神经元GAD和GABA—T的活性与癫痫发病的关系

目的：研究探索神经元内谷氨酸脱羧酶（GAD）和γ－氨基丁酸转氨酶（GABA－T）的活性与癫痫发病的关系。方法：用致痫剂马桑内酯（CL）和抗痫剂苯妥因钠（PHT）处理大鼠培养海马神经元,用液体闪烁计数仪,紫外分光光度计检测培养神经元内GAD和GABA－T的活性。结果：与对照组比较,CL致痫组培养神经元内GAD活性和GABA－T的活性显著增高,运用抗痫剂PHT处理致痫神经元后,与CL致痫组比较,神经元内GABA－T活性显著降低,与对照组比较差异不显著。结论：在癫痫发病中,痫性神经元GAD和GABA－T活性增高。     

Increased GAD67 mRNA expression in cerebellar interneurons in autism: implications for Purkinje cell dysfunction

It has been widely reported that in autism, the number of Purkinje cells (PCs) is decreased, and recently, decreased expression of glutamic acid decarboxylase 67 (GAD67) mRNA in Purkinje cells also has been observed. However, the autism literature has not addressed key GABAergic inputs into Purkinje cells. Inhibitory basket and stellate cell interneurons in the molecular layer of the cerebellar cortex provide direct key GABAergic input into Purkinje cells and could potently influence the output of Purkinje cells to deep cerebellar nuclei. We investigated the capacity for interneuronal synthesis of gamma-amino butyric acid (GABA) in both types of interneurons that innervate the remaining PCs in the posterolateral cerebellar hemisphere in autism. The level of GAD67 mRNA, one of the isoforms of the key synthesizing enzymes for GABA, was quantified at the single-cell level using in situ hybridization in brains of autistic and aged-matched controls. The National Institutes of Health imaging system showed that expression of GAD67 mRNA in basket cells was significantly up-regulated, by 28%, in eight autistic brains compared with that in eight control brains (mean +/- SEM pixels per cell, 1.03 +/- 0.05 versus 0.69 +/- 0.05, respectively; P < 0.0001 by independent t test). Stellate cells showed a trend toward a small increase in GAD67 mRNA levels, but this did not reach significance. The results suggest that basket cells likely provide increased GABAergic feed-forward inhibition to PCs in autism, directly affecting PC output to target neurons in the dentate nucleus and potentially disrupting its modulatory role in key motor and/or cognitive behaviors in autistic individuals.     

Amygdaloid kindling of rats increases preprosomatostatin mRNA and somatostatin without affecting glutamic acid decarboxylase (GAD) mRNA or GAD

The levels of preprosomatostatin (preproSS) mRNA, somatostatin-like immunoactivity (SS-LI) (also known as somatotropin-release inhibitory factor, or SRIF), glutamic acid decarboxylase (GAD) activity and GAD mRNA were determined in several brain regions of amygdaloid-kindled rats. SS mRNA and SS increased in the cortex and striatum, while only SS increased in the hippocampus. No changes were detected in either GAD activity or GAD mRNA in any brain region. The data suggest that somatostatin may be one of the factors involved in the chain of events leading to kindled seizures.     

GAD67 and GAD65 mRNA and protein expression in cerebrocortical regions of elderly patients with schizophrenia

Gamma-Aminobutyric acid (GABA), the principal inhibitory neurotransmitter of CNS, has been consistently implicated in the pathophysiology of schizophrenia. GABA is synthesized from glutamate by the enzyme glutamic acid decarboxylase (GAD). Two isoforms of GAD have been identified and have been named GAD65 and GAD67 based on their apparent molecular weights. In this study, GAD65 and GAD67 mRNA and protein levels were measured by using real-time RT-PCR and immunoblotting, respectively, in post-mortem brain tissue from the dorsolateral prefrontal cortex (DLPFC) and the occipital cortex of the elderly persons with schizophrenia and matched normal controls. In addition, the mRNA expression of GAT-1, one of the principal transporters of GABA, was also studied in the same subjects. Expression of GAD65 and GAD67 mRNA in the DLPFC and in the occipital cortex was significantly elevated in patients with schizophrenia, whereas the expression of the corresponding proteins and GAT-1 mRNA was unchanged. Although the levels of GAD65 and GAD67 messages were increased in schizophrenia subjects, the proportion of the two GAD isoforms remained constant in controls and schizophrenics. In the human DLPFC, GAD65 mRNA was found to be expressed significantly less than the message for GAD67, approximately 16% of that observed for GAD67. On the contrary, the abundance of GAD65 protein in the DLPFC was about 350% of that observed for GAD67. The results suggest a substantial dysregulation of GAD mRNA expression in schizophrenia and, taken together with the results of protein expression studies, raise the possibility that both cortical and subcortical GABA function may be compromised in the disease.     

Intra-accumbens infusions of antisense oligodeoxynucleotides to one isoform of glutamic acid decarboxylase mRNA,GAD65, but not to GAD67 mRNA,impairs sustained attention performance in the rat

The effects of bilateral infusions of antisense oligodeoxynucleotides (ODNs) for the two isoforms of glutamic acid decarboxylase (GAD₆₅; GAD₆₇) into the nucleus accumbens on the performance of intact rats in a task designed to assess sustained attention were tested. The task required the animals to discriminate between signal and non-signal events. Signals and non-signals were presented randomly and unpredictably. The task generated all four response types of a sustained attention task, i.e., hits, misses, correct rejections, false alarms. Infusions of the scrambled sequence ODNs did not affect performance. Likewise, infusions of the GAD₆₇ ODNs failed to produce any effect. However, infusions of the GAD₆₅ ODNs into the nucleus accumbens resulted in a robust and reliable decrease in the relative number of hits. Similarly, the combined infusion of GAD₆₅₊₆₇ ODNs impaired the hit rate but did not affect the animals' ability to reject non-signals. Following each treatment series, performance rapidly returned to baseline, further indicating the specificity and reversibility of the effects of the infusions of the ODNs. While these data suggest that translation arrest of specifically the GAD₆₅ isoform of the enzyme in the nucleus accumbens impairs attentional performance, the neuronal mechanisms mediating these effects remain unsettled.     

Colocalization of tyrosine hydroxylase and GAD65 mRNA in mesostriatal neurons

Although dopamine has been considered as the only neurotransmitter in the nigrostriatal pathway, studies carried out in the last two decades have suggested the existence of a nondopaminergic nigrostriatal projection, and more recently, gamma-aminobutyric acid (GABA) has been identified as its neurotransmitter. In this study, we used the combination of immunocytochemistry for tyrosine hydroxylase (TH; a marker of dopaminergic neurons), in situ hybridization (ISH) for two different isoforms of glutamic acid decarboxylase (GAD65 and GAD67, the rate-limiting enzyme in GABA synthesis) and retrograde tracing techniques to investigate the possible existence of nigrostriatal neurons containing both neurotransmitters (dopamine and GABA) in the rat. Our results revealed that approximately 10% of mesostriatal dopaminergic neurons, most of them lying in the medial region of the substantia nigra pars compacta (SNC) and neighbouring A10 region, contain GAD65 mRNA. These findings reveal a third nigrostriatal pathway formed by dopaminergic/GABAergic neurons. Contrasting with the idea that in the basal ganglia, dopamine and GABA are released from different cell populations, the results suggest a more complex dopamine/GABA interaction than previously assumed, probably including cotransmission.     

褪黑素对癫痫状态大鼠海马GAD、GABA-T活性和GABA、Glu含量的影响

目的从神经递质方面研究褪黑素(melatonin,Mel)抗癫痫作用的生化机制。方法采用匹罗卡品(pilocarpine,PILO)诱发大鼠癫痫持续状态(status epilepticus,SE)模型,观察大鼠SE后4个时相点即6、48、72h和7 d海马γ-氨基丁酸、谷氨酸含量和谷氨酸脱羧酶、γ-氨基丁酸转氨酶活性的变化,以及Mel对其变化的影响。结果PILO诱导大鼠SE后6 h~7 d海马谷氨酸含量显著升高,γ-氨基丁酸含量和谷氨酸脱羧酶活性显著降低,与对照组比较差异有统计学意义(P<0.05),γ-氨基丁酸转氨酶活性在SE后72 h才显著下降(P<0.05);给予Mel的大鼠在SE后6 h~7 d,海马γ-氨基丁酸含量和谷氨酸脱羧酶活性均显著高于仅予PILO处理的大鼠(P<0.05),而谷氨酸含量和γ-氨基丁酸转氨酶活性与仅予PILO处理的大鼠比较差异无统计学意义(P>0.05)。结论Mel通过增强SE大鼠海马谷氨酸脱羧酶活性及γ-氨基丁酸合成发挥抗癫痫作用。     

Alterations in prodynorphin, proenkephalin, and GAD67 mRNA levels in the aged human putamen: correlation with Parkinson's disease

A real-time quantitative PCR approach was used to quantify mRNA levels corresponding to the neuropeptides enkephalin, dynorphin, and the 67-kDa isoform of glutamic acid decarboxylase (GAD67) in the human putamen from young and aged individuals as well as from aged patients affected by Parkinson's disease (PD). cDNA-specific primers were designed to amplify GAD67, proenkephalin (pENK), prodynorphin (pDYN), and the housekeeping genes glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and guanine nucleotide binding protein, beta-peptide 2-like I (GNB2LI). GAPDH and GNB2LI mRNA levels were similarly expressed among the groups and were therefore used as endogenous reference genes. Normalized data showed that mRNA levels for both pENK and pDYN were reduced in the putamen of aged controls and aged individuals affected by PD, compared with young controls. In addition, we showed that GAD67 mRNA levels did not change during aging and PD. Further analyses showed no differences in mRNA levels, for pENK, pDYN, or GAD67 mRNA, between PD patients and aged matched controls. These findings contrast with animal models of parkinsonism, for which expression of pDYN, pENK, and GAD67 mRNA has been reported to change after striatal dopamine denervation. Compensatory mechanisms and regional differences within the human putamen as well as the severity index of the disease, clinical diagnosis, and response to phalmacological therapy are possible reasons for these results. The present study suggests that alteration of neuropeptide pathways in the human putamen may be involved in the functional deterioration of parts of the extrapyramidal system during aging.     

Markers of mRNA stabilization and degradation, and RNAi within astrocytoma GW bodies

GW bodies (GWBs) are unique cytoplasmic structures that contain the mRNA binding protein GW182 and other proteins involved in mRNA processing pathways. The rationale for this study arose from clinical studies indicating that 33% of patients with GWB autoantibodies have a motor/sensory neuropathy and/or ataxia. The novelty of this study is the identification of GWBs in astrocytes and astrocytoma cells within cell bodies and cytoplasmic projections. Astrocytoma GWBs exhibit complex heterogeneity with combinations of LSm4 and XRN1 as well as Ago2 and Dicer, key proteins involved in mRNA degradation and RNA interference, respectively. GWB subsets contained the mRNA transport and stabilization proteins SYNCRIP, hnRNPA1, and FMRP, not previously described as part of the GWB complex. Immunoprecipitation of astrocytoma GWBs suggested that Dicer, hDcp, LSm4, XRN1, SYNCRIP, and FMRP form a multiprotein complex. GWBs are likely involved in a number of regulatory mRNA pathways in astrocytes and astrocytoma cells.     

Effects of dopaminergic cell degeneration on electrophysiological characteristics and GAD65/GAD67 expression in the substantia nigra: different action on GABA cell subpopulations.

The motor disturbances occurring in Parkinson's disease have been partially attributed to a hyperactivity of gamma-aminobutyric acid (GABA)-ergic nigral cells largely in the substantia nigra pars reticulata (SNr) secondary to the degeneration of dopaminergic nigrostriatal neurons. However, some aspects of this response remain unclear. In this work, different electrophysiological and neurochemical parameters were studied in GABAergic cells of the SN after unilateral nigrostriatal dopaminergic lesion using 6-hydroxydopamine injection in rats. Our data showed that 1) the SN under normal conditions contains different subsets of GABAergic cells according to their firing pattern and glutamic acid decarboxylase mRNA levels, and 2) the response of these GABAergic cell subgroups was different after the ipsi- and contralateral dopaminergic cell degeneration. These findings indicate a complex regulation of nigral GABAergic activity after nigrostriatal dopaminergic degeneration that probably involves local mechanisms, the nigro-striato-nigral loop, as well as interhemispheric mechanisms whose anatomical basis remains unstudied.     

GABAergic axon terminals at perisomatic and dendritic inhibitory sites show different immunoreactivities against two GAD isoforms,GAD67 and GAD65, in the mouse hippocampus: A digitized quantitative analysis

Glutamic acid decarboxylase (GAD), the γ-aminobutyric acid (GABA)-synthetic enzyme, consists of two isoforms, GAD67 and GAD65. Although distributions of the two GAD isoforms at the somatic level are known to be heterogeneous among different subpopulations of GABAergic neurons, those at the synaptic level have not been investigated. In order to analyze quantitatively the two GAD-isoform immunoreactivities in axon terminals, we combined confocal laser scanning microscopy with digitized image analysis to measure the gray levels of immunofluorescent signals for the two GAD isoforms in a large number of individual boutons in each hippocampal and dentate layer of the mouse. Synaptic boutons exhibited lamina-specific immunoreactivities against the GAD isoforms. Boutons in the principal cell layers (stratum pyramidale of the hippocampus proper and the granule cell layer of the dentate gyrus) showed more intense immunoreactivity against GAD67 than those in the dendritic layers (strata lacunosum-moleculare, radiatum, and oriens of the hippocampus proper and the molecular layer of the dentate gyrus). By contrast, boutons in the dendritic layers showed more intense immunoreactivity against GAD65 than those in the principal cell layers. Such differential distributions could be correlated to the GAD-isoform immunoreactivities in the axon terminals originating from parvalbumin-containing neurons, a particular subpopulation of hippocampal GABAergic neurons mainly innervating the perisomatic domain of principal neurons. In addition to previously reported physiological and pharmacological differences between the GABAergic synapses on perisomatic domain and those on distal dendrites, the present results suggest a functional differentiation of GABAergic synapses between these two inhibitory sites. J. Comp. Neurol. 395:177–194, 1998. © 1998 Wiley-Liss, Inc.     

Decreased BDNF, trkB-TK+ and GAD67 mRNA expression in the hippocampus of individuals with schizophrenia and mood disorders

Background

Brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor (trkB-TK+) and glutamic acid decarboxylase (GAD₆₇) mRNA levels have previously been found to be reduced in the prefrontal cortex of patients with schizophrenia. To determine whether this reduction extends to other brain regions, we measured the expression levels of BDNF, trkB-TK+ and GAD₆₇ mRNA in regions of the hippocampus, including the dentate gyrus (DG), cornu ammonis subfields (CA1–4), subiculum and entorhinal cortex (EC) of individuals with schizophrenia, bipolar disorder, major depression and unaffected controls.

Methods

In situ hybridization was performed on postmortem brain tissue obtained from the Stanley Foundation Consortium and analyzed using film-based quantification.

Results

Analyses of covariance comparing the expression of mRNA among all groups revealed a significant decrease in BDNF mRNA in CA4 in the bipolar disorder group compared with controls (33%). We found trkB-TK+ mRNA levels to be significantly reduced in CA4 in the schizophrenia group (36%) and in layer II of the EC in the bipolar disorder and major depression groups (28%, 21%, respectively) compared with controls. In addition, GAD₆₇ mRNA levels were reduced in patients with schizophrenia in both the DG (23%) and CA4 (60%) compared with controls. Individuals with major depression also expressed significantly less GAD₆₇ mRNA (44%) compared with controls in CA4 of the hippocampus.

Limitations

It is necessary to account for factors that influence the molecular preservation in postmortem brain tissue, including pH, postmortem interval and tissue storage time. Moreover, there are limitations to the sensitivity of the film-based method of quantification.

Conclusion

Our findings show abnormal BDNF, trkB-TK+ and GAD₆₇ mRNA expression in the hippocampus of individuals with schizophrenia and mood disorders, indicating that fundamental properties of hippocampal signalling transmission, plasticity and circuitry may be affected in individuals with these major mental illnesses.     

Variations of Synaptotagmin I, Synaptotagmin IV, and Synaptophysin mRNA Levels in Rat Hippocampus during the Estrous Cycle

Periodic changes in ovarian steroid levels during fertility cycles affect learning both in humans and in rats in parallel with electrophysiological and morphological fluctuations in selective neuronal populations. In particular, during the estrous cycle of the female rat, hippocampal CA1 region undergoes cyclic modifications in synaptic density. To investigate the molecular mechanisms involved in synaptic remodeling during the estrous cycle, we analyzed the expression of three presynaptic markers, synaptotagmin I, synaptotagmin IV, and synaptophysin, in the female adult rat brain by in situ hybridization. Relative abundance in mRNA for these three markers was quantified at four phases of the estrous cycle: diestrus, proestrus (AM and PM), and estrus. mRNA levels for syt1 exhibited cyclic variations in pyramidal neurons of the CA3 region of hippocampus during the estrous cycle, while mRNA levels for syt4 and SYN were relatively invariant in this or other regions of the hippocampus. Because CA3 pyramidal neurons make synaptic contacts in CA1, modulation of syt1 expression in CA3 may participate in the changes in synaptic density observed in CA1 during the estrous cycle. Furthermore, both syt1 and SYN mRNA varied cyclically in layer II, but not in layer III of entorhinal cortex, while syt4 remained unchanged throughout the cycle. These data suggest that regular variations in steroid hormone levels during fertility cycles may alter the properties of several networks involved in information processing and learning and memory through altered levels of presynaptic proteins.