A critical level of protein synthesis is required for long-term potentiation

Long-term potentiation (LTP) of the hippocampal population spike in area CA1 was studied in the hippocampal slice before, during, and after inhibition of protein synthesis. LTP was tested 15 and 30 min following addition of the protein synthesis inhibitor cycloheximide (CXM) to the bathing medium. Stimulation-induced LTP of the population spike was reduced in slices incubated in CXM for 15 min and completely blocked if incubated for 30 min. Measures of protein synthesis inhibition under the same conditions showed 79% at 15 min and 85% after 30 min. Tests of the effects of repeated LTP treatments at these same time points in the absence of CXM and evaluation of the time course of recovery of LTP from CXM blockade were also conducted. The results confirm previous observations and indicate that a critical amount of protein synthesis inhibition is required to completely block LTP in the hippocampal slice.     

Developmental regulation of two isoforms of Ca/calmodulin-dependent protein kinase I β in rat brain

Subtractive hybridization analysis of region-specific gene expression in brain has demonstrated a mRNA species enriched in rat hypothalamus [K.M. Gautvik, L. de Lecea, V.T. Gautvik, P.E. Danielson, P. Tranque, A. Dopazo, F.E. Bloom, J.G. Sutcliffe, Proc. Natl. Acad. Sci. USA 93 (1996) 8733–8738.]. We here show that this mRNA encodes a Ca²⁺/calmodulin-dependent (CaM) kinase belonging in the CaM kinase I β subgroup. cDNA analysis showed that this enzyme was differentially spliced into two isoforms (designated β1 and β2) with distinct C-termini. The C-terminal of the translated CaM kinase I β2 protein (38.5 kDa molecular size), contained 25 amino acid residues not present in the β1 isoform. The two isoforms were differentially developmentally regulated, with the β1 isoform being present in rat embryos from day 18 and the β2 isoform being present from day 5 postnatally. In situ hybridization analysis of adult rat CNS showed CaM kinase I β2 mRNA being enriched in the hypothalamus and the hippocampal formation. Expression was also observed in a number of ventral limbic structures and in the thalamus. Northern blot analysis showed additional expression of multiple β2 isoforms in heart and skeletal muscle. The human mRNA showed a similar distribution. Our data suggest that the two isoforms of CaM kinase I β, created by a splicing process occurring within a week around birth, may have distinct pre- and postnatal functions in a distinct set of CNS neurons and excitable tissues.     

匹罗卡品致痫诱发大鼠齿状回颗粒细胞GAP-43 mRNA表达

目的 研究边缘系统癫痫发作后海马颗粒细胞生长相关蛋白（GAP-43）基因表达变化。方法 建立匹罗卡品急、慢性癫痫模型,用原位杂交方法定量检测不同时间点海马颗粒细胞GAP-43mRNA表达。结果 对照组颗粒细胞几乎不表达GAP-43mRNA,匹罗卡品致病后6～12h颗粒细胞表达GAP-43mRNA增高,15～30d呈现第2次高峰。结论 成年大脑海马颗粒细胞在致痫后发生可塑性变化,GAP-43mRNA表达是癫痫大鼠大脑结构性重组（颗粒细胞苔藓纤维出芽）的重要分子机制。     

Behavioral changes and expression of heat shock protein hsp-70 mRNA, brain-derived neurotrophic factor mRNA, and cyclooxygenase-2 mRNA in rat brain following seizures induced by systemic administration of kainic acid

Kainic acid-induced seizures in rats represent an established animal model for human temporal lobe epilepsy. However, it is well-known that behavioral responses to the systemic administration of kainic acid are inconsistent between animals. In this study, we examined the relationship between expression of genes, neuropathological damage, and behavioral changes (seizure intensity and body temperature) in rats after systemic administration of kainic acid. The considerable differences in the response to kainic acid-induced seizures were observed in rats after a single administration of kainic acid (12 mg/kg i.p.). There was no detection of the expression of heat shock protein hsp-70 mRNA and HSP-70 protein in brain of vehicle-treated controls and in animals exhibiting weak behavioral changes (stage 1–2). A moderate expression of hsp-70 mRNA was detected throughout all regions (the pyramidal cell layers of CA1–3 and dentate gyrus) of the hippocampus, the basolateral, lateral, central and medial amygdala, the piriform cortex, and the central medial thalamic nucleus of rats that developed moderate seizures (stage 3–4). Marked expression of hsp-70 mRNA was detected in the all regions (cingulate, parietal, somatosensory, insular, entorhinal, piriform cortices) of cerebral cortex and all regions of hippocampus, and the central medial thalamic nucleus of the rats that developed severe seizures (stage 4–5). In addition, marked HSP-70 immunoreactivity was detected in the pyramidal cell layers of CA1 and CA3 regions of hippocampus, all regions (cingulate, parietal, somatosensory, insular, piriform cortices) of cerebral cortex, and the striatum of rats that developed severe seizures (stage 4–5). Furthermore, a marked expression of cyclooxygenase-2 (COX-2) mRNA and brain-derived neurotrophic factor (BDNF) mRNA levels by kainic acid-induced behavioral seizures (stage 3–4 or stage 4–5) was detected in all hippocampal pyramidal cell layers, granule layers of dentate gyrus, piriform cortex, neocortex, and amygdala. The present study suggest that the behavioral changes (seizure intensity and body temperature) and neuropathological damage after systemic administration of kainic acid are inconsistent between animals, and that these behavioral changes (severity of kainic acid-induced limbic seizures) might be correlated with gene expression of hsp-70 mRNA, COX-2 mRNA, and BDNF mRNA in rat brain.     

Delayed kindling development after rapidly recurring seizures: relation to mossy fiber sprouting and neurotrophin, GAP-43 and dynorphin gene expression

Development of kindling and mossy fiber sprouting, and changes of gene expression were studied after 40 seizures produced during about 3 h by electrical stimulation every 5 min in the ventral hippocampus. As assessed by 5 test stimulations, enhanced responsiveness was present already after 6–24 h but from 1 week post-seizure increased gradually up to 4 weeks without additional stimuli. Sprouting of mossy fibers in the dentate gyrus was demonstrated only at 4 weeks with Timm's staining. In situ hybridization showed a transient increase (maximum at 2 h) of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), TrkB and TrkC mRNA levels and reduction (maximum at 12–24 h) of neurotrophin-3 (NT-3) mRNA expression in dentate granule cells after the seizures. In addition, BDNF mRNA levels were elevated in CAI and CA3 regions, amygdala and piriform cortex. Marked increases of mRNA for growth-associated protein (GAP-43), with maximum expression at 12–24 h, were observed in dentate granule cells and in amygdala-piri-form cortex. Dynorphin mRNA levels showed biphasic changes in dentate granule cells with an increase at 2 h followed by a decrease at 24 h. No long-term alterations of gene expression were observed. These findings indicate that increased responsiveness develops rapidly after recurring seizures but that the kindled state is reached gradually in about 4 weeks. Mossy fiber sprouting occurs in parallel to epileptogenesis and may play a causative role. Short-term changes of neurotrophin and Trk, GAP-43 and dynorphin mRNA levels and the assumed alterations of the corresponding proteins could trigger structural rearrangements underlying kindling but might also contribute to the initial increase of seizure susceptibility.     

The cross‐modal aspect of mouse visual cortex plasticity induced by monocular enucleation is age dependent

Monocular enucleation (ME) drastically affects the contralateral visual cortex, where plasticity phenomena drive specific adaptations to compensate for the unilateral loss of vision. In adult mice, complete reactivation of deprived visual cortex involves an early visually driven recovery followed by multimodal plasticity 3 to 7 weeks post ME (Van Brussel et al. [ 2011 ] Cereb. Cortex 21:2133–2146). Here, we specifically investigated the age dependence of the onset and the exact timing of both ME‐induced reactivation processes by comparing cortical activity patterns of mice enucleated at postnatal day (P) 45, 90, or 120. A swifter open‐eye potentiated reactivation characterized the binocular visual cortex of P45 mice. Nevertheless, even after 7 weeks, the reactivation remained incomplete, especially in the monocular cortex medial to V1. In comparison with P45, emergent cross‐modal participation was demonstrated in P90 animals, although robust reactivation similar to enucleated adults (P120) was not achieved yet. Concomitantly, at 7 weeks post ME, somatosensory and auditory cortex shifted from a hypoactive state in P45 to hyperactivity in P120. Thus, we provide evidence for a presensitive period in which gradual recruitment of cross‐modal recovery upon long‐term ME coincides with the transition from adolescence to adulthood in mice. J. Comp. Neurol. 522:950–970, 2014. © 2013 Wiley Periodicals, Inc.     

Neurotensin and neuromedin N brain levels after fornix transection: evidence for an efficient neurotensin precursor processing in subicular neurons

High levels of neurotensin/neuromedin N precursor mRNA, but few if any NT-positive perikarya have been detected in the dorsal subiculum of the adult rat or human hippocampus. This apparent discrepancy was tentatively ascribed to a lack of precursor mRNA translation or to a poor precursor positranslational processing in neurons of the hippocampus. Another hypothesis is that in long neuronal pathways, maturation of neuropeptide precursors and derived peptides occurs during axonal transport to terminals, a process which accounts for the lack of peptide detection in cell bodies. In order to test this hypothesis, we performed surgical transection of the fornix to interrupt axonal transport of putative NT/NN products arising from the dorsal hippocampus and measured NT and NN levels in different brain regions. In the mamillary bodies, the main projection area of the dorsal subiculum, NN and NT levels were highly reduced 4 or 14 days after the septo-hippocampus transection which was correlated with a slight increase in NN and NT levels in the dorsal hippocampus and the retrosplenial cortex of 4 days lesioned animals. An increase in hypothalamic NN levels was also detected 14 days after the lesion. These data suggest that the peptide precursor processing can take place during the axonal transport, as shown here for neurotensin and neuromedin N from subicular neurons to their efferent brain areas such as the mamillary bodies.     

A1 and A2A adenosine receptors and A1 mRNA in mouse brain: effect of long-term caffeine treatment

The effect of oral treatment with caffeine, in doses that are known to produce marked adaptive effects, was investigated on A₁ and A_2A receptors in the mouse brain. Caffeine (0.1, 0.3 or 1 g/l) was added to the drinking water and the animals were sacrificed after a 14-day treatment period. Ligand binding to A₁ receptors was studied, using quantitative autoradiography, with the agonist [³H]cyclohexyladenosine (CHA) and the antagonist [³H]1,3-dipropyl-8-cyclopentyl xanthine (DPCPX). Caffeine did not remain in the sections during the autoradiography experiments. Caffeine treatment (1 g/l, but not 0.1 or 0.3 g/l) tended to increase [³H]CHA binding to the CA3 subfield of the hippocampus, but in no other region studied. There was no change in the number of A₁ receptors since [³H]DPCPX binding to the CA3, cerebral and cerebellar cortex was not influenced by caffeine treatment. There was similarly no change in the ability of CHA to displace [³H]DPCPX binding, suggesting that there are no major changes in the proportion of A₁ receptors that are coupled to G-proteins. mRNA for the A₁ receptor, measured by in situ hybridization, did not differ significantly between caffeine-treated and control mice in the structures examined. Thus, higher doses of caffeine can cause an increase in A₁ agonist binding without a corresponding change in A₁ mRNA or in A₁ antagonist binding, suggesting that the adaptive changes seen upon prolonged caffeine treatment may be in sites different from A₁ receptors. Caffeine (1 g/l) increased A_2A receptors in the striatum measured as binding of the agonist [³H]CGS 21680 suggesting that up-regulation of A_2A receptors may be an adaptive effect of caffeine intake.     

Effects of age on messenger RNA expression of glucocorticoid, thyroid hormone, androgen, and estrogen receptors in postmortem human hippocampus

We studied messenger RNA (mRNA) expressions of receptors for glucocorticoid (GR), thyroid hormone (TR), androgen (AR), and estrogen (ER) and their changes with age in the hippocampal subregions in postmortem human brain. In situ hybridization was done with biotin-labeled antisense synthetic oligonucleotide probes. About 80% or more of the pyramidal neurons in the hippocampal subregions expressed mRNAs for individual receptors in the brains of subjects younger than 65. The ratio of mRNA-containing neuron density to total neuron density significantly decreased with age for GR in CA1 and CA3, and for AR in CA1. Non-significant trends in the reduction with age in the ratio of ER mRNA-containing neurons in CA1 and the ratio of GR mRNA-containing neurons in the hilus also were found. Age-related reductions in nuclear receptor protein mRNA expression in neurons in the hippocampal subfields may be important in the impairments of cognition, emotion, and responses to acute stress in the aged.     

Possible functions of a new genetic marker in central nervous system: the sulfated glycoprotein-2 (SGP-2).

This brief review discusses the recent characterization in the brain of a gene coding for a protein that may be involved in programmed cell death and/or brain plasticity. We will term it sulfated glycoprotein-2 (SGP-2), the name corresponding to the first cDNA characterized. Recent studies have demonstrated the overexpression of this sulfated glycoprotein in various CNS disorders, such as certain gliomas, Alzheimer's disease and epilepsy, as well as after experimental brain injury in animals where different cell types were undergoing tissue remodelling or cell death. In peripheral tissues, SGP-2 gene expression has been found to be strikingly increased following experimental manipulations in which cells of injured tissues were undergoing programmed cell death or apoptosis. The results reported thus far are intriguing and suggest the possible involvement of SGP-2 in apoptotic mechanisms as well as its interaction with components of the immune system possibly associated with cell death in neurodegenerative disorders.     

Effects of single or repeated restraint stress on GluR1 and GluR2 flip and flop mRNA expression in the hippocampal formation

The mRNAs encoding the flip and flop isoforms of the glutamate receptor subunits GluR1 and 2 were detected and quantified by in situ hybridization in the hippocampal formation of rats following acute (6h) or chronic (6h daily for 21 days) restraint stress. The GluR1 flip mRNA was slightly reduced in CA1 after chronic stress and the GluR2 flip mRNA was increased in the dentate gyrus (DG), CA4, and CA3 after acute stress. There were no changes in the mRNA encoding the flop isoforms of either GluR1 or 2 in the hippocampus. In entorhinal cortex, the GluR1 flip mRNA was significantly increased after both acute and chronic stress, while the flop isoform increased only after chronic stress. The GluR2 flip mRNA was slightly increased after acute and chronic stress. However, no changes were found for the flop isoform of GluR2. These results suggest that different assembly of AMPA receptors subunits and isoforms may underlie, in a different way, the neuronal plastic changes induced by specific type and intensity of stressful stimuli.     

Bursts of high-frequency stimulation trigger rapid delivery of pre-existing alpha-CaMKII mRNA to synapses: a mechanism in dendritic protein synthesis during long-term potentiation in adult awake rats

Messenger ribonucleic acid encoding the alpha-subunit of calcium/calmodulin-dependent protein kinase II (camkII) is abundantly and constitutively expressed in dendrites of pyramidal and granule cell neurons of the adult hippocampus. Recent evidence suggests that camkII messenger ribonucleic acid is stored in a translationally dormant state within ribonucleic acid storage granules. Delivery of camkII messenger ribonucleic acid from sites of storage to sites of translation may therefore be a key step in activity-driven dendritic protein synthesis and synaptic plasticity. Here we explored possible camkII trafficking in the context of long-term potentiation in the dentate gyrus of awake, adult rats. Long-term potentiation was induced by patterned high-frequency stimulation, synaptodendrosomes containing pinched-off dendritic spines were obtained from microdissected dentate gyrus, and messenger ribonucleic acid levels were determined by real-time polymerase chain reaction. High-frequency stimulation triggered a rapid 2.5-fold increase in camkII messenger ribonucleic acid levels in the synaptodendrosome fraction. This increase occurred in the absence of camkII upregulation in the homogenate fraction, indicating trafficking of pre-existing messenger ribonucleic acid to synaptodendrosomes. The elevation in camkII messenger ribonucleic acid was paralleled by an increase in protein expression specific to the synaptodendrosome fraction, and followed by depletion of camkII message. Activity-dependent regulation of camkII messenger ribonucleic acid and protein did not require N-methyl-d-aspartate receptor activation. In contrast, N-methyl-d-aspartate receptor activation was required for induction of the immediate early genes zif268 and activity-regulated cytoskeleton-associated protein in dentate gyrus homogenates. The results support a model in which locally stored camkII messenger ribonucleic acid is rapidly transported to dendritic spines and translated during long-term potentiation in behaving rats.     

SCG10 mRNA localization in the hippocampus: comparison with other mRNAs encoding neuronal growth-associated proteins (nGAPs)

SCG10 is a nerve growth factor (NGF)-inducible, neuron-specific protein whose expression is tightly correlated with axonal and/or dendritic growth. We have recently shown that the mRNA encoding SCG10 is expressed at significant levels in certain subsets of neurons in the adult rat brain, while its expression is undetectable or negligible in other non-neuronal tissues. Here we show that regional SCG10 mRNA expression in the adult mouse brain is comparable to that in the rat, however, in the hippocampus its expression profile is distinct. In the mouse, SCG10 mRNA is expressed at high levels in pyramidal cells of CA3–CA4 sub-fields of Ammon's horn and at low levels in the CA1–CA2 sub-fields, while it is found rather uniformly throughout the pyramidal cell layer of the rat hippocampus. SCG10 mRNA is not detectable in the dentate gyrus of the mouse hippocampus, although it is expressed in the rat dentate gyrus. Comparison with other mRNAs encoding neuronal growth-associated proteins (nGAPs) such as GAP-43, MAP2, α1-tubulin and stathmin suggests that dentate granule cells express a different repertoire of neuronal growth-associated genes in mouse and rat.     

The expression of mRNAs for hepatocyte growth factor/scatter factor, its receptor c-met, and one of its activators tissue-type plasminogen activator show a systematic relationship in the developing and adult cerebral cortex and hippocampus

The temporal and spatial expression in brain of the mRNAs for the pleiotropic cytokine hepatocyte growth factor/scatter factor (HGF/SF) and its receptor c-met were compared to those of a known HGF/SF activator, tissue-type plasminogen activator (tPA). In addition to the previously described expression in the developing and adult olfactory system [D.P. Thewke, N.W. Seeds, Expression of hepatocyte growth factor/scatter factor, its receptor, c-met, and tissue-type plasminogen activator during development of the murine olfactory system, J. Neurosci. 16 (1996) 6933–6944] two other regions of the mouse brain were found where the expression of tPA mRNA appeared to co-localized with HGF/SF and/or c-met mRNA. In the developing hippocampus, tPA mRNA was expressed coincident with HGF/SF and c-met mRNAs in the CA1 field. tPA mRNA was expressed in all areas of the adult hippocampus, while HGF/SF expression was restricted to the CA2 and CA3 fields, and c-met mRNA was seen primarily in the CA1 field. In the developing cerebral cortex, the expression of tPA mRNA was observed in the subplate and inner cortical plate between two layers of c-met expression, whereas HGF/SF mRNA was localized to the proliferative zone lining the lateral ventricle. Layer specific expression of both HGF/SF and c-met mRNA were observed in the adult cortex, where HGF/SF was expressed in layers IV and V and c-met in layers II–III, IV and V. The expression of tPA mRNA in the adult cortex was low and not layer specific, although homogenates of adult cortex did have detectable levels of tPA activity when subjected to zymography. Immunohistochemical analysis using HGF/SF and c-met antibodies on adult brain sections showed a distribution similar to the in situ hybridization results. C-met antibodies appeared to stain large neurons in the cortex and hippocampus. These results are consistent with the hypothesis that HGF/SF plays a role in the development and maintenance of both the cerebral cortex and hippocampus, and that tPA may act as a regulator of HGF/SF activity in these structures.     

Localization of cytochrome oxidase (COX) activity and COX mRNA in the hippocampus and entorhinal cortex of the monkey brain: correlation with specific neuronal pathways

Cytochrome oxidase (COX) activity and COX II mRNA expression were localized in the hippocampal formation and entorhinal cortex of the rhesus monkey brain by means of enzyme histochemistry and in situ hybridization, respectively. Within the hippocampal formation, the terminal field of the perforant pathway showed the highest levels of COX activity, whereas COX II mRNA was localized mainly in neuronal cell bodies. In the entorhinal cortex. COX II mRNA was detected in neuronal cell bodies of layers II and IV. These results indicate that the pattern of localization of COX and its mRNA in entorhinal cortex correlates with the input and output pathways of the hippocampus.     

Expression of androgen receptor mRNA in the brain of Gekko gecko: implications for understanding the role of androgens in controlling auditory and vocal processes

The neuroanatomical distribution of androgen receptor (AR) mRNA-containing cells in the brain of a vocal lizard, Gekko gecko, was mapped using in situ hybridization. Particular attention was given to auditory and vocal nuclei. Within the auditory system, the cochlear nuclei, the central nucleus of the torus semicircularis, the nucleus medialis, and the medial region of the dorsal ventricular ridge contained moderate numbers of labeled neurons. Neurons labeled with the AR probe were located in many nuclei related to vocalization. Within the hindbrain, the mesencephalic nucleus of the trigeminal nerve, the vagal part of the nucleus ambiguus, and the dosal motor nucleus of the vagus nerve contained many neurons that exhibited strong expression of AR mRNA. Neurons located in the peripheral nucleus of the torus in the mesencephalon exhibited moderate levels of hybridization. Intense AR mRNA expression was also observed in neurons within two other areas that may be involved in vocalization, the medial preoptic area and the hypoglossal nucleus. The strongest mRNA signals identified in this study were found in cells of the pallium, hypothalamus, and inferior nucleus of the raphe. The expression patterns of AR mRNA in the auditory and vocal control nuclei of G. gecko suggest that neurons involved in acoustic communication in this species, and perhaps related species, are susceptible to regulation by androgens during the breeding season. The significance of these results for understanding the evolution of reptilian vocal communication is discussed.     

Cellular localization and expression of the serotonin transporter in mouse brain

The high-affinity serotonin (5-HT) transporter (5-HTT) plays an important role in the removal of extracellular serotonin, thereby modulating and terminating the action of this neurotransmitter at various pre- and post-synaptic serotonergic receptors and heteroreceptors. In order to characterize the anatomical distribution of the 5-HTT in mouse brain, in situ hybridization histochemistry using ³⁵S-labeled riboprobes was performed. These results were compared with 5-HTT binding site distribution as evaluated by [¹²⁵I]RTI-55 autoradiography. High levels of 5-HTT mRNA were detected in all brain stem raphe nuclei, with variations in labeling among the various subnuclei. Those brain areas known to possess serotonergic cell bodies stained intensely for both 5-HTT mRNA and 5-HTT binding sites. In contrast to previous findings in rat brain, the highest densities of 5-HTT sites were found in areas outside the raphe complex, particularly in the substantia nigra, globus pallidus, and superior colliculi.