GAD67 Expression Research Articles

Relation between increased anxiety and reduced expression of alpha1 and alpha2 subunits of GABA A receptors in Wfs1-deficient mice

Mutations in the coding region of the WFS1 gene cause Wolfram syndrome, a rare multisystem neurodegenerative disorder of autosomal recessive inheritance. In clinical studies a relation between mutations in the Wfs1 gene and increased susceptibility for mood disorders has been established. According to our previous studies, mice lacking Wfs1 gene displayed increased anxiety in stressful environment. As the GABA-ergic system plays a significant role in the regulation of anxiety, we analyzed the expression of GABA-related genes in the forebrain structures of wild-type and Wfs1-deficient mice. Experimentally naïve Wfs1-deficient animals displayed a significant down-regulation of α1 (Gabra1) and α2 (Gabra2) subunits of GABA A receptors in the temporal lobe and frontal cortex. Exposure of wild-type mice to the elevated plus-maze decreased levels of Gabra1 and Gabra2 genes in the temporal lobe. A similar tendency was also established in the frontal cortex of wild-type animals exposed to behavioral test. In Wfs1-deficient mice the elevated plus-maze exposure did not induce further changes in the expression of Gabra1 and Gabra2 genes. By contrast, the expression of Gad1 and Gad2 genes, enzymes responsible for the synthesis of GABA, was not significantly affected by the exposure of mice to the elevated plus-maze or by the invalidation of Wfs1 gene. Altogether, the present study demonstrates that increased anxiety of Wfs1-deficient mice is probably linked to reduced expression of Gabra1 and Gabra2 genes in the frontal cortex and temporal lobe.

α1‐Adrenergic Receptors Regulate Neurogenesis and Differentiation of Interneurons, Dopaminergic, and/or Noradrenergic Neurons

Understanding the function of α1‐adrenergic receptors (α1‐ARs) in the brain has been limited due to a lack of specific ligands and antibodies. We approached this problem by generating transgenic mice engineered to over‐express either wild‐type receptor with enhanced green fluorescent protein tags or constitutively active mutant (CAM) α1‐ARs using isogenic promoters. We identified intriguing α1A‐AR expressing cells with a migratory morphology in the subventricular zone (SVZ) that co‐expressed markers of neural stem cells/progenitors. There was increased BrdU incorporation in vivo in the SVZ in CAM α1A mice while BrdU incorporation decreased in the migratory stream. Normal neonatal neurospheres expressed a mixture of α1A and α1B‐AR subtypes and α1‐AR stimulation resulted in increased neuronal precursors that increased the expression of tyrosine hydroxylase, dopamine‐β‐hydroxylase, and GAD 65/67. CAM‐derived neurospheres had a reduced ability to self‐renew, proliferate, and were composed of a greater number of neuronal, but reduced astrocytic precursors. CAM neurospheres also differentially increased the RNA of proneural transcription factors. Our results indicate that α1‐ARs can regulate neurogenesis and neuronal differentiation and may offer new therapies to treat neurodegenerative diseases. This work was supported by NIHRO1HL61438 (DMP) and NSF CAREER award 0347259 (VAD).

Dr. Perrone-Bizzozero and W. Michael Bullock Reply

Back to table of contents Previous article Next article Letters to the EditorFull AccessDr. Perrone-Bizzozero and W. Michael Bullock ReplyNORA PERRONE-BIZZOZERO Ph.D.W. MICHAEL BULLOCK,NORA PERRONE-BIZZOZERO Ph.D.Search for more papers by this authorW. MICHAEL BULLOCKSearch for more papers by this author,Published Online:1 Apr 2009https://doi.org/10.1176/appi.ajp.2009.09010011rAboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail To the Editor: We fully agree with Dr. Peedicayil that epigenetic mechanisms play an important role in the control of gene expression and that defects in these processes are associated with an increasing number of mental disorders. Moreover, recent reports suggest that reduced expression of GAD 67 (the GABA synthesizing enzyme encoded by the GAD1 gene) in schizophrenia patients is associated with both specific polymorphisms in the promoter region (1) and altered epigenetic regulation (2 , 3) , and we apologize for the oversight of not citing the latter in our article. However, we wish to clarify that it was not our intention to propose a mechanism for the decreases in GAD 67 in schizophrenia but to put these changes in the context of neuronal circuitry (4) . Specifically, our goal was to investigate gene expression alterations as they relate to a dysfunction in GABAergic and glutamatergic transmission in the cerebellum of individuals with schizophrenia. Our results strongly suggest that Golgi cells, which are the GABAergic interneurons that inhibit granule cell activity, are affected in schizophrenia. These results not only explain our previous observation of increased activity-dependent granule cell gene expression in schizophrenia (5) but also give further support to the hypothesis that some subtypes of GABAergic interneurons may be compromised in this illness. Along these lines, we recently found that Golgi cells are primarily affected by chronic intermittent exposure to relatively low doses of phencyclidine (data available upon request from W.M. Bullock et al.), suggesting that N -methyl- d -aspartic acid hypofunction could lead to decreases in GABA synthesis and neurotransmission in schizophrenia. Moreover, we feel that it is very important to determine the causes of the decrease in GAD 67 mRNA in postmortem cerebellar tissue, and we plan to address whether these deficits are associated with genetic factors and/or epigenetic dysregulation in future studies. Albuquerque, N.M.The authors’ disclosures accompany the original article.This letter (doi: 10.1176/appi.ajp.2009.09010011r) was accepted for publication in February 2009.

Homing of GAD65 specific autoimmunity and development of insulitis requires expression of both DQ8 and human GAD65 in transgenic mice

MHC-class II genes determine susceptibility in human type-1 diabetes. In their context, presentation of target antigen(s) results in autoimmunity and β-cell destruction. An animal model, in which human β-cell autoantigen(s) are presented to effector cells in the context of human MHC-class II diabetes-susceptibility genes, would be desirable for studying molecular mechanisms of disease and developing antigen-specific immune-interventions. We report the development of antigen-specific insulitis in double-transgenic mice carrying the HLA-DQ8 diabetes-susceptibility haplotype and expressing the human autoantigen GAD65 in pancreatic β-cells. Immunization with human GAD65 cDNA resulted in severe insulitis and low antibody levels in double-transgenic mice while control mice were mostly insulitis free. CFA/protein immunization resulted in high antibody levels and modest insulitis. Pancreatic lymphocytic infiltration progressed through stages (exocrine pancreas followed by peri- and intra-insulitis). Adoptive transfer of splenocytes from DNA-immunized mice resulted in development of insulitis in recipient transgenics. Our results show that immunization with a clinically relevant, type-1 diabetes human autoantigen, in a humanized genetic setting, results in the development of an immune response that homes to islets of Langerhans. This animal model will facilitate studies of autoimmunity to GAD65 in the context of HLA-DQ8, and development of methods to induce tolerance and prevent insulitis.

LMO4 Controls the Balance between Excitatory and Inhibitory Spinal V2 Interneurons

Multiple excitatory and inhibitory interneurons form the motor circuit with motor neurons in the ventral spinal cord. Notch signaling initiates the diversification of immature V2-interneurons into excitatory V2a-interneurons and inhibitory V2b-interneurons. Here, we provide a transcriptional regulatory mechanism underlying their balanced production. LIM-only protein LMO4 controls this binary cell fate choice by regulating the activity of V2a- and V2b-specific LIM complexes inversely. In the spinal cord, LMO4 induces GABAergic V2b-interneurons in collaboration with SCL and inhibits Lhx3 from generating glutamatergic V2a-interneuons. In LMO4;SCL compound mutant embryos, V2a-interneurons increase markedly at the expense of V2b-interneurons. We further demonstrate that LMO4 nucleates the assembly of a novel LIM-complex containing SCL, Gata2, and NLI. This complex activates specific enhancers in V2b-genes consisting of binding sites for SCL and Gata2, thereby promoting V2b-interneuron fate. Thus, LMO4 plays essential roles in directing a balanced generation of inhibitory and excitatory neurons in the ventral spinal cord.

Adult and Embryonic GAD Transcripts Are Spatiotemporally Regulated during Postnatal Development in the Rat Brain

BackgroundGABA (gamma-aminobutyric acid), the main inhibitory neurotransmitter in the brain, is synthesized by glutamic acid decarboxylase (GAD). GAD exists in two adult isoforms, GAD65 and GAD67. During embryonic brain development at least two additional transcripts exist, I-80 and I-86, which are distinguished by insertions of 80 or 86 bp into GAD67 mRNA, respectively. Though it was described that embryonic GAD67 transcripts are not detectable during adulthood there are evidences suggesting re-expression under certain pathological conditions in the adult brain. In the present study we systematically analyzed for the first time the spatiotemporal distribution of different GADs with emphasis on embryonic GAD67 mRNAs in the postnatal brain using highly sensitive methods.Methodology/Principal FindingsQPCR was used to precisely investigate the postnatal expression level of GAD related mRNAs in cortex, hippocampus, cerebellum, and olfactory bulb of rats from P1 throughout adulthood. Within the first three postnatal weeks the expression of both GAD65 and GAD67 mRNAs reached adult levels in hippocampus, cortex, and cerebellum. The olfactory bulb showed by far the highest expression of GAD65 as well as GAD67 transcripts. Embryonic GAD67 splice variants were still detectable at birth. They continuously declined to barely detectable levels during postnatal development in all investigated regions with exception of a comparatively high expression in the olfactory bulb. Radioactive in situ hybridizations confirmed the occurrence of embryonic GAD67 transcripts in the olfactory bulb and furthermore detected their localization mainly in the subventricular zone and the rostral migratory stream.Conclusions/SignificanceEmbryonic GAD67 transcripts can hardly be detected in the adult brain, except for specific regions associated with neurogenesis and high synaptic plasticity. Therefore a functional role in processes like proliferation, migration or synaptogenesis is suggested.

Non-overlapping expression of GAD forms in the developing mouse GnRH-1 system

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Adenovector GAD65 Gene Delivery into the Rat Trigeminal Ganglion Produces Orofacial Analgesia

BackgroundOur goal is to use gene therapy to alleviate pain by targeting glial cells. In an animal model of facial pain we tested the effect of transfecting the glutamic acid decarboxylase (GAD) gene into satellite glial cells (SGCs) of the trigeminal ganglion by using a serotype 5 adenovector with high tropisms for glial cells. We postulated that GABA produced from the expression of GAD would reduce pain behavior by acting on GABA receptors on neurons within the ganglion.ResultsInjection of adenoviral vectors (AdGAD65) directly into the trigeminal ganglion leads to sustained expression of the GAD65 isoform over the 4 weeks observation period. Immunohistochemical analysis showed that adenovirus-mediated GAD65 expression and GABA synthesis were mainly in SGCs. GABAA and GABAB receptors were both seen in sensory neurons, yet only GABAA receptors decorated the neuronal surface. GABA receptors were not found on SGCs. Six days after injection of AdGAD65 into the trigeminal ganglion, there was a statistically significant decrease of pain behavior in the orofacial formalin test, a model of inflammatory pain. Rats injected with control virus (AdGFP or AdLacZ) had no reduction in their pain behavior. AdGAD65-dependent analgesia was blocked by bicuculline, a selective GABAA receptor antagonist, but not by CGP46381, a selective GABAB receptor antagonist.ConclusionTransfection of glial cells in the trigeminal ganglion with the GAD gene blocks pain behavior by acting on GABAA receptors on neuronal perikarya.

The Reelin and GAD67 Promoters Are Activated by Epigenetic Drugs That Facilitate the Disruption of Local Repressor Complexes

The epigenetic down-regulation of genes is emerging as a possible underlying mechanism of the GABAergic neuron dysfunction in schizophrenia. For example, evidence has been presented to show that the promoters associated with reelin and GAD67 are down-regulated as a consequence of DNA methyltransferase (DNMT)-mediated hypermethylation. Using neuronal progenitor cells to study this regulation, we have previously demonstrated that DNMT inhibitors coordinately increase reelin and GAD67 mRNAs. Here, we report that another group of epigenetic drugs, histone deacetylase (HDAC) inhibitors, activate these two genes with dose and time dependence comparable with that of DNMT inhibitors. In parallel, both groups of drugs decrease DNMT1, DNMT3A, and DNMT3B protein levels and reduce DNMT enzyme activity. Furthermore, induction of the reelin and GAD67 mRNAs is accompanied by the dissociation of repressor complexes containing all three DNMTs, MeCP2, and HDAC1 from the corresponding promoters and by increased local histone acetylation. Our data imply that drug-induced promoter demethylation is relevant for maximal activation of reelin and GAD67 transcription. The results suggest that HDAC and DNMT inhibitors activate reelin and GAD67 expression through similar mechanisms. Both classes of drugs attenuate, directly or indirectly, the enzymatic and transcriptional repressor activities of DNMTs and HDACs. These data provide a mechanistic rationale for the use of epigenetic drugs, individually or in combination, as a potential novel therapeutic strategy to alleviate deficits associated with schizophrenia.

Differential alterations in the relations among GABAergic, catecholaminergic and calcium binding protein expression in the olfactory bulb of amphetamine-administered mouse

This study explores cellular responses of distinct layers of the main olfactory bulb (OB) to amphetamine (Amph), by examining the expression of glutamic acid decarboxylase 67 (GAD 67), calcium binding proteins (CaBP) and tyrosine hydroxylase (TH). Immunocytochemical analysis was performed on OB sections prepared from adult mice at 0.5 h or 4 h after receiving one intraperitoneal injection or multiple (2 doses/day, 7 doses in total) injections of saline or Amph, 5 mg/kg. In the glomerular layer, though the expression of TH and GAD 67 was unaltered by the single Amph injection, at 0.5 h post-repeated Amph exposure the levels of TH-immunopositive somata and processes/punctates, and GAD 67-somata/punctates were increased by 48–147%, compared with respective saline controls. By contrast, at 4 h post-repeated Amph GAD 67 levels were lower than saline, and TH similar to saline. For the repetitively saline-injected groups, TH and GAD 67 levels were higher at 4 h than 0.5 h, suggesting an injection-associated stress response. Double staining revealed that at 0.5 h post-repeated Amph exposure, the percentage of TH-soma number that expressed GAD 67 was raised to 46%, compared with 30% of the corresponding saline, and thus implies an activation of dopaminergic neurons to become GABAergic. In the external plexiform layer, the numbers of CaBP, parvalbumin or calretinin-somata were increased at 0.5 h/4 h or 4 h post-acute Amph injection; double staining disclosed that at 4 h post-acute Amph, 66% or 47% of GAD 67-somata contained parvalbumin or calretinin, being greater than 43% or 28% of the saline. In the granule somata, Amph probably inhibits expression of GAD 67 by decreasing phosphorylation of CREB (pCREB). The up-regulation of CaBPs, GAD 67 and TH at 0.5/4 h post-acute or 0.5 h post-repeated Amph could implicate protective roles and synaptic plasticity against Amph, whereas decreases of GAD 67 and pCREB at 4 h post-repeated Amph may indicate toxicity of Amph.

Uncoupling of neurodegeneration and gliosis in a murine model of juvenile cortical lesion

A small experimental cryolesion to the right parietal cortex of juvenile mice causes late-onset global brain atrophy with memory impairments, reminiscent of cognitive decline, and progressive brain matter loss in schizophrenia. However, the cellular events underlying this global neurodegeneration are not understood. Here we show, based on comprehensive stereological analysis, that early unilateral lesion causes immediate and lasting bilateral increase in the number of microglia in cingulate cortex and hippocampus, consistent with a chronic low-grade inflammatory process. Whereas the total number of neurons and astrocytes in these brain regions remain unaltered, pointing to a non- gliotic neurodegeneration (as seen in schizophrenia), the subgroup of parvalbumin-positive inhibitory GABAergic interneurons is increased bilaterally in the hippocampus, as is the expression of the GABA-synthesizing enzyme GAD67. Moreover, unilateral parietal lesion causes a decrease in the expression of synapsin1, suggesting impairment of presynaptic functions/neuroplasticity. Reduced expression of the myelin protein cyclic nucleotide phosphodiesterase, reflecting a reduction of oligodendrocytes, may further contribute to the observed brain atrophy. Remarkably, early intervention with recombinant human erythropoietin (EPO), a hematopoietic growth factor with multifaceted neuroprotective properties (intraperitoneal injection of 5000 IU/kg body weight every other day for 3 weeks), prevented all these neurodegenerative changes. To conclude, unilateral parietal lesion of juvenile mice induces a non- gliotic neurodegenerative process, susceptible to early EPO treatment. Although the detailed mechanisms remain to be defined, these profound EPO effects open new ways for prophylaxis and therapy of neuropsychiatric diseases, e.g. schizophrenia.

Neurobiology of stress-induced reproductive dysfunction in female macaques.

It is now well accepted that stress can precipitate mental and physical illness. However, it is becoming clear that given the same stress, some individuals are very vulnerable and will succumb to illness while others are more resilient and cope effectively, rather than becoming ill. This difference between individuals is called stress sensitivity. Stress sensitivity of an individual appears to be influenced by genetically inherited factors, early life (even prenatal) stress, and by the presence or absence of factors that provide protection from stress. In comparison to other stress-related diseases, the concept of sensitivity versus resilience to stress-induced reproductive dysfunction has received relatively little attention. The studies presented herein were undertaken to begin to identify stable characteristics and the neural underpinnings of individuals with sensitivity to stress-induced reproductive dysfunction. Female cynomolgus macaques with normal menstrual cycles either stop ovulating (stress sensitive) or to continue to ovulate (stress resilient) upon exposure to a combined metabolic and psychosocial stress. However, even in the absence of stress, the stress-sensitive animals have lower secretion of the ovarian steroids, estrogen and progesterone, have higher heart rates, have lower serotonin function, have fewer serotonin neurons and lower expression of pivotal serotonin-related genes, have lower expression of 5HT2A and 2C genes in the hypothalamus, have higher gene expression of GAD67 and CRH in the hypothalamus, and have reduced gonadotropin-releasing hormone transport to the anterior pituitary. Altogether, the results suggest that the neurobiology of reproductive circuits in stress-sensitive individuals is compromised. We speculate that with the application of stress, the dysfunction of these neural systems becomes exacerbated and reproductive function ceases.

Altered pharmacology and GABA-A receptor subunit expression in dorsal midline thalamic neurons in limbic epilepsy

The mediodorsal (MD) and paraventricular (PV) thalamic nuclei play a significant role in limbic epilepsy, and previous reports have shown changes in GABA-A receptor (GABAAR) mediated synaptic function. In this study, we examined changes in the pharmacology of GABAergic drugs and the expression of the GABAAR subunits in the MD and PV neurons in epilepsy. We observed nucleus specific changes in the sensitivity of sIPSCs to zolpidem and phenobarbital in MD and PV neurons from epileptic animals. In contrast, the magnitude of change in electrically evoked response (eIPSC) to zolpidem and phenobarbital were uniformly diminished in both MD and PV neurons in epilepsy. Immunohistochemical studies revealed that in epilepsy, there was a reduction in GAD65 expression and NeuN positive neurons in the MD neurons. Also, there was a decrease in immunoreactivity of the α1 and β2/3 subunit of GABAARs, but not the γ2 of the GABAAR in both MD and PV in epilepsy. These findings demonstrate significant alterations in the pharmacology of GABA and GABAARs in a key region for seizure generation, which may have implications for the physiology and pharmacology of limbic epilepsy.

Stimulation by cochlear implant in unilaterally deaf rats reverses the decrease of inhibitory transmission in the inferior colliculus

In the last decade, numerous studies have investigated synaptic transmission changes in various auditory nuclei after unilateral cochlear injury. However, few data are available concerning the potential effect of electrical stimulation of the deafferented auditory nerve on the inhibitory neurotransmission in these nuclei. We report here for the first time the effect of chronic electrical stimulation of the deafferented auditory nerve on alpha1 subunit of the glycinergic receptor (GlyRalpha1) and glutamic acid decarboxylase (GAD)67 expression in the central nucleus of inferior colliculus (CIC). Adult rats were unilaterally cochleectomized by intracochlear neomycin sulphate injection. Fifteen days later, the ipsilateral auditory nerve was chronically stimulated either 4, 8 or 22 h daily, for 5 days using intracochlear bipolar electrodes. GlyRalpha1 and GAD67 mRNA and protein were quantified in the CIC using in situ hybridization and immunohistofluorescence methods. Our data showed that as after surgical ablation, GlyRalpha1 and GAD67 expression were strongly decreased in the contralateral CIC after unilateral chemical cochleectomy. Most importantly, these postlesional down-modulations were significantly reversed by chronic electrical stimulation of the deafferented auditory nerve. This recovery, however, did not persist for more than 5 days after the cessation of the deafferented auditory nerve electrical stimulation. Thus, downregulations of GlyRalpha1 and GAD67 may be involved both in the increased excitability observed in the CIC after unilateral deafness and consequently in the tinnitus frequently observed in unilateral adult deaf patients. Electrical stimulation of the deafferented auditory nerve in patients may be a potential new approach for treating tinnitus with unilateral hearing loss.

SDF1α/CXCR4 Signaling, via ERKs and the Transcription Factor Egr1, Induces Expression of a 67-kDa Form of Glutamic Acid Decarboxylase in Embryonic Hippocampal Neurons

Stromal cell-derived factor alpha (SDF1alpha) and its cognate receptor CXCR4 play an important role in neuronal development in the hippocampus, but the genes directly regulated by SDF1alpha/CXCR4 signaling are unknown. To study the role of CXCR4 targeted genes in neuronal development, we used neuronal cultures established from embryonic day 18 rats. Hippocampal neurons express CXCR4 receptor proteins and are stimulated by SDF1alpha resulting in activation of extracellular signal-regulated kinase (ERK)1/2 and the transcription factor cAMP-response element-binding protein. SDF1alpha rapidly induces the expression of the early growth response gene Egr1, a transcription factor involved in activity-dependent neuronal responses, in a concentration-dependent manner. Gel-shift analysis showed that SDF1alpha enhances DNA binding activity to the Egr1-containing promoter for GAD67. Chromatin immunoprecipitation analysis using an Egr1 antibody indicated that SDF1alpha stimulation increases binding of Egr1 to a GAD67 promoter DNA sequence. SDF1alpha stimulation increases the expression of GAD67 at both the mRNA and protein levels, and increases the amount and neurite localization of gamma-aminobutyric acid (GABA) in neurons already expressing GABA. SDF1alpha-induced Egr1/GAD67 expression is mediated by the G protein-coupled CXCR4 receptor and activation of the ERK pathway. Reduction of Egr1 gene expression using small interfering RNA technology lowers the level of GAD67 transcripts and inhibits SDF1alpha-induced GABA production. Inhibition of CXCR4 activation in the developing mouse brain in utero greatly reduced Egr1 and GAD67 mRNA levels and GAD67 protein levels, suggesting a pivotal role for CXCR4 signaling in the development of GABAergic neurons in vivo. Our data suggest that SDF1alpha/CXCR4/G protein/ERK signaling induces the expression of the GAD67 system via Egr1 activation, a mechanism that may promote the maturation of GABAergic neurons during development.

Spectrum of neurological syndromes associated with glutamic acid decarboxylase antibodies: diagnostic clues for this association

The association of high levels of autoantibodies to glutamic acid decarboxylase (GAD-ab) and stiff-person syndrome (SPS) is well known. However, the full spectrum of neurological syndromes associated with GAD-ab is not well established. In addition, these patients usually present type 1 diabetes mellitus (DM1) that could justify the presence of high GAD-ab levels. To clarify these issues, we reviewed the clinical and immunological features of patients in whom high GAD-ab levels were detected in a reference centre for DM1 and for the detection of antineuronal antibodies in suspected paraneoplastic neurological syndromes (PNS). High GAD-ab levels were defined as values > or =2000 U/ml by radioimmunoassay. Intrathecal synthesis (IS) of GAD-ab was calculated in paired serum/CSF samples. Values higher than the IgG index were considered indicators for positive GAD-ab-specific IS. High GAD-ab levels were identified in 61 patients, 22 (36%) had SPS, 17 (28%) cerebellar ataxia, 11 (18%) other neurological disorders (epilepsy -- four, PNS -- four; idiopathic limbic encephalitis -- two; myasthenia gravis -- one), and 11 (18%) isolated DM1. Patients with SPS and cerebellar ataxia had the same frequency of female gender (86% vs 94%), DM1 (59% vs 53%), CSF oligoclonal bands (35% vs 69%). Three of the four PNS patients, with paraneoplastic encephalomyelitis, a predominant gait cerebellar ataxia, and limbic encephalitis, had neuroendocrine carcinomas. GAD expression was confirmed in the two tumours in which the study was done. The fourth patient presented with paraneoplastic cerebellar degeneration antedating a lung adenocarcinoma. The frequency of increased IS of GAD-ab was 85% in SPS, 100% in cerebellar ataxia, and 86% in other neurological disorders. In conclusion, our study emphasizes that high GAD-ab levels associate with other neurological disorders besides SPS. Cerebellar ataxia, the second most common syndrome associated with high GAD-ab levels, shares with SPS the same demographic, clinical and immunological features. The demonstration of an increased IS of GAD-ab is important to confirm that the GAD autoimmunity is related to the neurological syndrome particularly when there is a concomitant DM1 that could justify the presence of high GAD-ab levels. Lastly, in patients who develop neurological syndromes that suggest a PNS, the finding of GAD-ab does not rule out this possibility and appropriate studies should be done to confirm an underlying cancer.

Dopamine acting through D2 receptors modulates the expression of PSA-NCAM, a molecule related to neuronal structural plasticity, in the medial prefrontal cortex of adult rats

A “neuroplastic” hypothesis proposes that changes in neuronal structural plasticity may underlie the aetiology of depression and the action of antidepressants. The medial prefrontal cortex (mPFC) is affected by this disorder and shows an intense expression of the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), a plasticity-associated molecule, which is expressed mainly in interneurons. The monoamines serotonin, dopamine and noradrenaline are the principal targets of antidepressant action. Pharmacological manipulation of serotonin levels regulates synaptophysin and PSA-NCAM expression in the adult mPFC. However, the involvement of structural plasticity on the antidepressant effects of dopamine has not been well explored yet. Using immunohistochemistry, we have studied the relationship between dopaminergic fibers and PSA-NCAM expressing neurons in the mPFC and the expression of D2 receptors. In order to evaluate the effects of dopamine in neuronal structural plasticity and on inhibitory neurotransmission, we have analyzed the expression of synaptophysin, PSA-NCAM and GAD67 in the mPFC after cortical dopamine depletion with 6-OHDA and after chronic treatments with the D2 receptor antagonist haloperidol or the D2 receptor agonist PPHT. Many dopaminergic fibers were observed in close apposition to PSA-NCAM expressing neurons and 76% of these cells co-expressed D2 receptor. Both haloperidol treatment and 6-OHDA injection reduced significantly PSA-NCAM, synaptophysin and GAD67 expression in the mPFC. Conversely, PPHT treatment increased the expression of these molecules. Our results give support to the “neuroplastic” hypothesis of depression, suggesting that dopamine acting on D2 receptors may modulate neuronal structural plasticity and inhibitory neurotransmission through changes in PSA-NCAM expression.

The effect of serotonin on GABA synthesis in cultured rat spinal dorsal horn neurons

The spinal dorsal horn (SDH) is the first step in the integration of primary nociceptive information, which is controlled by the descending serotonin (5-HT) system as well as the principal inhibitory neurotransmitter γ-aminobutyric acid (GABA). However, the influence exerted by 5-HT on GABA synthesis remains poorly understood. The major pathway for GABA synthesis is the enzymatic decarboxylation of glutamate by glutamic acid decarboxylase (GAD) 65 and 67. In the present research, western blotting results show a time- and dose-dependent enhancement of GAD65 and GAD67 expression induced by 5-HT treatment and a concentration of 100 nM 5-HT applied for 3 days is shown to be the optimal condition for maximal expression of GAD67 and a significant expression of GAD65. Under the stimulation of such 5-HT application the phosphorylation of Akt and p42/p44 mitogen-activated protein (MAP) kinase is activated and specifically blocked by inhibitors of phosphatidylinositol 3-kinase (PI3-K) (LY294002) or the p42/p44 MAP kinase (PD98059 and U0126) pathways. Moreover, LY294002, or PD98059, or U0126 partially inhibit 5-HT-stimulated increases in GAD67 or GAD65 expression. Further, 5-HT application has no effect on the number of GAD65/GAD67-immunopositive neuronal cells; but it can induce an increase in the total area, process length and number of primary neurites of GAD65/67-positive neurons, an increase that appears to involve LY294002 and PD98059. The results of this study provide an in vitro model of the regulation of 5-HT on synthesis of GABA in the SDH that is putatively thought to occur in vivo as a result of excitatory neural activity.

Adenovirus-mediated Expression of GAD65 into the Trigeminal Ganglion Produces Long-lasting Analgesia

Adenovirus-mediated Expression of GAD65 into the Trigeminal Ganglion Produces Long-lasting Analgesia

Benefits and risks of intranigral transplantation of GABA-producing cells subsequent to the establishment of kindling-induced seizures

Neural transplantation has been investigated experimentally and clinically for the purpose of developing new treatment options for intractable epilepsy. In the present study we assessed the anticonvulsant efficacy and safety of bilateral allotransplantation of genetically engineered striatal GABAergic rat cell lines into the substantia nigra pars reticulata (SNr). Rats with previously-established seizures, induced by amygdala kindling, were used as a model of temporal lobe epilepsy. Three cell lines were transplanted: (1) immortalized GABAergic cells (M213-2O) derived from embryonic rat striatum; (2) M213-2O cells (CL4) transfected with human GAD67 cDNA to obtain higher GABA synthesis than the parent cell line; and (3) control cells (121-1I), also derived from embryonic rat striatum, but which did not show GAD expression. A second control group received injections of medium alone. Transplantation of M213-2O cells into the SNr of kindled rats resulted in significant but transient anticonvulsant effects. Neither control cells nor medium induced anticonvulsant effects. Strong tissue reactions were, however, induced in the host brain of kindled but not of non-kindled rats, and only in animals that received grafts of genetically modified CL4 cells. These tissue reactions included graft rejection, massive infiltration of inflammatory immune cells, and gliosis. The anticonvulsant effect of M213-2O cells emphasizes the feasibility of local manipulations of seizures by intranigral transplantation of GABA-producing cells. On the other hand, the present data suggest that kindling-induced activation of microglia in the SNr can enhance immune reactions to transplanted cells. In this case, under conditions of further immunological stimulation by CL4 cells, transfected with a human cDNA, substantial immune reactions occurred. Thus, it appears that the condition of the host brain and the production of foreign proteins by transplanted cells have to be considered in estimating the risks of rejection of transplants into the brain.