New Subregions of the Mouse Entopeduncular Nucleus Defined by the Complementary Immunoreactivities for Substance P and Cannabinoid Type-1 Receptor Combined with Distributions of Different Neuronal Types.

The entopeduncular nucleus (EPN) and substantia nigra pars reticulata (SNr) constitute the output nuclei of the basal ganglia, but studies on the EPN are limited compared with those on the SNr. Both nuclei receive projections from the striatum with axons containing substance P (SP) and cannabinoid type-1 receptor (CB1R), and immunoreactivities for these substances show complementary patterns in the striatum and SNr. In this study, we revealed a similar complementarity in the mouse EPN, combined it with region-specific neuronal distributions, and defined subregions of the EPN. First, the EPN was divided into two areas, one showing low SP and high CB1R (lSP/hCB1R) immunoreactivities, and the other showing high SP and low CB1R (hSP/lCB1R). The former received inputs from the dorsolateral striatum that are innervated by sensorimotor cortices, whereas the latter received inputs from the medial striatum that are innervated by limbic/association cortices. Then, the lSP/hCB1R area was further divided into the dorsolateral subregion in the rostral EPN and the core subregion in the caudal EPN, the latter characterized by the concentration of parvalbumin-positive neurons targeting the ventral anterior-ventral lateral thalamic nucleus. The hSP/lCB1R area was divided into the ventromedial subregion in the rostral EPN and the shell subregion in the caudal EPN, the former characterized by the concentration of nitric oxide synthase-positive neurons targeting the lateral habenula (LHb). Somatostatin-positive neurons targeting the LHb were located diffusely in three subregions other than the core. These findings illuminate structural organization inside the basal ganglia, suggesting mechanisms for sorting diverse information through parallel loops with differing synaptic modulation by CB1R.

Deep brain stimulation by optimized stimulators in a phenotypic model of dystonia: Effects of different frequencies.

Deep brain stimulation (DBS) of the globus pallidus internus (GPi, entopeduncular nucleus, EPN, in rodents) has become important for the treatment of generalized dystonia, a severe and often intractable movement disorder. It is unclear if lower frequencies of GPi-DBS or stimulations of the subthalamic nucleus (STN) are of advantage. In the present study, the main objective was to examined the effects of bilateral EPN-DBS at different frequencies (130 Hz, 40 Hz, 15 Hz) on the severity of dystonia in the dtsz mutant hamster. In addition, STN stimulations were done at a frequency, proven to be effective by the present EPN-DBS in dystonic hamsters. In order to obtain precise bilateral electrical stimuli with magnitude of 50 µA, a pulse width of 60 µs and defined frequencies, it was necessary to develop a new optimized stimulator prior to the experiments. Since the individual highest severity of dystonic episodes is known to be reached within three hours after induction in dtsz hamsters, the duration of DBS was 180 min. During DBS with 130 Hz the severity of dystonia was significantly lower within the third hour than without DBS in the same animals (p < 0.05). DBS with 40 Hz tended to exert antidystonic effects after three hours, while 15 Hz stimulations of the EPN and 130 Hz stimulations of the STN failed to show any effects on the severity. DBS of the EPN at 130 Hz was most effective against generalized dystonia in the dtsz mutant. The response to EPN-DBS confirms that the dtsz mutant is suitable to further investigate the effects of long-term DBS on severity of dystonia and neuronal network activities, important to give insights into the mechanisms of DBS.

Contribution of the Entopeduncular Nucleus and the Globus Pallidus to the Control of Locomotion and Visually Guided Gait Modifications in the Cat.

We tested the hypothesis that the entopeduncular (EP) nucleus (feline equivalent of the primate GPi) and the globus pallidus (GPe) contribute to both the planning and execution of locomotion and voluntary gait modifications in the cat. We recorded from 414 cells distributed throughout these two nuclei (referred to together as the pallidum) while cats walked on a treadmill and stepped over an obstacle that advanced towards them. Neuronal activity in many cells in both structures was modulated on a step-by-step basis during unobstructed locomotion and was modified in the step over the obstacle. On a population basis, the most frequently observed change, in both the EP and the GPe, was an increase in activity prior to and/or during the swing phase of the step over the obstacle by the contralateral forelimb, when it was the first limb to pass over the obstacle. Our results support a contribution of the pallidum, in concert with cortical structures, to the control of both the planning and the execution of the gait modifications. We discuss the results in the context of current models of pallidal action on thalamic activity, including the possibility that cells in the EP with increased activity may sculpt thalamo-cortical activity.

Inactivation of endopeduncular nucleus impaired fear conditioning and hippocampal synaptic plasticity in rats.

The internal globus pallidus (GPi) is one part of basal ganglion nucleuses which play fundamental role in motor function. Recent studies indicated that GPi could modulate emotional processing and learning, but the possible mechanism remains still unknown. In this study, the effects of endopeduncular nucleus (EP, a rodent homolog of GPi) on fear conditioning were tested in rats. GABAA receptor agonist muscimol was bilaterally delivered into the EP 15 min before or immediately after fear conditioning in rats. We found that EP inactivation impaired the acquisition but not consolidation of fear memory in rats. Furthermore, the long-term potentiation (LTP) in hippocampal CA1 area was impaired, and the learning related phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) subunit 1 (GluA1) at the Ser845 site in hippocampus was decreased in muscimol treated group. These results demonstrated that dysfunction of EP impaired hippocampal dependent learning and memory in rats.

Optogenetic inactivation of the entopeduncular nucleus improves forelimb akinesia in a Parkinson's disease model.

We performed optogenetic inactivation of rats' entopeduncular nucleus (EP, homologous to primates' globus pallidus interna (GPi)) and investigated the therapeutic effect in a rat model of PD. 6-Hydroxydopamine (6-OHDA)-induced hemiparkinsonian rats were injected with either a virus for halorhodopsin expression that is used to inactivate GABAergic neurons or a control virus injection and received optic fiber insertion. All the rats were illuminated by 590 nm of light. Each rat was then subjected to sequential sessions of stepping tests under controlled illumination patterns. The stepping test is a reliable evaluation method for forelimb akinesia. The number of adjusting steps was significantly higher in experimental (optogene with reporter gene expression) (5Hz - 10ms: 15.7 ±â€¯1.9, 5Hz - 100ms: 16.0 ±â€¯1.8, continuous: 21.6 ±â€¯1.9) than control rats (reporter gene expression) (5Hz-10ms: 1.9 ±â€¯1.1, 5Hz-100ms: 2.6 ±â€¯1.0, continuous: 2.5 ±â€¯1.2) (p < 0.001). Continuous EP illumination showed a significantly higher improvement of forelimb akinesia than other illumination patterns (p < 0.01). Optogene expression in the GABAergic neurons of the EP was confirmed by immunohistochemistry. Optogenetic inhibition of EP was effective to improve contralateral forelimb akinesia. However, further studies using prolonged illumination are needed to investigate the best illumination pattern for optogenetic stimulation.

Long-term plasticity of glutamatergic input from the subthalamic nucleus to the entopeduncular nucleus.

The hyperdirect pathway of the basal ganglia bypasses the striatum, and delivers cortical information directly to the subthalamic nucleus (STN). In rodents, the STN excites the two output nuclei of the basal ganglia, the entopeduncular nucleus (EP) and the substantia nigra reticulata (SNr). Thus, during hyperdirect pathway activation, the STN drives EP firing inhibiting the thalamus. We hypothesized that STN activity could induce long-term changes to the STN->EP synapse. To test this hypothesis, we recorded in the whole-cell mode from neurons in the EP in acute brain slices from rats while electrically stimulating the STN. Repetitive pre-synaptic stimulation generated modest long-term depression (LTD) in the STN->EP synapse. However, pairing EP firing with STN stimulation generated robust LTD that manifested for pre-before post-as well as for post- before pre-synaptic pairing. This LTD was highly sensitive to the time difference and was not detected at a time delay of 10 ms. To investigate whether post-synaptic calcium levels were important for LTD induction, we made dendritic recordings from EP neurons that revealed action potential back-propagation and dendritic calcium transients. Buffering the dendritic calcium concentration in the EP neurons with EGTA generated long term potentiation instead of LTD. Finally, mild LTD could be induced by post-synaptic activity alone that was blocked by an endocannabinoid 1 (CB1) receptor blocker. These results thus suggest there may be an adaptive mechanism for buffering the impact of the hyperdirect pathway on basal ganglia output which could contribute to the de-correlation of STN and EP firing.

Effect of optogenetic modulation on entopeduncular input affects thalamic discharge and behavior in an AAV2-α-synuclein-induced hemiparkinson rat model.

OBJECTIVE: Neuromodulation of the globus pallidus internus(GPi) alleviates Parkinson's disease symptoms. The primate GPi is homologous to the rat entopeduncular nucleus (EP). The aim of the present study was to determine if optogenetic modulation of the EP could alter parkinsonian behavior or thalamic discharge in a hemiparkinson rat model. METHODS: We injected an adeno-associated virus type-2 expressing α-synuclein (AAV2-α-syn) into the substantia nigra pars compacta (SNc) of the right hemisphere and confirmed parkinsonian behavior using an amphetamine-induced rotation test. Then we injected activated or inhibited neurons, using the channelrhodopsin2 (ChR2)/halorhodopsin (NpHR) system in the EP of the hemiparkinson rat model and examined downstream effects in vivo. We assessed alterations in parkinsonian behaviors using the stepping and cylinder tests before, during, and after optogenetic stimulation. RESULTS: Importantly, optogenetic inhibition of the EP improved parkinsonian motor behaviors. When we monitored thalamic neuronal activity following optogenetic neuromodulation in vivo, and we observed alterations in thalamic discharge The thalamic neuronal activity is increased for optogenetic inhibition stimulation, whereas decreased for optogenetic activation stimulation. CONCLUSIONS: Taken together, our data demonstrate that optical neuromodulation of the EP can successfully control contralateral forelimb movement and thalamic discharge in an AAV2-α-synuclein-induced hemiparkinson rat model.

Dopaminergic Modulation of Synaptic Integration and Firing Patterns in the Rat Entopeduncular Nucleus.

Dopamine is known to differentially modulate the impact of cortical input to the striatum between the direct and indirect pathways of the basal ganglia (BG). However, the role of extrastriatal dopamine receptors (DRs) in BG information processing is less clear. To investigate the role of extrastriatal DRs, we studied their distribution and function in one of the output nuclei of the BG of the rodent, the entopeduncular nucleus (EP). qRT-PCR indicated that all DR subtypes were expressed by EP neurons, suggesting that both D1-like receptors (D1LRs) and D2-like receptors (D2LRs) were likely to affect information processing in the EP. Whole-cell recordings revealed that striatal inputs to the EP were potentiated by D1LRs whereas pallidal inputs to the EP were depressed by D2LRs. Changes to the paired-pulse ratio of inputs to the EP suggested that dopaminergic modulation of striatal inputs is mediated by postsynaptic receptors, and that of globus pallidus-evoked inputs is mediated by presynaptic receptors. We show that these changes in synaptic efficacy changed the information content of EP neuron firing. Overall, the findings suggest that the dopaminergic system affects the passage of feedforward information through the BG by modulating input divergence in the striatum and output convergence in the EP.SIGNIFICANCE STATEMENT The entopeduncular nucleus (EP), one of the basal ganglia (BG) output nuclei, is an important station in information processing in BG. However, it remains unclear how EP neurons encode information and how dopamine affects this process. This contrasts with the well established role of dopamine in the striatum, which is known to redistribute cortical input between the direct and indirect pathways. Here we show that, in symmetry with the striatum, dopamine controls the rebalancing of information flow between the two pathways in the EP. Specifically, we demonstrate that dopamine regulates EP activity by differentially modulating striatal and pallidal GABAergic inputs. These results call for a reassessment of current perspectives on BG information processing by highlighting the functional role of extrastriatal dopamine receptors.

The anterior and posterior pedunculopontine tegmental nucleus are involved in behavior and neuronal activity of the cuneiform and entopeduncular nuclei.

Loss of cholinergic neurons in the mesencephalic locomotor region, comprising the pedunculopontine nucleus (PPN) and the cuneiform nucleus (CnF), is related to gait disturbances in late stage Parkinson's disease (PD). We investigate the effect of anterior or posterior cholinergic lesions of the PPN on gait-related motor behavior, and on neuronal network activity of the PPN area and basal ganglia (BG) motor loop in rats. Anterior PPN lesions, posterior PPN lesions or sham lesions were induced by stereotaxic microinjection of the cholinergic toxin AF64-A or vehicle in male Sprague-Dawley rats. First, locomotor activity (open field), postural disturbances (Rotarod) and gait asymmetry (treadmill test) were assessed. Thereafter, single-unit and oscillatory activities were measured in the non-lesioned area of the PPN, the CnF and the entopeduncular nucleus (EPN), the BG output region, with microelectrodes under urethane anesthesia. Additionally, ECoG was recorded in the motor cortex. Injection of AF64-A into the anterior and posterior PPN decreased cholinergic cell counts as compared to naive controls (P<0.001) but also destroyed non-cholinergic cells. Only anterior PPN lesions decreased the front limb swing time of gait in the treadmill test, while not affecting other gait-related parameters tested. Main electrophysiological findings were that anterior PPN lesions increased the firing activity in the CnF (P<0.001). Further, lesions of either PPN region decreased the coherence of alpha (8-12 Hz) band between CnF and motor cortex (MCx), and increased the beta (12-30 Hz) oscillatory synchronization between EPN and the MCx. Lesions of the PPN in rats had complex effects on oscillatory neuronal activity of the CnF and the BG network, which may contribute to the understanding of the pathophysiology of gait disturbance in PD.

Inhibitory short-term plasticity modulates neuronal activity in the rat entopeduncular nucleus in vitro.

The entopeduncular nucleus (EP) is one of the basal ganglia output nuclei integrating synaptic information from several pathways within the basal ganglia. The firing of EP neurons is modulated by two streams of inhibitory synaptic transmission, the direct pathway from the striatum and the indirect pathway from the globus pallidus. These two inhibitory pathways continuously modulate the firing of EP neurons. However, the link between these synaptic inputs to neuronal firing in the EP is unclear. To investigate this input-output transformation we performed whole-cell and perforated-patch recordings from single neurons in the entopeduncular nucleus in rat brain slices during repetitive stimulation of the striatum and the globus pallidus at frequencies within the in vivo activity range of these neurons. These recordings, supplemented by compartmental modelling, showed that GABAergic synapses from the striatum, converging on EP dendrites, display short-term facilitation and that somatic or proximal GABAergic synapses from the globus pallidus show short-term depression. Activation of striatal synapses during low presynaptic activity decreased postsynaptic firing rate by continuously increasing the inter-spike interval. Conversely, activation of pallidal synapses significantly affected postsynaptic firing during high presynaptic activity. Our data thus suggest that low-frequency striatal output may be encoded as progressive phase shifts in downstream nuclei of the basal ganglia while high-frequency pallidal output may continuously modulate EP firing.

Cholinergic mechanisms of high-frequency stimulation in entopeduncular nucleus.

Chronic, high-frequency (>100 Hz) electrical stimulation, known as deep brain stimulation (DBS), of the internal segment of the globus pallidus (GPi) is a highly effective therapy for Parkinson's disease (PD) and dystonia. Despite some understanding of how it works acutely in PD models, there remain questions about its mechanisms of action. Several hypotheses have been proposed, such as depolarization blockade, activation of inhibitory synapses, depletion of neurotransmitters, and/or disruption/alteration of network oscillations. In this study we investigated the cellular mechanisms of high-frequency stimulation (HFS) in entopeduncular nucleus (EP; rat equivalent of GPi) neurons using whole cell patch-clamp recordings. We found that HFS applied inside the EP nucleus induced a prolonged afterdepolarization that was dependent on stimulation frequency, pulse duration, and current amplitude. The high frequencies (>100 Hz) and pulse widths (>0.15 ms) used clinically for dystonia DBS could reliably induce these afterdepolarizations, which persisted under blockade of ionotropic glutamate (kynurenic acid, 2 mM), GABAA (picrotoxin, 50 µM), GABAB (CGP 55845, 1 µM), and acetylcholine nicotinic receptors (DHßE, 2 µM). However, this effect was blocked by atropine (2 µM; nonselective muscarinic antagonist) or tetrodotoxin (0.5 µM). Finally, the muscarinic-dependent afterdepolarizations were sensitive to Ca(2+)-sensitive nonspecific cationic (CAN) channel blockade. Hence, these data suggest that muscarinic receptor activation during HFS can lead to feedforward excitation through the opening of CAN channels. This study for the first time describes a cholinergic mechanism of HFS in EP neurons and provides new insight into the underlying mechanisms of DBS.

Dopamine D1 Receptor-Mediated Transmission Maintains Information Flow Through the Cortico-Striato-Entopeduncular Direct Pathway to Release Movements.

In the basal ganglia (BG), dopamine plays a pivotal role in motor control, and dopamine deficiency results in severe motor dysfunctions as seen in Parkinson's disease. According to the well-accepted model of the BG, dopamine activates striatal direct pathway neurons that directly project to the output nuclei of the BG through D1 receptors (D1Rs), whereas dopamine inhibits striatal indirect pathway neurons that project to the external pallidum (GPe) through D2 receptors. To clarify the exact role of dopaminergic transmission via D1Rs in vivo, we developed novel D1R knockdown mice in which D1Rs can be conditionally and reversibly regulated. Suppression of D1R expression by doxycycline treatment decreased spontaneous motor activity and impaired motor ability in the mice. Neuronal activity in the entopeduncular nucleus (EPN), one of the output nuclei of the rodent BG, was recorded in awake conditions to examine the mechanism of motor deficits. Cortically evoked inhibition in the EPN mediated by the cortico-striato-EPN direct pathway was mostly lost during suppression of D1R expression, whereas spontaneous firing rates and patterns remained unchanged. On the other hand, GPe activity changed little. These results suggest that D1R-mediated dopaminergic transmission maintains the information flow through the direct pathway to appropriately release motor actions.

High-Frequency Stimulation of the Rat Entopeduncular Nucleus Does Not Provide Functional or Morphological Neuroprotection from 6-Hydroxydopamine.

Deep brain stimulation (DBS) is the most common neurosurgical treatment for Parkinson's disease (PD). Whereas the globus pallidus interna (GPi) has been less commonly targeted than the subthalamic nucleus (STN), a recent clinical trial suggests that GPi DBS may provide better outcomes for patients with psychiatric comorbidities. Several laboratories have demonstrated that DBS of the STN provides neuroprotection of substantia nigra pars compacta (SNpc) dopamine neurons in preclinical neurotoxin models of PD and increases brain-derived neurotrophic factor (BDNF). However, whether DBS of the entopeduncular nucleus (EP), the homologous structure to the GPi in the rat, has similar neuroprotective potential in preclinical models has not been investigated. We investigated the impact of EP DBS on forelimb use asymmetry and SNpc degeneration induced by 6-hydroxydopamine (6-OHDA) and on BDNF levels. EP DBS in male rats received unilateral, intrastriatal 6-OHDA and ACTIVE or INACTIVE stimulation continuously for two weeks. Outcome measures included quantification of contralateral forelimb use, stereological assessment of SNpc neurons and BDNF levels. EP DBS 1) did not ameliorate forelimb impairments induced by 6-OHDA, 2) did not provide neuroprotection for SNpc neurons and 3) did not significantly increase BDNF levels in any of the structures examined. These results are in sharp contrast to the functional improvement, neuroprotection and BDNF-enhancing effects of STN DBS under identical experimental parameters in the rat. The lack of functional response to EP DBS suggests that stimulation of the rat EP may not represent an accurate model of clinical GPi stimulation.

Time and frequency-dependent modulation of local field potential synchronization by deep brain stimulation.

High-frequency electrical stimulation of specific brain structures, known as deep brain stimulation (DBS), is an effective treatment for movement disorders, but mechanisms of action remain unclear. We examined the time-dependent effects of DBS applied to the entopeduncular nucleus (EP), the rat homolog of the internal globus pallidus, a target used for treatment of both dystonia and Parkinson's disease (PD). We performed simultaneous multi-site local field potential (LFP) recordings in urethane-anesthetized rats to assess the effects of high-frequency (HF, 130 Hz; clinically effective), low-frequency (LF, 15 Hz; ineffective) and sham DBS delivered to EP. LFP activity was recorded from dorsal striatum (STR), ventroanterior thalamus (VA), primary motor cortex (M1), and the stimulation site in EP. Spontaneous and acute stimulation-induced LFP oscillation power and functional connectivity were assessed at baseline, and after 30, 60, and 90 minutes of stimulation. HF EP DBS produced widespread alterations in spontaneous and stimulus-induced LFP oscillations, with some effects similar across regions and others occurring in a region- and frequency band-specific manner. Many of these changes evolved over time. HF EP DBS produced an initial transient reduction in power in the low beta band in M1 and STR; however, phase synchronization between these regions in the low beta band was markedly suppressed at all time points. DBS also enhanced low gamma synchronization throughout the circuit. With sustained stimulation, there were significant reductions in low beta synchronization between M1-VA and STR-VA, and increases in power within regions in the faster frequency bands. HF DBS also suppressed the ability of acute EP stimulation to induce beta oscillations in all regions along the circuit. This dynamic pattern of synchronizing and desynchronizing effects of EP DBS suggests a complex modulation of activity along cortico-BG-thalamic circuits underlying the therapeutic effects of GPi DBS for conditions such as PD and dystonia.

The habenulo-interpeduncular and mammillothalamic tracts: early developed fiber tracts in the human fetal diencephalon.

PURPOSE: The habenulo-interpeduncular (HI) and mammillothalamic (MT) tracts are phylogenetically ancient. The clinical relevance of these tracts has recently received attention. In this work, we map the anatomy the developing HI and MT. METHODS: To investigate the topographical anatomy of developing fiber tracts in and around the diencephalon, we examined the horizontal, frontal, and sagittal serial paraffin sections of 28 human fetuses at 8-12 weeks of gestation. RESULTS: In all specimens, eosinophilic early fiber bundles were limited to the bilateral HI and MT tracts in contrast to pale-colored later developing fibers such as the thalamocortical projections and optic tract. The HI and MT tracts ran nearly parallel and sandwiched the thalamus from the dorsal and ventral sides, respectively. The nerve tract course appeared to range from 5-7 mm for the HI tract and 3-5 mm for the MT tract in 15 specimens at 11-12 weeks. The HI tract was embedded in, adjacent to, or distant from the developing parvocellular red nucleus. CONCLUSIONS: In early human fetuses, HI and MT tracts might be limited pathways for primitive cholinergic fiber connections between the ventral midbrain and epithalamic limbic system.

Effect of deep brain stimulation in rats selectively bred for reduced prepulse inhibition.

BACKGROUND: Sensorimotor gating, measured as prepulse inhibition (PPI) of the acoustic startle reaction (ASR), is disturbed in certain neuropsychiatric disorders, such as schizophrenia, obsessive compulsive disorder, and Tourette's syndrome (TS). Deep brain stimulation (DBS) of the centromedian-parafascicular complex (CM-Pf), globus pallidus internus (in rats the entopeduncular nucleus - EPN), and the ventral striatum (in rats the nucleus accumbens - NAC) has been used for treatment in TS. OBJECTIVE: We tested whether DBS of these regions would alleviate breeding-induced low PPI in rats. METHODS: Rats with breeding-induced low and high PPI were bilaterally implanted with electrodes in the CM-Pf, the EPN, or the NAC. After two weeks, they were stimulated or sham stimulated for epochs of 6 days (in the EPN with a current of 20% below the individual threshold for stimulation-induced side effects, in the NAC or CM-Pf with 100 µA and 150 µA). On the 6th day the rats were tested for PPI of ASR. RESULTS: Stimulation in the CM-Pf with 150 µA significantly alleviated PPI, while NAC stimulation was less effective. In PPI low rats electrode implantation in the EPN already improved PPI, while subsequent stimulation had no additional effect. Startle reaction of PPI low rats was not affected by stimulation of either region. CONCLUSION: The CM-Pf and the EPN are important for the modulation of sensorimotor gating in rats with breeding-induced low PPI. These rats may therefore be useful to further investigate the pathophysiological mechanisms of deficient sensorimotor gating and also mechanisms of action of DBS in these circumstances.

High-frequency electrical stimulation of the subthalamic nucleus excites target structures in a model using c-fos immunohistochemistry.

Deep-brain stimulation at high frequencies (HFS) directed to the subthalamic nucleus (STN) is used increasingly to treat patients with Parkinson's disease. However, the mechanism of action by which HFS of the STN achieves its therapeutic effects remains unresolved. Insofar as lesions of the STN have similar therapeutic benefit, a favored hypothesis is that HFS acts by suppressing neural activity in the STN. The purpose of the present study was to exploit prior observations that exposure to ether anesthesia in a rodent model evokes c-fos expression (a marker of neural activation) in the STN and its efferent structures, the globus pallidus, entopeduncular nucleus and substantia nigra. We showed first that exposure to ether induced a profound oscillatory pattern of neural activity in the STN and SNr, which could explain the marked induction of c-fos immunoreactivity in these structures. Secondly, inhibition of the STN by local injections of the GABA agonist, muscimol, suppressed ether-evoked c-fos expression in all target structures. This showed that excitation of target structures in the ether model originated, at least in part, from the STN. Thirdly, and contrary to expectation, HFS of the STN increased further the expression of c-fos in the STN target structures of animals treated with ether. Finally, we demonstrated, in the absence of ether treatment, that HFS and chemical stimulation of the STN with local injections of kainic acid both induced c-fos expression in the globus pallidus, entopeduncular nucleus and substantia nigra. Together these results suggest that the principal action of STN stimulation at high frequencies is to excite rather than inhibit its efferent targets. Given that Parkinsonism has been associated with increased levels of inhibitory output activity from the basal ganglia, it is unlikely that excitation of output structures revealed in this study provides a basis for deep-brain stimulation's therapeutic action.

Entopeduncular nucleus endocannabinoid system modulates sleep-waking cycle and mood in rats.

Since the pioneering work of Gadea-Ciria (Gadea-Ciria M, Stadler H, Lloyd KG, Bartholini G. Acetylcholine release within the cat striatum during the sleep-wakefulness cycle. Nature 1973; 243:518-519) indicating pointing to the involvement of acetylcholine and basal ganglia in sleep regulation; extensive literature has suggested that this brain complex participates in the control of the sleep-waking cycle (SWC). On the other hand, it has been demonstrated that the endocannabinoid system (eCBS) is prominently involved in the regulation of the SWC, mood and its related disorders. Since cannabinoid receptor 1 (CB1R) is highly expressed in basal ganglia, in particular in the entopeduncular nucleus (EP), we believe that it is important to know what the role of the EP CB1R is on SWC, depression, and anxiety. To provide insight into the role of the EP CB1R in the regulation of wakefulness (W), non-rapid eye movement sleep (NREMs) and rapid eye movement sleep (REMs), rats were recorded for 24h immediately after a single intra-EP administration of N-arachidonoylethanolamine (AEA) or 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide (AM251; CB1 inverse agonist). Likewise, the effect of these drugs on anxiety and depression was tested by means of the elevated plus maze (EPM) and forced swim test (FST), respectively. Results demonstrate that AEA increases NREMs expression, while AM251 increases W and decreases both NREMs and REMs. In addition, administration of AM251 decreases the time rats spent in the open arms and increases immobility time in the FST. It seems that activation of the CB1R in the EP is important to induce sleep, while its blockade promotes W, as well as anxiety and depression, somewhat resembling insomnia in humans. These results suggest that the EP CB1R is modulating sleep and mood.

Contribution of decreased serotonin release to the antidyskinetic effects of deep brain stimulation in a rodent model of tardive dyskinesia: comparison of the subthalamic and entopeduncular nuclei.

Mechanisms whereby deep brain stimulation (DBS) of the subthalamic nucleus (STN) or internal globus pallidus (GPi) reduces dyskinesias remain largely unknown. Using vacuous chewing movements (VCMs) induced by chronic haloperidol as a model of tardive dyskinesia (TD) in rats, we confirmed the antidyskinetic effects of DBS applied to the STN or entopeduncular nucleus (EPN, the rodent homolog of the GPi). We conducted a series of experiments to investigate the role of serotonin (5-HT) in these effects. We found that neurotoxic lesions of the dorsal raphe nuclei (DRN) significantly decreased HAL-induced VCMs. Acute 8-OH-DPAT administration, under conditions known to suppress raphe neuronal firing, also reduced VCMs. Immediate early gene mapping using zif268 in situ hybridization revealed that STN-DBS inhibited activity of DRN and MRN neurons. Microdialysis experiments indicated that STN-DBS decreased 5-HT release in the dorsolateral caudate-putamen, an area implicated in the etiology of HAL-induced VCMs. DBS applied to the EPN also suppressed VCMs but did not alter 5-HT release or raphe neuron activation. While these findings suggested a role for decreased 5-HT release in the mechanisms of STN DBS, further microdialysis experiments showed that when the 5-HT lowering effects of STN DBS were prevented by pretreatment with fluoxetine or fenfluramine, the ability of DBS to suppress VCMs remained unaltered. These results suggest that EPN- and STN-DBS have different effects on the 5-HT system. While decreasing 5-HT function is sufficient to suppress HAL-induced VCMs, 5-HT decrease is not necessary for the beneficial motor effects of DBS in this model.