c－fos基因在听源性惊厥易感大鼠脑的表达──原位杂交组织化学法研究

运用原位杂交技术，以遗传性听源性惊厥易感大鼠Ｐ７７ＰＭＣ为对象，发现听源性惊厥可诱导大鼠脑内ｃ－ｆｏｓ基因快速、大量、短暂性表达。ｃ－ｆｏｓｍＲＮＡ分布于大脑皮层、梨状皮层、杏仁复合体、海马齿状回、上丘脑、背侧丘脑、下丘脑部分核团、下丘、蜗神经核、蓝斑及小脑等处。惊厥后皮层下结构中ｃ－ｆｏｓ基因表达变化程度超过皮层的变化，尤其是下丘、蜗神经核与惊厥时程有明显关系。推测皮层下结构对听源性惊厥的发生有重要意义。Ｐ＜０．０１讨论本文结果说明听源性惊厥同其它因素诱导的惊厥一样［３］，可诱导大鼠脑内ｃ－ｆｏｓ基因的表达，表达涉及到大脑皮层、海马齿状回、丘脑、下丘、蜗神经核等结构，其中以皮层下结构如丘脑、下丘、蜗神经核表达变化最显著。原位杂交显示的ｃ－ｆｏｓ基因表达特征类似于Ｎｏｒｔｈｅｒｎ杂交结果即快速、大量和短暂性。由于不同部位在惊厥活动中的作用差别，因此用原位杂交可以显示每一结构内ｃ－ｆｏｓ基因表达特点。如在惊厥后３０ｍｉｎ，海马齿状回中７０％以上的神经元单位胞质面积上银粒计数超过２０个，而梨状皮层及运动皮层仅占５～１３．８％。有报告指出海马齿状回为钙离子通道和ＮＭＤＡ受体高密度区域［４］，推测Ｃａ２＋和ＮＭＤＡ?     

Redox proteomics identification of oxidized proteins in Alzheimer's disease hippocampus and cerebellum: an approach to understand pathological and biochemical alterations in AD

Alzheimer's disease (AD) is characterized by the presence of neurofibrillary tangles, senile plaques and loss of synapses. There is accumulating evidence that oxidative stress plays an important role in AD pathophysiology. Previous redox proteomics studies from our laboratory on AD inferior parietal lobule led to the identification of oxidatively modified proteins that were consistent with biochemical or pathological alterations in AD. The present study was focused on the identification of specific targets of protein oxidation in AD and control hippocampus and cerebellum using a redox proteomics approach. In AD hippocampus, peptidyl prolyl cis-trans isomerase, phosphoglycerate mutase 1, ubiquitin carboxyl terminal hydrolase 1, dihydropyrimidinase related protein-2 (DRP-2), carbonic anhydrase II, triose phosphate isomerase, alpha-enolase, and gamma-SNAP were identified as significantly oxidized protein with reduced enzyme activities relative to control hippocampus. In addition, no significant excessively oxidized protein spots were identified in cerebellum compared to control, consistent with the lack of pathology in this brain region in AD. The identification of oxidatively modified proteins in AD hippocampus was verified by immunochemical means. The identification of common oxidized proteins in different brain regions of AD brain suggests a potential role for these oxidized proteins and thereby oxidative stress in the pathogenesis of Alzheimer's disease.     

GABAA and GABAB receptor site distribution in the rat central nervous system

An autoradiographic procedure has been used to determine the quantitative distributions of gamma-aminobutyric acid (GABAA and GABAB) receptor subtypes in rat brain. Although the concentrations of both receptor binding sites were similar in some brain regions GABAA sites generally outnumbered GABAB sites. The highest concentration of GABAA sites were detected in the frontal cortex, the granule cell layer of the cerebellum, the olfactory bulb and the thalamic medial geniculate. The highest concentration of GABAB sites occurred in the molecular layer of the cerebellum, the interpeduncular nucleus, frontal cortex, anterior olfactory nucleus and thalamic nuclei. In addition the globus pallidus, temporal cortex, lateral posterior thalamus, superior colliculus, pontine nucleus, raphe magnus, spinal trigeminal tract and substantia gelatinosa contained significantly more GABAB sites than GABAA sites. The physiological and pharmacological significance of this heterogeneity has yet to be determined.     

Neurotransmitter and neuromodulator systems of the rat inferior colliculus and auditory brainstem studied by in situ hybridization

This study was concerned with the distribution of a variety of putative neuromodulator and neurotransmitter systems in auditory regions of the rat brainstem using in situ hybridization histochemistry. Serial brain sections were screened for the presence of mRNAs for (i) precursors of the neuroactive substances cholecystokinin, somatostatin, proenkephalin and substance P (preprotachykinin), (ii) glutamic acid decarboxylase, the key synthesizing enzyme for GABA, or (iii) subunits l, 2 and 3 of the GABA_A receptor. Detectable message for all of these probes was found in at least one auditory brainstem area. There were clear differences in the distribution of the various mRNAs in subregions of the inferior colliculus, superior olivary complex, lateral lemniscus and cochlear nucleus. Cells expressing mRNA for glutamic acid decarboxylase were most prominent in the inferior colliculus, but were also present in all lower auditory brainstem nuclei, except the medial superior olivary nucleus and medial nucleus of trapezoid body. The mRNA for GABA_A1 receptor subunits was detectable in all auditory regions investigated, although at different levels of expression. GABA_A2 and 3 mRNA signals were seen in inferior colliculus, lateral lemniscus and in almost all superior olivary complex regions, but in fewer cells and at lower levels than the GABA_A1 subtype. Moderate to high levels of preprocholecystokinin mRNA expression were seen in all subregions of the inferior colliculus. In other auditory brainstem areas, preprocholecystokinin mRNA levels were either low or absent. With regard to mRNAs for the neuroactive peptides somatostatin, preprotachykinin and preproenkephalin, all were expressed in the inferior colliculus but there were differences in their cellular distribution. For example, there were almost no preprotachykinin mRNA expressing cells in the central nucleus of inferior colliculus and levels of somatostatin mRNA were especially high in the dorsal cortex and in layer 3 of the external cortex of inferior colliculus. There were also differences in the pattern of expression of these mRNAs in the various brainstem auditory nuclei; there was no preprotachykinin mRNA in any part of the superior olivary complex, only somatostatin mRNA was found in the ventral cochlear nucleus, and expression of preproenkephalin mRNA was pronounced in the ventral nucleus of the trapezoid body and the rostral periolivary zone. The data are considered in light of the connectivity and functional organization of the auditory brainstem.     

Functional mapping of neural pathways in rodent brain in vivo using manganese-enhanced three-dimensional magnetic resonance imaging

This work presents three-dimensional MRI studies of rodent brain in vivo after focal and systemic administration of MnCl2. Particular emphasis is paid to the morphology and dynamics of Mn2+-induced MRI signal enhancements, and the physiological mechanisms underlying cerebral Mn2+ uptake and distribution. It turns out that intravitreal and intrahippocampal injections of MnCl2 emerge as useful tools for a delineation of major axonal connections in the intact central nervous system. Subcutaneous administrations may be exploited to highlight regions involved in fundamental brain functions such as the olfactory bulb, inferior colliculus, cerebellum and hippocampal formation. Specific insights into the processes supporting cerebral Mn2+ accumulation may be obtained by intraventricular MnCl2 injection as well as by pharmacologic modulation of, for example, hippocampal function. Taken together, Mn2+-enhanced MRI opens new ways for mapping functioning pathways in animal brain in vivo with applications ranging from assessments of transgenic animals to follow-up studies of animal models of human brain disorders.     

The distribution of motilin-like peptides in rhesus monkey brain as determined by radioimmunoassay

The distribution of motilin-like immunoreactivity (MLI) was determined by radioimmunoassay in the major regions of rhesus monkey brain. In agreement with previous studies, motilin concentration was higher in pituitary and pineal than brain. MLI was widely distributed in rhesus monkey brain and was particularly high in claustrum, inferior colliculus, and cerebellar floculus. Sephadex G50 chromatography of monkey cerebellar and cerebral cortical extracts revealed multiple forms of MLI. The presence of MLI in rhesus monkey brain confirms previous reports of localization of motilin-like peptides by immunocytochemistry in the human cerebellum and is suggestive of a possible role for MLI in the functioning of the human brain.     

Regional brain variations of cytochrome oxidase activity and motor coordination in Lurcher mutant mice

Lurcher mutant mice are characterized by massive degeneration of cerebellar Purkinje cells and granule cells and by deficits in motor coordination. Regional brain variations of cytochrome oxidase (CO) activity were analyzed to identify those brain regions with abnormal metabolic activity as a secondary consequence of the cerebellar atrophy and to establish the relationship between CO activity and motor deficits. Lurcher mutants had higher CO activity in all three cerebellar deep nuclei than normal littermate controls of the same background strain. Higher CO activity was also found in Lurcher mutants in brain regions directly connected to the cerebellum, such as the lateral vestibular nucleus, the cochlear nucleus, the red nucleus, the ventrolateral thalamus, the dorsal raphe, the interpeduncular nucleus, and the inferior colliculus. By contrast, there was a sharp decrease in CO activity in the inferior olive. As for brain regions not directly connected to the cerebellum, higher CO activity was observed in the trigeminal motor nucleus and the CA1 molecular layer of the hippocampus, which highlights probable transsynaptic alterations as a secondary consequence of cerebellar atrophy. A positive correlation between CO activity in the red nucleus and latencies before falling in two motor-coordination tests indicates that a compensatory increase of metabolic activity in a cerebellar efferent region is associated with improved behavior. Received: 15 September 1997 / Accepted: 3 March 1998     

Age-related changes in levels of tyrosine kinase B receptor and fibroblast growth factor receptor 2 in the rat inferior colliculus: implications for neural senescence

Brain-derived neurotrophic factor and fibroblast growth factor 2, and their respective binding sites, tyrosine kinase B receptor and fibroblast growth factor receptor 2, are known to regulate neurite outgrowth and antioxidant enzyme activity. Several studies suggest that brain-derived neurotrophic factor and fibroblast growth factor are contained in the inferior colliculus. Previous work in our laboratories revealed dendritic and synaptic losses in the inferior colliculus of aged Fischer-344 rats, along with coincident increases in lipid peroxidation possibly linked to a decrease in activity of antioxidant enzymes. In an effort to identify potential causal mechanisms underlying age-related synaptic and dendritic losses that occur in the inferior colliculus, the present study attempted to determine if inferior colliculus levels of tyrosine kinase B receptor and fibroblast growth factor receptor 2 expression are altered with age.Immunocytochemistry was performed in the inferior colliculus, hippocampus and cerebellum of 3-month-old F344 rats to study distributions of the full-length and truncated isoforms of tyrosine kinase B receptor, and fibroblast growth factor receptor 2. The latter two brain regions served as positive controls. For all three antigens, immunolabeling was localized primarily in somata and proximal dendrites in all subdivisions of the inferior colliculus, and in the dentate gyrus and Ammon's horn of the hippocampus. In the cerebellum, the somata and dendrites of the Purkinje cells were also immunolabeled.A significant reduction in levels of the full-length form of tyrosine kinase B receptor in 18- and 25-month-old rats (respectively, approximately 20% and 30% relative to 3-month-olds) was revealed using western blot analyses. Inferior colliculus and hippocampal levels of the truncated form were modestly decreased ( approximately 7%) as well in the two older age groups. In contrast, levels of fibroblast growth factor receptor 2 in the inferior colliculus and hippocampus were elevated by approximately 35% in the two older age groups when compared to 3-month-olds. Changes in cerebellar levels of tyrosine kinase B receptor and fibroblast growth factor receptor 2, while similar to those in the inferior colliculus and hippocampus among the age groups, did not achieve statistical significance in this study.These findings give rise to the possibility that age-related reductions in tyrosine kinase B receptor levels could be a causal factor in the degenerative changes observed in the inferior colliculus of aged animals, including mitochondrial damage and dendritic regression. The observed increases in fibroblast growth factor receptor 2 levels may be compensatory to the increased oxidative stress. The effectiveness of the fibroblast growth factor receptor 2 response is questionable given the damage that occurs in the inferior colliculus and hippocampus of aged animals. However, the deficits could worsen in the absence of an increase in fibroblast growth factor receptor 2.     

ADAM23在小鼠脑组织的表达

目的:观察整合素-金属蛋白水解酶23(ADAM23)在成年小鼠脑组织的表达及分布。方法:RT-PCR克隆ADAM23全长序列,体外逆转录合成地高辛标记的RNA探针;原位杂交法检测ADAM23在脑各组织的表达。结果:ADAM23 mRNA在小鼠大脑皮层区、海马CA3区、小脑的浦肯野细胞层(Purkinje cell)区、舌下神经核区高表达;而小脑、下丘的中央核、小脑核侧部区等弱表达。结论:ADAM23在成年小鼠脑组织的广泛表达尤其中枢神经系统的高表达,预示着ADAM23在中枢神经系统内有重要作用。     

Regional distribution of the 5-HT innervation in the brain of normal and lurcher mice as revealed by [H]citalopram quantitative autoradiography

The neurological cerebellar mutant lurcher is characterized by a primary degeneration of Purkinje cells as well as a retrograde secondary partial degeneration of cerebellar granule cells and inferior olivary neurons. Since serotonin (5-HT) has been implicated in the modulation of excitatory amino acid systems of the cerebellum, the 5-HT innervation of the normal and lurcher mice was examined by quantifying uptake sites using [³H]citalopram autoradiography, and by biochemical assays of the indoles 5-HT, 5-hydroxy- -tryptophan and 5-hydroxyindole-3-acetic acid using high-performance liquid chromatography. Comparable results were found between [³H]citalopram binding and 5-HT tissue concentrations in different brain regions. The highest [³H]citalopram labelling was observed in defined structures of the mesencephalic and upper pontine regions, in limbic structures, in hypothalamus and in discrete thalamic divisions, while the lowest labelling of uptake sites was documented in cerebellum and brainstem reticular formation. In lurcher mutants, the histology confirmed cell degeneration and the reduction in width, leading to 65%, 45% and 25% atrophies of total cerebellum, deep nuclei and inferior olivary nucleus, respectively. The [³H]citalopram labelling corrected for surface loss was 45% and 20% higher in cerebellar deep nuclei and red nucleus, respectively, but remained unchanged in the cerebellar cortex and inferior olivary nucleus. Moreover, higher labelling was found in nucleus raphe dorsalis, ventral tegmental area, inferior colliculus, locus coeruleus, pontine central grey and anterior thalamic nuclei, areas known to be part of cerebellar afferent and efferent systems. The present results indicate that in such pathological conditions as described for the lurcher mutant, the 5-HT system may modulate motor function not only at the level of the cerebellum, but also in other forebrain structures functionally related to the motor system.     

Neural substrates of vocalizations in gulls and pigeons

Summary Vocalizations were obtained by electrically stimulating the inferior colliculus, the auditory thalamic nucleus and a medial hypothalamic nucleus of awake, unrestrained herring gulls, lesser black-backed gulls and pigeons. The significance of the involvement of auditory centres in the motor control of avian calling is discussed. The wide gammut of calls and accompanying behaviour that was elicited is described and related to the normal behaviour, typical of the species concerned. A difference between immature gulls and adult pigeons regarding this relationship is attributed to their differing hormonal states. Attention is drawn to the heterogeneity of temporal characteristics associated with the stimulus induced responses even when elicited from virtually the same site. Incorporating earlier work on the central mechanism of avian vocalizations and based on anatomical, physiological and behavioural considerations it is tentatively concluded that the neural structures involved are linearly organized into a telencephalofugal, efferent system. It is suggested that the inferior colliculus incorporates the origin of a final common pathway to medular motor centres for all vocalization generating structures.Meinem hochverehrten Lehrer Professor Dr. G. Birukow, Göttingen, zu seinem 60. Geburtstag gewidmet.     

Vitamin transporters in mice brain with aging

Its high metabolic rate and high polyunsaturated fatty acid content make the brain very sensitive to oxidative damage. In the brain, neuronal metabolism occurs at a very high rate and generates considerable amounts of reactive oxygen species and free radicals, which accumulate inside neurons, leading to altered cellular homeostasis and integrity and eventually irreversible damage and cell death. A misbalance in redox metabolism and the subsequent neurodegeneration increase throughout the course of normal aging, leading to several age‐related changes in learning and memory as well as motor functions. The neuroprotective function of antioxidants is crucial to maintain good brain homeostasis and adequate neuronal functions. Vitamins E and C are two important antioxidants that are taken up by brain cells via the specific carriers αTTP and SVCT2, respectively. The aim of this study was to use immunohistochemistry to determine the distribution pattern of these vitamin transporters in the brain in a mouse model that shows fewer signs of brain aging and a higher resistance to oxidative damage. Both carriers were distributed widely throughout the entire brain in a pattern that remained similar in 4‐, 12‐, 18‐ and 24‐month‐old mice. In general, αTTP and SVCT2 were located in the same regions, but they seemed to have complementary distribution patterns. Double‐labeled cell bodies were detected only in the inferior colliculus, entorhinal cortex, dorsal subiculum, and several cortical areas. In addition, the presence of αTTP and SVCT2 in neurons was analyzed using double immunohistochemistry for NeuN and the results showed that αTTP but not SVCT2 was present in Bergmann's glia. The presence of these transporters in brain regions implicated in learning, memory and motor control provides an anatomical basis that may explain the higher resistance of this animal model to brain oxidative stress, which is associated with better motor performance and learning abilities in old age.     

Angiotensin II receptor subtype distribution in the rabbit brain

Previous work has reported that the distribution of AT(1) binding sites in the rabbit brain is similar to that in the rat, but AT(2) binding sites are confined to the septum and cerebellum of the rabbit brain. This receptor autoradiographic study was designed to enhance the detection of angiotensin II binding sites by using greater radioligand concentrations, and to survey the midbrain in more detail than in previous studies. Tissue sections from five rabbit forebrains, three midbrains, and three hindbrains were incubated with 520 pM (125)I-sar(1)ile(8) angiotensin II. The results confirm abundant AT(1) binding in regions involved in cardiovascular and drinking regulation: the nucleus of the solitary tract, ventrolateral medulla, subfornical organ, organum vasculosum of the lamina terminalis, median eminence, and several hypothalamic structures. Novel AT(1) binding sites were discovered in the pituitary, retrorubral field, periolivary region, dorsolateral nucleus of the lateral lemniscus, dorsal raphe, and laterodorsal tegmental nuclei. The distribution of AT(1) binding was similar to the distribution of monoaminergic neurons. AT(2) binding was moderately dense and well visualized in the cerebellum. In contrast to the rat, AT(2) binding was not detected in the inferior olive of the rabbit, but lobe 9 of the cerebellum exhibited a banding pattern of AT(2) binding reminiscent of the pattern of neuronal projections from the inferior olive. It is possible that AT(2) protein is observed at different stages of axonal transport between the inferior olive and the cerebellum in the two species. Our results did identify new AT(2) binding sites in the superior colliculus and cerebral cortex, but it is clear that AT(2) binding in the rabbit brain is weak and is not as widely distributed as in the rat.     

Role of the inferior colliculus in the inhibition of acoustic startle in the rat

Fifteen rats were tested for amplitude reduction of the acoustic startle response using auditory and visual prestimuli. Eight subjects then received large lesions of the inferior colliculus, and the remaining subjects served as normal controls. All animals were reassessed on a post-test identical to the pre-test. In addition, all subjects were tested for latency reduction of startle using auditory prestimuli. There were no significant differences between groups on the pre-test for startle amplitude, visual amplitude reduction, or auditory amplitude reduction, nor did the control group differ significantly on these measures from pre-test to post-test. After surgery, the lesion group displayed a large, significant increase in startle amplitude. Auditory prestimuli were no longer effective in reducing startle amplitude in this group, but visual prestimuli still produced reliable amplitude reduction. Both groups displayed reliable latency reduction to auditory prestimuli; the groups were not significantly different from each other on this measure. These data support the proposition that the inferior colliculus is part of a neural circuit for startle amplitude reduction by auditory prestimuli.     

The climbing fibers of the cerebellar cortex,their origin and pathways in cat

Summary The sources and pathways of the climbing fibers to the cerebellar posterior vermis were studied with combined electrophysiological and anatomical methods in cats.Recording from identified cerebellar Purkinje cells, monosynaptic climbing fiber (CF) responses have been obtained both for stimulation of the inferior olive (IO) and various parts of the brain stem (BS). CF responses were found to be of three types, IO only, BS only or both IO and BS. However the responses to BS stimulation were very few in number in comparison with IO or IO and BS types of responses. The latencies of the responses were shorter for the BS cases consistent with their distance from the cerebellum.A comparison of latencies and the relative responsiveness of the different area of the brain stem which were studied, indicate that part of the CF ascend through the pontine region and enter the cerebellum by way of the medium and superior peduncles. This finding is confirmed by the results of anatomical studies in which degenerating fibers were found in the molecular layer (using the Nauta technique) after lesion of the brachium pontis but not after lesions of the medial portion of the pons. Similarly, injection of radioactive leucine into the pontine nuclei failed to show any labeled fibers in the molecular layer.Horseradish peroxidase (HRP) was injected into localized regions of the posterior vermis after total bilateral destruction of the inferior peduncles. Large numbers of positive, marked cells were still found in the inferior olive.It is concluded that nearly all, if not all, the climbing fibers originate in the inferior olive and that they ascend to the cerebellum by way of all the peduncles.     

A possible collicular component of the auditory evoked potential and its relationship to brainstem and cerebellar auditory potentials.

Auditory evoked potentials were recorded from the rat using skull screw electrodes inserted over the inferior colliculus and the cerebellum. In addition, brainstem auditory evoked potentials (BAEPs) were also recorded. The response recorded from over the inferior colliculus consisted of a slow positive potential with one of two possible peak latencies. The mean latency of the earlier potential was 5.6 ms and that of the later potential was 6.4 ms. A hypothetical generator for the first collicular potential is the termination of the lateral lemniscus in the ventrolateral inferior colliculus, while the later collicular potential could have its origins within the brachium of the inferior colliculus. None of the principal nor minor BAEP waves corresponded to either of the collicular responses. Nor did the trough of negativity between BAEP waves IV and V which is often thought to reflect activity generated within the midbrain. The potential recorded over the cerebellum also consisted of a slow positivity but with a slightly sharper contour than that of the collicular response. The mean latency of the cerebellar potential was 4.9 ms. As there was no temporal relationship between collicular and cerebellar potentials, the present study provided no support for the theory that cerebellar auditory potentials are artefactual and simply far field reflections of activity generated in the inferior colliculus. Judging by the timing of the BAEP waves, it is also concluded that the afferent volley most likely projects to the cerebellum via a collateral pathway branching off the caudal part of the lateral lemniscus.     

GABAA receptor subtypes: autoradiographic comparison of GABA, benzodiazepine, and convulsant binding sites in the rat central nervous system

The regional distribution of radioactive ligand binding in rat brain for the different receptors of the gamma-aminobutyric acidA (GABAA)-benzodiazepine receptor/chloride channel complex was measured on tissue sections by autoradiography. Seven ligands were employed including [3H]muscimol for high-affinity GABA agonist sites; [3H]bicuculline methochloride and [3H]SR-95531 for the low-affinity GABA sites; [3H]flunitrazepam for benzodiazepine sites, and [3H]2-oxo-quazepam for the 'BZ1'-type subpopulation; and [35S]t-butyl bicyclophosphorothionate (TBPS) and [3H]t-butyl bicyclo-orthobenzoate (TBOB) for convulsant sites associated with the chloride channel. Allosteric interactions of benzodiazepine receptor ligands with [35S]TBPS binding also were examined in membrane homogenates. Comparison of 19 brain regions indicated areas of overlap between these ligands, but also significant lack of correspondence in some regions between any two ligands compared. In particular, the cerebellum, thalamus, hippocampus, substantia nigra and superior colliculus showed enrichment in the binding of some ligands compared to others, and other brain regions showed smaller discrepancies. In addition to the previously observed discrepancies between high-affinity GABA agonists binding and benzodiazepine receptor distribution, especially in the cerebellum, and the well-documented differences in 'BZ1'-selective versus non-selective ligands, significant differences were observed in comparing GABA agonists with antagonists, one antagonist with another, GABA ligands with benzodiazepine or convulsant sites, and even between the two convulsants TBPS and TBOB. The major factor in regional variations within one ligand and between ligands involves differences in binding site densities, although other factors such as endogenous ligands and conformational flexibility may contribute to these findings. The lack of correspondence between components of the GABAA-receptor complex is most consistent with the existence of subtypes that vary in their binding affinities or even binding capabilities. At least four such subtypes are required to explain the regional dissimilarities between ligands. It is likely that these subtypes based on binding alone correspond to different gene products demonstrated recently by molecular cloning and protein chemistry, indicating a pharmacological heterogeneity that might be exploited with subtype-specific drugs showing desirable clinical profiles.