the next highest level of steady-state evoked dopamine release in the striatum and also exhibited a gradual increase in the level of dopamine release with each bout of stimulation (figure 4, Group 2 dark line). Compared to the peak level of dopamine release attained within 60 seconds of stimulation by Group 1 , the highest level of evoked dopamine release with this stimulation parameter occurred within 40 seconds of stimulation (5^th bout). In marked contrast to Group 1 however, this peak increase in the level of dopamine release was followed by a more progressive and rapid decay achieving a significantly lower steady-state level of dopamine release within 200 seconds of stimulation.

Compared to Groups 1 and 2, Group 3 (3 seconds on, 4.2 seconds off) attained a significantly lower steady-state level of evoked striatal dopamine release (figure 4, Group 3 dark line). In addition, this stimulation parameter did not lead to an initial peak increase in dopamine release as was observed in Groups 1 and 2, but rather exhibited only a decline in the height of each evoked response immediately following the first bout of stimulation. This progressive decline was also significantly faster compared to Groups 1 and 2 with a steady-state level of evoked dopamine release attained within 64 seconds of stimulation (10^th bout).

Compared to all other groups, Group 4 exhibited the lowest steady-state level of evoked striatal dopamine release (figure 4, Group 4 black line). The pattern of dopamine release evoked by a continuous application of 100 Hz pulses led to an immediate exponential decline in the recorded amperometric signal reaching a steady- state level within 200 seconds of stimulation. As Groups 1, 2, and 4 did not attain steady-state levels of evoked dopamine release until after 200 seconds of stimulation (with only Group 3 reaching steady-state within 64 seconds of stimulation), statistical comparisons of significant differences between these groups were conducted on evoked dopamine levels between 250 and 300 seconds of stimulation (50 second bin). These data are described below.

Striatal Dopamine Release during the Period of 250-300 Seconds of Burst, Continuous Intermittent, and Continuous Stimulation Figure 5 shows the mean ± SEM steady-state levels of dopamine oxidation current recorded in the striatum during the period of 250 seconds to 300 seconds of medial forebrain bundle stimulation with each of the three test stimulation parameters (Gl : Burst-like; G2: Continuous 3 seconds on, 7 seconds off; and G3: Continuous 3 seconds on, 4.2 seconds off) and control stimulation parameter (G4: Continuous). As noted above, steady-state levels of evoked dopamine release were attained within 200 seconds of stimulation for Groups 1, 2, and 4 and within 64 seconds for Group 3. Group 1 elicited the highest level of steady-state striatal dopamine release, with Group 2 the second highest, followed by Group 3, and Group 4 the lowest level. In addition to Group 1 having the highest overall level of dopamine release, this group also exhibited relatively higher patterns of release during each bout of stimulation superimposed on the basal increase in dopamine in response to burst-like stimulation (figure 5 Group 1 , dark line (a single bout of stimulation is indicated by a black bar for each of the test stimulation parameters)). By comparison, continuous application of 300 second pulses at 100 Hz yielded significantly lower patterns of dopamine release for both Group 2 and 3 and correspondingly lower overall levels of dopamine release compared to Group 1 (figure 5 Group 2, Group 3). In contrast, continuous stimulation at 100 Hz (figure 5 Group 4, black line) exhibited similar patterns of dopamine release as observed in Group 3, but attained a significantly lower overall level of dopamine release.

Steady-State Striatal Dopamine Release Levels Attained with Burst, Continuous Intermittent, and Continuous Stimulation

The average (n=4 per group) overall levels of striatal dopamine release evoked by stimulation of the medial forebrain bundle for each of the three test stimulation parameters, and control stimulation parameter, were assessed by summing dopamine oxidation current over the period of 250-300 seconds after electrical stimulation had begun (figures 6-7). Under these conditions, the average (mean ± SEM throughout) value of summed dopamine oxidation current for Group 1 (burst stimulation), Group 2 (3 seconds on, 7 seconds off), Group 3 (3 seconds on, 4.2 seconds off), and Group 4 (continuous) were 79.4 ± 2.8, 46.9 ± 2.1, 36.3 ± 2.1, and 28.7 ± 1.4 nAmps, respectively (figure 8). All groups were found to be significantly different from one another according to multiple t-test analyses between Groups 1 and 2 (p < 0.0001) (figure 9), Groups 1 and 3 (p < 0.0001) (figure 10), Groups 1 and 4 (p < 0.0001) (figure 11), Groups 2 and 3 (p = 0.006) (figure 12), Groups 2 and 4 (p < 0.0001) (figure 13), and Groups 3 and 4 (p = 0.013) (figure 14).

Discussion The results provided herein suggest that utilizing burst-like stimulation parameters for DBS in the treatment of PD can allow for greater range of dopamine release. More flexibility for the electrical stimulation system can modulate the level of dopamine in the basal ganglia to effectively alleviate symptoms of PD. Burst- like stimulation can evoke the greatest amount of dopamine release both initially and over an extended period of stimulation, compared to all other stimulation parameters.

Burst stimulation can also reduce the overall duty cycle of the stimulator, and extend the battery life of the stimulator. For example, if a battery presently lasts for 2.5 years on a continuous stimulation schedule, the increase in battery life using a burst stimulation schedule (i.e. 0.2 seconds on/off train for 5.8 seconds with 4.2 seconds off) can be calculated to result in a battery life of approximately 9.6 years. This would constitute provide a much longer period between surgical replacement of the battery or eliminate the need altogether, for some patients.

Example 2 - High frequency stimulation of the subthalamic nucleus evokes striatal dopamine release in a large animal model of human DBS neurosurgery Using electrochemical techniques, including fast scan cyclic voltammetry (FSCV), high frequency stimulation (HFS) of the subthalamic nucleus (STN) is capable of evoking striatal dopamine release in the intact rat (Lee et al., Eur. J. Neurosci., 23:1005-1014 (2006) and Covey et al., Monitoring subthalamic nucleus- evoked dopamine release in the striatum using fast-scan cyclic voltammetry in vivo. In: P. E. M. Phillips, S. G. Sandberg, S. Ahn, A. G. Phillips (Eds.), Monitoring Molecules in Neuroscience. University of British Columbia, Vancouver, BC, 2008, pp. 398-400). In addition, significant striatal dopamine release evoked by HFS of the STN was detected in a well-recognized animal model of PD (dopamine nigrostriatal pathway lesions with 6-OHDA) (Blaha et al., Striatal dopamine release evoked by subthalamic stimulation in intact and 6-OHDA-lesioned rats: Relevance to deep brain stimulation in Parkinson's Disease. In: P. E. M. Phillips, S. G. Sandberg, S. Ahn, A. G. Phillips (Eds.), Monitoring Molecules in Neuroscience. University of British Columbia, Vancouver, BC, 2008, pp. 395-397).

Using a large animal (pig) model of human STN DBS neurosurgery, FSCV was applied in combination with a carbon-fiber microelectrode (CFM) stereotactically implanted into the striatum to monitor dopamine release evoked by a human DBS electrode unilaterally implanted into the STN. Several characteristics make the pig a useful animal model to investigate STN DBS mechanisms in that it has face validity with human DBS surgery, and rivals that of DBS studies in non-human primate (Zhao et al, Brain Res., 1286:230-238 (2009)). Adult pig brains (-160 g) are comparable in size to that of rhesus monkeys (-100 g) and baboons (-140 g), the volume of the pig STN (50±7 mm³) is comparable to that of the rhesus monkey (34±6 mm3) (Hardman et al., J. Comp. Neurol., 445:238-255 (2002)), and a pig brain atlas is available with significant similarities with human and non-human primates (Felix et al., Brain Res. Bull., 49:1-137 (1999)).

The results provided herein demonstrate that the magnitude and temporal pattern of dopamine release evoked by STN electrical stimulation in pig was both dependent upon stimulation frequency and intensity. Moreover, maximal dopamine release was obtained with stimulation parameters that are typically used with therapeutic DBS in PD patients.

The following experiments were performed in accordance with NIH guidelines (publication 86-23) and approved by the Mayo Clinic Institutional Animal Care and Use Committee. Four male pigs, weighing 26 - 30 kg, were initially sedated with Telazol (5 mg/kg i.m.) and xylazine (2 mg/kg i.m.), then intubated with endotracheal tube and ventilated with a artificial ventilator, then maintained with isoflurane (1%) for the remaining experimental procedure. In the prone position, the pig was placed in a MRI-compatible stereotactic head frame. A localizer box was then attached onto the head frame to create nine fiducials to enable localization of MR images in stereotactic space. For preoperative targeting of the STN, MRI was performed with a General Electric Signa 3.0 T system. The DICOM image data were then transferred to a stereotactic planning computer, and the anterior commissure - posterior commissure line identified on the MR images. Using COMPASS navigational software, MRI data was then merged with a pig atlas (Felix et al., Brain Res. Bull., 49:1-137 (1999)) and stereotactic coordinates for the DBS electrode implantation trajectory defined (Figure 15A and B).

Thereafter, in the operating room, a large midline incision of the skin was made to expose the cranial landmarks of bregma and lambda. This was followed by a burr hole made on the skull in line with the trajectory coordinates. A tungsten extracellular microelectrode (0.5-1.0 MΩ), mounted onto a microdrive, was then lowered using the same trajectory for the DBS electrode obtained by the navigation software to identify the final dorsoventral coordinates of the STN. Following electrophysiologic confirmation of the STN coordinates, a Medtronic 3389 human DBS electrode was then implanted into the STN target. The pig was then returned to the MRI scanner for postoperative confirmation of the placement of the DBS electrode (Figure 15D). For FSCV measurements, the recording CFM was prepared by aspirating a single carbon-fiber into a glass capillary, pulling to a fine taper on a pipette puller (Model PE-2, Narashige, Tokyo, Japan), and sealing with nonconducting epoxy (Cahill et al., Anafyt. Chem., 68:3180-3186 (1996)). Borosilicate capillaries containing the carbon fiber were pulled by a laser-based micropipette puller (P-2000, Sutter Instrument Co., Navato, CA). FSCV recordings of STN DBS evoked striatal dopamine release were performed using the wireless instantaneous neurotransmitter concentration system (WINCS) as described elsewhere (Bledsoe et al., J. Neurosurg., I l l :712-723 (2009) and Shon et al., Co-monitoring of adenosine and dopamine using the Wireless Instantaneous Neurotransmitter Concentration System (WINCS): proof of principle, J. Neurosurg., E-Pub Ahead of Print (2009)). FSCV not only supports sub-sec measurements at a CFM, but additionally provides a chemical signature in the form of a background-subtracted voltammogram to identify the chemical origin of the signal (Robinson et al., Clin. Chem., 49:1763-1773 (2003)). FSCV parameters for dopamine measurements consisted of a -0.4 V rest potential, 1.5 V peak potential, 400 V/s scan rate, and 10 Hz scan application. In addition, to complete the circuit necessary for the FSCV a 14 mm burr hole was drilled on the contralateral side of the skull to allow the placement of an Ag/ AgCl reference electrode. In a similar fashion to the DBS electrode, preoperative MRI data combined with the pig atlas were used to define the trajectory and final stereotactic coordinates for implantation of the CFM electrode into the center of the head of the caudate (striatum; Figure 15C).

Post-m vzvo calibrations of the CFMs utilized in each experiment were performed using a flow injection analysis system (Kristensen et al., Analyt. Chem., 58:986-988 (1986) and Bledsoe et al., J. Neurosurg., 111 :712-723 (2009)). Briefly, individual CFMs were placed in a flowing stream of solution in which various concentrations of dopamine (1 to 10 μM) were injected sequentially over a 10 second period using an electronic loop injector. The flowing solution consisted of buffered physiological saline (150 mM sodium chloride and 25 mM Hepes at a pH 7.4) pumped at a rate of 4 niL/minute across the CFM, positioned in the center of the inflow tubing to a Plexiglas reservoir. An Ag/ AgCl reference electrode was placed in the bottom of the flow cell reservoir immersed in the buffer solution. Injection of each dopamine concentration permitted the establishment of a dopamine calibration curve to assess the relationship between stimulation-evoked increases in dopamine oxidation current and dopamine extracellular concentration. Using a hand held stimulator (Medtronic screener model 3628, Medtronic Inc.,

MN), a series of two-second duration biphasic, charge-balanced, voltage-isolated stimulations were delivered between lead 0 (most distal) and lead +1 of the Medtronic 3389 electrode (Medtronics Inc.) implanted in the STN. Electrical stimulation parameters included various intensities of stimulation at 3, 4, 5, and 7 volts with frequency held constant at 120 Hz, while frequency dependency was tested at 60, 90, 120, 150, 180, and 240 Hz with intensity held constant at 3 V, all at 0.5 msec pulse width. To maximize the reproducibility of each STN-evoked dopamine response, these stimulations were repeated on the basis of the number of pulses applied (a 5 minute interstimulus interval was incrementally added to the total interstimulus period for every 120 pulses delivered).

Comparison of the pre- and post-operative MRI confirmed the accurate placement of the DBS electrodes within the STN of each of the four pigs tested (see Figures 15A and D). As shown in Figure 16A (Inset), STN DBS (2 s, 120Hz, 0.5 ms pulse width and 5 V) results in a voltammogram, a plot of measured dopamine oxidation (ox.) and reduction (rd.) current versus the applied potential, that provides a signature to identify the recorded chemical. For dopamine, oxidation during the positive scan and reduction of the electro formed dopamine ortho-quinone (DOQ) back to dopamine during the negative scan results in two distinct current peaks. These peaks are clearly observed when voltammograms recorded in the striatum are plotted sequentially, with oxidation and reduction current magnitude designated by a pseudo-color plot (Figure 16B; x-axis: time [sec], y-axis: applied scanning potential [V], z-axis: ox. and rd. current [nA]) or with only oxidation current magnitude plotted with respect to time (Figure 16A: x-axis: time [sec], y-axis: current [nA]) during and after electrical stimulation of the STN (Bergstrom et al., J. Neurosci. Methods, 87:201-208 (1999)). Changes in the amplitude of the dopamine oxidation peak recorded by FSCV thus provide a quantitative concentration measurement of the temporal effects of electrical stimulation of the STN on dopamine release.

As shown in Figure 17A for one representative pig, while holding the STN stimulation frequency constant (120 Hz), increasing voltage intensity from 3 to 7 V resulted in a progressive increase in the magnitude of striatal dopamine release and duration of recovery to pre-stimulation baseline levels. A plot of the maximal increases in dopamine release observed at each of the stimulation intensities revealed clear sigmoidal-like intensity-dependent responses for three of the pigs tested (Figure 17B). In contrast, with stimulation intensity held constant (3 V), increasing stimulation frequency from 60 to 240 Hz resulted in a linear increase in the peak magnitude of striatal dopamine release within 60 to 120 Hz and a plateau at and above 120 Hz (Figure 17C). The duration of post-stimulation recovery to pre-stimulation baseline levels for 60 to 240 Hz ranged from 4 to 10 sec (data not shown). In summary, the results provided herein demonstrate that electrical stimulation of the STN in the large animal (pig) model of DBS surgery elicited a stimulation time-locked increase in striatal dopamine release that was dependent on both stimulation intensity and frequency. In the case of stimulation intensity, the increase in evoked dopamine release exhibited a sigmoidal pattern in all three pigs tested, attaining a plateau at around 7 V of stimulation with a fixed frequency of 120 Hz. However, the maximal concentration in extracellular dopamine attained by these applied stimulus intensities exhibited a relatively high degree of variance between animals (1-4 μM). The variance observed in intensity evoked dopamine responses between animals are not unexpected given the heterogeneity of dopaminergic innervation of the striatum where the recording electrode was located (Nomoto et al., J. Neurol., 247:16-22 (2000)) and the precise location of the stimulating electrode within the STN. In contrast to the stimulation intensity dependency, over a range of stimulation frequencies (60 to 240 Hz) and fixed intensity (3 V), striatal dopamine release exhibited a linear increase up to 120 Hz and thereafter attained a plateau. Anatomically, STN glutamatergic neurons project to dendrites of SNc dopaminergic cells (Albin et al., Trends Neurosci., 12:366-375 (1989) and Kita and Kitai, J. Comp. Neurol., 260:435-452 (1987)), suggesting that STN stimulation evoked increases in dopamine release in the ipsilateral striatum likely results from STN glutamatergic activation of SNc dopaminergic neurons (Lee et al., Eur. J. Neurosci., 23:1005-1014 (2006) and Shimo and Wichmann, Eur. J. Neurosci., 29: 104-113 (2009)). In the rat, similar STN electrical stimulation resulted in excitatory postsynaptic potentials in SNc dopaminergic neurons in vitro (Lee et al., Stereotactic. Fund. Neurosurg., 80:32-36 (2003) and Lee et al., J. Neurosurg., 101 :511-517 (2004)) and increases in striatal dopamine release in vivo (Lee et al., Eur. J. Neurosci., 23:1005-1014 (2006)). Alternatively, given the placement of the DBS electrodes, it is also conceivable that the evoked dopamine release may be due to direct stimulation of dopaminergic fibers of passage which course within the medial forebrain bundle lying immediately dorsal to the STN (Lee et al., Eur. J. Neurosci., 23:1005-1014 (2006)).

Optimal clinical results in PD patients have been routinely obtained using stimulation intensities of 2-5 V and frequencies of 120-180 Hz delivered in the STN (Garcia et al., Trends Neurosci., 28:209-216 (2005) and Moro et al., Neurology, 59:706-713 (2002)). Thus, maximal dopamine release observed here in striatum of the pig was obtained only with STN stimulation parameters that fell within the range of human therapeutic DBS parameters. In the rat, striatal dopamine release could be evoked using low to high frequency stimulation (5-300 Hz) of the STN (Lee et al., Eur. J. Neurosci., 23:1005-1014 (2006)). In contrast to the present results, a peak dopamine release response in the rat was seen at 50 Hz with the response falling off at 75 Hz and greater. As such, these present findings highlight the importance of examining neurochemical mechanisms of STN DBS in a large animal model (pig) that more closely represents human neuroanatomy and DBS electrode configuration. Taken together, these results demonstrate that dopamine neuronal activation can be studied in pigs, a versatile and economically affordable animal model of human STN DBS. These results in combination with the results presented in Example 1 demonstrate that human STN DBS also results in dopamine release.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for increasing dopamine release from the basal ganglia of a mammal, said method comprising applying an electrical stimulus to the medial forebrain bundle of the brain of said mammal, wherein said electrical stimulus comprises an intermittent stimulation.

2. The method of claim 1, wherein said stimulus is delivered by an electrode connected to an implanted neurostimulator.

3. The method of claim 1 , wherein the frequency of said electrical stimulus is from about 100 to about 250 Hz.

4. The method of claim 1 , wherein the current intensity of said electrical stimulus is about 800 μA.

5. The method of claim 1, wherein said intermittent stimulus comprises a rest period of between about 4 seconds and about 7 seconds subsequent to said electrical stimulus.

6. The method of claim 5, wherein said intermittent stimulus cycles continuously.

7. The method of claim 6, wherein said electrical stimulus comprises a burst-like stimulation.

8. The method of claim 7, wherein said burst- like stimulation comprises an alternating cycle of a 0.2 second current pulse followed by a 0.2 second inter-stimulus rest.

9. The method of claim 8, wherein said intermittent stimulus comprises about twenty alternating cycles.

10. A method for treating a human having Parkinson's disease, said method comprising applying an intermittent electrical stimulus to the medial forebrain bundle of the brain of said human, wherein said stimulus evokes dopamine release in the striatum of said brain, thereby treating said human.

11. The method of claim 10, wherein said stimulus is delivered by an electrode connected to an implanted neurostimulator.

12. The method of claim 10, wherein the frequency of said electrical stimulus is from about 100 to about 250 Hz.

13. The method of claim 10, wherein the current intensity of said electrical stimulus is about 800 μA.

14. The method of claim 10, wherein said intermittent stimulus comprises a rest period of between about 4 seconds and about 7 seconds subsequent to said electrical stimulus.

15. The method of claim 14, wherein said intermittent stimulus cycles continuously.

16. The method of claim 15, wherein said electrical stimulus comprises a burst- like stimulation.

17. The method of claim 16, wherein said burst-like stimulation comprises an alternating cycle of a 0.2 second current pulse followed by a 0.2 second inter-stimulus rest.

18. The method of claim 17, wherein said intermittent stimulus comprises about twenty alternating cycles.