Activation of group III mGluRs inhibits GABAergic and glutamatergic transmission in the substantia nigra pars reticulata

The GABAergic projection neurons of the substantia nigra pars reticulata (SNr) exert an important influence on the initiation and control of movement. The SNr is a primary output nucleus of the basal ganglia (BG) and is controlled by excitatory inputs from the subthalamic nucleus (STN) and inhibitory inputs from the striatum and globus pallidus. Changes in the output of the SNr are believed to be critically involved in the development of a variety of movement disorders. Anatomical studies reveal that metabotropic glutamate receptors (mGluRs) are highly expressed throughout the BG. Interestingly, mRNA for group III mGluRs are highly expressed in STN, striatum, and globus pallidus, and immunocytochemical studies have shown that the group III mGluR proteins are present in the SNr. Thus it is possible that group III mGluRs play a role in the modulation of synaptic transmission in this nucleus. We performed whole cell patch-clamp recordings from nondopaminergic SNr neurons to investigate the effect of group III mGluR activation on excitatory and inhibitory transmission in the SNr. We report that activation of group III mGluRs by the selective agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4, 100 microM) decreases inhibitory synaptic transmission in the SNr. Miniature inhibitory postsynaptic currents studies and paired-pulse studies reveal that this effect is mediated by a presynaptic mechanism. Furthermore we found that L-AP4 (500 microM) also reduces excitatory synaptic transmission at the STN-SNr synapse by action on presynaptically localized group III mGluRs. The finding that mGluRs modulate the major inputs to SNr neurons suggests that these receptors may play an important role in motor function and could provide new targets for the development of pharmacological treatments of movement disorders.     

Functional changes of the basal ganglia circuitry in Parkinson's disease

The basal ganglia circuitry processes the signals that flow from the cortex, allowing the correct execution of voluntary movements. In Parkinson's disease, the degeneration of dopaminergic neurons of the substantia nigra pars compacta triggers a cascade of functional changes affecting the whole basal ganglia network. The most relevant alterations affect the output nuclei of the circuit, the medial globus pallidus and substantia nigra pars reticulata, which become hyperactive. Such hyperactivity is sustained by the enhanced glutamatergic inputs that the output nuclei receive from the subthalamic nucleus. The mechanisms leading to the subthalamic disinhibition are still poorly understood. According to the current model of basal ganglia organization, the phenomenon is due to a decrease in the inhibitory control exerted over the subthalamic nucleus by the lateral globus pallidus. Recent data, however, suggest that additional if not alternative mechanisms may underlie subthalamic hyperactivity. In particular, given the reciprocal innervation of the substantia nigra pars compacta and the subthalamic nucleus, the dopaminergic deficit might influence the subthalamic activity, directly. In addition, the increased excitatory drive to the dopaminergic nigral neurons originating from the hyperactive subthalamic nucleus might sustain the progression of the degenerative process. The identification of the role of the subthalamic nucleus and, more in general, of the glutamatergic mechanisms in the pathophysiology of Parkinson's disease might lead to a new approach in the pharmacological treatment of the disease. Current therapeutic strategies rely on the use of L-DOPA and/or dopamine agonists to correct the dopaminergic deficit. Drugs capable of antagonizing the effects of glutamate might represent, in the next future, a valuable tool for the development of new symptomatic and neuroprotective strategies for therapy of Parkinson's disease.     

A unilateral 6-hydroxydopamine lesion decreases the expression of metabotropic glutamate receptors in rat substantia nigra

Loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) in Parkinson's disease (PD) leads to augmentation of glutamatergic activity in the subthalamic nucleus (STN). Moreover, antagonizing excitotoxicity has yielded mostly symptomatic improvements in experimental animals of PD. Therefore, we used immunocytochemistry to examine the effect of unilateral 6-hydroxydopamine lesions of SNc on the expression of metabotropic glutamate receptors (mGluR1a, 2/3, 5, 4, 8) and tyrosine hydroxylase (TH). 6-Hydroxydopamine causes a fall in the number of mGluRs and TH in the lesioned lateral substantia nigra. Pharmacological activation of group II or III mGluRs or blockade of group I mGluRs for 1 week significantly increased the expression of the same group receptors. The alteration in the receptor expression may be a compensatory mechanism developed after mGluRs ligands neuroprotective treatment.     

Effect of a functional impairment of corticostriatal transmission on cortically evoked expression of c-Fos and zif 268 in the rat basal ganglia.

The activity-dependent induction of immediate-early genes is commonly used to map activated neuronal networks. In a previous analysis of the cortico-basal ganglia circuits, we have shown that a cortical stimulation produces Fos protein expression in the striatum and the subthalamic nucleus, with a pattern which conforms to the anatomical organization of cortical projections [Sgambato V. et al. (1996) Neuroscience 81, 93-112]. In the present study, we examined the effects of a unilateral blockade of the corticostriatal transmission on c-fos and zif 268 messenger RNA expression evoked in the substantia nigra pars reticulata and the subthalamic nucleus following stimulation of the ipsilateral motor cortex. The blockade of the corticostriatal pathway was performed either by an excitotoxic striatal lesion or by an application of the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione within the striatum. After application of the glutamate receptor antagonist, which prevented the cortical stimulation activating the GABAergic striatonigral pathway, the induction of both c-fos and zif 268 messenger RNAs was facilitated in the ipsilateral substantia nigra pars reticulata. In the subthalamic nucleus ipsilateral to the application of 6-cyano7-nitroquinoxaline-2,3-dione, the cellular discharges evoked by stimulation of the cortex were considerably shortened as a result of the blockade of the disinhibitory striato-pallido-subthalamic circuit. However, a strong expression of immediate-early genes was still induced by the cortical stimulation. By contrast, after unilateral kainate lesion of the striatum, the cortical stimulation was no longer able to induce c-fos and zif 268 messenger RNA expression in the ipsilateral subthalamic nucleus and in the substantia nigra pars reticulata bilaterally. The lack of immediate-early gene induction strongly contrasted with the neuronal discharges evoked in these nuclei by the cortical stimulation. Comparison between the cortically evoked neuronal activities and the pattern of immediate-early gene expression suggests that the induction of immediate-early genes in the basal ganglia mainly reflects the level of synaptic activity rather than the frequency of discharge of the postsynaptic neurons. Moreover, the results stress that modifications of immediate-early gene expression observed in the basal ganglia after an acute or a chronic interruption of the corticostriatal transmission are not superimposable.     

Dopamine agonist-mediated rotation in rats with unilateral nigrostriatal lesions is not dependent on net inhibitions of rate in basal ganglia output nuclei.

Current models of basal ganglia function predict that dopamine agonist-induced motor activation is mediated by decreases in basal ganglia output. This study examines the relationship between dopamine agonist effects on firing rate in basal ganglia output nuclei and rotational behavior in rats with nigrostriatal lesions. Extracellular single-unit activity ipsilateral to the lesion was recorded in awake, locally-anesthetized rats. Separate rats were used for behavioral experiments. Low i.v. doses of D1 agonists (SKF 38393, SKF 81297, SKF 82958) were effective in producing rotation, yet did not change average firing rate in the substantia nigra pars reticulata or entopeduncular nucleus. At these doses, firing rate effects differed from neuron to neuron, and included increases, decreases, and no change. Higher i.v. doses of D1 agonists were effective in causing both rotation and a net decrease in rate of substantia nigra pars reticulata neurons. A low s.c. dose of the D1/D2 agonist apomorphine (0.05 mg/kg) produced both rotation and a robust average decrease in firing rate in the substantia nigra pars reticulata, yet the onset of the net firing rate decrease (at 13-16 min) was greatly delayed compared to the onset of rotation (at 3 min). Immunostaining for the immediate-early gene Fos indicated that a low i.v. dose of SKF 38393 (that produced rotation but not a net decrease in firing rate in basal ganglia output nuclei) induced Fos-like immunoreactivity in the striatum and subthalamic nucleus, suggesting an activation of both inhibitory and excitatory afferents to the substantia nigra and entopeduncular nucleus. In addition, D1 agonist-induced Fos expression in the striatum and subthalamic nucleus was equivalent in freely-moving and awake, locally-anesthetized rats. The results show that decreases in firing rate in basal ganglia output nuclei are not necessary for dopamine agonist-induced motor activation. Motor-activating actions of dopamine agonists may be mediated by firing rate decreases in a small subpopulation of output nucleus neurons, or may be mediated by other features of firing activity besides rate in these nuclei such as oscillatory firing pattern or interneuronal firing synchrony. Also, the results suggest that dopamine receptors in both the striatum and at extrastriatal sites (especially the subthalamic nucleus) are likely to be involved in dopamine agonist influences on firing rates in the substantia nigra pars reticulata and entopeduncular nucleus.     

Effect of high-frequency stimulation of the subthalamic nucleus on the neuronal activities of the substantia nigra pars reticulata and ventrolateral nucleus of the thalamus in the rat

Electrophysiological recordings were made in anaesthetized rats to investigate the mode of function of high-frequency stimulation of the subthalamic nucleus used as a therapeutic approach for Parkinson's disease. High-frequency electrical stimulation of the subthalamic nucleus (130 Hz) induced a net decrease in activity of all cells recorded around the site of stimulation in the subthalamic nucleus. It also caused an inhibition of the majority of neurons recorded in the substantia nigra pars reticulata in normal rats (94%) and in rats with 6-hydroxydopamine lesions of the substantia nigra pars compacta (90%) or with ibotenic acid lesions of the globus pallidus (79.5%). The majority of cells recorded in the ventrolateral nucleus of the thalamus responded with an increase in their activity (84%).These results show that high-frequency stimulation of the subthalamic nucleus induces a reduction of the excitatory glutamatergic output from the subthalamic nucleus which results in deactivation of substantia nigra pars reticulata neurons. The reduction in tonic inhibitory drive of nigral neurons induces a disinhibition of activity in the ventrolateral motor thalamic nucleus, which should result in activation of the motor cortical system.     

Subthalamic stimulation-induced synaptic responses in substantia nigra pars compacta dopaminergic neurons in vitro.

The subthalamic nucleus (STN) is one of the principal sources of excitatory glutamatergic input to dopaminergic neurons of the substantia nigra, yet stimulation of the STN produces both excitatory and inhibitory effects on nigral dopaminergic neurons recorded extracellularly in vivo. The present experiments were designed to determine the sources of the excitatory and inhibitory effects. Synaptic potentials were recorded intracellularly from substantia nigra pars compacta dopaminergic neurons in parasagittal slices in response to stimulation of the STN. Synaptic potentials were analyzed for onset latency, amplitude, duration, and reversal potential in the presence and absence of GABA and glutamate receptor antagonists. STN-evoked depolarizing synaptic responses in dopaminergic neurons reversed at approximately -31 mV, intermediate between the expected reversal potential for an excitatory and an inhibitory postsynaptic potential (EPSP and IPSP). Blockade of GABA(A) receptors with bicuculline caused a positive shift in the reversal potential to near 0 mV, suggesting that STN stimulation evoked a near simultaneous EPSP and IPSP. Both synaptic responses were blocked by application of the glutamate receptor antagonist, 6-cyano-7-nitroquinoxalene-2,3-dione. The confounding influence of inhibitory fibers of passage from globus pallidus and/or striatum by STN stimulation was eliminated by unilaterally transecting striatonigral and pallidonigral fibers 3 days before recording. The reversal potential of STN-evoked synaptic responses in dopaminergic neurons in slices from transected animals was approximately -30 mV. Bath application of bicuculline shifted the reversal potential to approximately 5 mV as it did in intact animals, suggesting that the source of the IPSP was within substantia nigra. These data indicate that electrical stimulation of the STN elicits a mixed EPSP-IPSP in nigral dopaminergic neurons due to the coactivation of an excitatory monosynaptic and an inhibitory polysynaptic connection between the STN and the dopaminergic neurons of substantia nigra pars compacta. The EPSP arises from a direct monosynaptic excitatory glutamatergic input from the STN. The IPSP arises polysynaptically, most likely through STN-evoked excitation of GABAergic neurons in substantia nigra pars reticulata, which produces feed-forward GABA(A)-mediated inhibition of dopaminergic neurons through inhibitory intranigral axon collaterals.     

Endocannabinoid-mediated short-term suppression of excitatory synaptic transmission to medium spiny neurons in the striatum

Medium spiny neurons in the dorsal striatum receive glutamatergic excitatory synaptic inputs from the cerebral cortex. These synapses undergo long-term depression that requires release of endocannabinoids from medium spiny neurons and activation of cannabinoid CB1 receptors. However, it remains unclear how cortico-striatal synapses exhibit endocannabinoid-mediated short-term suppression, which has been found in various brain regions including the hippocampus and cerebellum. Endocannabinoids are released from postsynaptic neurons by strong depolarization and resultant Ca2+ elevation or activation of postsynaptic Gq/11-coupled receptors such as group I metabotropic glutamate receptors (mGluRs) and M1/M3 muscarinic acetylcholine receptors. Moreover, endocannabioids are effectively released when weak depolarization is combined with Gq/11-coupled receptor activation. We found that muscarinic activation induced transient suppression of excitatory synaptic transmission to medium spiny neurons, which was independent of retrograde endocannabinoid signaling but was mediated directly by presynaptic muscarinic receptors. Neither postsynaptic depolarization alone nor depolarization and muscarinic activation caused suppression of cortico-striatal synapses. In contrast, activation of group I mGluRs readily suppressed cortico-striatal excitatory synaptic transmission. Furthermore, postsynaptic depolarization induced clear suppression when combined with group I mGluR activation. These results indicate that group I mGluRs but not muscarinic receptors contribute to endocannabinoid-mediated short-term suppression of cortico-striatal excitatory synaptic transmission.     

Structural correlates of heterogeneous in vivo activity of midbrain dopaminergic neurons

Dopaminergic neurons of the substantia nigra pars compacta (SNc) exhibit functional heterogeneity that likely underpins their diverse roles in behavior. We examined how the functional diversity of identified dopaminergic neurons in vivo correlates with differences in somato-dendritic architecture and afferent synaptic organization. Stereological analysis of individually recorded and labeled dopaminergic neurons of rat SNc revealed that they received approximately 8,000 synaptic inputs, at least 30% of which were glutamatergic and 40-70% were GABAergic. The latter synapses were proportionally greater in number and denser on dendrites located in the substantia nigra pars reticulata (SNr) than on those located in SNc, revealing the existence of two synaptically distinct and region-specific subcellular domains. We also found that the relative extension of SNc neuron dendrites into the SNr dictated overall GABAergic innervation and predicted inhibition responses to aversive stimuli. We conclude that diverse wiring patterns determine the heterogeneous activities of midbrain dopaminergic neurons in vivo.     

Morphological study of the tegmental pedunculopontine nucleus, substantia nigra and subthalamic nucleus, and their interconnections in rat organotypic culture

The morphological organization of the tegmental pedunculopontine nucleus, midbrain extrapyramidal area, substantia nigra and subthalamic nucleus and their interrelationships were studied in rat organotypic culture using immunohistochemistry and NADPH-diaphorase histochemistry. Three coronal sections, one containing the tegmental pedunculopontine nucleus/midbrain extrapyramidal area, another with the substantia nigra and the third with the subthalamic nucleus, were obtained from postnatal 1-2-day-old rats. These sections were co-cultured for 3-4 weeks using the roller-tube technique. In the tegmental pedunculopontine nucleus/midbrain extrapyramidal area, the distribution pattern of cholinergic neurons was similar to that found in the in vivo study. We could, therefore, identify the subdivisions of the tegmental pedunculopontine nucleus (i.e., pars compacta and pars dissipata) and the midbrain extrapyramidal area. As in the in vivo situation, glutamate immunoreactive neurons were also located in these areas. Approximately 10% of NADPH-diaphorase positive neurons in the tegmental pedunculopontine nucleus, were glutamate immunoreactive. In the substantia nigra, as in the in vivo, tyrosine hydroxylase immunoreactive (putative dopaminergic) neurons were identified predominantly in the substantia nigra pars compacta, and parvalbumin immunoreactive neurons (putative GABAergic) mainly in the substantia nigra pars reticulata. The subthalamic nucleus was ladened with glutamate immunoreactive neurons. NADPH-diaphorase stained axons originating from the tegmental pedunculopontine nucleus were traced into the substantia nigra and subthalamic nucleus. They were often in close apposition to tyrosine hydroxylase immunoreactive neurons in the substantia nigra. Parvalbumin immunoreactive fibers from the substantia nigra projected heavily to the midbrain extrapyramidal area, but only sparsely to the tegmental pedunculopontine nucleus and the subthalamic nucleus. These findings indicate that the tegmental pedunculopontine nucleus/midbrain extrapyramidal area, substantia nigra and subthalamic nucleus in the organotypic culture have retained a basic morphological organization and connectivity similar to those seen in the in vivo situation. Therefore, this preparation could be a useful model to conduct further studies to investigate functional circuits among the structures represented.     

Synaptic localization of ionotropic glutamate receptors in the rat substantia nigra

Glutamatergic neurotransmission in the substantia nigra pars compacta and pars reticulata is mediated through N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxaline propionic acid/kainate (AMPA) type receptors as well as other glutamate receptors and is critical for basal ganglia functioning. A major glutamatergic input to the substantia nigra originates in the subthalamic nucleus, and the long-lasting stimulation of the dopaminergic cells of the substantia nigra pars compacta by the subthalamic neurons has been implicated in the pathophysiology of Parkinson's disease. The objectives of the present study were to determine the subcellular and subsynaptic localization of subunits of the N-methyl-D-aspartate and AMPA receptors in the substantia nigra, and also to determine whether co-localization of N-methyl-D-aspartate and AMPA receptor subunits occur at individual synapses. To achieve this, pre-embedding and post-embedding immunocytochemistry was applied to sections of substantia nigra using antibodies that recognize the NR1 and NR2A/B subunits of the N-methyl-D-aspartate receptor, and GluR2/3 subunits of the AMPA receptor.In both regions of the substantia nigra, immunolabelling for each of the subunits was observed in numerous perikarya and proximal dendrites. At the subcellular level, silver-intensified immunogold particles localizing N-methyl-D-aspartate and AMPA receptor subunits were most commonly present within dendrites where they were associated with a variety of intracellular organelles and with the internal surface of the plasma membrane. Post-embedding immunogold labelling revealed immunoparticles labelling for NR1, NR2A/B and GluR2/3 to be enriched at asymmetric synaptic specializations, although a large proportion of asymmetric synapses were immunonegative. Double immunolabelling revealed, in addition to single-labelled synapses, the co-localization of subunits of the N-methyl-D-aspartate receptor and subunits of the AMPA receptor at individual asymmetric synapses. Similarly, double immunolabelling also revealed the co-localization of the NRl and NR2A/B subunits of the N-methyl-D-aspartate receptor at individual asymmetric synapses. Labelling for NR1 and GluR2/3 was, on average, relatively evenly distributed across the width of the synapse with a gradual reduction towards the periphery when analysed in single sections.In summary, the present results demonstrate that AMPA and N-methyl-D-aspartate receptors are selectively localized at a subpopulation of asymmetric synapses in the substantia nigra pars compacta and reticulata and that the two receptor types, at least partially co-localize at individual synapses. It is concluded that glutamatergic transmission in the substantia nigra pars compacta and pars reticulata occurs primarily at asymmetric synapses and, at least in part, is mediated by both N-methyl-D-aspartate and AMPA receptors.     

Pre- and postsynaptic localization of GABA(B) receptors in the basal ganglia in monkeys

GABAergic neurotransmission involves ionotropic GABA(A) and metabotropic GABA(B) receptor subtypes. Although fast inhibitory transmission through GABA(A) receptors activation is commonly found in the basal ganglia, the functions as well as the cellular and subcellular localization of GABA(B) receptors are still poorly known. Polyclonal antibodies that specifically recognize the GABA(B)R1 receptor subunit were produced and used for immunocytochemical localization of these receptors at the light and electron microscope levels in the monkey basal ganglia. Western blot analysis of monkey brain homogenates revealed that these antibodies reacted specifically with two native proteins corresponding to the size of the two splice variants GABA(B)R1a and GABA(B)R1b. Preadsorption of the purified antiserum with synthetic peptides demonstrated that these antibodies recognize specifically GABA(B)R1 receptors with no cross-reactivity with GABA(B)R2 receptors. Overall, the distribution of GABA(B)R1 immunoreactivity throughout the monkey brain correlates with previous GABA(B) ligand binding studies and in situ hybridization data as well as with recent immunocytochemical studies in rodents. GABA(B)R1-immunoreactive cell bodies were found in all basal ganglia nuclei but the intensity of immunostaining varied among neuronal populations in each nucleus. In the striatum, interneurons were more strongly stained than medium-sized projection neurons while in the substantia nigra, dopaminergic neurons of the pars compacta were much more intensely labeled than GABAergic neurons of the pars reticulata. In the subthalamic nucleus, clear immunonegative neuronal perikarya were intermingled with numerous GABA(B)R1-immunoreactive cells. Moderate GABA(B)R1 immunoreactivity was observed in neuronal perikarya and dendritic processes throughout the external and internal pallidal segments. At the electron microscope level, GABA(B)R1 immunoreactivity was commonly found in neuronal cell bodies and dendrites in every basal ganglia nuclei. Many dendritic spines also displayed GABA(B)R1 immunoreactivity in the striatum. In addition to strong postsynaptic labeling, GABA(B)R1-immunoreactive preterminal axonal segments and axon terminals were frequently encountered throughout the basal ganglia components. The majority of labeled terminals displayed the ultrastructural features of glutamatergic boutons and formed asymmetric synapses. In the striatum, GABA(B)R1-containing boutons resembled terminals of cortical origin, while in the globus pallidus and substantia nigra, subthalamic-like terminals were labeled. Overall, these findings demonstrate that GABA(B) receptors are widely distributed and located to subserve both pre- and postsynaptic roles in controlling synaptic transmission in the primate basal ganglia.     

Dizocilpine maleate, MK-801, but not 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline, NBQX, prevents transneuronal degeneration of nigral neurons after neurotoxic striatal-pallidal lesion

Unilateral neurotoxin lesion of rat caudate-putamen and globus pallidus resulted in delayed, transneuronal degeneration of GABAergic substantia nigra pars reticulata neurons. To explore whether the disinhibition of endogenous glutamate excitatory input played a role in the degeneration of substantia nigra pars reticulata neurons, animals with unilateral striatal-pallidal lesions received three daily intraperitoneal injections of either dizocilpine maleate (MK-801, 1 or 10 mg/kg), an N-methyl-D-aspartate glutamate receptor blocker, or 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX, 30 mg/kg), an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor blocker, that began 24 h after the striatal-pallidal neurotoxin lesion. Drug treatment affected neither the volume of the initial lesion nor the volume of striatal-pallidal glial fibrillary acidic protein immunoreactivity. Neuron number in the substantia nigra pars reticulata ipsilateral to the lesioned striatopallidum was reduced on average by 37% in untreated control rats, in low dose MK-801, and NBQX-treated rats (P<0.0001). However, in animals treated with high doses of MK-801 there was no difference in the number of neurons in the substantia nigra pars reticulata ipsilateral or contralateral to the neurotoxin lesion. These data demonstrate that dose-related treatment with N-methyl-D-aspartate glutamate receptor blockers protects substantia nigra pars reticulata neurons, and suggests that glutamatergic mechanisms play a role in delayed transneuronal degeneration.     

Bilateral cerebral metabolic effects of pharmacological manipulation of the substantia nigra in the rat: Unilateral intranigral application of the putative excitatory neurotransmitter substance P

The metabolic activity of several anatomically distinct brain areas was investigated by means of the quantitative autoradiographic 2-deoxy-d[1-¹⁴C]glucose method in awake rats following unilateral intranigral application of the putative excitatory neurotransmitter substance P. The primary goal was to determine the metabolic effects of substance P on the substantia nigra and its targets. Intranigral injection of 1 mM substance P (1.5 μl) induced metabolic activation locally in the substantia nigra reticulata by 117% and substantia nigra compacta by 35%, as well as distally in the contralateral substantia nigra reticulata by 22% and contralateral substantia nigra compacta by 21%. All the basal ganglia components, the striatum, pallidum, entopeduncular, subthalamic nucleus and nucleus accumbens displayed bilateral metabolic activations after unilateral intranigral substance P injection. Among the principal reticulata efferent projections, the ventromedial, ventrolateral, parafascicular, mediodorsal and centrolateral thalamic nuclei, as well as the pedunculopontine nucleus displayed bilateral metabolic activations after intranigral substance P application. Moreover, unilateral intranigral substance P injection elicited metabolic activations in the thalamic and cortical components of the reticular, intralaminar, limbic and prefrontal systems, mostly bilateral.

It is suggested that substance P applied into one substantia nigra reticulata activates the compacta nigrostriatal dopaminergic and the reticulata nigrothalamic GABAergic outputs inducing distal metabolic effects, similar to those elicited by unilateral nigral electrical stimulation [Savaki et al. (1983) J. comp. Neurol.213, 46–65] and, opposite to several of those induced by intranigral injection of the inhibitory GABA_A agonist muscimol [Savaki et al. (1992) Neuroscience50, 781–794]. Furthermore, it is suggested that the ipsilateral basal ganglia effects induced by intranigral substance P application are mediated via both the compacta dopaminergic nigrostriatal projection and the reticulata GABAergic nigro-thalamo-cortico-striatal loop, whereas the contralateral basal ganglia and associated thalamocortical effects are due to the activation of the GABAergic reticulata efferents and are mediated via an interthalamic circuitry involving the motor, reticular and intralaminar thalamic nuclei.     

Distribution of divalent metal transporter-1 in the monkey basal ganglia

An accumulation of iron occurs in the brain with age, and it is thought that this may contribute to the pathology of certain neurodegenerative diseases, including Parkinson's disease. In this study, we elucidated the distribution of divalent metal transporter-1 (DMT1) in the monkey basal ganglia by immunocytochemistry, and compared it with the distribution of ferrous iron in these nuclei by Turnbull's Blue histochemical staining. We observed a general correlation between levels of DMT1, and iron staining. Thus, regions such as the caudate nucleus, putamen, and substantia nigra pars reticulata contained dense staining of DMT1 in astrocytic processes, and were also observed to contain large numbers of ferrous iron granules. The exceptions were the globus pallidus externa and interna, which contained light DMT1 staining, but large numbers of ferrous iron granules. The thalamus, subthalamic nucleus, and substantia nigra pars compacta contained neurons that were lightly stained for DMT1, but few or no iron granules. The high levels of DMT1 expression in some of the nuclei of the basal ganglia, particularly the caudate nucleus, putamen, and substantia nigra pars reticulata, may account for the high levels of iron in these regions.     

Distribution and synaptic localisation of the metabotropic glutamate receptor 4 (mGluR4) in the rodent CNS

Group III metabotropic glutamate receptors (mGluRs) are selectively activated by L-2-amino-4-phosphonobutyrate (L-AP4), which produces depression of synaptic transmission. The relative contribution of different group III mGluRs to the effects of L-AP4 remains to be clarified. Here, we assessed the distribution of mGluR4 in the rat and mouse brain using affinity-purified antibodies raised against its entire C-terminal domain. The antibodies reacted specifically with mGluR4 and not with other mGluRs in transfected COS 7 cells. No immunoreactivity was detected in brains of mice with gene-targeted deletion of mGluR4. Pre-embedding immunocytochemistry for light and electron microscopy showed the most intense labelling in the cerebellar cortex, basal ganglia, the sensory relay nuclei of the thalamus, and some hippocampal areas. Immunolabelling was most intense in presynaptic active zones. In the basal ganglia, both the direct and indirect striatal output pathways showed immunolabelled terminals forming mostly type II synapses on dendritic shafts. The localisation of mGluR4 on GABAergic terminals of striatal projection neurones suggests a role as a presynaptic heteroreceptor. In the cerebellar cortex and hippocampus, mGluR4 was also localised in terminals establishing type I synapses, where it probably operates as an autoreceptor. In the hippocampus, mGluR4 labelling was prominent in the dentate molecular layer and CA1-3 strata lacunosum moleculare and oriens. Somatodendritic profiles of some stratum oriens/alveus interneurones were richly decorated with mGluR4-labelled axon terminals making either type I or II synapses. This differential localisation suggests a regulation of synaptic transmission via a target cell-dependent synaptic segregation of mGluR4.Our results demonstrate that, like other group III mGluRs, presynaptic mGluR4 is highly enriched in the active zone of boutons innervating specific classes of neurones. In addition, the question of alternatively spliced mGluR4 isoforms is discussed.     

The pedunculopontine nucleus: its role in the genesis of movement disorders

The pedunculopontine nucleus (PPN) is located in the dorso-lateral part of the ponto-mesencephalic tegmentum. The PPN is composed of two groups of neurons: one containing acetylcholine, and the other containing non-cholinergic neurotransmitters (GABA, glutamate). The PPN is connected reciprocally with the limbic system, the basal ganglia nuclei (globus pallidus, substantia nigra, subthalamic nucleus), and the brainstem reticular formation. The caudally directed corticolimbic-ventral striatal-ventral pallidal-PPN-pontomedullary reticular nuclei-spinal cord pathway seems to be involved in the initiation, acceleration, deceleration, and termination of locomotion. This pathway is under the control of the deep cerebellar and basal ganglia nuclei at the level of the PPN, particularly via potent inputs from the medial globus pallidus, substantia nigra pars reticulata and subthalamic nucleus. The PPN sends profuse ascending cholinergic efferent fibers to almost all the thalamic nuclei, to mediate phasic events in rapid-eye-movement sleep. Experimental evidence suggests that the PPN, along with other brain stem nuclei, is also involved in anti-nociception and startle reactions. In idiopathic Parkinson's disease (IPD) and parkinson plus syndrome, overactive pallidal and nigral inhibitory inputs to the PPN may cause sequential occurrences of PPN hypofunction, decreased excitatory PPN input to the substantia nigra, and aggravation of striatal dopamine deficiency. In addition, neuronal loss in the PPN itself may cause dopamine-resistant parkinsonian deficits, including gait disorders, postural instability and sleep disturbances. In patients with IPD, such deficits may improve after posteroventral pallidotomy, but not after thalamotomy. One of the possible explanations for such differences is that dopamine-resistant parkinsonian deficits are mediated to the PPN by the descending pallido-PPN inhibitory fibers, which leave the pallido-thalamic pathways before they reach the thalamic targets.     

The parafascicular thalamic complex and basal ganglia circuitry: further complexity to the basal ganglia model

This work is focused on the study of neuronal circuits arising from the rodent caudal intralaminar nuclei and their presumed role on basal ganglia function. Emphasis was placed on the analysis of the architecture of thalamostriatal and thalamo-subthalamic projections in albino rats. Our major interest was to elucidate whether thalamic inputs were related to projection neurons or local circuit neurons within targeted structures (striatum and subthalamic nucleus). Projections coming from the parafascicular nucleus (PF) to the striatum displayed a patchy organization throughout the matrix compartment. These patches are composed by dense terminal axonal arborizations, often containing striatal neurons projecting to the entopeduncular nucleus (ENT) (medial globus pallidus in primates) and neurons projecting to the substantia nigra reticulata (SNR). The thalamostriatal projections under scrutiny were also seen to be in register with all the major classes of striatal interneurons (nitrergic neurons, neurons containing the calcium binding protein parvalbumin (PV) and cholinergic interneurons). Subthalamic neurons projecting to the ENT are the presumed postsynaptic target for fibers coming from the sensorimotor part (dorsolateral) of the PF. In summary, glutamatergic axons arising from the PF might exert a dual control of the striatal output, either by directly exciting striatal projection neurons or indirectly by means of a previous synaptic contact onto an striatal interneuron which in turn modulates the activity of projection neurons. Furthermore, thalamic inputs can also gain access to basal ganglia output nuclei via subthalamo-pallidal projecting neurons, neurons receiving glutamatergic thalamo-subthalamic projections. Thus, activation of either circuit has an opposite physiological effect on the basal ganglia output nucleus. Taken together, these data suggest that the PF may influence neuronal activity in the direct and indirect circuits and could be considered as an additional component of the basal ganglia motor loops.     

Intrasubthalamic nucleus metabotropic glutamate receptor activation: a behavioral, Fos immunohistochemical and [14C]2-deoxyglucose autoradiographic study

Metabotropic glutamate receptors are a major class of excitatory amino acid receptors. Eight metabotropic glutamate receptor subtypes have been cloned, and are classified into three groups (I, II and III) based on amino acid sequence identity, effector systems and pharmacological profile. Previous results have shown that unilateral stimulation of metabotropic glutamate receptors in the subthalamic nucleus with the non-subtype-selective metabotropic glutamate receptor agonist 1S,3R-1-amino-1,3-cyclopentane dicarboxylate results in contralateral rotation in rats and Fos expression in the subthalamic nucleus. This suggests that metabotropic glutamate receptor stimulation results in altered subthalamic nucleus activity with consequent altered basal ganglia activity on the injected side. We sought to determine the metabotropic glutamate receptor subtype(s) involved and the functional neuroanatomy underlying the rotational behavior. Unilateral intrasubthalamic nucleus injection of group II or group III metabotropic glutamate receptor agonists induced contralateral rotation. In addition to producing rotation, group II and group III metabotropic glutamate receptor agonists induce toxicity in the subthalamic nucleus and overlying thalamus. Following group II or group III subthalamic nucleus metabotropic glutamate receptor stimulation, there is Fos-like immunoreactivity in the globus pallidus, subthalamic nucleus, substantia nigra pars reticulata and entopeduncular nucleus, suggesting altered activity in subthalamic nucleus target regions. However, examination of [14C]2-deoxyglucose uptake suggests that the alterations in basal ganglia activity are different following group II versus group III metabotropic glutamate receptor stimulation, suggesting that rotation is occurring via different mechanisms. It appears that stimulation of subthalamic nucleus group II metabotropic glutamate receptors induces rotation by increasing subthalamic nucleus activity. These results suggest that group II metabotropic glutamate receptor antagonists may be useful for alleviating subthalamic nucleus overactivity in Parkinson's disease.     

Correlated multisecond oscillations in firing rate in the basal ganglia: modulation by dopamine and the subthalamic nucleus

Previous studies from this laboratory have shown that many neurons in the basal ganglia have multisecond (<0.5 Hz) periodicities in firing rate in awake rats. The frequency and regularity of these oscillations are significantly increased by systemically injected dopamine (DA) agonists. Because oscillatory activity should have greater functional impact if shared by many neurons, the level of correlation of multisecond oscillations was assessed by recording pairs of neurons in the globus pallidus and substantia nigra pars reticulata in the same hemisphere, or pairs of globus pallidus neurons in opposite hemispheres in awake, immobilized rats. Cross-correlation (90-180 s lags) and spectral analysis were used to characterize correlated oscillations. Thirty-eight percent of pairs recorded in baseline (n=50) demonstrated correlated multisecond oscillations. Phase relationships were near 0 or 180 degrees. DA agonist injection significantly increased the incidence of correlation (intra- and interhemispheric) to 94% (n=17). After DA agonist injection, phase relationships of globus pallidus/substantia nigra neuron pairs were exclusively concentrated near 180 degrees, and phases of interhemispheric pairs of globus pallidus neurons were concentrated near 0 degrees. After subthalamic nucleus lesion (n=8), the incidence of correlated multisecond oscillations (or of multisecond oscillations per se) was not changed, although the consistent phase relationship between the globus pallidus and substantia nigra pars reticulata was disrupted. Subthalamic lesion also blocked apomorphine-induced decreases in oscillatory period and increases in oscillation amplitude, and significantly attenuated apomorphine-induced changes in mean firing rate. The data demonstrate that multisecond oscillations in the basal ganglia can be correlated between nuclei, and that DA receptor activation increases the level of correlation and organizes internuclear phase relationships at these multisecond time scales. While the subthalamic nucleus is not necessary for generating or transmitting these slow oscillations, it is involved in DA agonist-induced modulation of mean firing rate, oscillatory period, and internuclear phase relationship. These data further support a role for DA in modulating coherent oscillatory activity in the basal ganglia, and for the subthalamic nucleus in shaping the effects of DA receptor stimulation on basal ganglia output.