K.K.L. Yung

Neuroprotective effects of ginsenoside-Rg1 in primary nigral neurons against rotenone toxicity

Ginsenoside-Rg1, the pharmacologically active component isolated from ginseng, demonstrated neuroprotective effects on primary cultured rat nigral neurons against rotenone toxicity. Rotenone, a common household pesticide known for its specific and irreversible mitochondria complex I inhibition, has been suggested to be the causal agent of Parkinsons disease (PD) by inducing degeneration of cells in the substantial nigra. The present study demonstrated that co-treatment of rotenone and Rg1 could reduce rotenone-induced cell death by 58% (SEM=+/-5.60; N=3). Rotenone-induced mitochondria membrane potential (MMP, DeltaPsim) depletion was restored and elevated by at least 38% (SEM=+/-2.15; N=3) by Rg1. In addition, Rg1 prevented cytochrome c release from the mitochrondrial membrane and increased the phosphorylation inhibition of the pro-apoptotic protein Bad through activation of the PI3K/Akt pathway. The protective effects of Rg1 was blocked by glucocorticoid receptor antagonist RU486, indicating that the action of Rg1 is mediated through glucocorticoid receptor (GR). In conclusion, Rg1 inhibits the mitochondrial apoptotic pathway and increases the survival chance of the primary cultured nigral neurons against rotenone toxicity. Thus, Rg1 and its related compounds may be developed as protective agents against neurodegenerative diseases induced by mitochondrial toxins.

Differential expression of GABABR1 and GABABR2 receptor immunoreactivity in neurochemically identified neurons of the rat neostriatum

Gamma‐aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the neostriatum. Functions of GABA are known to mediate GABAA and GABAB receptors. A functional GABAB receptor is known to compose of heteromeric subunits, namely the GABABR1 and GABABR2 subunits. Our previous report (Yung et al. [1999] Brain Res. 830:345–352) has demonstrated that all major subpopulations of striatal neurons express GABABR1 immunoreactivity. The cellular localization of the second subunit of GABAB receptor protein, i.e., GABABR2 immunoreactivity, in the rat neostriatum is not yet known. By using a new commercially available specific antibody against GABABR2, immunofluorescence was performed to investigate the cellular expression of GABABR2 in neurochemically identified subpopulations of neurons in the rat neostriatum. Immunoreactivity for GABABR2 was primarily found in the neuropil of the rat neostriatum. Double labeling revealed that those perikarya that expressed immunoreactivity for parvalbumin, choline acetyltransferase, nitric oxide synthase, glutamate receptor two, N‐methyl‐D‐aspartate receptor one, or GABAAα1 receptor, respectively, did not express GABABR2 immunoreactivity. In addition, perikarya and most of the neuropilar elements in the neostriatum that expressed glutamic acid decarboxylase 67 immunoreactivity were found to be GABABR2‐negative. In contrast, immunoreactivity for GABABR1 was found to be expressed by all of the above neuronal subpopulations. Moreover, a vast number of SV2‐immunoreactive profiles and a number of tyrosine hydroxylase‐immunoreactive profiles in the neuropil of the neostriatum were found to display GABABR2 immunoreactivity. The present results indicate that there is a differential expression of GABABR2 and GABABR1 immunoreactivity in different subpopulations of striatal neurons that are identified by their specific neurochemical markers. Immunoreactivity for GABABR2 is likely to localize in neuropilar elements of the neostriatum that may belong to non‐GABAergic elements. These findings provide anatomical evidence of GABABR2 receptor localization in the neostriatum that may have an important functional implication of the GABAB‐mediated functions in neurons of the neostriatum. J. Comp. Neurol. 433:458–470, 2001.

Cellular localization of GluR1, GluR2/3 and GluR4 glutamate receptor subunits in neurons of the rat neostriatum.

Glutamate excitocytotoxicity is implied in the cause of neuronal degeneration in the neostriatum, in which the toxicity may be mediated by different families of glutamate receptors. The precise cellular localization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate receptor subunits (GluR1-4), one of the major family that involves in the mechanisms of glutamate excitocytotoxicity, in different populations of striatal neurons is therefore of special interest. Immunoreactivity for GluR2/3 subunits was detected in the medium-sized spiny neurons. By double labelling experiments, immunoreactivity for GluR1 and GluR4 was detected only in aspiny striatal neurons that display parvalbumin immunoreactivity, but not in the other neuron populations that display choline acetyltransferase or muscarinic m2 receptor immunoreactivity, nor neurons that display nitric oxide synthase immunoreactivity or nicotinamide adenine dinucleotide phosphate-diaphorase activity. These results indicate that GluR1 and GluR4 immunoreactivity is displayed only in the GABAergic interneurons in the neostriatum. In addition, almost all of the GluR1-immunoreactive neurons were found to display GluR4 immunoreactivity. This finding indicates for the first time that the striatal GABAergic interneurons co-express GluR1 and GluR4 subunits. The results of the present study indicate that there is a differential localization of AMPA-type glutamate receptor subunits in different populations of striatal neurons and they may have a different susceptibility to glutamate excitocytotoxicity.

Subpopulations of neurons in the rat neostriatum display GABABR1 receptor immunoreactivity.

Immunoreactivity for gamma aminobutyric acid BR1 receptor (GABABR1) was detected in the neuropilar elements as well as in the perikarya of neurons in the neostriatum. Many of the GABABR1-immunoreactive perikarya were medium-sized with a thin rim of cytoplasm. They resembled the morphology of medium spiny neurons, the projection neurons of the neostriatum. In addition, some GABABR1-immunoreactive neurons were densely labeled and were of medium to large in size. These neurons were characterized by double immunofluorescence using their neurochemicals as markers. Over 90% of the parvalbumin- and choline acetyltransferase-immunoreactive neurons and about 80% of the nitric oxide synthase-immunoreactive neurons displayed GABABR1 immunoreactivity. The present results show for the first time that the major four subpopulations of striatal neurons express GABABR1 receptor and may have a functional implication in the GABA neurotransmission in the microcircuitry of the neostriatum.

Localization of ionotropic and metabotropic glutamate receptors in distinct neuronal elements of the rat substantia nigra

The localization of glutamate receptors in the substantia nigra is of critical importance since glutamate receptor-mediated excitotoxicity is implied in the cause for the neuronal degeneration in Parkinsons disease. The major glutamatergic synaptic inputs to the substantia nigra originate in the subthalamic nucleus, in which hyperactivity is reported in Parkinsons disease. In order to compare directly the localization of different ionotropic and metabotropic glutamate receptors in the substantia nigra of the same animals, rats were perfuse-fixed under deep anesthesia. Sections of the substantia nigra were obtained and receptor immunocytochemistry was performed using commercially available antibodies (against subunits of ionotropic glutamate receptors: GluR1, GluR2/3, GluR4, NMDAR1, NMDAR2A/B; and subtypes of metabotropic glutamate receptors: mGluR1alpha, mGluR2/3). When compared to the localization of tyrosine hydroxylase immunoreactivity, immunoreactivity for GluR1, GluR2/3 and NMDARI was mainly localized in the perikarya and proximal dendrites of the compacta neurons and only in a few reticulata neurons. In contrast, GluR4 immunoreactivity was only detected in the reticulata neurons. Consistent results were obtained by double labeling experiments that revealed tyrosine hydroxylase and GluR1, GluR2/3, GluR4 or NMDAR1 immunoreactivity in the same sections. Immunoreactivity for NMDAR2A/B, mGluR1alpha. and mGluR2/3 was detected in the neuropil of the substantia nigra pars reticulata. No NMDAR2A/B- and mGluR2/3-immunoreactive perikarya were detected. However, a few neurons in the reticulata were found to be mGluR1alpha-immunoreactive. The present results indicate there is a differential localization of different subunits and subtypes of glutamate receptors in the substantia nigra and there may be functional implications in different neuronal elements in the substantia nigra in normal and in Parkinsons disease.

Changes in expression of N-methyl-d-aspartate receptor subunits in the rat neostriatum after a single dose of antisense oligonucleotide specific for N-methyl-d-aspartate receptor 1 subunit

In order to investigate the process of gene expression of N-methyl-D-aspartate glutamate receptor (NMDAR) subunits in the rat neostriatum and how this relates to motor behaviors, a single dose of antisense phosphodiester oligodeoxynucleotide specific for NMDAR1 was unilaterally applied in the neostriatum in a stereotaxic apparatus. After one day of antisense treatment, ipsilateral rotation behaviors that were induced by apomorphine were found in the treated animals. Reductions in the levels of expression of NMDAR1 and NMDAR2A messenger RNAs (NMDAR1: 20.6%; NMDAR2A: 19.7%) were found in the antisense-treated striatal tissues by reverse transcriptase-polymerase chain reaction. There was no change in the levels of NMDAR2B, NMDAR2C and NMDAR2D messenger RNAs. After two days, western blotting experiments showed that there were decreases in the levels of expression of NMDAR1 (decreased 27.6%) and NMDAR2A (decreased 19.2%) proteins in the NMDAR1 antisense-treated striatal tissues. In addition, NMDAR1 immunoreactivity was found to decrease in intensity in the NMDAR1 antisense-treated neostriatum. At the cellular level, the intensity of NMDAR1 immunoreactivity in perikarya of presumed medium spiny neurons was found to decrease. These results indicate that a single dose of NMDAR1 antisense modifies the expression of NMDAR1 messenger RNA and protein in neurons in the neostriatum. The modification in the expression of NMDAR1 has differential effects in the expression of NMDAR2 subunits. Gene expression of the native NMDAR subunits is likely to be a dynamic process. The change in gene expression of the NMDAR subunits in the neostriatum may have a profound effect on the motor behaviors of rats.

Neurokinin Peptides and Neurokinin Receptors as Potential Therapeutic Intervention Targets of Basal Ganglia in the Prevention and Treatment of Parkinsons Disease

Parkinsons disease (PD) is a serious motor disorder and it is the second most common brain degenerative disease in human. PD is known to be caused by degeneration of dopamine neurons in the substantia nigra but the cause of cell death is largely unknown. Mammalian neurokinins [NKs] are a group of neuropeptides that include substance P (SP; neurokinin-1, NK-1), substance K (SK; NK-2; neurokinin A), and neuromedin K (NK; NK-3; neurokinin B). Their biological effects as neurotransmitters, neuromodulators, or neurotrophic-like factors are mediated by three distinct neurokinin receptors, namely SP receptor (SPR: NK-1 receptor, NK-1R), SKR (NK-2R), and NKR (NK-3R). Several lines of evidence have indicated that neurokinins are implicated in the pathogenesis of PD. First, decreases of SP level and SP-immunoreactivity have been found in nigral and striatal tissues of animals with PD and postmortem PD patients. Second, NKs exert neuroprotective effects on neurons. In addition, NK receptors, namely NK-1 and NK-3 receptors, are abundantly localized in dopaminergic and cholinergic neurons of the basal ganglia, indicating that these neurons are under the physiological regulation of NKs. Moreover, modulation in motor activity occurred in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, PD animal model, after systemic administration of NK receptor agonists. NKs and NK receptors, therefore, might be important molecules that are associated with functions and survival of neurons in the basal ganglia, in particular the dopamine neurons. Further studies should be devoted to elucidate the functional roles of NK systems in (a) the neuropathogenesis and neuroprotection during the course of PD, (b) the efficacy of NK receptor drugs towards PD, and (c) potential therapeutic intervention that targets at the prevention or treatment of PD.

Gene expression of glutamate receptors GluR1 and NR1 is differentially modulated in striatal neurons in rats after 6-hydroxydopamine lesion

In the present study, we attempted to address the modulation of the gene expression of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) glutamate receptors in the neostriatum of the 6-hydroxydopamine (6-OHDA)-lesioned rat, an animal model of Parkinsons disease. After 2 weeks of lesion, reverse transcriptase-polymerase chain reactions (RT-PCRs) revealed significant reduction in GluR1 mRNA expression but a significant enhancement of NR1 mRNA expression in the striatal tissues of the lesioned side. No modulation in the mRNA expression of GluR2, GluR3, GluR4 and NR2B were found. Immunofluorescence with digital imaging analysis also demonstrated a significant reduction in GluR1 immunoreactivity in the lesioned neostriatum. Interestingly, the reduction in GluR1 immunoreactivity was primarily observed in presumed striatal medium spiny neurons but not in parvalbumin-labeled striatal GABAergic interneurons. Immunoreactivity for GluR2, GluR2/3, GluR4, NR1 and NR2B was unchanged in neurons of the neostriatum of the lesioned side. The present results indicate that there is an opposite trend in modulation in the gene expressions of GluR1 and NR1 in the neostriatum of 6-OHDA-lesioned rats after dopamine denervation. Modulation of GluR1 mRNA and immunoreactivity is likely to be limited in the striatal projection neurons. These findings have implications for the use of NMDA and AMPA receptor antagonists in the treatment of Parkinsons disease.

Differential expression of N-methyl-D-aspartate receptor subunit messenger ribonucleic acids and immunoreactivity in the rat neostriatum during postnatal development.

The present study was performed to investigate the patterns of gene expression of N-methyl-D-aspartate (NMDA) receptors (NRs) in the rat neostriatum during postnatal development. Reverse transcriptase-polymerase chain reactions (RT-PCR) indicated that levels of NR1, NR2A and NR2D mRNAs reached peak levels between postnatal days 7 (PND 7) and PND 14. The levels of NR2B and NR2C mRNAs were low at PND 1 and their levels increased at PND 7 and remained high in adults. Immunofluorescence combined with image analysis revealed that the levels of NR1 immunoreactivity rose to its maximum at PND 14. In contrast, NR1 immunoreactivity rose progressively in perikarya of striatal neurons. Levels of NR2A immunoreactivity in the neostriatum were highest in adults. However, levels of NR2A immunoreactivity were higher in striatal neurons at PND 1 and PND 7. Levels of NR2B immunoreactivity were highest at PND 7. In the perikarya of striatal neurons however, the highest levels of NR2B immunoreactivity were detected at PND 14 and adult striatal neurons. In addition, double immunofluorescence revealed that the levels of NR1 immunoreactivity increased but the levels of NR2A immunoreactivity were the same in parvalbumin (PV)-positive striatal interneurons of PND 14 and adult rats. NR2B immunoreactivity was not detected in PV-positive neurons of PND 14 rats, but intense NR2B labeling was seen in PV-positive neurons of adult rats. Last but not least, in choline acetyltransferase (ChAT)-positive striatal interneurons of PND 14 and adult rats, levels of NR1 and NR2A immunoreactivity was seen to increase. Level of NR2B immunoreactivity remained the same in ChAT-positive neurons of PND 14 and adult rats. The present results indicate that there are differential patterns of expression of NR mRNAs and immunoreactivity in the neostriatum during different stages of postnatal development. Different combinations of NR have been found in different subpopulations of striatal neurons at different developmental stages. NR1, NR2A and NR2B are the major NMDA receptor subunits expressed during development. The change in patterns of expression of NR may be related to the functional maturation of neurons in the neostriatum.

Distinct cellular distribution of GABABR1 and GABAAα1 receptor immunoreactivity in the rat substantia nigra

Abstract GABA is one of the most important inhibitory neurotransmitters in the substantia nigra. Functions of GABA are mediated by two major types of GABA receptors, namely the GABA A and GABA B receptors. Subunits of both the GABA A and GABA B receptors have been cloned and functional characteristics of the receptors depend on their subunit compositions. In order to characterize the cellular localization of GABA B R1 and GABA A α1 subunit immunoreactivity in subpopulations of neurons in the rat substantia nigra, double and triple immunofluorescence was employed. Over 90% of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra pars compacta were found to display immunoreactivity for GABA B R1. In contrast, immunoreactivity for GABA A α1 was found to be primarily displayed by neurons in the substantia nigra pars reticulata. Around 85% of the GABA A α1-immunoreactive reticulata neurons were found to display parvalbumin immunoreactivity and some GABA A α1-positive reticulata neurons were found to be parvalbumin negative. In addition, triple-labeling experiments revealed that at the single cell level, the tyrosine hydroxylase-positive, i.e. the dopaminergic neurons in the compacta displayed intense immunoreactivity for GABA B R1 but not GABA A α1 receptors. The parvalbumin-positive neurons in the reticulata displayed intense immunoreactivity for GABA A α1 but not GABA B R1 receptors. The present results demonstrate in the same sections that there is a distinct pattern of localization of GABA B R1 and GABA A α1 receptor immunoreactivity in different subpopulations of the rat substantia nigra and provide anatomical evidence for GABA neurotransmission in the subpopulations of nigral neurons.