成年大鼠脑内ATP敏感性钾通道亚型mRNA表达的研究

为探讨ATP敏感性钾通道(KATP)亚型在大鼠脑组织中的表达,本研究采用半定量逆转录聚合酶链反应 (RT PCR)检测了大鼠小脑、大脑皮质、海马、纹状体、黑质的KATP亚型mRNA的表达。结果显示Kir6. 1、Kir6. 2、Sur1、Sur2B在小脑、大脑皮质、海马、纹状体、黑质中均有表达,Sur2A在脑组织中未见表达。Kir6. 1mRNA在海马和黑质的相对表达水平明显高于小脑、大脑皮质和纹状体(P<0.01);Kir6. 2和Sur1mRNA在黑质的相对表达水平明显高于小脑、大脑皮质、海马和纹状体 (P<0.01 );Sur2BmRNA在黑质、海马和纹状体的相对表达水平明显高于小脑和大脑皮质(P<0.01 )。以上结果提示KATP在脑内具有广泛表达,其表达水平在不同部位存在着差异性。     

Repeated methamphetamine treatment increases expression of TRPV1 mRNA in the frontal cortex but not in the striatum or hippocampus of mice

The transient receptor potential vanilloid type 1 (TRPV1) is a non-selective ligand-gated cationic channel. The distribution of TRPV1 mRNA in various regions of the brain has been successfully established. Methamphetamine (MAP) is a psychostimulant and a major drug of abuse in many parts of the world. The powerful rewarding properties of MAP are attributed to multiple pharmacological actions, but the mechanistic association between TRPV1 expression and MAP-induced drug addiction has not established. In the present study, we conducted a time-course analysis of TRPV1 mRNA levels in the frontal cortex, striatum, and hippocampus of mouse brain following repeated MAP (2 mg/kg, i.p.) treatment. Our results demonstrate that expression of TRPV1 mRNA is significantly increased 1, 2, 6, 24, 48 h, and 1 week after the last MAP treatment in the frontal cortex but not in the striatum or hippocampus. These data support a potential role for TRPV1 in the treatment of MAP-induced drug addiction.     

Cellular localisation of somatostatin mRNA and neuropeptide Y mRNA in foetal striatal tissue grafts

Using in situ nucleic acid hybridisation histochemistry, we have studied the expression of somatostatin mRNA and neuropeptide Y mRNA in grafts of embryonic striatal neurones implanted into the ibotenic acid-lesioned rat neostriatum. Tissue sections of the grafted striatum were incubated with either 32P- or 35S-labelled complementary oligodeoxyribonucleotide probes specific for somatostatin mRNA and neuropeptide Y mRNA, exposed with X-ray film and dipped in Ilford K-5 emulsion. Neither somatostatin mRNA nor neuropeptide Y mRNA was detectable in the ibotenic acid-lesioned striatum indicating a pronounced degeneration of somatostatin- and neuropeptide Y-containing neurones. However, in the striatal grafts the levels of somatostatin mRNA and neuropeptide Y mRNA were substantially increased over those in the control intact striata. The results suggest that in the grafts, somatostatin mRNA and neuropeptide Y mRNA were expressed in a higher proportion of primordial striatal neurones and there was also an increased level of expression of each neuropeptide gene per individual neurone (reflecting a higher synthetic activity of such neurones) compared to the intact mature striatum. These data demonstrate the sensitivity of the in situ hybridisation technique to study patterns of gene expression in developing neuronal tissues after transplantation.     

Gene expression in striatal grafts--I. Cellular localization of neurotransmitter mRNAs

This study utilized the technique of in situ hybridization histochemistry to identify cells expressing neurotransmitter mRNAs in embryonic striatal tissue grafts implanted into the ibotenic acid-lesioned rat neostriatum. Synthetic 32P- or 35S-labelled oligodeoxyribonucleotide probes specific for prosomatostatin, proneuropeptide Y. proenkephalin, prodynorphin and preprotachykinin mRNAs and a 32P-labelled cRNA probe specific for glutamate decarboxylase mRNA were used to study the regional and cellular changes in these mRNA levels in the normal, lesioned and grafted neostriatum. The levels of neuropeptide Y mRNA and somatostatin mRNA were substantially increased in the striatal grafts compared with the intact control striata. The levels of glutamate decarboxylase mRNA in the grafts also appeared to be slightly elevated over those in the control striata. However, the levels of proenkephalin mRNA, prodynorphin mRNA and preprotachykinin mRNA were significantly lower in the grafts. The increased levels of neuropeptide Y mRNA and somatostatin mRNA in the grafts were due both to an increase in the number of labelled cells and to an increase in the cellular levels of each neuropeptide mRNA. In contrast, the cellular levels of proenkephalin mRNA, prodynorphin mRNA and preprotachykinin mRNA in the grafts were comparable, or elevated relative, to those in the intact striata but the density of cells expressing each of these mRNAs was reduced. Since neuropeptide Y and somatostatin are known to be present in medium to large aspiny striatal neurons (interneurons) and enkephalin, dynorphin and tachykinin peptides and GABA are localized in medium spiny striatal projection neurons, the above findings would indicate that there is a divergence in the levels of activity between these two neuronal populations in the striatal grafts. Our data suggest that the levels of gene expression and hence the functional neurotransmitter-synthesizing and releasing activity in the grafted neuron are different from those in the normal mature striatum.     

Kainic acid induced seizures: changes in somatostatin, substance P and neurotensin

The neuropeptides somatostatin, neurotensin and substance P were investigated in rats during and after limbic seizures induced by systemic injection of kainic acid (10 mg/kg, i.p.). Three hours after injection of the toxin, pronounced decreases (40-50%) in somatostatin-like immunoreactivity in frontal cortex, striatum, dorsal hippocampus and amygdala/pyriform cortex were observed. Concomitantly, neurotensin-like and substance P-like immunoreactivities were also reduced in the frontal cortex and the hippocampus. These early decreases in peptide levels may result from increased release and subsequent inactivation of the peptides during acute seizures. At later time intervals, 3, 10 and 30 days after injection of kainic acid, the initially decreased peptide levels were partially normalized. However, the reduction in somatostatin-like immunoreactivity in amygdala/pyriform cortex and striatum persisted up to 30 days. Neurotensin-like immunoreactivity remained decreased in the frontal cortex. On the other hand, neurotensin- and substance P-like immunoreactivities were increased in the striatum and substantia nigra 10-30 days after injection of kainic acid. These late changes in peptide levels may suggest destruction of peptidergic neurons or adaptive changes induced by the convulsions. Pretreatment of rats with cysteamine (100 mg/kg, i.p.), an agent which decreases brain somatostatin levels, had no effect on the intensity of kainic acid induced convulsions, although a slightly earlier onset of seizures was observed. The changes in peptide levels, especially the marked decreases in somatostatin content after systemic injection of kainic acid, suggest considerable acute and chronic alterations in peptidergic systems caused by limbic convulsions.     

Brain regional neuropeptide changes resulting from social defeat

Past work has demonstrated robust brain changes in cholecystokinin (CCK-8) following social defeat. Here the authors analyzed brain regional, CCK-8, substance P, corticotropin releasing factor (CRF), and neuropeptide Y levels in adult male Long-Evans rats defeated in a resident-intruder social aggression paradigm, as indexed by elevated bites received, freezing, and emission of 20-kHz calls. Brains harvested 6 hr after social defeat were dissected into 12 regions (olfactory bulbs, 3 cortical regions [frontal cortex, cortex above the basal ganglia, cortex above the diencephalon], caudate-putamen, basal forebrain, hypothalamus, hippocampus, thalamus, tectum, tegmentum, and lower brain stem). Neuropeptide radioimmunoassays demonstrated the following statistically significant regional changes in defeated rats as compared with nondefeated rats: CCK-8 was reduced in frontal cortex and cortex overlying diencephalon, the olfactory bulbs, caudate-putamen, hippocampus, tectum, and lower brainstem. Neuropeptide Y was elevated in the caudate-putamen. Substance P was elevated in the cortex over the basal ganglia and decreased in basal forebrain. CRF was diminished in the hippocampus. The results highlight more robust CCK modulation by social defeat as compared with 3 other neuropeptide systems involved in brain emotional regulation.     

Neurochemical characteristics of aluminum-induced neurofibrillary degeneration in rabbits

Aluminum-induced neurofibrillary degeneration in rabbits is known to affect particular populations of neurons. The neurotransmitter alterations which accompany aluminum neurofibrillary degeneration were examined in order to assess how closely they mimic those of Alzheimer's disease. There was a significant reduction in choline acetyltransferase activity in entorhinal cortex and hippocampus as well as significant reductions in cortical concentrations of serotonin and norepinephrine in the aluminum-treated rabbits. Significant reductions in glutamate, aspartate and taurine were found in frontoparietal and posterior parietal cortex. Concentrations of GABA were unchanged in cerebral cortex. Both substance P and cholecystokinin immunoreactivity were significantly reduced in entorhinal cortex but there were no significant changes in somatostatin, neuropeptide Y and vasoactive intestinal polypeptide. The five neuropeptides were unaffected in striatum, thalamus, cerebellum and brainstem. Neurochemical changes were found in the regions with the most neurofibrillary degeneration while regions with little or no neurofibrillary degeneration were unaffected. The reductions in choline acetyltransferase activity, serotinin and noradrenaline suggest that some neuronal populations preferentially affected in Alzheimer's disease are also affected by aluminum-induced neurofibrillary degeneration; however, the cortical somatostatin deficit which is a feature of Alzheimer's disease is not replicated in the aluminum model.     

The effect of neuropeptide Y on striatal catecholamines

Neuropeptide Y and peptide YY were injected into rat striatum and their effects on dopamine, serotonin and their metabolites were examined at 1 h. Neuropeptide Y induced a dose-dependent increase in dopamine turnover in the ipsilateral striatum with no effect on serotonin turnover. When neuropeptide Y was coinjected with somatostatin there was an additive effect in increasing dopamine turnover. There was no alteration in striatal concentrations of gamma-aminobutyric acid, glutamate, or aspartate with either neuropeptide Y or somatostatin injections. These results suggest that neuropeptide Y may play a role with somatostatin in regulating striatal dopaminergic transmission.     

The effect of 6-hydroxydopamine, reserpine and cold stress on the neuropeptide Y content of the rat central nervous system

Neuropeptide Y has previously been detected in neurons throughout the rat brain and spinal cord. On histochemical grounds, the neuropeptide Y-containing cell bodies have been subdivided into two groups: those in the brain stem in which colocalization with noradrenaline and adrenaline has been demonstrated and those in other brain regions where no catecholamine coexistence is found. In this paper the regional distribution of neuropeptide Y has been investigated in the rat brain by a specific neuropeptide Y radioimmunoassay, before and after the destruction of catecholaminergic nerve terminals by the administration of intraventricular 6-hydroxydopamine. Despite massive reductions in brain catecholamines, the neuropeptide Y level was unchanged in the cerebral cortex, striatum, spinal cord and hippocampus. A minor reduction in neuropeptide Y was found in the hypothalamus. Reserpine treatment, which is known to deplete brain nerve terminal stores of catecholamines, likewise did not result in any loss of neuropeptide Y. Cold stress which increases noradrenergic turnover in the rat brain stem had no effect on neuropeptide Y levels. These results suggest that the bulk of neuropeptide Y in the rat brain and spinal cord may not be stored in catecholaminergic nerve terminals.     

Lack of asymmetrical distribution of receptor binding sites and of neurally active peptides within rat brain

The high affinity binding of 5 tritiated (naloxone, spiroperidol, serotonin, quinuclidinylbenzilate, dihydroalprenolol) ligands to left and right frontal cortex, hippocampus and striatum has been compared in 8-week-old male rats. No lateralization of specific binding was observed between paired brain regions. Also, no regional asymmetry of protein content was found. No significant differences in met-enkephalin and substance P contents were found between paired striata, frontal cortex, and hippocampi. These data suggest that several neurotransmitter and neuromodulator indices are symmetrically distributed within the rat brain.     

Perforant path kindling induces differential alterations in the mRNA levels coding for prodynorphin and proenkephalin in the rat hippocampus

The effect of perforant path kindling on the levels of mRNAs coding for proenkephalin and prodynorphin in hippocampus and frontal cortex of rats was measured using RNA blot analysis. In rats showing stage 3 kindled seizures, after consecutive stimulation of the right perforant path, a decrease in the level of prodynorphin mRNA and an increase in levels of proenkephalin mRNA in the ipsilateral hippocampus was found. In addition, the levels of prodynorphin were also decreased in the contralateral hippocampus. No changes in the opioid peptide mRNAs were found in the frontal cortex of the animals. The altered mRNA levels in the hippocampus returned to normal 8 days following cessation of the electrical stimulation. However, at that time a single stimulus was still effective in producing stage 3 kindling seizures. These findings indicate that (1) the opioid peptide gene expression in the hippocampus can be transynaptically altered by kindling of the perforant path and (2) that the opioid peptides may play a role in the development, but not in the maintenance of kindling.     

Electroconvulsive shock increases serotonin transporter in the rat frontal cortex

The antidepressive action of electroconvulsive shock (ECS) is thought to involve the alteration in serotonin (5-HT) neurotransmission, including the increase in 5-HT release and uptake. In our previous study, 5-HT transporter (5-HTT) mRNA expression was decreased after single and repetitive ECS in rat raphe nucleus. In the present study, we investigated the effects of single and repetitive ECS on the protein levels of 5-HTT in the frontal cortex, hippocampus and raphe nucleus of rat brain using quantitative Western blot analysis. Single ECS did not alter 5-HTT protein expression in any brain regions examined. Repetitive ECS stably increased 5-HTT protein in the frontal cortex, but not in the hippocampus and raphe nucleus. Because ECS is known to facilitate the release of neurotransmitters, our results suggest that the increased 5-HTT protein expression in the frontal cortex might be a compensatory change against the enhanced 5-HT release by ECS in presynaptic terminals.     

A survey of substance P, somatostatin, and neurotensin levels in aging in the rat and human central nervous system

Levels of the neuropeptides substance P, somatostatin, and neurotensin were measured by radioimmunoassay in regions of the rat and human central nervous system (CNS) in aging. Somatostatin levels were significantly lower only in the corpus striatum of older rats. Substance P levels and neurotensin levels were generally stable with aging as were levels of somatostatin in regions other than the corpus striatum. In post-mortem human CNS tissues, no significant negative correlations of levels of the three peptides were observed with time to refrigeration or time to freezer for the samples. In the human CNS, there were no significant age-related alterations in substance P levels in frontal cortex, thalamus, hypothalamus, caudate nucleus, globus pallidus, or substantia nigra. There was a significant age-related decrease in substance P levels in the human putamen. This age-related decrease was not present in tissues from victims of Huntington's disease nor was there any striking difference in substance P levels as a function of duration of the disease. There were no significant age-related changes in somatostatin levels in human frontal cortex, caudate nucleus, putamen, medial globus pallidus, or substantia nigra. Among these same regions, there was a significant age-related decrease in neurotensin levels only in the pars compacta and pars reticulata of the human nigra. These results implicate neuropeptides in aging processes in certain regions of the CNS. There are differences between rats and humans with respect to neuropeptides in the aging process in the CNS. Deterioration of some neuropeptide pathways in and to human basal ganglia may be involved in the suspected functional deterioration of parts of the extrapyramidal system in aging.     

A survey of substance P, somatostatin, and neurotensin levels in aging in the rat and human central nervous system

Levels of the neuropeptides substance P, somatostatin, and neurotensin were measured by radioimmunoassay in regions of the rat and human central nervous system (CNS) in aging. Somatostatin levels were significantly lower only in the corpus striatum of older rats. Substance P levels and neurotensin levels were generally stable with aging as were levels of somatostatin in regions other than the corpus striatum. In post-mortem human CNS tissues, no significant negative correlations of levels of the three peptides were observed with time to refrigeration or time to freezer for the samples. In the human CNS, there were no significant age-related alterations in substance P levels in frontal cortex, thalamus, hypothalamus, caudate nucleus, globus pallidus, or substantia nigra. There was a significant age-related decrease in substance P levels in the human putamen. This age-related decrease was not present in tissues from victims of Huntington's disease nor was there any striking difference in substance P levels as a function of duration of the disease. There were no significant age-related changes in somatostatin levels in human frontal cortex, caudate nucleus, putamen, medial globus pallidus, or substantia nigra. Among these same regions, there was a significant age-related decrease in neurotensin levels only in the pars compacta and pars reticulata of the human nigra. These results implicate neuropeptides in aging processes in certain regions of the CNS. There are differences between rats and humans with respect to neuropeptides in the aging process in the CNS. Deterioration of some neuropeptide pathways in and to human basal ganglia may be involved in the suspected functional deterioration of parts of the extrapyramidal system in aging.     

Neurotransmitters in neocortex of aged rhesus monkeys.

The effects of aging on levels of neurotransmitters were determined in two regions of the cerebral cortex in rhesus monkeys (Macaca mulatta). Choline acetyltransferase (ChAT) activity as well as somatostatin, neuropeptide Y, and substance P immunoreactivities were analyzed in the right caudal cingulate gyrus and in the left and right inferior occipital poles in five age groups: 4-6 years; 8-11 years; 20-25 years; 26-29 years; and 31-34 years. Neuroactive amino acids and markers for monoamine transmitters were analyzed only in the youngest (4-6 years) and oldest (31-34 years) animals. Across the five age groups studied. ChAT activity as well as somatostatin and neuropeptide Y immunoreactivities were significantly decreased bilaterally in occipital poles of the 31- to 34-year-old group. There were no significant age-related differences in substance P immunoreactivity. In 4-6-year-old vs. 31-34-year-old monkeys, levels of amino acid neurotransmitters were unchanged. However, there were significant reductions in norepinephrine, serotonin and its metabolites, kynurenine, and 4-hydroxyphenyllactic acid in occipital poles of the 31- to 34-year-old monkeys. No significant neurochemical changes were detected in the cingulate cortex. These findings demonstrate that aged nonhuman primates show reductions in cortical markers for a variety of neurotransmitters, including acetylcholine, somatostatin, neuropeptide Y, norepinephrine, and serotonin but that these changes do not occur uniformly in the neocortex.     

Differential distribution of somatostatin receptor subtypes in rat brain revealed by newly developed somatostatin analogs.

Somatostatin receptor subtypes were labeled with the somatostatin analogs [125I]CGP 23996 and [125I]MK 678 and the distribution of these receptors in rat brain was investigated using quantitative autoradiographic techniques. [125I]CGP 23996 and [125I]MK 678 specifically label different populations of somatostatin receptors in rat brain. In a number of brain regions striking differences in the distribution of the somatostatin receptor subtypes labeled by each peptide were observed. High levels of binding sites for both [125I]CGP 23996 and [125I]MK 678 were present in the cerebral cortex, CA1 region and subiculum of the hippocampus. In contrast, high levels of [125I]MK 678 binding were found in the dentate gyrus of the hippocampus while few [125I]CGP 23996 binding sites were observed in this brain region. [125I]CGP 23996 binding was detected in the central region of the interpeduncular nucleus whereas the dorsal and lateral subnuclei of this brain area expressed mainly somatostatin receptors with high affinity for MK 678. The locus coeruleus and regions of the superior colliculus and hypothalamus selectively express [125I]MK 678-sensitive somatostatin receptors. Furthermore, limbic structures such as the lateral septum, the nucleus accumbens and ventromedial striatum had much higher levels of [125I]MK 678 binding sites than [125I]CGP 23996 binding sites. Differences in the expression of the somatostatin receptor subtypes were also detected in the substantia nigra. [125I]CGP 23996 binding was present in the pars reticulata but not the pars compacta whereas the reverse distribution for [125I]MK 678 binding sites was observed. The differential distribution of [125I]CGP 23996 and [125I]MK 678 binding sites in rat brain supports the hypothesis that these peptides selectively label different somatostatin receptor subtypes in the central nervous system.     

Coexistence of somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivity in some forebrain neurons

The indirect immunofluorescence technique was used to demonstrate the coexistence of somatostatin together with avian pancreatic polypeptide-like immunoreactivity within certain neurons of the rat forebrain. Numerous neurons containing these peptides were observed in the neocortex, hippocampus, olfactory tubercle, striatum, nucleus accumbens and lateral septum. In studies of serial sections stained alternately for these two peptides, and in restaining experiments, It could be determined that in many neurons in these areas these two peptides coexisted. In other brain areas such as the anterior periventricular hypothalamus, somatostatin cells were never found to contain avian pancreatic polypeptide-like immunoreactivity. Also, within the pancreas these two peptides were never found to coexist in the same cells. The findings represent a further example of the coexistence of more than one neuropeptide within a single neuron.     

Characterization of NPY mRNA-expressing cells in the human brain: co-localization with Y2 but not Y1 mRNA in the cerebral cortex, hippocampus, amygdala, and striatum

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system. Its effects on, for example, cognition, memory and motor functions are thought to be mediated mainly via its interactions with the NPY Y1 and Y2 receptor subtypes. We had previously described the neuroanatomical organization of the Y1 and Y2 mRNA expression in humans. However, in view of the lack of information regarding the overall detailed distribution of NPY mRNA expression in the human brain, a complete picture of the anatomical organization of the NPY-related genes was still missing. Thus, in the present study, the regional distribution of NPY mRNA-expressing cells was analyzed in the post-mortem human brain. In addition, double labeling in situ hybridization was performed to characterize the NPY neuronal populations in relation to the Y1 and/or Y2 receptor mRNA localization in the human cerebral cortex, striatum, and amygdala. NPY mRNA was found to be abundant in layers II and VI of the neocortex, polymorphic layer of the dentate gyrus, basal ganglia, and amygdala. Double labeling in situ hybridization showed the co-expression of NPY mRNA with the Y2, but not with the Y1, mRNA in the human cerebral cortex, hippocampus, amygdala, striatum, and nucleus accumbens, and the existence of co-expression of the Y1 and Y2 mRNAs in the cerebral cortex and amygdala. Overall, these results suggest a role for the Y2, but not Y1, as an autoreceptor in the NPY neuronal populations of the human brain.     

Ovariectomy up-regulates neuronal neurofilament light chain mRNA expression with regional and temporal specificity

Estrogens can influence the survival, plasticity and function of many adult neurons. Many of these effects, such as neurite outgrowth and increased dendritic spine density, are mediated by changes in neuronal cytoskeletal architecture. Since neurofilament proteins play a key role in the maintenance and remodeling of the neuronal cytoskeleton, we postulated that changes in neurofilament light chain mRNA may parallel some of the alterations in neuronal architecture which follow bilateral ovariectomy. We measured neurofilament light chain mRNA levels using a ribonuclease protection assay at two time-points after ovariectomy in mature female rats. One week after ovariectomy, neurofilament light chain mRNA levels (corrected for glucose-6-phosphate dehydrogenase mRNA) did not differ from sham-operated animals in the five brain regions examined (hypothalamus, striatum, hippocampus, frontal cortex and occipital cortex). Four months after ovariectomy, neurofilament light chain mRNA levels were similarly unchanged in the hypothalamus and striatum. In contrast, statistically significant increases in neurofilament light chain mRNA expression were observed in the three regions receiving basal forebrain projections (hippocampus, frontal cortex and occipital cortex). In situ hybridization demonstrated increases in neurofilament light chain mRNA expression involving subpopulations of smaller medial septal neurons. There also appeared to be an increased number of larger septal neurons following long-term ovariectomy.We propose that atrophic changes involving basal forebrain projection fibers are followed by compensatory axonal growth by other 'intact' basal forebrain neurons. Increased neurofilament light chain mRNA expression and somatic hypertrophy in medial septal neurons may both be reflective of the need to sustain an axonal network which is larger and more complex. In contrast, increased neurofilament light chain mRNA expression observed in basal forebrain targets following long-term ovariectomy may be reflective of compensatory changes taking place in local neurons.