Metabotropic glutamate receptor 4 in the basal ganglia of parkinsonian monkeys: ultrastructural localization and electrophysiological effects of activation in the striatopallidal complex

Abstract

Group III metabotropic glutamate receptors (mGluR4,7,8) are widely distributed in the basal ganglia. Injection of group III mGluR agonists into the striatopallidal complex alleviates parkinsonian symptoms in 6-hydroxydopamine-treated rats. In vitro rodent studies have suggested that this may be partly due to modulation of synaptic transmission at striatopallidal and corticostriatal synapses through mGluR4 activation. However, the in vivo electrophysiological effects of group III mGluRs activation upon basal ganglia neurons activity in nonhuman primates remain unknown. Thus, in order to examine the anatomical substrates and physiological effects of group III mGluRs activation upon striatal and pallidal neurons in monkeys, we used electron microscopy immunohistochemistry to localize mGluR4, combined with local administration of the group III mGluR agonist L-AP4, or the mGluR4 positive allosteric modulator VU0155041, to assess the effects of group III mGluR activation on the firing rate and pattern of striatal and pallidal neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonian monkeys. At the ultrastructural level, striatal mGluR4 immunoreactivity was localized in pre- (60%) and post-synaptic (30%) elements, while in the GPe, mGluR4 was mainly expressed pre-synaptically (90%). In the putamen, terminals expressing mGluR4 were evenly split between putative excitatory and inhibitory terminals, while in the GPe, most labeled terminals displayed the ultrastructural features of striatal-like inhibitory terminals, though putative excitatory boutons were also labeled. No significant difference was found between normal and parkinsonian monkeys. Extracellular recordings in awake MPTP-treated monkeys revealed that local microinjections of small volumes of L-AP4 resulted in increased firing rates in one half of striatal cells and one third of pallidal cells, while a significant number of neurons in both structures showed either opposite effects, or did not display any significant rate changes following L-AP4 application. VU0155041 administration had little effect on firing rates. Both compounds also had subtle effects on bursting and oscillatory properties, acting to increase the irregularity of firing. The occurrence of pauses in firing was reduced in the majority (80%) of GPe neurons after L-AP4 injection. Our findings indicate that glutamate can mediate multifarious physiological effects upon striatal and pallidal neurons through activation of pre-synaptic group III mGluRs at inhibitory and excitatory synapses in parkinsonian monkeys. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Figure 1

Light micrographs of coronal monkey brain sections showing immunostaining for mGluR4 at various levels of a normal (A-D) and an MPTP-treated monkey (E,F). The approximate interaural coordinate for each section is designated in the lower left of each panel. Scale bar in A equals 5 mm and applies to all panels. AC – anterior commissure, CN – caudate nucleus, CTX – cortex, GPe – external globus pallidus, GPi – internal globus pallidus, HIP – hippocampus, IC – internal capsule, PUT – putamen, SNc – substantia nigra pars compacta, SNr – substantia nigra pars reticulata, TH - thalamus.

Figure 2

Immunolabeling for mGluR4 at the electron microscopic level in the monkey external globus pallidus. A, B) Labeled terminals form symmetric synapses on dendrites in the GPe. C, D) Labeled terminals form asymmetric synapses on dendrites in the GPe. E) Histogram showing the breakdown of the proportions of each type of labeled element found in the GPe of normal and MPTP-treated monkeys. n=3 normal, 3 MPTP-treated monkeys. Error bars represent SEM. No significant difference was found between normal and MPTP-treated monkeys. Synapses are identified with arrowheads. Immunoperoxidase labeling is identified with arrows. a – labeled axon, g – labeled glial process, Te – labeled terminal, u.Te – unlabeled terminal.

Figure 3

Prevalence of mGluR4-labeled terminal subtypes in the putamen and GPe. A) The percentage of mGluR4-positive terminals that formed symmetric synapses in normal and MPTP-treated monkeys. (N total labeled terminals=Normal: 52 in putamen, 128 in GPe; MPTP: 56 in putamen, 120 in GPe) B) The percentage of the total population of GPe terminals, forming asymmetric or symmetric synapses, that contained mGluR4 immunoreactivity. (N total GPe terminals labeled and unlabeled=Normal: 26 asymmetric, 412 asymmetric; MPTP: 33 asymmetric, 351 symmetric) Error bars represent SEM. No significant difference was found between normal and MPTP-treated monkeys. (N monkeys=3 normal, 3 MPTP-treated).

Figure 4

Immunolabeling for mGluR4 at the electron microscopic level in the monkey putamen. A) An unlabeled terminal arising from a labeled axon and forming a symmetric synapse on a dendrite. B) A labeled terminal forming a symmetric synapse on a spine. C) A labeled and an unlabeled terminal forming asymmetric synapses on spines. D) A labeled dendrite and a labeled terminal forming a symmetric synapse on a dendrite. E) Histogram showing the breakdown of the proportions of each type of labeled element found in the putamen of normal and MPTP-treated monkeys. n=3 normal, 3 MPTP-treated monkeys. Error bars represent SEM. No significant difference was found between normal and MPTP-treated monkeys. Synapses are identified with arrowheads. Immunoperoxidase labeling is identified with arrows. a – labeled axon, d – labeled dendrite, s - spine, Te – labeled terminal, u.Te – unlabeled terminal.

Figure 5

An example trace of a GPe neuron that increased its firing rate in response to L-AP4. The green bar represents the control period. The red bar represents the drug injection. The blue bar represents the window of time during which a drug effect may begin, in order to be considered as such. The solid horizontal line indicates the median firing rate at baseline, and the dashed lines show the 90^th and 10^th percentiles. The red vertical dotted line represents the center of the 60 sec period analyzed for drug effect.

Figure 6

Responses of GPe neurons in MPTP-treated monkeys to infusion of aCSF, L-AP4, or VU0155041 (VU). A) Changes in firing rate (expressed as firing rate ratios, effect/baseline) of GPe neurons in response to drug infusion. The proportions of cells showing increased, decreased, or no change in firing rate were statistically different between the L-AP4 and aCSF groups (chi-squared, p=0.02). B) Changes in firing pattern (expressed as coefficient of variance ratios) of GPe neurons in response to drug infusion. Solid horizontal line represents the mean of aCSF data. Upper and lower dashed lines represent 90^th and 10^th percentiles of aCSF data, respectively. aCSF n=14, L-AP4 n=27, VU n=10.

Figure 7

Responses of striatal neurons in MPTP-treated monkeys to infusion of aCSF, L-AP4, or VU0155041 (VU). A) Changes in firing rate (expressed as firing rate ratios, effect/baseline) of striatal neurons in response to drug infusion. B) Changes in firing pattern (expressed as coefficient of variance ratios) of striatal neurons in response to drug infusion. Blue circles represent PANs, green circles represent TANs, and tan circles represent striatal cells that were not classified (NC). Solid horizontal line represents the mean of aCSF data. Upper and lower dashed lines represent 90^th and 10^th percentiles of aCSF data, respectively. aCSF n=9, L-AP4 n=24, VU n=13.