Developmental changes in KCC1, KCC2, and NKCC1 mRNA expressions in the rat brain

We investigated the expressions of KCC1, KCC2 and NKCC1 mRNAs in the developing rat brain. The neuroepithelium showed abundant KCC1 and NKCC1 mRNA expressions, while KCC2 mRNA was not detected there. In contrast, KCC2 mRNA was preferentially expressed in postmitotic mature neurons. These results suggest that the appearance of KCC2 expression mainly depends on the maturation of individual neurons.     

Differential functional expression of cation-Cl- cotransporter mRNAs (KCC1, KCC2, and NKCC1) in rat trigeminal nervous system

GABA is the main inhibitory neurotransmitter in the adult brain, which causes Cl- influx into the cell via GABAA receptors. The direction of Cl- inflow is dependent on the Cl- gradient across the membrane. Cation-Cl- cotransporters have been considered to play pivotal roles in controlling intracellular Cl- concentration ([Cl-]i) of neurons; hence, they modulate the GABAergic function. To elucidate how these cotransporters are distributed in the trigeminal nuclei, we investigated the expressions of K+-Cl- cotransporters (KCC1 and KCC2) and Na+-K+-2Cl- cotransporter (NKCC1) mRNAs by using in situ hybridization histochemistry. KCC2 mRNA was expressed in the motor trigeminal nucleus (Mo5), the principal trigeminal nucleus (Pr5), and the spinal trigeminal nucleus (Sp5), but not in the trigeminal ganglion (TG) and the mesencephalic trigeminal nucleus (Me5). On the other hand, KCC1 and NKCC1 mRNAs were expressed in all the trigeminal nuclei. The resting [Cl-]i of Me5 neurons was significantly higher than that of Mo5 neurons. Thus, in primary sensory neurons such as the TG and the Me5, [Cl-]i would be higher than those in the other trigeminal nuclei because of the lack of KCC2 mRNA expression. Since Me5 neurons, but not Mo5 neurons, responded to GABA by depolarization, GABA would have differential physiological functions among trigeminal nuclei and TG.     

NKCC1 and KCC2 prevent hyperexcitability in the mouse hippocampus

During postnatal development of the central nervous system (CNS), the response of GABA_A receptors to its agonist undergoes maturation from depolarizing to hyperpolarizing. This switch in polarity is due to the developmental decrease of the intracellular Cl concentration in neurons. Here we show that absence of NKCC1 in P9–P13 CA3 pyramidal neurons, through genetic manipulation or through bumetanide inhibition, results in a significant increase in cell excitability. Furthermore, the pro-convulsant agent 4-aminopyridine induces seizure-like events in NKCC1-null mice but not in wild-type mice. Measurements of muscimol responses in the presence and absence of NKCC1 shows that the Na–K–2Cl cotransporter only marginally affects intracellular Cl⁻ in P9–P13 CA3 principal neurons. However, large increases in intracellular Cl⁻ are observed in CA3 pyramidal neurons following increased hyperexcitability, indicating that P9–P13 CA3 pyramidal neurons lack robust mechanisms to regulate intracellular Cl⁻ during high synaptic activity. This increase in the Cl⁻ concentration is network-driven and activity-dependent, as it is blocked by the non-NMDA glutamate receptor antagonist DNQX. We also show that expression of the outward K–Cl cotransporter, KCC2, prevents the development of hyperexcitability, as a reduction of KCC2 expression by half results in increased susceptibility to seizure under control and 4-AP conditions.     

Ketogenic diet does not change NKCC1 and KCC2 expression in rat hippocampus

In control rats, we examined the effects of ketogenic diet on NKCC1 and KCC2 expression levels in hippocampus. Neither the number of NKCC1 immunoreactive cells nor the intensity of labeling of KCC2 was found to modify in hippocampus of the rats after ketogenic diet treatment. These results indicate that ketogenic diet by itself does not modify the expression of these cation chloride cotransporters.     

NKCC1、KCC2及mTOR相关蛋白4E-BP1在外伤性癫痫中的表达及意义

目的探讨主要表达于神经元细胞膜上的NA⁺-K⁺-2CL^-转运体（NKCC1）、K⁺-CL^-转运体（KCC2）及表达于神经元细胞质中mTOR通路信号蛋白4E结合蛋白1（4E-BP1）在外伤性癫痫（PTE）癫痫灶中的表达及临床意义。 方法收集2010年1月至2015年12月福建医科大学附属第一医院神经外科外伤性癫痫患者术后脑组织标本14例作为实验组;选取脑外伤患者行减压或清创手术获取的和各种病变患者手术入路不可避免要切除的正常脑组织8例作为对照组。用Western blot和实时定量荧光PCR（RT-PCR）检测14例PTE癫痫脑组织、8例对照组"正常脑组织"NKCC1、KCC2、4E-BP1的表达。 结果Western blot显示PTE病灶脑组织中4E-BP1、NKCC1相对灰度值（0.61±0.12、0.92±0.19）高于正常脑组织（0.27±0.05、0.67±0.66）,差异有统计学意义（P<0.05）。PTE病灶脑组织中KCC2相对灰度值（0.58±0.99）低于正常脑组织（0.72±0.06）,差异有统计学意义（P<0.05）。RT-PCR结果显示,PTE病灶脑组织中4E-BP1、NKCC1相对灰度值（30.84±1.32、27.81±1.92）高于正常脑组织（26.94±1.24、23.52±0.74）,差异有统计学意义（P<0.05）。PTE病灶脑组织中KCC2相对灰度值（21.55±1.01）低于正常脑组织（24.59±1.02）,差异有统计学意义（P<0.05）。PTE组中NKCC1/KCC2比值（1.29±0.11）高于对照组（0.96±0.26）,差异有统计学意义（P<0.05）。 结论NKCC1、KCC2和mTOR通路信号蛋白4E-BP1的异常改变,可能是外伤后脑组织组织学改变及反复癫痫发作的重要分子机制。     

Developmental changes of EAA metabotropic receptor activity in rat cerebellum.

The potency but not the efficacy of t-ACPD stimulation of phosphatydil inositide hydrolysis changes in developing rat cerebellum. This suggests that the excitatory amino-acid-stimulated metabotropic receptors and/or their coupling are ontogenically regulated. In this, cerebellum differs from other CNS regions where only efficacy changes were described. Differently from hippocampus, the t-ACPD effect, at all ages, is independent of the activation of the NMDA receptor.     

Developmental changes in PSD-95 and Narp mRNAs in the rat olfactory bulb.

Glutamate is the main neurotransmitter in the olfactory bulb. Recently, postsynaptic-density 95 (PSD-95) and neuronal activity-regulated pentraxin (Narp) have been reported to be pivotal for targeting and clustering of NMDA receptors and AMPA receptors, respectively. We thus investigated the expressions of PSD-95 and Narp mRNAs in the rat developing olfactory bulb. PSD-95 mRNA was already expressed in most neurons on the first postnatal day (P1). On the other hand, Narp mRNA expression was weakly seen only in mitral cells on P1. Thereafter, we found initial expression of Narp mRNA on P7 in periglomerular cells, and on P14 in granular cells, indicating that in the developing olfactory bulb PSD-95 mRNA expression precedes Narp mRNA expression, and that the expression pattern of Narp mRNA seems to be well correlated with the maturation of the neurons. These results indicate that PSD-95 and Narp play important roles in making efficient excitatory synapses in the developing rat olfactory bulb, and suggest that olfactory neurons might first express PSD-95 for making efficient NMDA receptors and thereafter express Narp for efficient AMPA receptors.     

Developmental differences in alternative splicing of the NR1 protein in rat cortex and cerebellum

Expression of the C-terminal cassettes of the NR1 protein was examined using a quantitative Western blot method with cassette-specific antibodies. Measurements were made of the percent of total NR1 protein that contained a specific cassette in both the cerebellum and cortex over development. In the cortex, the C1 cassette was shown to be present in about half of total NR1 protein with no change over development. While about half of total NR1 in the cerebellum at postnatal day 42 (P42) contained the C1 cassette, little NR1 protein with this cassette was seen at young ages. In both the cerebellum and cortex, the C2 and C2' cassettes showed opposite developmental patterns, with the C2 cassette decreasing and the C2' cassette increasing over age. Together with previous data on the expression of the N1 cassette, this study describes the alternatively spliced forms of NR1 protein that are dominant at different ages. In the young cerebellum, the NR1(001) form appeared dominant, while in the young cortex there appeared to be a mix of NR1(001) and NR1(011). The most common splice forms of NR1 protein in the adult cerebellum appeared to be NR1(111) and NR1(100). In the adult cortex, there appeared to be a mix of NR1(001) and NR1(011). These data on the expression of the alternatively spliced forms of NR1 allow predictions on the possible characteristics of NMDA receptors in different regions at specific ages.     

Differential expression of Na,K-ATPase α-isoform mRNAs in aging rat cerebellum

Age-dependent changes in the expression of Na,K-ATPase α1- and α3-mRNAs were analyzed in the rat cerebellum by in situ hybridization. In young rats, α1-mRNA showed prominent labeling in the granular layer (GL) with moderate fine distribution in the molecular layer (ML), Purkinje cell layer (PCL), and white matter (WM) but no clusters over Purkinje cells (PCs). In old rats, α1-mRNA remained unchanged in ML and PCL, but declined by 43% (P < 0.0001) in GL and increased by 624% (P < 0.0001) in WM. α3-mRNA in young rats showed large clusters of label on stellate, basket, Golgi, and PCs and fine grains diffusely in ML, GL, and WM. In old rats, α3-mRNA declined by 87% in ML, 83% in PCL, 84% per PC, and 89% in GL and increased by 111% in WM (all values P < 0.0001) relative to young rats. PC numbers were reduced by 30%, but the average area of PC profiles did not change significantly. In old rats, the specific cluster-like label related to α3-mRNA on PCs, stellate, basket, and Golgi cells was lost. Immunocytochemistry of cerebellum and hippocampus showed no age-related change in the distribution and density of total catalytic polypeptide. Thus, the discordance between changes in the levels of mRNAs in neuronal layers and WM in the face of constant polypeptide levels indicates age-related changes in polypeptide turnover. Cell- and isoform-specificity of α-isoform mRNAs in aging rat cerebellum may reflect differential regulation underlying age-related impairments in signal transduction and motor learning. J. Neurosci. Res. 47:287–299, 1997. © 1997 Wiley-Liss, Inc.     

Regulation of KCC2 and NKCC during development: membrane insertion and differences between cell types

The developmental switch of GABA's action from excitation to inhibition is likely due to a change in intracellular chloride concentration from high to low. Here we determined if the GABA switch correlates with the developmental expression patterns of KCC2, the chloride extruder K+-Cl- cotransporter, and NKCC, the chloride accumulator Na+-K+-Cl- cotransporter. Immunoblots of ferret retina showed that KCC2 upregulated in an exponential manner similar to synaptophysin (a synaptic marker). In contrast, NKCC, which was initially expressed at a constant level, upregulated quickly between P14 and P28, and finally downregulated to an adult level that was greater than the initial phase. At the cellular level, immunocytochemistry showed that in the inner plexiform layer KCC2's density increased gradually and its localization within ganglion cells shifted from being primarily in the cytosol (between P1-13) to being in the plasma membrane (after P21). In the outer plexiform layer, KCC2 was detected as soon as this layer started to form and increased gradually. Interestingly, however, KCC2 was initially restricted to photoreceptor terminals, while in the adult it was restricted to bipolar dendrites. Thus, the overall KCC2 expression level in ferret retina increases with age, but the time course differs between cell types. In ganglion cells the upregulation of KCC2 by itself cannot explain the relatively fast switch in GABA's action; additional events, possibly KCC2's integration into the plasma membrane and downregulation of NKCC, might also contribute. In photoreceptors the transient expression of KCC2 suggests a role for this transporter in development.     

Developmental dyslexia, learning and the cerebellum

Theoretical frameworks for dyslexia must explain how the well-established phonological deficits and the literacy deficits arise. Our longstanding research programme has led to a distinctive 'twin level' framework that proposes, first, that the core deficits are well described in terms of poor skill automaticity. Second, these 'cognitive level' symptoms are attributed to abnormal cerebellar function--a 'brain-level' analysis. The evidence includes data from behavioural, imaging, neuroanatomical and learning studies. The frame-work leads to an 'ontogenetic' analysis that links cerebellar deficit at birth, via problems in articulation and working memory, to the known phonological, speed and literacy difficulties. Differences in locus of cerebellar impairment, experience and/or links to other brain regions may account for subtypes of dyslexia and possibly other developmental disorders. The automaticity/ cerebellar deficit framework provides an explicit demonstration that it is possible to explain motor, speed and phonological deficits within a unified account, integrating previously opposed approaches.     

Age-related electrophysiological changes in rat cerebellum

The spontaneous discharge of cerebellar Purkinje neurons was studied in 3-, 12-, 15- and 20-month-old rats. Fluphenazine and haloperidol administered intraperitoneally produced a dose-dependent increase in the spontaneous discharge in 3-month-old animals, but were ineffective in 12-, 15- and 20-month-old rats. Intraperitoneal administration of amphetamine resulted in a dose-dependent decrease in spontaneous Purkinje neuron discharge in 3-month-old rats without affecting neurons from the older animals. Disruption of norepinephrine afferents by 6-OHDA or reserpine increased spontaneous discharge rate in 3-month-old animals. This treatment did not affect Purkinje neurons in 12-, 15- and 20-month-old rats. Local application of drug with the same multibarreled electrode revealed that neurons from older rats were significantly less sensitive to inhibition by norepinephrine than were Purkinje neurons from 3-month-old animals. Similar application of GABA did not reveal a differential sensitivity. For locus coeruleus activation to produce 50% inhibition in Purkinje neuron discharge, significantly higher stimulation currents were required in 15-month-old rats than in 3-month-old animals. Our results suggest a decreased postsynaptic sensitivity to norepinephrine in 12-, 15- and 20-month-old rats as compared to 3-month-old animals.     

Senescent microstructural changes in rat cerebellum

Senescent changes in Sprague-Dawley rat cerebellar microstructure have been quantified, focusing on the dominant element of cerebellar information processing, the Purkinje cell. In Golgi-Kopsch sections, many 26-month-old Purkinje cells appear defoliated, with small distal dendrites and spiny branchlets being most affected. The mean Purkinje cell area (soma plus dendrites) in computer-oriented sagittal sections is significantly decreased from 20,675 +/- 1,355 micron2/cell in 6-month-old rats to 17,088 +/- 1,107 micron2/cell in 26-month rats. These morphologic changes may be the hallmark of dying cells: in hematoxylin and eosin (H + E)-stained sections from the same rats we also observe a significant senescent decrease in Purkinje neuron density in every vermis lobule examined (lobules II-VII). Overall, the mean number of Purkinje cells/mm of Purkinje cell layer (measured in 10 micron thick sagittal sections) declines from 16.6 +/- 0.8 cells/mm in young rats to 12.5 +/- 0.2 cells/mm in old rats. As Purkinje cells are lost, so too are ethanolic phosphotungstic acid-stained (EPTA) synapses in the upper molecular layer of the cerebellar vermis (lobules VIII-X), and there is a highly significant within-subjects correlation between Purkinje cell density and synaptic density. Overall, synaptic density (in sagittal, 842 micron2 thin sections) decreases significantly from an average of 150,485 +/- 3,641 synapses/mm2 in 6-month rats to an average of 125,000 +/- 4,849 synapses/mm2 in 26-month rats. These changes are consistent with previous electrophysiologic and biochemical data showing age pathology of the cerebellum.     

Developmental up-regulation of KCC2 in the absence of GABAergic and glutamatergic transmission

Postsynaptic gamma-aminobutyric acid (GABA)A-mediated responses switch from depolarizing to hyperpolarizing during postnatal development of the rodent hippocampus. This is attributable to a decrease in the concentration of intracellular chloride set by the expression of the neuron-specific K+-Cl- co-transporter, KCC2. A recent in vitro study [Ganguly et al. (2001) Cell, 105, 521-532] showed that KCC2 expression may be under the trophic control of GABAA receptor-mediated transmission. Here we have studied the developmental expression of KCC2 protein in mouse hippocampal dissociated cultures as well as organotypic cultures. A low somatic expression level was found in neurons prior to the formation of the first synapses, as detected by synaptophysin immunoreactivity. Thereafter, KCC2 expression was strongly up-regulated during neuronal maturation. The developmental up-regulation of KCC2 expression was not altered by a chronic application (throughout the culturing period; 2-15 days in vitro) of the action-potential blocker TTX or the N-methyl-d-aspartate (NMDA) and non-NMDA antagonists APV and NBQX. Blockade of GABAA-mediated transmission with picrotoxin did not affect the expression levels of KCC2 protein either. These data show that neither neuronal spiking nor ionotropic glutamatergic and GABAergic transmission are required for the developmental expression of KCC2 in mouse hippocampal neurons in vitro.     

Developmental changes of synaptojanin expression in the human cerebrum and cerebellum.

Synaptojanin is a highly abundant polyphosphoinositide phosphatase in nerve terminals, and has been thought to play roles in clathrin-mediated synaptic vesicle endocytosis and signaling. In order to determine the broader role of synaptojanin in the central nervous system, we examined synaptojanin expression in the cerebrum and cerebellum from the fetal to the adult period by means of immunohistochemical and Western blot analyses. Immunohistochemistry consistently revealed the localization of synaptojanin in Cajal--Retzius cells, cortical plate neurons, subplate neurons, intermediate neurons, germinal matrix cells and the ventricular neuroepithelium of the fetal cerebrum. In the fetal cerebellum, synaptojanin immunoreactivity was localized in the external granular cell layer, Purkinje cell layer neuropil, cytoplasm of Purkinje cells and internal granular cells. The immunoreactivity in these structures was decreased around birth. After birth, the synaptojanin immunoreactivity of cortical neurons in the cerebrum, Purkinje cell layer neuropil, and internal granular cells and Purkinje cells in the cerebellum increased and reached a plateau after 11 years of age. These results were consistent with the intensity observed on Western blot analysis. These developmental changes of synaptojanin suggest a broader role in not only synaptic vesicle recycling, but also the regulation of neuronal migration and synaptogenesis in the fetal cerebrum and cerebellum.     

Developmental changes in distribution of death-associated protein kinase mRNAs

Death-associated protein kinase (DAP-kinase) is Ca(2+)/calmodulin-dependent serine/threonine kinase that contains ankyrin repeats and the death domain. It has been isolated as a positive mediator of interferon-gamma-induced apoptotic cell death of HeLa cells. In order to reveal the physiological role of DAP-kinase, the tissue distribution and developmental changes in mRNA expression of DAP-kinase were investigated by Northern blot and in situ hybridization analyses. DAP-kinase mRNA was predominantly expressed in brain and lung. In brain, DAP-kinase mRNA had already appeared at embryonic day 13 (E13) and was, thereafter, detected throughout the entire embryonic period. High levels of expression were detected in proliferative and postmitotic regions within cerebral cortex, hippocampus, and cerebellar Purkinje cells. These findings suggest that DAP-kinase may play an important role in neurogenesis where a physiological type of cell death takes place. The overall expression of DAP-kinase mRNA in the brain gradually declined at postnatal stages, and the expression became restricted to hippocampus, in which different expression patterns were observed among rostral, central, and caudal coronal sections, suggesting that DAP-kinase may be implicated in some neuronal functions. Furthermore, it was found that the expression of DAP-kinase mRNA was increased prior to a certain cell death induced by transient forebrain ischemia, indicating a possible relationship between DAP-kinase and neuronal cell death.