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.

Serotonin Receptor Gene Expression And Insulin Function In Streptozotocin Induced Diabetic Rats

Recent studies have established a fimctional correlation of serotonergic and adrenergic function in the brain regions with insulin secretion in diabetic rats (Vahabzadeh et al., 1995). Administration of 5-HT”. agonist 8-OH-DPAT to conscious rats caused an increase in blood glucose level. This increase in blood glucose is due to inhibition of insulin secretion by increased circulating EPI (Chaouloff et al., 1990a; Chaouloff et al., 1990d; Chaoulo1T& Jeanrenaud, 1987). The increase in EPI is brought about by increased sympathetic stimulation. This increase can lead to increased sympatho-medullary stimulation thereby inhibiting insulin release (Bauhelal & Mir, 1993, Bauhelal & Mir, 1990a; Chaouloffet al., 1990d). Also, studies have shown that Gi protein in the liver has been decreased in diabetes which will increase gluconeogenesis and glycogenolysis thereby causing hyperglycaemia (Pennington, 1987). Serotonergic control is suggested to exert different effects on insulin secretion according to the activation of different receptor subclasses (Pontiroli et al., 1975). In addition to this mechanism, the secretion of insulin is dependent on the turnover ratio of endogenous 5-hydroxy tryptophan (5-HTP) to 5-HT in the pancreatic islets (Jance er al., 1980). The reports so far stated does not explain the complete mechanism and the subclass of 5-HT receptors whose expression regulate insulin secretion in a diabetic state. Also, there is no report of a direct regulation of insulin secretion by 5-HT from the pancreatic islets even though there are reports stating that the pancreatic islets is a rich source of 5-HT (Bird et al., 1980). Therefore, in the present study the mechanism by which 5-HT and its receptors regulate insulin secretion from pancreatic [3-cells was investigated. Our results led to the following hypotheses by which 5-HT and its receptors regulate the insulin secretion.

ALPHA2 Adrenergic and High Affinity Serotonergic Receptor Changes in the Brain Stem of Streptozotocin induced Diabetic Rats

The brain stems (13S) of streptozotocin (STZ)-diabetic rats were studied lo see the changes in neurotransmitter content and their receptor regulation. The norepinephrine (NE) content determined in the diabetic brain stems did ^ control. an E showed la while PI turnover hri content increased significantly compared N^r eNveFa o the recep significant increase. The alpha2 adrenergic receptor IneP utisoulinntreat d ratsetheNE contentt dec^ sled was significantly reduced during diabetes. in versedcto reanorm sed ulcrea e tK reatment the state. while EPI content remained increased as in die diabetic B,, for a]pha2 adrenergic receptors slw^nificantly while Unlabelled clonidine inhibited [31-I]NE binding in BS of control, diabetic and insulin treated ulations bindi diabetic rats showed that alpha2 adrenergicre^ punks cojnidiabetic animal the ligand bound sites with Hill slopes significantly away from unity. weaker to the low affinity site than in controls. Insulin treatment reversed[ this allumbmn to control levels. The displacement analysis using (-)-epinephrine age in control and diabetic animals revealed two populations of receptor affinidtyo=tat ss. In control animals, when GTP analogue added with epinephrine, the curve nagnlde caofnfitnroit yS model; but in the diabetic BS this effect `not aobserved. In bintact oth the diabetic data thus showlthat the effects of monovalent cations on affinity alphaz adrenergic receptors have a reduced affinity v due in stem ialtered Itscppeomson(5- regulation. The serotonin (5-HT) coat hydroxy) tryptophan (5-HTP) showed an increase and its breakdown metabolite (5-hydroxy) indoleacetic acid (5-I{IAA) showed a significant decrease. This showed that in serotonergic which l nerves there is a disturbance in both synthetic and breankduomwnbers pretma'med ana increased 5-HT. The high affinity serotonin receptor um ese serotonerg decrease in the receptor affinity. The insulin ^treatmentsturtiy showsha decreased serotonergic receptor kinetic parameters to control level. receptor function. These changes in adrenergic and serotonergic receptor function were suggested to be important in insulin function during STZ diabetes.

Dopamine Receptor Subtypes,IP3, cAMP and cGMP Functional Regulation in Parkinsonism Induced by Unilateral Infusion of Rotenone in Rats: Recovery with Bone Marrow Cells,Serotonin and GABA Supplementation

In the present study, the effects of 5-HT, GABA and Bone Marrow Cells infused intranigrally to substantia nigra individually and in combinations on unilateral rotenone infused Parkinsonism induced rats. Scatchard analysis of DA, DA D1 and D2 receptors in the corpus striatum, cerebral cortex, cerebellum, brain stem and hippocampus showed a significant increase in the Brain regions of rotenone infused rat compared to control. Real Time PCR amplification of DA D1, D2, Bax and ubiquitin carboxy-terminal hydrolase were up regulated in the brain regions of rotenone infused rats compared to control. Gene expression studies of -Synuclien, cGMP and Cyclic AMP response element-binding protein showed a significant down regulation in Rotenone infused rats compared to control. Behavioural studies were carried out to confirm the biochemical and molecular studies.Our study demonstrated that BMC administration alone cannot reverse the above said molecular changes occurring in PD rat. 5-HT and GABA acting through their specific receptors in combination with bone marrow cells play a crucial role in the functional recovery of PD rats. 5-HT, GABA and Bone marrow cells treated PD rats showed significant reversal to control in DA receptor binding and gene expression. 5-HT and GABA have co-mitogenic property. Proliferation and differentiation of cells re-establishing the connections in Parkinson's disease facilitates the functional recovery. Thus, it is evident that 5-HT and GABA along with BMC to rotenone infused rats renders protection against oxidative, related motor and cognitive deficits which makes them clinically significant for cellbased therapy. The BMC transformed to neurons when co-transplanted with 5-HT and GABA which was confirmed with PKH2GL and nestin. These newly formed neurons have functional significance in the therapeutic recovery of Parkinson’s disease.

Increased 5-HT2C Receptor Binding in the Brain Stem And Cerebral Cortex During Liver Regeneration And Hepatic Neoplasia In Rats

In the present study, serotonin 2C (5-HT2c) receptor binding parameters in the brainstem and cerebral cortex were investigated during liver generation after partial hepatectomy (PH) and N-nitrosodiethylamine (NDEA) induced hepatic neoplasia in male Wistar rats. The serotonin content increased significantly (p<0.01) in the cerebral cortex after PH and in NDEA induced hepatic neoplasia. Brain stem serotonin content increased significantly (p<0.05) after PH and (p<0.001) in NDEA induced hepatic neoplasia. The number and affinity of the 5-HT2c receptors in the crude synaptic membrane preparations of the brain stem showed a significant (p<0.001) increase after PH and in NDEA induced hepatic neoplasia. The number and affinity of 5-HT2c receptors increased significantly (p<0.001) in NDEA induced hepatic neoplasia in the crude synaptic membrane preparations of the cerebral cortex. There was a significant (p<0.01) increase in plasma norepinephrine in PH and (p<0.001) in NDEA induced hepatic neoplasia, indicating sympathetic stimulation. Thus, our results suggest that during active hepatocyte proliferation 5-HT2c receptor in the brain stem and cerebral cortex are up-regulated which in turn induce hepatocyte proliferation mediated through sympathetic stimulation.

Brain 5HT2A Receptor Regulation by Tryptophan Supplementation in Streptozotocin Diabetic Rats

5-HT2A receptor binding parameters were studied in the cerebral cortex and brain stem of control, diabetic, insulin, insulin + tryptophan and tr3yptophan treated streptozotocin diabetic rats. Scatchard analysis using selective antagonist, [-H](±)2,3-dimethoxyphenyl-l-[2-(4-piperidine)- methanol] ([3H]MDL100907) in cerebral cortex of diabetic rats showed a significant decrease in dissociation constant (Kd) without any change in maximal binding (Bm). Competition binding studies in cerebral cortex using ketanserin against [3H]MDL100907 showed the appearance of an additional site in the low affinity region during diabetes. In the brain stem, Scatchard analysis showed a significant increase in Bmax and Kd. Displacement studies showed a shift in the receptor affinity towards a low affinity state. All these altered parameters in diabetes were reversed to control level by insulin, insulin + tryptophan and tryptophan treatments. Tryptophan treatment is suggested to reverse the altered 5-HT2Abinding and blood glucose level to control status by increasing the brain 5-HT content.

Effect of Pyridoxine Deficiency in Young-Rats on High-Affinity Serotonin and Dopamine Receptors

The high-affinity bindings of [3H]-5-hydroxytryptamine to serotonin S-1 receptors, [3H]-ketanserin to serotonin S-2 receptors in the cerebral cortex, [3H]- fluphenazine to dopamine D-1 receptors, and [3H]-spiroperidol to dopamine D-2 receptors in the corpus striatum were studied in pyridoxine-deficient rats and compared to pyridoxine-supplemented controls. There was a significant increase in the maximal binding (Bmax) of serotonin S-1 and S-2 receptors with a significant decrease in their binding affinities (Kd). However, there were no significant changes either in the maximal binding or binding affinity of striatal dopamine D- 1 and D-2 receptors. Receptor sensitivity seems to correlate negatively with the corresponding neurotransmitter concentrations in the pyridoxine-deficient rats.

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.

Dopamine D2 and 5-HT2A receptor gene expression

Neuronal dopamine and serotonin receptors are widely distributed in the central and the peripheral nervous systems at different levels. Dopaminergic and serotonergic systems have crucial role in aldehyde dehydrogenase regulation Stimulation of autonomic nervous system during ethanol treatment is suggested to be an important factor in regulating the ALDH function. The ALDH enzyme activity was increased in plasma, cerebral cortex, and liver but decreased in cerebellum. The ALDH enzyme affinity was decreased in plasma, brainstem and liver and increased in cerebral cortex and cerebellum. Dopamine and serotonin content decreased in liver and brain regions - cerebral cortex, corpus striatum of ethanol treated rats with an increased HVA/DA, 5-HIAA/5-HT tumover rate. Dopamine content decreased in brainstem with an increased HVA/DA turnover rate and serotonin content decreased with an increased 5-HIAA/5-HT turnover rate in the brainstem of ethanol treated rats compared to control. Serotonin content increased in hypothalamus with a decreased 5-HIAA/5—HT turnover rate where as dopamine content decreased in hypothalamus with an increased HVA/DA tumover rate of ethanol treated rats compared to control.alterations of DA D2 and 5-HTQA receptor function and gene expression in the cerebellum, hypothalamus, corpus striatum, cerebral cortex play an important role in the sympathetic regulation of ALDH enzyme in ethanol addiction. There is a serotonergic and dopaminergic functional regulation of ALDH activity in the brain regions and liver of ethanol treated rats. Gene expression studies of DA D2 and 5'HT2A studies confirm these observations. Perfusion studies using DA, 5-HT and glucose showed ALDH regulatory function. Brain activity measeurement using EEG showed a prominentfrontal brain wave difference. This will have immense clinical significance in the management of ethanol addiction.