Neurobiology of Disease High-Frequency Deep Brain Stimulation of the Nucleus Accumbens Region Suppresses Neuronal Activity and Selectively Modulates Afferent Drive in Rat Orbitofrontal Cortex In Vivo Clinton B. McCracken and Anthony A. Grace Departments of Neuroscience, Psychiatry, and Psychology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260 High-frequency deep-brain stimulation (DBS) of the nucleus accumbens (NAc) region is an effective therapeutic avenue for patients with treatment-resistant obsessive���compulsive disorder (OCD). Imaging studies suggest that DBS acts by suppressing the aberrant metabo- lism in the orbitofrontal cortex (OFC) that is a hallmark of OCD however, little is known about the mechanisms by which this occurs. We examined the effects of 30 min NAc DBS at 130 Hz on spontaneously active OFC neurons and local field potentials (LFPs) in addition to evoked responses elicited by single-pulse stimulation of the NAc or mediodorsal thalamus (MD) in urethane-anesthetized rats. NAc DBS reduced the mean firing rate of OFC neurons, although neurons receiving monosynaptic input from MD were less affected and some putative interneurons were excited by DBS. Single-pulse stimulation of the NAc produced a robust inhibition in OFC neurons that was attenuated after DBS, whereas excitatory responses were unchanged. In contrast, after DBS inhibitory responses evoked from MD were unchanged, whereas excitatory responses were enhanced. NAc-evoked LFP responses were potentiated after DBS, whereas MD-evoked LFP responses were unchanged. NAc DBS also enhanced OFC spontaneous LFP oscillatory activity in the slow (0.5���4 Hz) frequency band. These results suggest that DBS of the NAc region may alleviate OCD symptoms by reducing activity in subsets of OFC neurons, potentially by driving recurrent inhibition though antidromic activation of corticostriatal axon collaterals. Moreover, selective potentiation of input to these inhibitory circuits may also contribute to the therapeutic effects produced by DBS in OCD patients. Key words: electrical stimulation nucleus accumbens prefrontal thalamus electrophysiology obsessive���compulsive Introduction High-frequency (HF) electrical stimulation of specific subcorti- cal structures, known as deep-brain stimulation (DBS), has at- tracted substantial attention for treatment of severe neurological and psychiatric disorders that fail to respond to pharmacothera- peutic intervention. Thus, DBS of thalamic and subthalamic nu- clei is a relatively safe and efficacious alternative to ablative neu- rosurgery for Parkinson���s disease and other movement disorders (Benabid et al., 1991, 1994 Krack et al., 2003 Rehncrona et al., 2003 Rodriguez-Oroz et al., 2005 Deuschl et al., 2006 Perlmut- ter and Mink, 2006). More recently, DBS of the ventral anterior internal capsule and ventral striatum has proven to be an effective therapeutic approach for treatment-resistant obsessive���compul- sive disorder (OCD) (Nuttin et al., 1999 Anderson and Ahmed, 2003 Gabriels et al., 2003 Nuttin et al., 2003 Sturm et al., 2003 Abelson et al., 2005 Aouizerate et al., 2005 Greenberg et al., 2006). However, the neuronal mechanisms underlying the ther- apeutic actions of DBS in OCD remain unclear. Imaging studies have provided insight into the brain systems involved in OCD and its treatment. Metabolic hyperactivity in the striatum, medial thalamus, and, in particular, the orbitofron- tal cortex (OFC) has been consistently associated with OCD pa- tients at rest (Baxter et al., 1988 Swedo et al., 1989) and is accen- tuated after symptom provocation (McGuire et al., 1994 Rauch et al., 1994 Breiter et al., 1996). Moreover, successful pharmaco- logical (Swedo et al., 1992), cognitive-behavioral (Schwartz et al., 1996), and neurosurgical (Mindus et al., 1986) treatment of OCD have all been associated with reductions in activity in these same regions. Significantly, studies using DBS for OCD have also shown that successful treatment correlates with lowered activity in frontal cortical regions (Nuttin et al., 2003 Abelson et al., 2005 Van Laere et al., 2006). The cellular mechanisms by which DBS produces therapeutic effects in OCD are not known. Drawing from the Parkinson���s disease and movement disorders literature, DBS is proposed to act by decreasing neuronal activity within the stimulated nucleus, which is postulated to occur either by depolarization blockade or increase in local GABAergic transmission (Boraud et al., 1996 Benazzouz et al., 2000 Dostrovsky et al., 2000 Beurrier et al., Received June 26, 2007 revised Sept. 21, 2007 accepted Sept. 22, 2007. This work was supported by National Institutes of Health Grants MH57440, DA15408, and MH45156 (A.A.G.) and a by National Alliance for Research on Schizophrenia and Depression Young Investigator Award (C.B.M.). We thank N. Macmurdo and C. Smolak for technical assistance, B. Lowry for data acquisition software, and W. Lipski for assistance with MATLAB. Correspondence should be addressed to Dr. Clinton B. McCracken, Department of Neuroscience, A210 Langley Hall, University of Pittsburgh, Pittsburgh, PA 15260. E-mail: cbm12@pitt.edu. DOI:10.1523/JNEUROSCI.3750-07.2007 Copyright �� 2007 Society for Neuroscience 0270-6474/07/2712601-10$15.00/0 The Journal of Neuroscience, November 14, 2007 ��� 27(46):12601���12610 ��� 12601

2001 Kiss et al., 2002 Magarinos-Ascone et al., 2002 Lian et al., 2003). However, given that electrical stimulation preferentially activates axons as opposed to cell bodies (Nowak and Bullier, 1998a,b McIntyre and Grill, 1999), it has been proposed that DBS may produce inhibition of the stimulated area but activation of efferent and afferent axons (Vitek, 2002 McIntyre et al., 2004a,b). Thus, characterizing the effects of HF stimulation on nuclei upstream and downstream from the stimulation site is necessary for clarifying the system-level actions of DBS. The pur- pose of the present study was to investigate the effects of HF electrical stimulation of the nucleus accumbens (NAc) region on spontaneous and evoked neuronal and local field potential (LFP) activity in the OFC in vivo. Materials and Methods Animals and surgery. Male Sprague Dawley rats (275���400 g) were anes- thetized with urethane (1.5 g/kg, i.p.) and placed in a stereotaxic frame. Body temperature was maintained at 37��C with a temperature- controlled heating pad. In all surgical preparations, the scalp was ex- posed, and burr holes were drilled in the skull overlying the lateral OFC, the NAc core, and the mediodorsal thalamus (MD). Concentric bipolar stimulating electrodes (NEX-100 David Kopf Instruments, Tujunga, CA) were placed according to the following coordinates: NAc electrode: anteroposterior (AP), 1.2 mm (from bregma) mediolateral (ML), 2.0 mm dorsoventral (DV), 6.9 mm (from skull) MD electrode: AP, 3.2 mm ML, 0.7 mm DV, 5.5 mm. All procedures were performed in accordance with the guidelines outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were ap- proved by the Institutional Animal Care and Use Committee of the Uni- versity of Pittsburgh. Single-unit recordings. Extracellular recording microelectrodes were constructed from borosilicate glass tubing using a vertical microelec- trode puller (Narishige, Tokyo, Japan), with the tip broken back under microscopic control and filled with 2% pontamine sky blue dye dissolved in 2 M NaCl (impedance measured in situ ranged between 6 and 14 M ). A burr hole was drilled over the OFC, the dura was resected, and a recording electrode was lowered slowly into the OFC (AP, 3.2 mm ML, 3.5 mm DV, 4.7���6.2 mm from skull) using a hydraulic micromanip- ulator. Signals from the recording electrode were amplified by a head stage connected to a preamplifier before being amplified and filtered (0.05���16 kHz) by a window discriminator/amplifier unit (Fintronics, Orange, CT). Signals were also sent to an audio monitor (AM8 Grass Instruments, Quincy, MA) and displayed on an oscilloscope (Tektronics, Wilsonville, OR). The data were acquired, stored, and analyzed using custom-designed computer software (Neuroscope) with a data acquisi- tion board interface (Microstar Laboratories, Bellevue, WA). To identify OFC neurons responsive to single-pulse stimulation of the NAc or MD electrode, the microelectrode was lowered slowly through the OFC while stimuli were delivered alternately to the NAc and MD electrodes (1000 A) at 2 s intervals (i.e., each area was stimulated at 0.25 Hz). Cathodal constant current pulses (0.2 ms duration) were given us- ing a Grass Instruments S88 stimulator and a Grass Instruments photo- electric stimulus isolation unit. When a responsive neuron was identi- fied, 5 min of baseline activity were recorded, after which peristimulus time histograms were constructed for 50 stimulations from each elec- trode. Two seconds of data were collected before and after the stimulus for each stimulus ���sweep.��� Both inhibitory and excitatory responses were observed. Single-pulse stimulation of the NAc electrode frequently re- sulted in antidromic spikes recorded in OFC however, because our aim in this study was to characterize the effects of DBS on spontaneous activ- ity, the responses of antidromically activated OFC neurons to NAc DBS were not examined because of potential confounds as a consequence of this activation. Responsive neurons were classified as either putative py- ramidal neurons or putative projection neurons based on the spike du- ration frequency histogram constructed from all OFC neurons recorded. Spike duration has proven to be a reliable measure for classifying neuron types in the frontal cortex in vivo (Tierney et al., 2004 Tseng et al., 2006). In this study, neurons with spike durations of 0.9 ms were classified as putative interneurons, and neurons with spike durations 1.1 ms were classified as putative pyramidal neurons. Inhibitory responses before and after DBS were characterized accord- ing to the following: (1) the latency until inhibition onset (2) duration of inhibition and (3) changes in the magnitude of inhibition after DBS. The change in magnitude of inhibition after DBS was evaluated in two ways: (1) for a raw measure, the number of spikes discharged in the first 200 ms after stimulus was determined and (2) for a normalized measure, the average percentage of baseline firing in the first 200 ms was calculated using 25 ms bins. Excitatory responses were considered monosynaptic and presumed orthodromic if the response occurred within 15 ms of the stimulus, dis- played spike ���jitter��� of at least 2 ms and a shift in spike latency with increasing current amplitude, and failed to follow 400 Hz paired-pulse stimulation (otherwise characterized as antidromic). Excitation was also quantified using complementary measures, including calculation of spike probability (i.e., the percentage of stimulations that result in an evoked spike), the number of spikes occurring within the first 25 ms after stimulus, and the percentage of baseline firing in the first 25 ms when calculated using 25 ms bins. With regard to MD-evoked responses, it has been suggested that some longer-latency excitatory responses in the pre- frontal cortex (PFC) may represent antidromic activation of corticotha- lamic afferents that invades local PFC recurrent collaterals (Pirot et al., 1994). Because the OFC has reciprocal connections with MD in a manner analogous to the medial PFC (mPFC) (Krettek and Price, 1977 Groe- newegen, 1988), latencies longer than 15 ms for MD3OFC excitatory responses were not included in the analysis to rule out this class of re- sponse (Pirot et al., 1994). After recording evoked activity under baseline conditions, neurons were allowed to equilibrate for 3���5 min. An additional 5 min of baseline activity was then collected before recording with DBS-on for 30 min. The DBS parameters used (0.1���0.4 mA, 100 S pulse duration, 130 Hz) have been shown to be efficacious in rat Parkinson���s models (Chang et al., 2003 Degos et al., 2005) and are reported to cause negligible trauma to the stimulated area. This DBS protocol produced no visible lesions in the NAc under 25 light microscopy with cresyl violet staining. After 30 min of recording with DBS-on, baseline activity was recorded for an addi- tional 5 min, after which evoked activity was reassessed. LFPs. When filter settings were adjusted to allow preferential passage of low-frequency (LF) signals (i.e., 0.1���1000 Hz), single-pulse stimula- tion of either the NAc or MD resulted in large-amplitude LFP responses in the OFC. To determine the relative contributions of GABA and gluta- mate to these evoked LFP responses, evoked LFPs were recorded using a chemotrode (Plastics One, Roanoke, VA) that also allowed local infusion of drugs. The chemotrode consisted of two individually polyimide- insulated stainless steel wires (0.2 mm diameter) attached to an insulated stainless steel 26 gauge guide cannula. The wires extended 1 mm past the end of the guide cannula when inserted, the tip of the injection cannula used was flush with the recording surface of the electrode. LFP responses were assessed after infusion of the GABAA receptor antagonist ( )- bicuculline methiodide or the broad-spectrum ionotropic glutamate re- ceptor antagonist kynurenic acid. Drugs or vehicle [Dulbecco���s PBS (DPBS) Sigma, St. Louis, MO) were administered over 2 min using a 30 gauge injection cannula connected to a Hamilton syringe (bicuculline, 0.2 g in 0.5 l of DPBS kynurenic acid, 10 g in 0.5 l of DPBS vehicle, 0.5 l of DPBS). In a separate set of animals, using low-impedance glass microelectrodes (1���4 M ), baseline input���output curves were con- structed for these responses (0.2���1.0 mA, 10 stimulations at each current intensity, stimulation at 0.4 Hz), then reassessed 5 and 90 min after HF (130 Hz) or LF (10 Hz) DBS. Spontaneous LFP activity was recorded immediately before and after 30 min DBS and occasionally during the first 5 min and last 5 min of DBS-on. We also examined the effects of the NMDA receptor antagonist ( )-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate (MK-801) on evoked LFPs before and after DBS. MK-801 (Sigma) was dissolved in vehicle (DPBS) and administered intraperitoneally. Data analysis. Data are presented as mean SEM. Spontaneous unit activity was assessed in terms of firing rate and the proportion of spikes 12602 ��� J. Neurosci., November 14, 2007 ��� 27(46):12601���12610 McCracken and Grace ��� Accumbens DBS and Orbitofrontal Neuronal Activity