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Our lab recently showed that N-methyl-D-aspartate (NMDA) evokes ATP-sensitive K(+) (K-ATP) currents in subthalamic nucleus (STN) neurons in slices of the rat brain. Both K-ATP channels and 5'-adenosine monophosphate-activated protein kinase (AMPK) are considered cellular energy sensors because their activities are influenced by the phosphorylation state of adenosine nucleotides. Moreover, AMPK has been shown to regulate K-ATP function in a variety of tissues including pancreas, cardiac myocytes, and hypothalamus. We used whole-cell patch clamp recordings to study the effect of AMPK activation on K-ATP channel function in STN neurons in slices of the rat brain. We found that bath or intracellular application of the AMPK activators A769662 and PT1 augmented tolbutamide-sensitive K-ATP currents evoked by NMDA receptor stimulation. The effect of AMPK activators was blocked by the AMPK inhibitor dorsomorphin (compound C), and by STO609, an inhibitor of the upstream AMPK activator CaMKKβ. AMPK augmentation of NMDA-induced K-ATP current was also blocked by intracellular BAPTA and by inhibitors of nitric oxide synthase and guanylyl cyclase. However, A769662 did not augment currents evoked by the K-ATP channel opener diazoxide. In the presence of NMDA, A769662 inhibited depolarizing plateau potentials and burst firing, both of which could be antagonized by tolbutamide or dorsomorphin. These studies show that AMPK augments NMDA-induced K-ATP currents by a Ca(2+)-dependent process that involves nitric oxide and cGMP. By augmenting K-ATP currents, AMPK activation would be expected to dampen the excitatory effect of glutamate-mediated transmission in the STN., (Published by Elsevier Ltd.)

In the present work we analyzed a possibility of interaction of protein p53, family members of the ERK1/2 signaling cascade, and the transcription factor CREB in regulation of functional activity of dopaminergic neurons. There were considered neurons of Substantia nigra and Zona incerta of control rats and of rats injected intraperitoneally with chemical inhibitor of p53 Pifithrin-alpha blocking transcription activity ofproapoptotic protein p53. We have shown the p53 inactivation to lead to an increase in the content of tyrosine'hydroxylase both in cell bodies and in terminal parts of axons. At the same time, activity of the transcription factor CREB is enhanced in the brain dopaminergic neurons. No significant differences in the content of phospho-ERK1/2 kinases were revealed in the cell bodies at use of Pifithrin-alpha as compared with control group. Thus, we have shown that action of p53 on biosynthesis of tyrosine hydroxylase is of inhibitory character and seems to be mediated by the transcription factor CREB.

Unlabelled: We have shown previously that electrical stimulation of the motor cortex reduces spontaneous painlike behaviors in animals with spinal cord injury (SCI). Because SCI pain behaviors are associated with abnormal inhibition in the inhibitory nucleus zona incerta (ZI) and because inactivation of the ZI blocks motor cortex stimulation (MCS) effects, we hypothesized that the antinociceptive effects of MCS are due to enhanced inhibitory inputs from ZI to the posterior thalamus (Po)-an area heavily implicated in nociceptive processing. To test this hypothesis, we used a rodent model of SCI pain and performed in vivo extracellular electrophysiological recordings in single well-isolated neurons in anesthetized rats. We recorded spontaneous activity in ZI and Po from 48 rats before, during, and after MCS (50 μA, 50 Hz; 300-ms pulses). We found that MCS enhanced spontaneous activity in 35% of ZI neurons and suppressed spontaneous activity in 58% of Po neurons. The majority of MCS-enhanced ZI neurons (81%) were located in the ventrorateral subdivision of ZI-the area containing Po-projecting ZI neurons. In addition, we found that inactivation of ZI using muscimol (GABAA receptor agonist) blocked the effects of MCS in 73% of Po neurons. Although we cannot eliminate the possibility that muscimol spread to areas adjacent to ZI, these findings support our hypothesis and suggest that MCS produces antinociception by activating the incertothalamic pathway., Perspective: This article describes a novel brain circuit that can be manipulated, in rats, to produce antinociception. These results have the potential to significantly impact the standard of care currently in place for the treatment of patients with intractable pain., (Copyright © 2013. Published by Elsevier Inc.)

The zona incerta (ZI) is a subthalamic nucleus connected to several structures, some of them known to be involved with antinociception. The ZI itself may be involved with both antinociception and nociception. The antinociceptive effects of stimulating the ZI with glutamate using the rat tail-flick test and a rat model of incision pain were examined. The effects of intraperitoneal antagonists of acetylcholine, noradrenaline, serotonin, dopamine, or opioids on glutamate-induced antinociception from the ZI in the tail-flick test were also evaluated. The injection of glutamate (7 μg/0.25 μl) into the ZI increased tail-flick latency and inhibited post-incision pain, but did not change the animal performance in a Rota-rod test. The injection of glutamate into sites near the ZI was non effective. The glutamate-induced antinociception from the ZI did not occur in animals with bilateral lesion of the dorsolateral funiculus, or in rats treated intraperitoneally with naloxone (1 and 2 m/kg), methysergide (1 and 2 m/kg) or phenoxybenzamine (2 m/kg), but remained unchanged in rats treated with atropine, mecamylamine, or haloperidol (all given at doses of 1 and 2 m/kg). We conclude that the antinociceptive effect evoked from the ZI is not due to a reduced motor performance, is likely to result from the activation of a pain-inhibitory mechanism that descends to the spinal cord via the dorsolateral funiculus, and involves at least opioid, serotonergic and α-adrenergic mechanisms. This profile resembles the reported effects of these antagonists on the antinociception caused by stimulating the periaqueductal gray or the pedunculopontine tegmental nucleus., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Siberian hamsters provide a useful model to define mechanisms underlying obesity reversal as they naturally transition from their extreme seasonal obesity in long 'summer-like' days (LDs) to a leaner state in short 'winter-like' days (SDs). These day length changes are coded into durational melatonin (MEL) signals by the pineal gland resulting in stimulation of MEL receptors (MEL(1a)-Rs). MEL(1a)-R mRNA is colocalized centrally in sympathetic nervous system (SNS) outflow neurons comprising a chain of neurons that ultimately innervates white adipose tissue (WAT). Neural components in this circuit include the subzona incerta (subZI), dorsomedial hypothalamic nucleus (DMH) and thalamic reuniens nucleus (ReN). SD, long-duration MEL signals induce gonadal regression and increase WAT SNS drive triggering lipolysis and thereby reversing LD obesity. We attempted to block the reversal of SD MEL signal-induced obesity by making electrolytic or sham lesions of the subZI, ReN or DMH in LD-housed hamsters. To create SD-like, long-duration MEL signals, we injected MEL 3 h before lights out, thereby lengthening the naturally occurring nocturnal duration of circulating MEL. ReN and subZI lesions did not block SD-like MEL signal-induced decreases in body, WAT, testicular masses or food intake; by contrast, DMH lesions blocked decreases in WAT and testicular mass. This nonresponsiveness was not due to lesion-induced inappropriate nocturnal LD MEL secretion that would have altered our creation of SD-like signals. Therefore, the DMH appears to participate in the control of both SD energy and reproductive responses, and joins the suprachiasmatic nucleus as sites necessary for SD responses in this species., (Copyright © 2010 S. Karger AG, Basel.)

The peptide melanin-concentrating hormone (MCH), produced mainly by cells in the lateral hypothalamus (LH), perifornical area (PF) and zona incerta (ZI), is suggested to have a role in the consumption of rewarding substances, such as ethanol, sucrose and palatable food. However, there is limited information on the specific brain sites where MCH acts to stimulate intake of these rewarding substances and on the feedback effects that their consumption has on the expression of endogenous MCH. The current study investigated MCH in relation to ethanol consumption, in Sprague-Dawley rats. In Experiment 1, chronic consumption of ethanol (from 0.70 to 2.7 g/kg/day) dose-dependently reduced MCH gene expression in the LH. In Experiments 2-4, the opposite effect was observed with acute oral ethanol, which stimulated MCH expression specifically in the LH but not the ZI. In Experiment 5, the effect of MCH injection in brain-cannulated rats on ethanol consumption was examined. Compared to saline, MCH injected in the paraventricular nucleus (PVN) and nucleus accumbens (NAc) selectively stimulated ethanol consumption without affecting food or water intake. In contrast, it reduced ethanol intake when administered into the LH, while having no effect in the ZI. These results demonstrate that voluntary, chronic consumption of ethanol leads to local negative feedback control of MCH expression in the LH. However, with a brief exposure, ethanol stimulates MCH-expressing neurons in this region, which through projections to the feeding-related PVN and reward-related NAc can promote further drinking behavior., (Copyright © 2010 Elsevier Inc. All rights reserved.)

This study explores whether near-infrared (NIr) light treatment neuroprotects dopaminergic cells in the substantia nigra pars compacta (SNc) and the zona incerta-hypothalamus (ZI-Hyp) from degeneration in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. BALB/c albino mice were divided into four groups: 1) Saline, 2) Saline-NIr, 3) MPTP, 4) MPTP-NIr. The injections were intraperitoneal and they were followed immediately by NIr light treatment (or not). Two doses of MPTP, mild (50 mg/kg) and strong (100 mg/kg), were used. Mice were perfused transcardially with aldehyde fixative 6 days after their MPTP treatment. Brains were processed for tyrosine hydroxylase (TH) immunochemistry. The number of TH(+) cells was estimated using the optical fractionator method. Our major finding was that in the SNc there were significantly more dopaminergic cells in the MPTP-NIr compared to the MPTP group (35%-45%). By contrast, in the ZI-Hyp there was no significant difference in the numbers of cells in these two groups. In addition, our results indicated that survival in the two regions after MPTP insult was dose-dependent. In the stronger MPTP regime, the magnitude of loss was similar in the two regions ( approximately 60%), while in the milder regime cell loss was greater in the SNc (45%) than ZI-Hyp ( approximately 30%). In summary, our results indicate that NIr light treatment offers neuroprotection against MPTP toxicity for dopaminergic cells in the SNc, but not in the ZI-Hyp.

Coadministration of entacapone with levodopa attenuates motor complications in experimental models of Parkinson's disease. The mechanisms underlying entacapone effects are unknown. We investigated the effect of entacapone, on: long-duration response (LDR) to levodopa, levodopa-induced postsynaptic pharmacodynamic mechanisms and molecular changes in hemiparkinsonian rats. 6-Hydroxydopamine-unilaterally lesioned rats were treated with levodopa (25 mg/kg)+vehicle; levodopa+entacapone (30 mg/kg) or saline, twice daily for 22 days. The LDR and the apomorphine-induced rotations were measured. In situ hybridization was performed measuring the expression of striatal preproenkephalin, preprodynorphin and dopamine D-3 receptor mRNAs, subthalamic cytochrome oxidase mRNA and nigral glutamic acid decarboxylase mRNA. Entacapone potentiated the LDR but did not modify either the apomorphine-induced rotational behavior or the molecular changes. Our results suggest that the effects of entacapone on levodopa-induced motor response are not mediated by postsynaptic mechanisms and that administration of entacapone is not able to normalize the molecular alterations induced by levodopa in the basal ganglia.

The zona incerta (ZI) is at the crossroad of almost all major ascending and descending fiber tracts and targets numerous brain centers from the thalamus to the spinal cord. Effective ascending drive of ZI cells has been described, but the role of descending cortical signals in patterning ZI activity is unknown. Cortical control over ZI function was examined during slow cortical waves (1-3 Hz), paroxysmal high-voltage spindles (HVSs), and 5-9 Hz oscillations in anesthetized rats. In all conditions, rhythmic cortical activity significantly altered the firing pattern of ZI neurons recorded extracellularly and labeled with the juxtacellular method. During slow oscillations, the majority of ZI neurons became synchronized to the depth-negative phase ("up state") of the cortical waves to a degree comparable to thalamocortical neurons. During HVSs, ZI cells displayed highly rhythmic activity in tight synchrony with the cortical oscillations. ZI neurons responded to short epochs of cortical 5-9 Hz oscillations, with a change in the interspike interval distribution and with an increase in spectral density in the 5-9 Hz band as measured by wavelet analysis. Morphological reconstruction revealed that most ZI cells have mediolaterally extensive dendritic trees and very long dendritic segments. Cortical terminals established asymmetrical synapses on ZI cells with very long active zones. These data suggest efficient integration of widespread cortical signals by single ZI neurons and strong cortical drive. We propose that the efferent GABAergic signal of ZI neurons patterned by the cortical activity can play a critical role in synchronizing thalamocortical and brainstem rhythms.

The effects of activating dopaminergic D(2/3) and D(4) receptors during activation of the subthalamic projection to the globus pallidus (GP) were explored in rat brain slices using the whole cell patch-clamp technique. Byocitin labeling and both orthodromic and antidromic activation demonstrated the integrity of some subthalamopallidal connections in in vitro parasagittal brain slices. Excitatory postsynaptic currents (EPSCs) that could be blocked by CNQX and AP5 were evoked onto pallidal neurons by local field stimulation of the subthalamopallidal pathway in the presence of bicuculline. Bath application of dopamine and quinpirole, a dopaminergic D(2)-class receptor agonist, reduced evoked EPSCs by about 35%. This effect was only partially blocked by sulpiride, a D(2/3) receptor antagonist. The sulpiride-sensitive reduction of the subthalamopallidal EPSC was associated with an increase in the paired-pulse ratio (PPR) and a reduction in the frequency but not the mean amplitude of spontaneous EPSCs (sEPSCs), indicative of a presynaptic site of action, which was confirmed by variance-mean analysis. The sulpiride-resistant EPSC reduction was mimicked by PD 168,077 and blocked by L-745,870, selective D(4) receptor agonist and antagonist, respectively, suggesting the involvement of D(4) receptors. The reduction of EPSCs produced by PD 168,077 was not accompanied by changes in PPR or the frequency of sEPSCs; however, it was accompanied by a reduction in mean sEPSC amplitude, indicative of a postsynaptic site of action. These results show that dopamine modulates subthalamopallidal excitation by presynaptic D(2/3) and postsynaptic D(4) receptors. The importance of this modulation is discussed.

In this study, we explore Fos expression (a measure of cell activity) in three nuclei associated with locomotion, namely the zona incerta, pedunculopontine tegmental nucleus and cuneiform nucleus (the latter two form the mesencephalic locomotor region) in hemiparkinsonian rats. Sprague-Dawley rats had small volumes of either saline (control) or 6 hydroxydopamine (6OHDA) injected into the medial forebrain bundle, the major tract carrying dopaminergic nigrostriatal axons. After various post-lesion survival periods, ranging from 2 h to 28 days, rats were perfused with formaldehyde and their brains processed for routine tyrosine hydroxylase and Fos immunocytochemistry. Our results showed a significant increase (P < 0.05) in the number of strongly labelled Fos+ cells in the cuneiform nucleus in the 6OHDA-lesioned cases compared to the controls after 7 and 28 days survival periods. By contrast, there were no significant differences (P > 0.05) in the number of strong-labelled Fos+ cells in the zona incerta and pedunculopontine nucleus of 6OHDA-lesioned rats compared to controls at any survival period. Many of the Fos+ cells within the pedunculopontine and cuneiform nuclei were glutamatergic (35-60%), while none or very few were nitric oxide synthase+. In conclusion, we reveal an increase in the number of strongly labelled Fos+ cells within the cuneiform nucleus of the so-called defensive locomotive system in 6OHDA-lesioned rats. In relation to Parkinson disease, we suggest that this increase is associated with the akinesia or lack of movement seen in patients.

Previous studies have shown that N-methyl-D-aspartate (NMDA) receptor stimulation evokes Ca2+- and Na+-dependent burst firing in subthalamic nucleus (STN) neurons. Using whole-cell patch pipettes to record currents under voltage-clamp, we identified a time-dependent depolarization-activated inward current (DIC) that may underlie NMDA-induced burst firing in STN neurons in rat brain slices. Continuous superfusion with NMDA (20 microM) elicited a marked TTX-insensitive inward current when the membrane was depolarized to the level of -70 or -50 mV, from a holding potential of -100 mV. This current had a long duration, and its peak amplitude occurred at a test potential of -60 mV. DIC could not be evoked using the non-NMDA receptor agonist D,L-alpha-amino-3-hydroxy-5-methylisoxalone-4-propionic acid (AMPA). DIC was blocked by either intracellular BAPTA or by removal of extracellular Ca2+, but selective blockers of T-type (mibefradil), L-type (nifedipine) and N-type (omega-conotoxin GVIA) Ca2+ channels did not. Perfusing slices with a low extracellular concentration of sodium abolished the NMDA-induced DIC, implying that both Ca2+ and Na+ are necessary for the expression of DIC. Transient receptor potential (TRP) channel blockers flufenamic acid and SKF96365 severely reduced DIC amplitude, whereas NMDA-gated currents were either increased or were unchanged. These results suggest that the activation of NMDA receptors enhances a Ca2+-activated non-selective cation current that may be mediated by a member of the TRP channel family in STN neurons.

In view of the recent focus on the zona incerta (and surrounding regions) as a target for deep brain stimulation in patients with Parkinson Disease, we have explored incertal cyto and chemoarchitecture in normal and MPTP (methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated macaque monkeys. Brains were processed for routine tyrosine hydroxylase (TH), nitric oxide synthase (NOs), parvalbumin (Pv) and calbindin D 28k (Cal) immunocytochemistry, as well as for Nissl staining. We show four main sectors in the zona incerta, namely rostral, dorsal, ventral and caudal, each with a largely distinct cytoarchitecture. Each of the antibodies screened had signature distribution patterns across the zona incerta; TH+ cells were localised within the rostral sector, NOs+ cells were concentrated in the dorsal sector, Pv+ cells were found mainly in the ventral sector and Cal+ cells were distributed uniformly across all sectors. These patterns match closely those reported in non primates. We found no major differences in the distribution and shape of labelled cells in the zona incerta of MPTP-treated monkeys when compared to control. In conclusion, we report that the primate zona incerta shows considerable cyto and chemoarchitectonic heterogeneity; that it forms a nucleus with distinct sectors presumably associated with diverse functions--from generating arousal to shifting attention, and from controlling visceral activity to influencing posture and locomotion. These functions have been proposed for the zona incerta of non primates. Our results have clinical implications, in that deep brain stimulation of the zona incerta (or parts thereof) could manifest in signs and symptoms other than those associated with the motor system. Such clinical stimulations could well involve other systems, including those of arousal, attention and visceral control.

1. The aim of the present work is to demonstrate the interaction between the glutamatergic/NMDA and dopaminergic systems in the medial zona incerta on the control of luteinizing hormone and prolactin secretion and the influence of reproductive hormones. 2. Proestrus and ovariectomized rats were primed with estrogen and progesterone to induce high or low levels of luteinizing hormone and prolactin. 2-Amino-7-phosphonoheptanoic acid, an NMDA receptor antagonist, and dopamine were injected in the medial zona incerta. Blood samples were withdrawn every hour between 1,600 and 2,000 hours or 2,200 hours via intracardiac catheter from conscious rats. Additional groups of animals injected with the NMDA receptor antagonist were killed 1 or 4 h after injection. Dopamine and its metabolite 3,4-dihydroxyphenylacetic acid were measured in different hypothalamic regions. 3. 2-Amino-7-phosphonoheptanoic acid blocked the ovulatory luteinizing hormone surge in proestrus rats. 2-Amino-7-phosphonoheptanoic acid also blocked the increase in luteinizing hormone induced by ovarian hormones in ovariectomized rats, an effect that was partially reversed by dopamine injection. Conversely, the increased release of luteinizing hormone and prolactin induced by dopamine was prevented by 2-amino-7-phosphonoheptanoic acid. We found that the NMDA antagonist injection decreased the dopaminergic activity--as evaluated by the 3,4-dihydroxyphenylacetic acid/dopamine ratio--in the medio basal hypothalamus and increased in the preoptic area. 4. Our results show an stimulatory role of NMDA receptors on the ovulatory luteinizing hormone release and on luteinizing hormone release induced by sexual hormones and demonstrate that the stimulatory effect of dopamine on luteinizing hormone and prolactin is mediated by the NMDA receptors. These results suggest a close interaction between the glutamatergic and dopaminergic incertohypothalamic systems on the control of luteinizing hormone and prolactin release.

5-Hydroxytryptamine (5-HT) and the 5-HT(1A/7) receptor agonist (+)-8-hydroxy-2-(di-n-propylamino) tetralinHBr (8-OH-DPAT), injected into the zona incerta (an area in the dorsal hypothalamus) of the female rat, inhibit the release of luteinizing hormone (LH) and the effects of both are blocked by the 5-HT(2/7) receptor antagonist, ritanserin. As both 8-OH-DPAT and ritanserin have moderate activity at the 5-HT7 receptor subtype, the possibility that this subtype might mediate their effects in the zona incerta has been investigated. Ovariectomised rats were primed with 5 microg oestradiol benzoate followed at 48 h by 0.5 mg progesterone, which induces an LH surge. 5-Carboxamidotryptamine (5-CT), a potent but non-selective agonist at 5-HT7 receptors, like 5-HT and 8-OH-DPAT, inhibited the LH surge at 5 and 1.25 nmol injected bilaterally into the zona incerta. The non-selective 5-HT(2/7) receptor antagonist ritanserin and the selective 5-HT7 receptor antagonist, (R)-3-(2-(2-(4-methyl-piperidin-1-yl)-pyrrolidine-1-sulfonyl)-phenol (SB-269970-A) at 0.5 microg/side blocked all three receptor agonists when injected concurrently into the zona incerta. However, lower (0.2 microg) and higher doses (2 and 5 microg) of SB-269970-A were less effective, indicating a bell-shaped dose-response curve. SB-269970-A was also inhibitory when administered systemically (1 mg/kg intraperitoneally (i.p.)). When LH release was suppressed by 5 microg oestradiol benzoate, SB-269970-A (0.5 and 2 microg) did not elevate levels, indicating it is unlikely that 5-HT7 receptors mediate a tonic inhibition on release but rather are involved in terminating the pre-ovulatory LH surge. These data demonstrate that 5-HT7 receptors play a role in the regulation of LH by the zona incerta in rat brain.

Our recent results showed that angiotensin II or III (AII, AIII) microinjected into the zona incerta (ZI) significantly increased water intake. The most effective doses of AII and AIII were also defined. The two neuropeptides had their effects differently on drinking via different receptors. AII bound to AT(1) that was blocked by AT(1) receptor antagonist Losartan and the effect of AIII was eliminated by prior application of AT(2) receptor antagonist PD 123319. After different hydrational challenges, the effects of AII and AIII in the ZI have never been experimented, however. In the present experiments, the previously defined effective doses of AII (100 ng) or AIII (200 ng) were microinjected into the ZI after different types of challenges: (1). lowered thirst motivation when animals ingested approximately 40% of their daily fluid need during the consequent 60-min-daily-drinking period before the injection, (2). 48-h water deprivation, (3). intracellular dehydration and (4). extracellular dehydration. In all of the cases, incertally injected AII increased the animals' water ingestion. While Losartan could block these effects, PD 123319 was ineffective. Experiments were repeated by AIII, but in none of the cases differences were experienced between the groups. The finding that following hydrational challenges water intake increased only after AII injections and it could be blocked only by Losartan suggests that AII and AT(1) receptor play a pivotal role in the ZI in maintaining the body water balance.

Different doses of angiotensin II (AII) or angiotensin III (AIII) microinjections into the zona incerta have been studied on drinking of rats in separate experiments during the consequent 60-min-daily-drinking period. Also, the dipsogen power of only the effective dose of AII and AIII was compared to vehicle treated rats. After, angiotensin receptor (AT(1), AT(2)) antagonists on AII or AIII induced drinking were tested. In the first and second experiments only the 100 ng AII and the 200 ng AIII increased water intake significantly. In the third experiment the AII started its dipsogen effect earlier, at the 5 min measuring time, compared to the AIII. Both effects kept on lasting parallel from the 10 min on. Considering the antagonist pre-treatments in the fourth experiment, animals were injected with 90 ng losartan, an AT(1) antagonist, 180 ng PD 123319 or 200 ng CGP 42112, both AT(2) antagonists, respectively. Both AII and AIII increased water consumption. The effect of AII could be blocked by losartan, but not by PD 123319 or CGP 42112. On the other hand, the effect of AIII could not be blocked by losartan, but by both the PD 123319 and CGP 42112. Since, the effects of AII, AIII and angiotensin antagonists have not been tested in the zona incerta, the finding that water intake increased after AII or AIII injections and it could be blocked only by either of the antagonists suggests that AT(1) and AT(2) receptors play partially different roles in the regulation of water intake.

Behavioral and electrophysiological methods were used to determine whether a transplant of dopamine-rich fetal tissue in striatum combined with an electrolytic lesion of the entopeduncular nucleus have additive effects in the unilaterally lesioned rat model for Parkinson's disease. The subjects were rats with the left substantia nigra lesioned with 6-hydroxydopamine (6-OHDA) and responding to systemic amphetamine with rotation toward the side of the lesion (ipsilateral rotation). The motor response to amphetamine was fractionated into six aspects, half reflecting the unilateral deafferentation in striatum and half representing those aspects of the response evoked in normal rats. After collection of baseline values, 25 rotators received a transplant of fetal ventral mesencephalic tissue in the left striatum and 20 received a transplant and, at the same time, an electrolytic lesion of the left entopeduncular nucleus. Testing for the motor response to amphetamine resumed after 4 weeks of recovery and continued at weekly intervals for 5 weeks. Upon completion of these tests, each rotator was implanted with multiple electrodes in the subthalamic nucleus. After recovery, multiunit responses to amphetamine and apomorphine were recorded from several electrodes in parallel during the motor response to the drugs. In rotators with transplant only, treatment with amphetamine evoked oral stereotypy and an attenuated ipsilateral rotation response. In rotators with combined transplant and entopeduncular lesion, ipsilateral rotation did not change or increased. Subthalamic responses to amphetamine and apomorphine were larger in rotators with combined transplant and entopeduncular lesion than in rotators with transplant alone. These findings indicate that the combination of transplant and pallidotomy in the 6-OHDA rat model for parkinsonism does not lead to additive benefits, an effect that may have been due to the nonselectivity of the electrolytic damage and/or of the lesion extending beyond the entopeduncular nucleus into the lateral hypothalamus., (Copyright 2003 Wiley-Liss, Inc.)

The subthalamic nucleus (STN) plays a crucial role in basal ganglia functions and has been shown to be hyperactive in parkinsonian syndromes. The zona incerta (ZI), located dorsally to the STN, is also reported to be overactive after nigrostriatal denervation. In this study, we examined the behavioral consequences of an increased activity of the STN or the ZI in awake, freely moving rats. Unilateral microinjections of a GABA(A) receptor antagonist (bicuculline; 25, 50, and 100 microg/microl) were performed in the STN or in the ZI of rats, and locomotor activity, spontaneous behaviors, and the occurrence of abnormal movements were quantified. Microinjection of bicuculline (50 and 100 microg/microl) into the STN did not modify spontaneous locomotor activity, whereas it induced an increase in locomotion when injected into the ZI. Furthermore, when injected into the STN or ZI, these same doses of bicuculline produced changes in spontaneous behaviors (sniffing and grooming decreased whereas chewing and rearing increased) and the appearance of abnormal movements directed contralaterally to the injection side. Application of a lower dose of bicuculline (25 ng/microl) in the STN or ZI did not modify behavior. This study suggests that the subthalamic region including the ZI, and not the STN per se, might be involved in the induction of abnormal movements. In addition, these data suggest that the hyperactivity of neurons in this region may have different consequences in the normal state and in the pathological state.

The effects of systemic administration of the serotonin (5-HT)(1A/1B) agonist 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl)1H-indole (RU 24969) on locomotor and investigatory behavior in rats have been well characterized using the behavioral pattern monitor (BPM). To elucidate the neural circuitry underlying this behavioral profile, intracerebral dose--response studies were conducted at two sites with high densities of 5-HT(1B) receptors, the subthalamic nucleus (STN) and substantia nigra. Infusion of RU 24969 into the STN produced systemic RU 24969-like changes in locomotor activity and patterns but an uncharacteristic increase in investigatory holepokes. Intra-STN administration of the selective 5-HT(1A) receptor agonist 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) produced RU 24969-like changes in locomotor patterns only, while the 5-HT(1B) receptor agonist 3(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one dihydrochloride (CP-93,129) increased locomotor activity, produced no change in locomotor patterns and nonsignificantly increased holepokes. Intranigral infusion of RU 24969 produced systemic and intra-STN RU 24969-like increases in locomotor activity. Intranigral RU 24969, however, failed to produce any changes in locomotor patterns or investigatory holepokes. Intranigral infusions of CP-93,129 or 8-OH-DPAT had no effects on locomotor activity, locomotor patterns or investigatory holepokes. These results provide evidence for multiple-site mediation of the locomotor-activating effects of RU 24969 and for a dissociation of the neural substrates underlying locomotor and investigatory components of the RU 24969-induced behavioral profile.

Recent experiments have suggested that the zona incerta might be regarded as a highly sensitive structure for seizure induction. This sensitivity has been linked to this structure's abundant expression of cholinergic receptors. Here we have decided to investigate the participation of the GABAergic system of the zona incerta, one of its major neurotransmitters with widespread projections to the neocortex, in the pilocarpine (Pilo) model of epilepsy. Stereotaxic administration of a GABA(A) agonist (muscimol), antagonist (bicuculline) or saline (controls) bilaterally into the zona incerta of adult male Wistar rats was performed 30 min prior to the systemic injection of pilocarpine. Animals were electroencephalographically and behaviorally monitored for seizure activity. Administration of muscimol had a pro-convulsant effect characterised by a higher percentage of animals developing SE with a shorter latency. Conversely, administration of bicuculline had a dose dependent anticonvulsant effect, with no animals displaying SE. Our results contribute to the further characterisation of the regulatory role of the zona incerta in seizure-related phenomena, suggesting that its modulation might be a relevant target for anticonvulsant strategies.

Extracellular single-unit recordings were made from neurons in the lateral hypothalamus (LH) or zona incerta (ZI) of conscious sheep. A small population of neurons (12/83) were found which responded with increased firing rate when the animal looked at food but did not respond when the sheep looked at water. The effects of rapidly inducing intense thirst by the intracerebroventricular (i.c.v.) injection of hypertonic (0.85 M) saline or 200 ng of angiotensin II, or a mixture of the two dipsogenic stimuli, on the response of neurons initially responding only to the sight of food were investigated. Following i.c.v. injection of the dipsogenic stimuli the neurons began to respond strongly to the sight of water. The results demonstrated that changing the animal's motivational state alters the response of some neurons in the LH and ZI and suggests that the neuronal response is influenced by the animal's dominant need at the time of testing.

Objectives: Previous studies suggested that the hypo-activity of the external pallidus (GPe) might drive the hyper-activity of subthalamic neurons, which underlies the cardinal symptoms of Parkinson's disease. We have challenged this view, based on the so-called 'indirect pathway', by recording apomorphine effects from both structures of parkinsonian patients, at rest and during passive movements., Methods: We performed single-unit recordings from external pallidus (GPe), internal pallidus (GPi) and subthalamic nucleus (STN) during the stereotactic neurosurgery aimed to implant deep brain stimulating electrodes in GPi or STN. First, we verified the firing frequency of each structure in off-state conditions. Then, therapeutic, subdyskinetic concentrations of the dopaminergic agonist apomorphine was delivered to assess each nucleus response., Results: The firing rate of STN averaged about 40 Hz; a large proportion (75%) of STN units exhibited marked responsiveness to passive movements. Apomorphine reduced the firing discharge of parkinsonian STN in all cells, although electrophysiological recovery was usually incomplete. Movement-related activity was also dramatically reduced. In contrast, apomorphine failed to modify the firing frequency of GPe, despite the amelioration of hypo-kinetic symptoms and the simultaneous inhibition of GPi firing discharge., Conclusions: We demonstrate that part of the models on basal ganglia circuitry needs to be revised. The re-balancing of STN hyper-activity, when patients benefit from dopaminergic therapy, is not due to an increased input from GPe, but, instead, due to changes in STN intrinsic firing properties and/or modulation of glutamatergic inputs.

We investigated whether stimulation of the functionally discrete subthalamic region, subthalamic cerebrovasodilator area (SVA), which increases cerebral blood flow (CBF) when excited, would, like stimulation of cerebellar fastigial nucleus (FN), produce central neurogenic neuroprotection. A 1-h electrical stimulation of SVA or FN reduced infarctions triggered by permanent occlusion of middle cerebral artery (MCA) by 48-55% in Sprague-Dawley rats and by 59% in Fisher rats. The salvaging effect of SVA stimulation, similar to FN, was long lasting and reduced the volume of infarctions placed 72 h or 10 days later by 58 and 26%, respectively, in Fisher rats. Bilateral lesioning of FN neurons by the microinjection of ibotenic acid 5 days before SVA stimulation did not affect SVA-evoked neuroprotection. Bilateral lesions of SVA neurons administered 5 days before FN stimulation had no effect on FN-induced neuroprotection but reversed the stimulus-locked increase in CBF accompanying FN stimulation. This study demonstrates that (1) excitation of neurons and/or fibers projecting through the SVA reduces ischemic infarctions as substantially as excitation of FN neurons; (2) the effects are long-lasting and not attributable to increases in cerebral blood flow, changes in blood gases or brain temperature, or rat strain; (3) the neuroprotective effects of SVA and FN stimulation are mutually independent and (4) FN-evoked cerebrovasodilation is mediated by SVA neurons. The SVA and FN are part of a neuronal system in CNS, which is distributed and, when excited, acts to protect the brain from ischemic injury.

The subthalamic nucleus (STN) is a key nucleus in the basal ganglia motor circuit that provides the major glutamatergic excitatory input to the basal ganglia output nuclei. The STN plays an important role in the normal motor function, as well as in pathological conditions such as Parkinson's disease. Development of a complete understanding of the role of the STN in motor control will require a detailed understanding of the mechanisms involved in the regulation of excitatory and inhibitory synaptic transmission in this nucleus. Here, we report that activation of groups I or III metabotropic glutamate (mGlu) receptors, but not group II, causes a depression of excitatory transmission in the STN. In contrast, mGlu receptor activation has no effect on the inhibitory transmission in this nucleus. Further characterization of the group I mGlu receptor-induced effect on EPSCs suggests that this response is mediated by mGlu1 and not mGlu5. Further, paired pulse studies suggest that both the mGlu1 receptor and the group III mGlu receptor-mediated effects are due to a presynaptic mechanism. If these receptors are involved in endogenous synaptic transmission in the STN, these results raise the exciting possibility that selective agents targeting mGlu receptors may provide a novel approach for the treatment of motor disorders involving the STN.

Sympathoexcitatory reticulospinal neurons of the rostral ventrolateral medulla (RVLM) are oxygen detectors excited by hypoxia to globally elevate regional cerebral blood flow (rCBF). The projection, which accounts for >50% of hypoxic cerebral vasodilation, relays through the medullary vasodilator area (MCVA). However, there are no direct cortical projections from the RVLM/MCVA, suggesting a relay that diffusely innervates cortex and possibly originates in thalamic nuclei. Systematic mapping by electrical microstimulation of the thalamus and subthalamus revealed that elevations in rCBF were elicited only from a limited area, which encompassed medial pole of zona incerta, Forel's field, and prerubral zone. Stimulation (10 sec train) at an active site increased rCBF by 25 +/- 6%. Excitation of local neurons with kainic acid mimicked effects of electrical stimulation by increasing rCBF. Stimulation of the subthalamic cerebrovasodilator area (SVA) with single pulses (0.5 msec; 80 microA) triggered cortical EEG burst-CBF wave complexes with latency 24 +/- 5 msec, which were similar in shape to complexes evoked from the MCVA. Selective bilateral lesioning of the SVA neurons (ibotenic acid, 2 microg, 200 nl) blocked the vasodilation elicited from the MCVA and attenuated hypoxic cerebrovasodilation by 52 +/- 12% (p < 0.05), whereas hypercarbic vasodilation remained preserved. Lesioning of the vasodilator site in the basal forebrain failed to modify SVA-evoked rCBF increase. We conclude that (1) excitation of intrinsic neurons of functionally restricted region of subthalamus elevates rCBF, (2) these neurons relay signals from the MCVA, which elevate rCBF in response to hypoxia, and (3) the SVA is a functionally important site conveying vasodilator signal from the medulla to the telencephalon.

The adipose hormone, leptin, not only restrains appetite, but also influences energy expenditure. One such influence is to promote sexual maturation and fertility. The neuromodulatory circuits that mediate this effect are not well known but the present study suggests that one mediator could be melanin-concentrating hormone (MCH). We show that the long-form receptor (Ob-Rb) is expressed in the zona incerta of the rat and that administration of leptin (both 0.5 microg and 1.0 microg/side) into this area of ovariectomized, oestrogen-primed rats stimulated the release of luteinizing hormone (LH) within 1 h, the effect enduring for a further 1 h. Injections of leptin into the arcuate nucleus induced a smaller, transient rise in LH while injections into the paraventricular and ventromedial nuclei were without effect. MCH neurones are present in the zona incerta and administration of this hormone into the medial preoptic area (mPOA) stimulates LH release, therefore we investigated the possibility that MCH might mediate this effect of leptin. An injection of MCH antiserum into mPOA prevented the rise in LH normally induced by leptin injected into the zona incerta. In addition, melanocortin receptor antagonists ([D-Arg8]ACTH(4-10) and [Ala6]ACTH(4-10)), previously shown to inhibit the stimulatory effect of MCH on LH release, also inhibited the effect of leptin. We propose that one route by which leptin may promote reproductive activity is by enhancing MCH release from fibres within the mPOA. Speculative mechanisms for the action of MCH include the following possibilities: MCH may be acting on the specific MCH receptor which in turn interacts with a melanocortin or melanocortin-like receptor; MCH may bind directly to one of the melanocortin receptors; or melanocortin antagonists may interact with the MCH receptor.

The zona incerta (ZI), an area in the dorsal hypothalamus, contains neuronal systems that appear to control gonadotropin release. Previous findings show that there is an inverse relationship between serotonin (5-HT) activity in the ZI and plasma luteinizing hormone (LH) levels, indicating that the 5-HT system in this area has an inhibitory effect on LH release. Employing anaesthetised, ovariectomised rats primed with 5 microg oestradiol benzoate followed at 48 h by 0.5 mg progesterone, we have shown that 2 microg/side 5-HT in the ZI inhibits the LH surge that normally occurs 4 h after the progesterone treatment. This effect was mimicked by 2 microg/side 8-OH-DPAT, a 5-HT1A agonist, but not by DOI, a 5-HT2 agonist, BMY7378, a presynaptic 5-HT1A agonist or MCPP, a 2B & 2C agonist. The inhibitory effect of 5-HT and 8-OH-DPAT was prevented by pretreatment, 1 h before, with either 2 mg/kg i.p. WAY100135, a 5-HT1A antagonist or 0.25 mg/kg i.p. ritanserin, a 5-HT2 antagonist. These results indicate that 5-HT in the ZI exerts its inhibitory effect on LH release via 5-HT1A receptors but that another 5-HT subtype may also be involved.

In mammals, the subthalamic nucleus (STN) is a glutamatergic diencephalic cell group that develops in the caudal hypothalamus and migrates to a position above the cerebral peduncle. By its input from the external pallidal segment and projection to the internal pallidal segment, STN plays a critical role in basal ganglia functions. Although the basal ganglia in birds is well developed, possesses the same major neuron types as in mammals, and plays a role in movement control similar to that in mammals, it has been uncertain whether birds possess an STN. We report here evidence indicating that the so-called anterior nucleus of the ansa lenticularis (ALa) is the avian homolog of mammalian STN. First, the avian ALa too develops within the mammillary hypothalamic area and migrates to a position adjacent to the cerebral peduncle. Second, ALa specifically receives input from dorsal pallidal neurons that receive input from enkephalinergic striatal neurons, as is true of STN. Third, ALa projects back to avian dorsal pallidum, as also the case for STN in mammals. Fourth, the neurons of ALa contain glutamate, and the target neurons of ALa in dorsal pallidum possess AMPA-type glutamate receptor profiles resembling those of mammalian pallidal neurons. Fifth, unilateral lesions of ALa yield behavioral disturbances and movement asymmetries resembling those observed in mammals after STN lesions. These various findings indicate that ALa is the avian STN, and they suggest that the output circuitry of the basal ganglia for motor control is similar in birds and mammals.

Reciprocally connected glutamatergic subthalamic and GABAergic globus pallidus neurons have recently been proposed to act as a generator of low-frequency oscillatory activity in Parkinson's disease. To determine whether GABA(A) receptor-mediated synaptic potentials could theoretically generate rebound burst firing in subthalamic neurons, a feature that is central to the proposed oscillatory mechanism, we determined the equilibrium potential of GABA(A) current (E(GABA(A))) and the degree of hyperpolarization required for rebound firing using perforated-patch recording. In the majority of neurons that fired rebounds, E(GABA(A)) was equal to or more hyperpolarized than the hyperpolarization required for rebound burst firing. These data suggest that synchronous activity of pallidal inputs could underlie rhythmic bursting activity of subthalamic neurons in Parkinson's disease.