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N J Tillakaratne

Publications and source records attributed to N J Tillakaratne.

At least 19 recordsLinked to original sources
PubMed2001

Retraining the injured spinal cord.

The present review presents a series of concepts that may be useful in developing rehabilitative strategies to enhance recovery of posture and locomotion following spinal cord injury. First, the loss of supraspinal input results in a marked change in the functional efficacy of the remaining synapses and neurons of intraspinal and peripheral afferent (dorsal root ganglion) origin. Second, following a complete transection the lumbrosacral spinal cord can recover greater levels of motor performance if it has been exposed to the afferent and intraspinal activation patterns that are associated with standing and stepping. Third, the spinal cord can more readily reacquire the ability to stand and step following spinal cord transection with repetitive exposure to standing and stepping. Fourth, robotic assistive devices can be used to guide the kinematics of the limbs and thus expose the spinal cord to the new normal activity patterns associated with a particular motor task following spinal cord injury. In addition, such robotic assistive devices can provide immediate quantification of the limb kinematics. Fifth, the behavioural and physiological effects of spinal cord transection are reflected in adaptations in most, if not all, neurotransmitter systems in the lumbosacral spinal cord. Evidence is presented that both the GABAergic and glycinergic inhibitory systems are up-regulated following complete spinal cord transection and that step training results in some aspects of these transmitter systems being down-regulated towards control levels. These concepts and observations demonstrate that (a) the spinal cord can interpret complex afferent information and generate the appropriate motor task; and (b) motor ability can be defined to a large degree by training.

Animals↗
PubMed2000

Increased expression of glutamate decarboxylase (GAD(67)) in feline lumbar spinal cord after complete thoracic spinal cord transection.

To determine changes in gamma-aminobutyric acid (GABA) in the spinal cord in response to a complete transection, we examined the cellular and tissue changes of the two forms of GABA synthetic enzyme glutamate decarboxylase (GAD(65) and GAD(67)). In situ hybridization, immunohistochemistry, and Western blot analyses show that spinal cord transection between thoracic segments 12 and 13 results in an increase of GAD(67), but not GAD(65), protein and mRNA in the lumbar spinal cord. This increase occurs mainly in the dorsal horn and persists for at least 12 months. In addition, there was relatively high GAD(67)-immunoreactivity around the central canal, with dorsolateral GAD(67)-immunoreactive fibers extending toward the ependyma and into the central canal in the transected animals. We suggest that an increase in GAD(67) leads to increased GABA production in spinal neurons below the injury site, resulting in altered inhibition and trophic support during posttrauma recovery and adaptation. Increased GABA synthesis around the central canal, in the vicinity of ependymal cells, may represent part of a regenerative process in the mammalian spinal cord, reminiscent of that observed in lower vertebrates.

Animals↗
PubMed1998

Alterations in GABAA receptor alpha 1 and alpha 4 subunit mRNA levels in thalamic relay nuclei following absence-like seizures in rats.

Modification of GABAA receptor mRNA levels by seizure activity can regulate general neuronal excitability. The possibility of absence seizure-induced alteration in GABAA receptor alpha 1, alpha 4, beta 2, and gamma 2 subunit gene expression in thalamic relay nuclei was studied in a rat model of absence seizures induced by gamma-hydroxybutyric acid (GHB). We observed a marked increase in alpha 1 mRNA and a corresponding decrease in alpha 4 mRNA in thalamic relay nuclei 2-4 h after the onset of GHB-induced absence seizures (when the seizures were terminating). These changes were selective to these alpha isoforms as neither beta 2 nor gamma 2 mRNA changed following seizures and occurred only in thalamic relay nuclei but not in hippocampus, a structure from which absence seizures do not evolve. The alterations in alpha 1 and alpha 4 mRNA persisted until about 12 h, and by 24 h after the seizure-onset the mRNA levels normalized. Blocking GHB-seizures produced no change in the levels of alpha 1 and alpha 4 mRNA in thalamic relay nuclei, suggesting that seizures themselves were responsible for mRNA alterations. In order to determine if absence seizure-induced changes in alpha 1 and alpha 4 mRNA had any physiological significance, GHB was readministered in rats 6 and 24 h after the onset of seizures. The total duration of GHB-seizures was found to be significantly decreased when GHB was readministered at 6 h but not 24 h after the seizure-onset. These results suggest that absence seizures regulate GABAA receptor alpha 1 and alpha 4 gene expression in thalamic relay nuclei as a compensatory mechanism by which absence seizures are terminated.

4-Butyrolactone↗
PubMed1998

Absence seizures decrease steroid modulation of t-[35S]butylbicyclophosphorothionate binding in thalamic relay nuclei.

Interaction of gamma-aminobutyric acid (GABA), pentobarbital and two neuroactive steroids on t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABAA receptors in thalamus was studied during absence seizures. In control brain sections, the steroids alphaxalone and tetrahydrodeoxycorticosterone (at low 0.1-1 microM concentrations) increased [35S]TBPS binding in thalamic relay nuclei. Both GABA and pentobarbital dose-dependently decreased [35S]TBPS binding in these nuclei. A significant decrease in the ability of steroids to increase [35S]TBPS binding in thalamic relay nuclei was observed during absence seizures induced by gamma-hydroxybutyric acid (GHB). This loss of steroid effect on binding was 1) selective to steroids only as GABA and pentobarbital modulation of [35S]TBPS binding in these nuclei did not change significantly and 2) not causally related to the generation of GHB-induced absence seizures as it was not observed at the onset of GHB-seizures but developed 30 min after the seizure-onset. We tested whether absence seizures were critical for the development of this loss of steroid effect on [35S]TBPS binding in thalamic relay nuclei. The ability of the steroids to increase [35S]TBPS binding in relay nuclei was preserved when GHB-seizures were blocked. When the duration of GHB-seizures was prolonged, the loss of steroid effect on [35S]TBPS binding in thalamus persisted throughout the seizure-duration. These findings suggest that absence seizures cause a rapid loss of steroid effect on [35S]TBPS binding to GABAA receptors in thalamic relay nuclei.

Action Potentials↗
PubMed1995

gamma-Aminobutyric acid (GABA) metabolism in mammalian neural and nonneural tissues.

4-Aminobutyric acid (GABA), a major inhibitory neurotransmitter of mammalian central nervous system, is found in a wide range of organisms, from prokaryotes to vertebrates. GABA is widely distributed in nonneural tissue including peripheral nervous and endocrine systems. GABA acts on GABAA and GABAB receptors. GABAA receptors are ligand-gated chloride channels modulated by a variety of drugs. GABAB receptors are essentially presynaptic, usually coupled to potassium or calcium channels, and they function via a GTP binding protein. In neural and nonneural tissues, GABA is metabolized by three enzymes--glutamic acid decarboxylase (GAD), which produces GABA from glutamic acid, and the catabolic enzymes GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). Production of succinic acid by SSADH allows entry of the GABA carbon skeleton into the tricarboxylic acid cycle. Alternate sources of GABA include putrescine, spermine, spermidine and ornithine, which produce GABA via deamination and decarboxylation reactions, while L-glutamine is an additional source of glutamic acid via deamination. GAD from mammalian brain occurs in two molecular forms, GAD65 and GAD67 (referring to subunit relative molecular weight (Mr) in kilodaltons). These different forms of GAD are the product of different genes, differing in nucleotide sequence, immunoreactivity and subcellular localization. The presence and characteristics of GAD have been investigated in a wide variety of nonneural tissues including liver, kidney, pancreas, testis, ova, oviduct, adrenal, sympathetic ganglia, gastrointestinal tract and circulating erythrocytes. In some tissues, one form (GAD65 or GAD67) predominates. GABA-T has been located in most of the same tissues, primarily through histochemical and/or immunochemical methods; GABA-T is also present in a variety of circulating cells, including platelets and lymphocytes. SSADH, the final enzyme GABA catabolism, has been detected in some of the tissues in which GAD and GABA-T have been identified, although the presence of this enzyme has not been in mammalian pancreas, ova, oviduct, testis or sympathetic ganglia.

Animals↗
PubMed1994

Differential localization of two glutamic acid decarboxylases (GAD65 and GAD67) in adult monkey visual cortex.

Adult monkey primary visual cortex contains a diverse population of stellate neurons that utilize the neurotransmitter gamma aminobutyric acid (GABA). Two glutamic acid decarboxylase (GAD) enzymes that synthesize GABA, GAD65 and GAD67, were localized within these stellate neurons by in situ hybridization of 35S or digoxigenin (DIG) labeled riboprobes. Double labels were done by using 35S GAD67 riboprobe and GABA immunocytochemistry on the same section to verify that the neuronal population identified by immunocytochemistry was the same one studied in the in situ hybridization experiments. We find that GAD65 mRNA and GAD67 mRNA are widely distributed in the cortex, with four bands of heavily labeled neurons in upper layer 2, lower 3, 4C, and 6. GAD67 labeled neurons were more obvious in layer 4C beta, while GAD65 containing neurons were common in layer 1 and white matter. Northern blots and in situ hybridization on sections with both 35S and DIG riboprobes indicate that cortical neurons typically contain more GAD67 mRNA. Cell counts show that 18% of all cortical neurons contain GAD67 mRNA and 13% contain GAD65 mRNA, suggesting that a small population of GABA neurons might lack GAD65. Cell bodies that contain high amounts of GAD65 mRNA are prominent in layers deep 3, 4B, 4C alpha, and 6 and often are the largest cells in their respective layers. Double labels demonstrate that 96% of all GABA+ neurons contain GAD67 mRNA. Neurons heavily labeled for GABA tend to have smaller cell bodies and contain less GAD67 mRNA, while lightly labeled GABA neurons are larger and contain more GAD67 mRNA. These data indicate that most GABA neurons in monkey striate cortex contain both GAD enzymes. Although the differences in GABA content, cell size, laminar distribution, and GAD mRNA concentration suggest different requirements for GAD67 and GAD65 in cortical circuits, our experiments do not reveal what different roles these two enzymes subserve within GABAergic stellate neurons.

Animals↗
PubMed1994

A rat brain cDNA encodes enzymatically active GABA transaminase and provides a molecular probe for GABA-catabolizing cells.

cDNAs encoding gamma-aminobutyric acid aminotransferase (GABA-T) were isolated from a lambda ZAP rat hippocampal cDNA expression library by two independent cloning methods, immunological screening with an antimouse GABA-T antibody and plaque hybridization with a GABA-T cDNA probe derived by polymerase chain reaction. We have produced enzymatically active GABA-T from a rat brain cDNA containing the full-length GABA-T coding region. Our rat brain GABA-T cDNAs hybridize to mRNAs in brain and peripheral tissues, including liver, kidney, and testis. We have also detected GABA-T mRNA in GABAergic cells of rat cerebellar cortex by in situ hybridization. Our rat brain GABA-T probe hybridizes to Purkinje, basket, stellate, and Golgi II cells, the same GABAergic neurons previously shown to contain glutamate decarboxylase GAD65 and GAD67.

4-Aminobutyrate Transaminase↗
PubMed1994

Cyclic AMP decreases the expression of a neuronal marker (GAD67) and increases the expression of an astroglial marker (GFAP) in C6 cells.

C6 cells express proteins and mRNAs that are characteristic of both glia and neurons. Agents that increase intracellular levels of cyclic AMP (cAMP) decrease the enzymatic activity of glutamate decarboxylase (GAD), a neuronal marker, and the mRNA levels for one of the two GAD isoenzymes, GAD67. This reduction is accompanied by increased levels of glial fibrillary acidic protein (GFAP) mRNA, an astrocyte marker. Transient transfection assays, in which a 2-kb upstream regulatory region of the human GFAP gene was linked to a reporter gene, indicate that at least some of the cAMP-mediated increase of GFAP mRNA levels is due to increased transcription. Increases in intracellular cAMP appear to induce differentiation of C6 cells toward a more mature astrocyte phenotype.

Actins↗
PubMed1994

Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms.

Two isoforms of glutamic acid decarboxylase (GAD67 and GAD65) and their mRNAs were localized in the rat brain by immunohistochemistry and nonradioactive in situ hybridization methods with digoxigenin-labeled cRNA probes. In most brain regions, both GAD isoforms were present in neuronal cell bodies as well as axon terminals. A few populations of neurons, such as those in the reticular nucleus of the thalamus, exhibited similar cell body labeling for both GADs. However, in many brain regions, the cell bodies that were immunoreactive for GAD67 were often more numerous than those that were immunoreactive for GAD65. In contrast, the density (quantity) of GAD65-immunoreactive axon terminals was higher than that of GAD67-immunoreactive terminals. Strong parallels were observed between the intensity of immunohistochemical labeling of cell bodies and the levels of mRNA labeling for both GAD isoforms. Many groups of GAD-containing cell bodies were distinctly labeled for GAD67, and these same groups of neurons were heavily labeled for GAD67 mRNA. Such neurons included Purkinje cells of the cerebellar cortex, nonpyramidal cells in the cerebral cortex, and neurons of the reticular nucleus of the thalamus. Similar parallels in labeling were observed for GAD65 and its mRNA. Distinct cell body labeling for the protein and associated high levels of GAD65 mRNA were found in neurons of the reticular nucleus of the thalamus and periglomerular cells in the olfactory bulb. However, many cell bodies were not readily labeled for GAD65 with immunohistochemical methods. Such absence or weakness of cell body labeling for the protein was associated with low or moderate levels of GAD65 mRNA. Even though light cell body staining was frequently observed for GAD65 and its mRNA, strong axon terminal labeling for GAD65 was present. Thus, in the deep cerebellar nuclei to which the Purkinje cells of the cerebellar cortex project, strong terminal labeling was observed for both GAD isoforms even though only light cell body labeling of the Purkinje cells was obtained for GAD65 and its mRNA. The findings suggest that the two isoforms of GAD are present in most classes of GABA neurons but that they are not similarly distributed within the neurons. GAD67 is present in readily detectable amounts in many GAD-containing cell bodies whereas GAD65 is particularly prominent in many axon terminals. In addition, neurons that express either form of GAD mRNA also express the corresponding protein. Levels of labeling for the GAD mRNAs suggest that, under normal conditions, the synthesis of GAD65 is frequently lower than that of GAD67.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Comparative localization of mRNAs encoding two forms of glutamic acid decarboxylase with nonradioactive in situ hybridization methods.

Nonradioactive in situ hybridization methods with digoxigenin-labeled cRNA probes were used to localize two glutamic acid decarboxylase (GAD) mRNAs in rat brain. These mRNAs encode two forms of GAD that both synthesize GABA but differ in a number of characteristics including their molecular size (65 and 67 kDa). For each GAD mRNA, discrete neuronal labeling with high cellular resolution and low background staining was obtained in most populations of known GABA neurons. In addition, the current methods revealed differences in the intensity of labeling among neurons for each GAD mRNA, suggesting that the relative concentrations of each GAD mRNA may be higher in some groups of GABA neurons than in others. Most major classes of GABA neurons were labeled for each GAD mRNA. In some groups of GABA neurons, the labeling for the two mRNAs was virtually identical, as in the reticular nucleus of the thalamus. In other groups of neurons, although there was substantial labeling for each GAD mRNA, labeling for one of the mRNAs was noticeably stronger than for the other. In most brain regions, such as the cerebellar cortex, labeling for GAD67 mRNA was stronger than for GAD65 mRNA, but there were a few brain regions in which labeling for GAD65 mRNA was more pronounced, and these included some regions of the hypothalamus. Finally, some groups of GABA neurons were predominantly labeled for one of the GAD mRNAs and showed little or no detectable labeling for the other GAD mRNA, as, for example, in neurons of the tuberomammillary nucleus of the hypothalamus where labeling for GAD67 mRNA was very strong but no labeling for GAD65 mRNA was evident. The findings suggest that most classes of GABA neurons in the central nervous system (CNS) contain mRNAs for at least two forms of GAD, and thus, have dual enzyme systems for the synthesis of GABA. Higher levels of one or the other GAD mRNA in certain groups of GABA neurons may be related to differences in the functional properties of these neurons and their means of regulating GABA synthesis.

Animals↗
PubMed1992

Expression of GABAA receptor polypeptides in clonal rat cell lines.

The neuronal-like cell lines, B35, B65, B103, and B104, previously reported to possess high affinity GABA binding, were analyzed for various cellular properties. They possessed peripheral but lacked central benzodiazepine binding. Only B65 cells possessed [3H-]muscimol binding; none bound [35S]TBPS. None of the cells exhibited GABA-stimulated chloride conductance with patch clamp recordings. By Western blots the cells possessed alpha subunits. Northern blot and polymerase chain reaction analysis showed that out of alpha 1, alpha 4, beta 1 and gamma 2 subunits, only alpha 1 subunit mRNA was present. Thus, GABAA-receptor binding without associated central benzodiazepine receptor sites and without functional chloride channels appears to result from expression of an incomplete subunit composition.

Animals↗
PubMed1992

Two human glutamate decarboxylases, 65-kDa GAD and 67-kDa GAD, are each encoded by a single gene.

We report the isolation and sequencing of cDNAs encoding two human glutamate decarboxylases (GADs; L-glutamate 1-carboxy-lyase, EC 4.1.1.15), GAD65 and GAD67. Human GAD65 cDNA encodes a Mr 65,000 polypeptide, with 585 amino acid residues, whereas human GAD67 encodes a Mr 67,000 polypeptide, with 594 amino acid residues. Both cDNAs direct the synthesis of enzymatically active GADs in bacterial expression systems. Each cDNA hybridizes to a single species of brain mRNA and to a specific set of restriction fragments in human genomic DNA. In situ hybridization of fluorescently labeled GAD probes to human chromosomes localizes the human GAD65 gene to chromosome 10p11.23 and the human GAD67 gene to chromosome 2q31. We conclude that GAD65 and GAD67 each derive from a single separate gene. The cDNAs we describe should allow the bacterial production of test antigens for the diagnosis and prediction of insulin-dependent diabetes mellitus.

Amino Acid Sequence↗
PubMed1992

Transient increase in expression of a glutamate decarboxylase (GAD) mRNA during the postnatal development of the rat striatum.

We recently reported that the mammalian brain has two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD, E.C. 4.1.1.15), which are the products of two genes. The two forms, which we call GAD65 and GAD67, differ from each other in sequence, molecular size, subcellular distribution, and interactions with the cofactor pyridoxal phosphate (PLP), with GAD65 activity more dependent than that of GAD67 on the continued presence of exogenous PLP. The existence of two GAD genes suggests that individual GABA neurons may be subject to differential regulation of GABA production. We have examined the expression of these two forms of GAD during postnatal development of the rat striatum to determine whether different classes of GABA neurons selectively express different amounts of the two GAD mRNAs. Here we present evidence for a dramatic developmental difference in the expression of the two mRNAs during postnatal development of the rat striatum. Using in situ hybridization to the two GAD mRNAs, we observed a selective increase in GAD65 mRNA during the second postnatal week, at the time when striatal matrix neurons innervate the substantia nigra (SN). PLP-dependent enzyme activity in the midbrain increases in parallel with increased expression of GAD65 mRNA in the striatum. We hypothesize that the innervation of the SN by striatal neurons triggers an increase in GAD65. The changing ratios of GAD65 and GAD67 in the striatum may contribute to the well-documented changes in seizure susceptibility that occur in early life.

Animals↗
PubMed1992

Glutamate decarboxylases in nonneural cells of rat testis and oviduct: differential expression of GAD65 and GAD67.

gamma-Aminobutyric acid (GABA) and its synthetic enzyme, glutamate decarboxylase (GAD), are not limited to the nervous system but are also found in nonneural tissues. The mammalian brain contains at least two forms of GAD (GAD67 and GAD65), which differ from each other in size, sequence, immunoreactivity, and their interaction with the cofactor pyridoxal 5'-phosphate (PLP). We used cDNAs and antibodies specific to GAD65 and GAD67 to study the molecular identity of GADs in peripheral tissues. We detected GAD and GAD mRNAs in rat oviduct and testis. In oviduct, the size of GAD, its response to PLP, its immunoreactivity, and its hybridization to specific RNA and DNA probes all indicate the specific expression of the GAD65 gene. In contrast, rat testis expresses the GAD67 gene. The GAD in these two reproductive tissues is not in neurons but in nonneural cells. The localization of brain GAD and GAD mRNAs in the mucosal epithelial cells of the oviduct and in spermatocytes and spermatids of the testis shows that GAD is not limited to neurons and that GABA may have functions other than neurotransmission.

Animals↗
PubMed1991

Postnatal expression of glutamate decarboxylases in developing rat cerebellum.

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD67, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD67 mRNA predominates in the cerebellum. The mRNA for GAD65, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD65 is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD67 mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.

Aging↗
PubMed1991

Independent cellular and ontogenetic expression of mRNAs encoding three alpha polypeptides of the rat GABAA receptor.

Previous studies have shown that several distinct but related polypeptides can serve as alpha subunits of functional GABAA receptors. Furthermore, the diversity of these polypeptides at least partially accounts for the functional heterogeneity of GABAA receptors. In this paper, we report the results of in situ hybridization studies using probes derived from our recently reported cDNAs for alpha 1, alpha 2, and alpha 4 GABAA receptor polypeptides. We show that the mRNAs that encode these isoforms have distinct regional and cellular distributions and are present at widely varying levels within the rat brain. In addition, our Northern blot analyses indicate that each of these three alpha mRNAs has a distinct pattern of ontogenetic regulation. Differential regulation of alpha polypeptide isoforms may lead to changes in GABAA receptor function during ontogeny as well as to distinct cellular responses to GABA and GABA-related drugs.

Animals↗
PubMed1991

Two genes encode distinct glutamate decarboxylases.

gamma-Aminobutyric acid (GABA) is the most widely distributed known inhibitory neurotransmitter in the vertebrate brain. GABA also serves regulatory and trophic roles in several other organs, including the pancreas. The brain contains two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD), which differ in molecular size, amino acid sequence, antigenicity, cellular and subcellular location, and interaction with the GAD cofactor pyridoxal phosphate. These forms, GAD65 and GAD67, derive from two genes. The distinctive properties of the two GADs provide a substrate for understanding not only the multiple roles of GABA in the nervous system, but also the autoimmune response to GAD in insulin-dependent diabetes mellitus.

Amino Acid Sequence↗