Muscarinic M1, M3, Nicotinic,GABAA and GABAB Receptor Subtypes Gene Expression in Insulin Induced Hypoglycemic Rat Brain Regions: Functional Regulation through Phospholipase C and CREB Protein

In the present study, a detailed investigation on the alterations of muscarinic M1, M3, α7 nicotinic acetylcholine receptor (α7 nAchR), GABA receptors and its subtypes; GABAAα1 and GABAB in the brain regions of streptozotocin induced diabetic and insulin induced hypoglycemic rats were carried out. Gene expression of acetylcholine esterase (AChE), choline acetyltransferase (ChAT), GAD, GLUT3, Insulin receptor, superoxide dismutase (SOD), Bax protein, Phospholipase C and CREB in hypoglycemic and hyperglycemic rat brain were studied. Muscarinic M1, M3 receptors, AChE, ChAT, GABAAα1, GABAB, GAD, Insulin receptor, SOD, Bax protein and Phospholipase C expression in pancreas was also carried out. The molecular studies on the CNS and PNS damage will elucidate the therapeutic role in the corrective measures of the damage to the brain during hypoglycemia and hyperglycemia.

Bone marrow cells differentiation to neurons in the rat brain using Serotonin and GABA:Their role on glutamate receptors, IP3, cAMP and cGMP functional regulation in unilateral Parkinsonism induced by 6-hydroxydopamine

Parkinson's disease is a chronic progressive neurodegenerative movement disorder characterized by a profound and selective loss of nigrostriatal dopaminergic neurons. Our findings demonstrated that glutamatergic system is impaired during PD. The evaluations of these damages have important implications in understanding the molecular mechanism underlying motor, cognitive and memory deficits in PD. Our results showed a significant increase of glutamate content in the brain regions of 6- OHDA infused rat compared to control. This increased glutamate content caused an increase in glutamatergic and NMDA receptors function. Glutamate receptor subtypes- NMDAR1, NMDA2B and mGluR5 have differential regulatory role in different brain regions during PD. The second messenger studies confirmed that the changes in the receptor levels alter the IP3, cAMP and cGMP content. The alteration in the second messengers level increased the expression of pro-apoptotic factors - Bax and TNF-α, intercellular protein - α-synuclein and reduced the expression of transcription factor - CREB. These neurofunctional variations are the key contributors to motor and cognitive abnormalities associated with PD. Nestin and GFAP expression study confirmed that 5-HT and GABA induced the differentiation and proliferation of the BMC to neurons and glial cells in the SNpc of rats. We also observed that activated astrocytes are playing a crucial role in the proliferation of transplanted BMC which makes them significant for stem cell-based therapy. Our molecular and behavioural results showed that 5-HT and GABA along with BMC potentiates a restorative effect by reversing the alterations in glutamate receptor binding, gene expression and behaviour abnormality that occur during PD. The therapeutic significance in Parkinson’s disease is of prominence.

Cholinergic receptor subtypes functional regulation in spinal cord injured monoplegic rats

The present study deals with the Cholinergic Receptor subtypes functional regulation in spinal cord injured monoplegic rats: Effect of 5-HT GABA and bone marrow cells.Spinal cord injury causes permanent and irrevocable motor deficits and neurodegeneration. Disruption of the spinal cord leads to diminished transmission of descending control from the brain to motor neurons and ascending sensory information. Behavioural studies showed deficits in motor control and coordination in SCI rats. Cholinergic system plays an important role in SCI, the evaluation of which provides valuable insight on the underlying mechanisms of motor deficit that occur during SCI. The cholinergic transmission was studied by assessing the muscarinic and nicotinic receptors; cholinergic enzymes- ChAT and AChE; second messenger enzyme PLC; transcription factor CREB and second messengers - IP3, cAMP and cGMP. We observed a decrease in the cholinergic transmission in the brain and spinal cord of SCI rats. The disrupted cholinergic system is the indicative of motor deficit and neuronal degeneration in the spinal cord and brain regions. SCI mediated oxidative stress and apoptosis leads to neuronal degeneration in SCI rats. The decreased expression of anti oxidant enzymes – SOD, GPx and neuronal cell survival factors - BDNF, GDNF, IGF-1, Akt and cyclin D2 along with increased expression of apoptotic factors – Bax, caspase-8, TNFa and NF-kB augmented the neuronal degeneration in SCI condition. BMC administration in combination with 5-HT and GABA in SCI rats showed a reversal in the impaired cholinergic neurotransmission and reduced the oxidative stress and apoptosis. It also enhanced the expression of cell survival factors in the spinal cord region. In SCI rats treated with 5-HT and GABA, the transplanted BMC expressed NeuN confirming that 5-HT and GABA induced the differentiation and proliferation of BMC to neurons in the spinal cord. Neurotrophic factors and anti-apoptotic elements in SCI rats treated with 5-HT and GABA along with BMC rendered neuroprotective effects accompanied by improvement in behavioural deficits. This resulted in a significant reversal of altered cholinergic neurotransmission in SCI. The restorative and neuro protective effects of BMC in combination with 5-HT and GABA are of immense therapeutic significance in the clinical management of SCI.

Serotonin receptor subtypes functional regulation and oxidative stress mediated apoptosis in 6-hydroxydopamine lesioned Parkinsonian rats

Parkinson’s disease is a chronic progressive neurodegenerative disorder characterized by the selective loss of dopaminergic neurons in the SNpc resulting in severe motor impairments. Serotonergic system plays an important regulatory role in the pathophysiology of PD in rats, the evaluation of which provides valuable insight on the underlying mechanisms of motor, cognitive and memory deficits in PD. We observed a decrease in 5-HT content in the brain regions of 6-OHDA infused rat compared to control. The decreased 5-HT content resulted in a decrease of total 5-HT, 5-HT2A receptors and 5-HTT function and an increase of 5-HT2C receptor function. 5-HT receptor subtypes - 5-HT2A and 5-HT2C receptors have differential regulatory role on the modulation of DA neurotransmission in different brain regions during PD. Our observation of impaired serotonergic neurotransmission in SNpc, corpus striatum, cerebral cortex, hippocampus, cerebellum and brain stem demonstrate that although PD primarily results from neurodegeneration in the SNpc, the associated neurochemical changes in other areas of the brain significantly contributes to the different motor and non motor symptoms of PD. The antioxidant enzymes – SOD, CAT and GPx showed significant down regulation which indicates increased oxidative damage resulting in neurodegeneration. We also observed an increase in the level of lipid peroxidation. Reduced expression of anti-apoptotic Akt and enhanced expression of NF-B resulting from oxidative stress caused an activation of caspase-8 thus leading the cells to neurodegeneration by apoptosis. BMC administration in combination with 5-HT and GABA to PD rats showed reversal of the impaired serotonergic neurotransmission and oxidative stress mediated apoptosis. The transplanted BMC expressed NeuN confirming that 5-HT and GABA induced the differentiation and proliferation of BMC to neurons in the SNpc along with an increase in DA content and an enhanced expression of TH. Neurotrophic factors – BDNF and GDNF rendered neuroprotective effects accompanied by improvement in behavioural deficits indicating a significant reversal of altered dopaminergic and serotonergic neurotransmission in PD. The restorative and neuroprotective effects of BMC in combination with 5-HT and GABA are of immense therapeutic significance in the clinical management of PD.

Evidence that GABA rho subunits contribute to functional ionotropic GABA receptors in mouse cerebellar Purkinje cells

Ionotropic gamma-amino butyric acid (GABA) receptors composed of heterogeneous molecular subunits are major mediators of inhibitory responses in the adult CNS. Here, we describe a novel ionotropic GABA receptor in mouse cerebellar Purkinje cells (PCs) using agents reported to have increased affinity for rho subunit-containing GABA(C) over other GABA receptors. Exogenous application of the GABA(C)-preferring agonist cis-4-aminocrotonic acid (CACA) evoked whole-cell currents in PCs, whilst equimolar concentrations of GABA evoked larger currents. CACA-evoked currents had a greater sensitivity to the selective GABA(C) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) than GABA-evoked currents. Focal application of agonists produced a differential response profile; CACA-evoked currents displayed a much more pronounced attenuation with increasing distance from the PC soma, displayed a slower time-to-peak and exhibited less desensitization than GABA-evoked currents. However, CACA-evoked currents were also completely blocked by bicuculline, a selective agent for GABA(A) receptors. Thus, we describe a population of ionotropic GABA receptors with a mixed GABA(A)/GABA(C) pharmacology. TPMPA reduced inhibitory synaptic transmission at interneurone-Purkinje cell (IN-PC) synapses, causing clear reductions in miniature inhibitory postsynaptic current (mIPSC) amplitude and frequency. Combined application of NO-711 (a selective GABA transporter subtype 1 (GAT-1) antagonist) and SNAP-5114 (a GAT-(2)/3/4 antagonist) induced a tonic GABA conductance in PCs; however, TPMPA had no effect on this current. Immunohistochemical studies suggest that rho subunits are expressed predominantly in PC soma and proximal dendritic compartments with a lower level of expression in more distal dendrites; this selective immunoreactivity contrasted with a more uniform distribution of GABA(A) alpha 1 subunits in PCs. Finally, co-immunoprecipitation studies suggest that rho subunits can form complexes with GABA(A) receptor alpha 1 subunits in the cerebellar cortex. Overall, these data suggest that rho subunits contribute to functional ionotropic receptors that mediate a component of phasic inhibitory GABAergic transmission at IN-PC synapses in the cerebellum.

Effect of a nanostructured dendrimer-naloxonazine complex on endogenous opioid peptides mu(1) receptor-mediated post-ictal antinociception

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Natriorexigenic effect of baclofen is reduced by AT(1) receptor blockade in the lateral parabrachial nucleus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The bradycardic and hypotensive responses to serotonin are reduced by activation of GABA A receptors in the nucleus tractus solitarius of awake rats

We investigated the effects of bilateral injections of the GABA receptor agonists muscimol (GABA A) and baclofen (GABA B) into the nucleus tractus solitarius (NTS) on the bradycardia and hypotension induced by iv serotonin injections (5-HT, 2 µg/rat) in awake male Holtzman rats. 5-HT was injected in rats with stainless steel cannulas implanted bilaterally in the NTS, before and 5, 15, and 60 min after bilateral injections of muscimol or baclofen into the NTS. The responses to 5-HT were tested before and after the injection of atropine methyl bromide. Muscimol (50 pmol/50 nl, N = 8) into the NTS increased basal mean arterial pressure (MAP) from 115 ± 4 to 144 ± 6 mmHg, did not change basal heart rate (HR) and reduced the bradycardia (-40 ± 14 and -73 ± 26 bpm at 5 and 15 min, respectively, vs -180 ± 20 bpm for the control) and hypotension (-11 ± 4 and -14 ± 4 mmHg, vs -40 ± 9 mmHg for the control) elicited by 5-HT. Baclofen (12.5 pmol/50 nl, N = 7) into the NTS also increased basal MAP, but did not change basal HR, bradycardia or hypotension in response to 5-HT injections. Atropine methyl bromide (1 mg/kg body weight) injected iv reduced the bradycardic and hypotensive responses to 5-HT injections. The stimulation of GABA A receptors in the NTS of awake rats elicits a significant increase in basal MAP and decreases the cardiac Bezold-Jarisch reflex responses to iv 5-HT injections.

AT(1) receptor blockade in the lateral parabrachial nucleus reduces the effects of muscimol on sodium intake

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hypnotic, anticonvulsant and muscle relaxant effects of Rubus brasiliensis. Involvement of GABA(A)-system

Rubus brasiliensis hexanic fraction induced anxiolysis in rodents, which was reversed by flumazenil, a specific GABA(A)-benzodiazepine receptor antagonist (Nogueira et al., 1998a,b). Then, we investigated if this hexanic fraction was able to induce hypnotic, anticonvulsant and muscle relaxant effects, and the involvement of GABA(A)-system. The hexanic fraction (50, 100, 150 and 300 mg/kg, vo) was administered to male Swiss mice, 30 min before the tests. Only the dose of 300 mg/kg of this fraction decreased the latency and increased sleeping time in the barbituric-hypnosis test (sodium pentobarbital, 30 mg/kg, ip), prevented the pentylenetetrazol seizures (70 mg/kg, ip) and induced muscle relaxant (inclined plane) in 100% of animals. These effects were reversed by flumazenil (3 mg/kg, ip). In conclusion: (1) R. brasiliensis hexanic fraction induced hypnotic, anticonvulsant and muscle relaxant effects, in mice, and the GABA(A)-benzodiazepine receptor may play an important role in the effects of this fraction; (2) it is strongly suggested that this fraction contains a benzodiazepine-like principle. (C) 2000 Elsevier B.V. Ireland Ltd. All rights reserved.

Similar anxiolytic-like effects following intra-amygdala infusions of benzodiazepine receptor agonist and antagonist: Evidence for the release of an endogenous benzodiazepine inverse agonist in mice exposed to elevated plus-maze test

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cocaine effects on mouse incentive-learning and human addiction are linked to alpha 2 subunit-containing GABA(A) receptors

Because GABA(A) receptors containing alpha 2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with alpha 2 gene deletion showed reduced synaptic GABA(A) receptor-mediated responses. Behaviorally, the deletion abolished cocaine`s ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of alpha 2-GABA(A) receptors (alpha 2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In alpha 2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of alpha 2-GABA(A) receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction.

Study on the regulation of mRNA expression of GABA transporters (GAT-1 and GAT-3) in rat brain

The submitted work concentrated on the study of mRNA expression of two distinct GABA transporters, GAT-1 and GAT-3, in the rat brain. For the detection and quantification of the chosen mRNAs, appropriate methods had to be established. Two methods, ribonuclease protection assay (RPA) and competitive RT-PCR were emloyed in the present study. Competitive RT-PCR worked out to be 20 times more sensitive as RPA. Unlike the sensitivity, the fidelity of both techniques was comparable with respect to their intra- and inter-assay variability.The basal mRNA levels of GAT-1 and GAT-3 were measured in various brain regions. Messenger RNAs for both transporters were detected in all tested brain regions. Depending on the region, the observed mRNA level for GAT-1 was 100-300 higher than for GAT-3. The GAT-1 mRNA levels were similar in all tested regions. The distribution of GAT-3 mRNA seemed to be more region specific. The strongest GAT-3 mRNA expression was detected in striatum, medulla oblongata and thalamus. The lowest levels of GAT-3 were in cortex frontalis and cerebellum.Furthermore, the mRNA expression for GAT-1 and GAT-3 was analysed under altered physiological conditions; in kindling model of epilepsy and also after long-term treatment drugs modulating GABAergic transmission. In kindling model of epilepsy, altered GABA transporter function was hypothesised by During and coworkers (During et al., 1995) after observed decrease in binding of nipecotic acid, a GAT ligand, in hippocampus of kindled animals. In the present work, the mRNA levels were measured in hippocampus and whole brain samples. Neither GAT-1 nor GAT-3 showed altered transcription in any tested region of kindled animals compared to controls. This leads to conclusion that an altered functionality of GABA transporters is involved in epilepsy rather than a change in their expression.The levels of GAT-1 and GAT-3 mRNAs were also measured in the brain of rats chronically treated with diazepam or zolpidem, GABAA receptor agonists. Prior to the molecular biology tests, behavioural analysis was carried out with chronically and acutely treated animals. In two tests, open field and elevated plus-maze, the basal activity exploration and anxiety-like behaviour were analysed. Zolpidem treatment increased exploratory activity. There were observed no differencies between chronically and acutely treated animals. Diazepam increased exploratory activity and decresed anxiety-like behaviour when applied acutely. This effect disappeard after chronic administration of diazepam. The loss of effect suggested a development of tolerance to effects of diazepam following long-term administration. Double treatment, acute injection of diazepam after chronic diazepam treatment, confirmed development of a tolerance to effects of diazepam. Also, the mRNAs for GAT-1 and GAT-3 were analysed in cortex frontalis, hippocampus, cerebellum and whole brain samples of chronically treated animals. The mRNA levels for any of tested GABA transporters did not show significant changes in any of tested region neither after diazepam nor zolpidem treatment. Therefore, changes in GAT-1 and GAT-3 transcription are probably not involved in adaptation of GABAergic system to long-term benzodiazepine administration and so in development of tolerance to benzodiazepines.

Charakterisierung funktioneller Domänen für GABA und Furosemid auf GABA A-Rezeptoren

ZUSAMMENFASSUNGDie schnelle inhibitorische Neurotransmission im Säugerhirn ist wesentlich GABA-erg vermittelt.Neben GABA binden u.a. Picrotoxinin und TBPS (tert-Butylbicyclophosphorothionat) am GABAA-Rezeptor. Die Bindung von [35S]TBPS wird durch alle am GABAA-Rezeptor bindenden Substanzen moduliert. Zur Untersuchung der GABAA-Rezeptor-Funktionen wurden TBPS-Bindungsstudien an rekombinant exprimierten Rezeptoren in vitro und nativen Rezeptoren in situ verwendet.Die alpha-Untereinheiten spielen bei der gehirnarealspezifischen Auswirkungen verschiedener GABA-Mimetika, der Charakterisierung subtypspezifischer Substanzen und der Ausprägung der GABA-Sensitivitäten eine große Rolle. Für die Detaillierung der höheren GABA-Sensitivität alpha6-enthaltender Rezeptoren wurden Chimären und Punktmutationen zwischen den Untereinheiten alpha1 und alpha6 hergestellt. Nach Austausch des Asparagins 188 in der alpha1-Untereinheit durch das alpha6-entsprechende Lysin zeigten Rezeptoren in Kombination mit den Untereinheiten beta3 und gamma2 eine erhöhte GABA-Sensitivität gegenüber dem Wildtyp. Dementsprechend wiesen alpha6-enthaltende Rezeptoren mit der umgekehrten Punktmutation L187N eine geringere GABA-Sensitivität auf. Furosemid wirkt ausschließlich auf alpha6beta2/3-enthaltende GABAA-Rezeptors GABA-agonistisch. [35S]TBPS-Bindungsstudien an chimären alpha1/alpha6-Rezeptoren weisen auf eine niedrigpotente Bindungsstelle für Furosemid im extrazellulären Sequenzabschnitt zwischen der Aoc I-Schnittstelle und der TM3-Region hin. Die Substanz 4 PIOL zeigte subtypspezifischen Charakter am GABAA-Rezeptor. In den [35S]TBPS-Autoradiographien und den Bindungsstudien an Membranen wirkte 4 PIOL auf alpha6-enthaltende Rezeptoren schwach GABA-mimetisch bzw. agonistisch, in den [35S]TBPS-Bindungsstudien an alpha6-enthaltenden rekombinanten Rezeptoren schwach negativ modulatorisch oder antagonistisch.

Neue 18 F-markierte Liganden zur Visualisierung des GABA A, alpha 5-Rezeptorstatus mittels PET

Die heutige Verfügbarkeit der molekularen Bildgebung ermöglicht einen signifikanten Einfluss auf die Diagnostik und die Therapiekontrolle von neurodegenerativen Erkrankungen, die unter anderem durch Fehlsteuerungen im GABAergen System auftreten können. Die Visualisierung und Quantifizierung des GABAA-alpha5-Subtyps durch PET könnte dabei zu einem besseren Verständnis von Erkrankungen wie Alzheimer und traumatischen Neurosen (emotionales Langzeitgedächtnis) beitragen. Ferner eröffnen GABAA/alpha5-subtypselektive Liganden die Möglichkeit, wesentliche Grundlagen der elementaren Vorgänge von Lernen und Erinnern zu untersuchen. 7,8,9,10-Tetrahydro-(7,10-ethan)-1,2,4-triazol[3,4-alpha]phthalazine stellen sich als vielverspre-chende Leitstrukturen zur Entwicklung neuer 18F-markierter alpha5-subtypselektiver GABAA-Rezeptorliganden für die PET dar. Um diese neuartigen Substanzen hinsichtlich ihrer Potenz als GABAA-alpha5-subtypselektive Radioliganden zu verifizieren, wurden zunächst die entsprechenden 19F-Derivate TC07-TC12 synthetisiert. Diese Referenzverbindungen wurden in Rezeptor-bindungsassays und in Autoradiographien mit [3H]Ro 15-4513 als zu verdrängender Radioligand evaluiert. In beiden Experimenten als auch in in vivo-Verdrängungsexperimenten an Ratten konnte eine hohe Affinität im nanomolaren Bereich als auch eine hohe Selektivität bezüglich der GABAA/alpha5-Untereinheit für einige der dargestellten Referenzverbindungen nachgewiesen werden. Gemäß diesen vielversprechenden Ergebnissen wurden verschiedene Markie-rungsvorläufer für eine 18F-Direktmarkierung der relevantesten Substanz TC07 in einer mehrstufigen organischen Synthese dargestellt. Die anschließende 18F-Markierung erfolgte über eine nukleophile Substitution mit [18F]Fluorid. Die Reaktionsparameter wurden hinsichtlich Reaktionstemperatur und dauer, Markierungsvorläuferkonzentration, Basenabhängigkeit und verschiedenen Markierungsmethoden optimiert. Daraus resultierend konnte [18F]TC07 mit bis zu 45 % radiochemischer Ausbeute erhalten werden. Die zerfallskorrigierte, gesamtradiochemische Ausbeute von nca [18F]TC07 in isotonischer NaCl-Lösung betrug 15 %. Basierend auf den bisher erhaltenen Ergebnissen wurde der Radioligand in in vitro-, ex vivo- und in vivo µPET-Experimenten evaluiert. Die zunächst durchgeführten in vitro-Experimente deuteten auf eine homogene Verteilung der Aktivität hin und zeigten keine spezifische Anreicherung. Diese Ergebnisse wurden sowohl in ex vivo- als auch in in vivo-µPET-Studien bestätigt. Auch hier konnte nur eine niedrige Aktivitätsanreicherung, eine homogene Verteilung im gesamten Gehirn und keine Übereinstimmung mit der bekannten GABAA/alpha5-Subtypverteilung gefunden werden. Eine im Anschluss durchgeführte Metabolismusstudie zeigte eine langsame Metabolisierungsrate des [18F]TC07 und auch eine Organverteilungsstudie zeigte keine außergewöhnlichen Anreicherungen. Aus den erhaltenen Ergebnissen kann geschlossen werden, dass der Radioligand [18F]TC07 kein geeigneter Tracer zur in vivo-Visualisierung der alpha5-Untereinheit des GABAA-Rezeptors ist.