Colocalization of somatostatin,neuropeptide Y,and NADPH-diaphorase in the caudate-putamen of the rat

Somatostatin, neuropeptide Y, and nicotinamide adenine dinucleotide phosphatediaphorase are colocalized within a small population of medium aspiny neurons in the caudate-putamen of the rat. The extent of colocalization, however, appears to be in dispute. In order to examine the question of colocalization between these three neuroactive substances, a series of double-labelling experiments was performed. This was accomplished by combining immunocytochemistry for somatostatin or neuropeptide Y or enzyme histochemistry for nicotinamide adenine dinucleotide phosphate-diaphorase with in situ hybridization for somatostatin and/or neuropeptide Y mRNA. The results of such analysis indicate that nicotinamide adenine dinucleotide phosphate-diaphorase and somatostatin mRNA are 100% colocalized throughout the caudate-putamen, except for the area bordering the globus pallidus. All neurons that contain neuropeptide Y contain somatostatin message. Only 84% of the neurons that contain somatostatin mRNA, however, also contain neuropeptide Y. Neurons that contain somatostatin 28 but not neuropeptide Y are found throughout the caudate-putamen. These results indicate that the somatostatin neuron population in the rat caudate-putamen is not homogeneous. Instead, the medium aspiny neuron population is actually composed of several subpopulations based on the content of neuroactive substances. © 1995 Willy-Liss, Inc.     

Effect of cysteamine on somatostatin and neuropeptide Y in rat striatum and cortical synaptosomes

Local injection of cysteamine into rat striatum results in a rapid but reversible reduction in somatostatin-like immunoreactivity (SLI). Since somatostatin and neuropeptide Y are co-localized in striatal and cortical neurons, we examined the effects of cysteamine in these areas. SLI and neuropeptide Y-like immunoreactivity (NPYLI) were measured following local injection of cysteamine into the striatum. In addition, we examined the effects of cysteamine on SLI and NPYLI in cortical synaptosomes. SLI was significantly reduced in both experiments, but NPYLI was unaffected. These results suggest that the mechanism by which somatostatin is depleted by cysteamine is one of specific biochemical modification, probably affecting the somatostatin disulfide bond, rather than one affecting neuronal metabolism.     

A comparison of regional somatostatin and neuropeptide Y distribution in rat striatum and brain

Somatostatin-like immunoreactivity (SLI) and neuropeptide Y-like immunoreactivity (NPYLI) were detected using specific radioimmunoassays in extracts from rat brain. Since we have previously found a topographic distribution of SLI in rat striatum the distribution of NPYLI was examined in the same regions. NPYLI showed an identical distribution to SLI in rat striatum and levels were significantly correlated (r = 0.93, P less than 0.01). Concentrations of both neuropeptides were consistently highest in ventromedial striatum and nucleus accumbens while they were lowest in dorsolateral striatum. These findings provide further evidence of neurochemical heterogeneity in the striatum. Concentrations of NPYLI and SLI were also significantly correlated in cerebral cortex (r = 0.99, P less than 0.01). Concentrations of NPYLI were generally higher than SLI and showed a similar predilection for limbic system nuclei. The present findings support the concept that somatostatin and neuropeptide Y may be co-localized in both striatal and cortical neurons.     

Distribution and relative abundance of neurons in the pigeon forebrain containing somatostatin, neuropeptide Y, or both

Immunohistochemical studies in several mammalian species and in red-eared turtles have shown that somatostatin (SS) and neuropeptide Y (NPY) co-occur in a substantial proportion of the telencephalic neurons containing either. To explore further the possibility that telencephalic neurons co-containing SS and NPY may be evolutionarily conserved among amniotes, we determined the distribution and co-occurrence of SS and NPY in forebrain neurons in pigeons. Single-label immunohistochemical studies revealed the presence of overlapping populations of SS+ neurons and NPY+ neurons in most of the major subdivisions of the telencephalon. Double-label immunofluorescence studies revealed that in subdivisions of the telencephalon that are comparable to mammalian cortex (i.e., those dorsal and lateral to the basal ganglia), the vast majority of NPY+ neurons were also SS+, whereas a major and regionally variable percentage of the SS+ neurons were not NPY+. In contrast, within the basal telencephalon (including the basal ganglia and several other structures) neurons labeled only for NPY or only SS were more abundant than those containing both neuropeptides. Outside the telencephalon, the only forebrain cell group containing neurons in which SS and NPY were co-localized was in the lateral hypothalamus. A series of double- and triple-label immunohistochemical studies was undertaken to determine the extent of co-occurrence of SS and NPY in striatal neurons and the relationship of these neurons to striatal neurons containing other neuropeptides. In addition, immunohistochemical single- and double-label techniques were employed in conjunction with retrograde-labeling by fluorogold to determine the projections of SS+ and NPY+ striatal neurons. The results indicate that: 1) a population of striatal interneurons containing both SS and NPY exists in pigeons and constitutes approximately the same fraction of all striatal neurons as reported in mammals, 2) neurons containing NPY (but not SS) form a second, larger population of striatal interneurons, 3) neurons containing SS (but not NPY) form a third population of striatal interneurons that is approximately half as abundant as the NPY+ interneuron population, and 4) one-third of the substance P-containing striatonigral projection neurons also contain SS. The existence in pigeons of a major population of neurons containing both SS and NPY throughout the telencephalon, the existence of a population of neurons containing only SS in cortex-equivalent parts of the telencephalon, and the existence of a population of interneurons containing only NPY in the striatum is consistent with findings in mammals and turtles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Colocalization of somatostatin, neuropeptide Y, neuronal nitric oxide synthase and NADPH-diaphorase in striatal interneurons in rats

The neuropeptides somatostatin (SS), neuropeptide Y (NPY), the enzyme neuronal nitric oxide synthase (nNOS) and enzymatic activity for NADPH diaphorase (NADPHd) are extensively colocalized in striatal interneurons, which has led to the widespread tendency to operationally treat all four substances as being completely colocalized within a single class of striatal interneurons. We have explored the validity of this assumption in rat striatum using multiple-labeling methods. Conventional epi-illumination fluorescence microscopy was used to examine tissue triple labeled for SS, NPY and nNOS, or double-labeled for SS and nNOS or for SS and NPY. In tissue double-labeled for SS and nNOS, confocal laser scanning microscopy (CLSM) images of SS and nNOS labeling were compared to subsequent NADPHd labeling. We found that SS, NPY and nNOS co-occurred extensively, but a moderately abundant population of neurons containing SS and nNOS but not NPY was also observed, as were small populations of SS only and nNOS only neurons. About 80% of SS + neurons contained NPY, and no NPY neurons were devoid of SS or nNOS. All neurons containing nNOS in rat striatum were found to contain NADPHd. Combining our various quantitative observations, we found that of those striatal neurons containing any combination of SS, NPY, nNOS and NADPHd in rats, about 73% contained all four, 16% contained SS, nNOS and NADPHd, 5% contained SS only, and 6% contained only nNOS and NADPHd. These results indicate that while there is a large population of striatal neurons in which SS, NPY, nNOS and NADPHd are colocalized in rats, there may be smaller populations of striatal neurons devoid of NPY in which SS or nNOS/NADPHd are found individually or together.     

Effects of aging on quinolinic acid lesions in rat striatum

Several neurologic illnesses in which excitotoxic mechanisms may play a role increase in prevalence with age. In the present study we examined the susceptibility of rats to quinolinic acid striatal lesions at 1, 4 and 20 months of age, and susceptibility to N-methyl-d-aspartate (NMDA) at 1 and 4 months of age. The extent of the lesions was quantitated with measurements of substance P-like immunoreactivity (SPLI) and γ-aminobutyric acid (GABA). The lesions in the 4- and 20-month-old age groups showed significantly smaller depletions of SPLI and GABA than those in 1-month-old animals. Neuropeptide Y-like immunoreactivity (NPYLI) and somatostatin-like immunoreactivity (SLI) were unchanged in the lesioned striata. NMDA lesions were also attenuated in 4-month- and 12-month-old animals as compared with 1-month-old animals. Uric acid concentrations showed marked dose-dependent increases in the lesioned striatum, and to a lesser extent in the overlying cerebral cortex, in all 3 age groups. There were no changes of SLI, NPYLI or SPLI with aging in the cerebral cortex or hippocampus. Kynurenine and kynurenic acid concentrations showed significant increases with aging in frontal cortex. The present results show a reduced susceptibility of animals to striatal quinolinic acid and NMDA lesions with normal aging. The delayed onset of several neurodegenerative illnesses is therefore unlikely to be due to an increasing susceptibility to excitotoxin lesions with aging.     

C-PON containing neurons in the rat striatum are also positive for NADPH-diaphorase activity. A light microscopic study

The indirect immunofluorescence technique to detect endogenous C-flanking peptide of neuropeptide Y (NPY), C-PON, in combination with NADPH-diaphorase (NADPH-d) histochemistry, were applied to the same sections to establish whether C-PON containing cell bodies in the rat striatum can be labelled by their content of NADPH-diaphorase activity. NADPH-diaphorase activity proved to be a reliable marker for these positive C-PON neurons. Our results suggest that, in the rat striatum, C-PON, SOM and NPY co-exist in the same cells.     

Chronic exposure to aluminium decreases NADPH-diaphorase positive neurons in the rat cerebral cortex

Aluminium (Al) exposure is neurotoxic and is considered a possible etiological factor for many neurodegenerative disorders. Since it is known that Al impairs the glutamate-nitric oxide-cGMP pathway in neurons, this study was carried out to monitor the expression of NADPH-d in some central nervous system areas of rats after chronic administration of Al in drinking water. We tested three different nervous areas known to contain NADPH-diaphorase positive neurons: two cortical area (somatosensory cerebral cortex and cerebral cortex), a deep brain area (dorsolateral periaqueductal gray matter) and a spinal area (lumbar enlargement of the spinal cord). Our data showed that Al significantly decreased NADPH-d positive neurons in the cerebral cortex and the NADPH-d staining of many granular neurons in the cerebellum. We also found that Al did not cause neuron loss or apoptosis in the cerebral cortex. These findings suggest that the cortical nitroxidergic neurons and granule cells were a specific target of Al neurotoxicity.     

Effect of quinolinic acid on endogenous antioxidants in rat corpus striatum

The response of endogenous antioxidants to the N-methyl-D-aspartate (NMDA) receptor agonist and excitotoxin, quinolinic acid (QUIN), was investigated in rat corpus striatum. Animals treated with QUIN (240 nmol/microl), were sacrificed at 120 min after a single intrastriatal injection to examine the alterations in the levels of both reduced (GSH) and oxidized (GSSG) glutathione, and the activities of the antioxidant enzymes, superoxide dismutase (SOD) and glutathione peroxidase (Gpx). Changes in the rate of lipid peroxidation (LP) were also measured after exposure to different doses of QUIN (60, 120, 240 and 480 nmol/microl) as an index of oxidative stress. When compared to control, lipid peroxidation was increased at QUIN doses of 240 and 480 nmol/microl. Striatal levels of GSH and GSSG were decreased and increased, respectively, after QUIN injection; whereas GPx activity was unchanged. Cytosolic copper/zinc SOD (CuZn-SOD) activity decreased after treatment, while mitochondrial manganese SOD (Mn-SOD) was unchanged. The alterations observed on these antioxidant systems suggest that QUIN toxicity is mediated by specific mechanisms leading to oxidative stress.     

Effect of quinolinic acid on endogenous antioxidants in rat corpus striatum

Considering that magnesium and non-competitive NMDA receptor antagonists inhibit the opening of the channel linked to the NMDA receptor, we assessed their effects on mechanical hyperalgesia in two animal models of neuropathic pain (rats with a sciatic nerve ligature and diabetic rats). Our data show that magnesium reverses the hyperalgesia, as does MK-801. These results suggest that magnesium could be an alternative for the treatment of neuropathic pain in patients.     

Parvalbumin-containing neurons in the cerebral cortex of the lizard Podarcis hispanica: Morphology,ultrastructure, and coexistence with GABA,somatostatin, and neuropeptide Y

The morphology, fine structure, and degree of colocalization with GABA, somatostatin, and neuropeptide Y of parvalbumin-containing cells were studied with immunocytochemistry in the cerebral cortex of the lizard Podarcis hispanica. Parvalbumin-containing cells make up a morphologically heterogeneous population of spine-free neurons, displaying the morphological features of nonprincipal cells previously described in Golgi studies. Electron microscopically, parvalbumin-immunoreactive cell bodies are similar in all cortical areas and layers. The perisomatic input is moderate in number, and boutons with either round clear vesicles or flattened vesicles were observed making asymmetric or symmetric synaptic contacts, respectively. Parvalbumin-immunoreactive dendrites are smooth and almost completely covered with synaptic boutons of different types, most of which establish asymmetric contacts. Parvalbumin-immunoreactive boutons are concentrated around cell bodies of principal cells. They are large, containing abundant mitochondria and small pleomorphic vesicles, and establishing symmetric synaptic contacts with somata, proximal dendritic shafts, and axon initial segments of principal cells. Colocalization studies revealed that all the parvalbumin-containing cells are GABA-immunoreactive, representing only a fraction of the GABA-immunopositive cell population, and that parvalbumin- and peptide- (somatostatin and neuropeptide Y) containing cells show a negligible overlap. These results demonstrate that in the cerebral cortex of the lizard Podarcis hispanica, parvalbumin-containing cells represent a subset of nonprincipal GABAergic neurons largely involved in perisomatic inhibition, which are different from the peptide-containing cells, and suggest that they may include both axosomatic and axoaxonic cells. © 1993 Wiley-Liss, Inc.     

Cortical somatostatin, neuropeptide Y, and NADPH diaphorase neurons: normal anatomy and alterations in Alzheimer's disease

Somatostatin and neuropeptide Y are two neuropeptides that are of particular interest in Alzheimer's disease because they are reported to be depleted in cerebral cortex. In the present study we examined somatostatin, neuropeptide Y, and nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase neurons in nine cortical regions in both normal and Alzheimer's disease brains. These three neurochemical markers show a high degree of co-localization (greater than 90%) in nonpyramidal neurons that are primarily distributed in cortical layers II-III, V-VI, and, most prominently, in infracortical white matter. The highest cell density was in temporal and parietal association cortex. The major morphological abnormality in Alzheimer's disease brains was a marked pruning and distortion of fiber plexuses with an apparent reduction in fiber density. In contrast, perikaryal density was preserved except for a reduction in parietal association cortex. Approximately 10 to 15% of senile plaques in the inferior temporal gyrus contained abnormal neurites. Additional abnormal collections of neurites without plaque cores were frequently found in layers II-III and V-VI. Neuropeptide Y and somatostatin were co-localized in abnormal neurites, suggesting an origin from local intrinsic neurons in which the two peptides are co-localized. Double immunofluorescence staining for both tau protein, a major antigenic component of paired helical filaments, and either somatostatin or neuropeptide Y showed that these neurons do not contain tau-immunoreactive neurofibrillary tangles. The morphological correlate of reduced somatostatin and neuropeptide Y content in Alzheimer's disease brain therefore appears to be a distortion and reduction in fiber plexuses. In addition, it is apparent that these neurons can develop widespread morphological abnormalities in the absence of neurofibrillary tangle formation.     

Coexistence of somatostatin with neuropeptide Y, but not with cholecystokinin or vasoactive intestinal peptide, in neurons of the rat amygdala

A two-color immunoperoxidase procedure was used to determine whether somatostatin (SOM) containing neurons in the amygdala also contain neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), or cholecystokinin (CCK). There was no evidence that SOM-containing neurons in any of the amygdaloid nuclei contain VIP or CCK. In contrast, there was extensive colocalization of SOM and NPY in all of the amygdaloid nuclei with the exception of the intercalated masses and the lateral subdivision of the central nucleus. The greatest number of SOM-NPY double-labeled cells was observed in the medial nucleus, lateral nucleus, and intra-amygdaloid portion of the bed nucleus of the stria terminalis. The morphology of these SOM-NPY neurons was similar in all nuclei. Most exhibited fusiform or avoid cell bodies with one or two sparsely branched dendrites emerging from each pole of the cell. The extensive coexistence of SOM and NPY in non-pyramidal neurons of the basolateral amygdala is similar to that seen in the cerebral cortex and supports the concept that these brain regions share many important characteristics. The extensive colocalization of SOM and NPY in the medial amygdala, in conjuction with the results of previous studies, suggests that some of these cells may project to the bed nucleus of the stria terminalis and hypothalamus.     

Upregulation of brain somatostatin and neuropeptide Y following lidocaine-induced kindling in the rat

Male Sprague-Dawley rats received a daily injection of 60 mg/kg of lidocaine (> 30 days). Twenty percent of rats developed convulsions (kindled rats) and remaining rats did not show convulsions (non-kindled rats). The level of immunoreactive somatostatin (IR-SRIF) in kindled rats was significantly increased in amygdala than that in non-kindled rats and control rats. Immunoreactive neuropeptide Y (IR-NPY) contents in kindled rats were significantly increased in amygdala, hippocampus, cortex and striatum compared to non-kindled and control rats. The expression of SRIF mRNA in kindled rats produced a significant increase in amygdala, while NPY mRNA in kindled rats showed an elevated expression in both amygdala and hippocampus. These results coincide with the previous findings with the elevated expression of SRIF and NPY mRNA in electrically and pharmacologically kindled models, suggesting the important role of these peptides in the kindling phenomenon.     

Differential loss of neurochemical markers following quinolinic acid-induced lesions of rat striatum.

Twenty-one days following bilateral striatal injections of the excitotoxin quinolinic acid (QA) (75, 100, or 150 nmol) or vehicle there were differential losses of various neurochemical markers. Choline acetyltransferase was relatively resistant to QA-induced lesions while glutamate decarboxylase activity was more sensitive. The binding of [3H]glutamate to the N-methyl-D-aspartate receptor was very sensitive to QA-induced lesions while the loss of [3H]MK801 binding was less sensitive. The differential loss of [3H]glutamate and [3H]MK801 binding indicated that these sites may represent distinct molecules which are differentially located or differentially regulated. The binding of [3H]SCH23390 to the D1 dopamine receptor was also very sensitive to QA-induced lesions. [3H]SCH23390 binding may represent a relatively simple and sensitive neurochemical assay of QA-induced neurotoxicity.     

Effects of quinolinic acid on messenger RNAs encoding somatostatin and glutamic acid decarboxylases in the striatum of adult rats.

The level of expression of mRNAs encoding somatostatin and two isoforms of glutamic acid decarboxylase (Mr 65,000, GAD65 and 67,000, GAD67) was examined by quantitative in situ hybridization histochemistry in the striatum of adult rats after local injections of quinolinic acid. After a 2-week survival period, Nissl strains showed a profound loss of neurons in the injected striata. With a dose of 120 nmol quinolinic acid, the lesioned area was completely devoid of somatostatin mRNA-positive neurons but contained cells expressing nicotinamide adenine dinucleotide-diaphorase activity (a marker of somatostatinergic interneurons in striatum). After 60 nmol of quinolinic acid, the number of neurons expressing somatostatin mRNA in the lesioned area was similar to controls but the level of labeling per neuron was increased. In the lesioned area, labeling for GAD65 mRNA was abolished and labeling for GAD67 mRNA markedly reduced. However, scattered neurons expressing GAD67 mRNA could still be detected. The majority of surviving GABA-ergic neurons expressed immunoreactivity to parvalbumin, a marker for striatal GABA-ergic interneurons. The results show that quinolinic acid induces dose-dependent alterations in the expression of striatal somatostatin mRNA and reveal a relative sparing of GABA-ergic interneurons in the quinolinic acid-lesioned rat striatum.     

Behavioral and anatomical effects of quinolinic acid in the striatum of the hemiparkinsonian rat

Parkinson's disease (PD), a hypokinetic disorder, and Huntington's disease (HD), a hyperkinetic disorder, share the fact that in the motor pathways the dysfunction starts in the striatum. In PD the projection neurons are overactive due to decreased inhibitory regulation by lost dopamine afferents, while in HD the output from the striatum is insufficient due to loss of projection neurons. This study aimed to determine whether the introduction of a mild HD condition in the PD striatum can counter the hypokinetic condition. The experiment was carried out in the 6-OHDA rat model for PD in which amphetamine, 5 mg/kg, evokes an asymmetric rotation response toward the side of the 6-OHDA lesion (ipsilateral rotation). The response to amphetamine in this study was fractionated into multiple components and measured automatically. After baseline measurements, the subjects were divided into four groups. Group I was unilaterally sham-lesioned in medial, central, and lateral striatum. Group II was injected quinolinic acid (QA) 20 nM in medial, central, and lateral striatum. Group III was injected QA 60 nM in central striatum. Group IV was injected QA 120 nM in central striatum. The effects of QA were measured weekly. The sham lesions in Group I had no effects. In Group II, ipsilateral rotation was reduced and replaced by oral stereotypy, a competitive behavior. In Group III, ipsilateral rotation decreased, but to a lesser degree than in Group II. In Group IV, QA had no effects. Histological findings show a unilateral loss of tyrosine immunoreactive (TH) neurons in substantia nigra and of fibers in striatum in all subjects. In addition, in Group II the striatum was atrophied. These findings suggest that the shift in Group II from ipsilateral rotation to oral stereotypy after QA was due to reduced striatal output caused by a loss of projection neurons, a loss insufficient to induce HD symptoms, but sufficient to counter the PD condition.     

Synergistic effects of chronic exposure to subthreshold concentrations of quinolinic acid and malonate in the rat striatum

Adult rats received chronic intrastriatal dialytic exposure to quinolinic acid (QUIN), malonate, or a combination of QUIN and malonate. The combination of subthreshold concentrations of QUIN (4 mM) and malonate (400 mM) produced lesions larger than did either QUIN or malonate alone. The neurotoxic effect of QUIN combined with malonate was subsequently blocked by co-administration of the NMDA receptor antagonist MK-801 (1 mM). These findings indicate that malonate synergistically enhances NMDA receptor mediated excitotoxicity.     

Dissociation of neuropeptide Y and somatostatin in Parkinson's disease

The concentration of neuropeptide Y has been determined in the cortex and hippocampus of subjects with Parkinson's disease and compared to changes of activity of dopamine β-hydroxylase and concentration of somatostatin. Despite a marked reduction in the concentration of somatostatin in the severely demented subject, in both cortex and hippocampus, no significant change in concentration of NPY was found in either region. This finding therefore suggests that the majority of NPY within the cortex is independent of somatostatin. This study provides some further evidence of neurochemical similarities between the dementia of Parkinson's disease and Alzheimer's disease.