Unilateral stereotaxic injection of small amounts of the cholinotoxin, AF64A, caused minimal nonselective tissue damage and resulted in a significant loss of the presynaptic cholinergic markers ...3Hhemicholinium-3 (45% reduction) and choline acetyltransferase (27% reduction). No significant change from control was observed in tyrosine hydroxylase or tryptophan hydroxylase activity; presynaptic neuronal markers for dopamine- and serotonin-containing neurons, respectively. The AF64A lesion resulted in a significant reduction of dopamine D2 receptors as evidenced by a decrease in 3Hsulpiride binding (42% reduction) and decrease of muscarinic non-M1 receptors as shown by a reduction in 3HQNB binding in the presence of 100 nM pirenzepine (36% reduction). Saturation studies revealed that the change in 3Hsulpiride and 3HQNB binding was due to a change in Bmax not Kd. Intrastriatal injection of AF64A failed to alter dopamine D1 or muscarinic M1 receptors labeled with 3HSCH23390 and 3Hpirenzepine, respectively. In addition, no change in 3Hforskolin-labeled adenylate cyclase was observed. These results demonstrate that a subpopulation of muscarinic receptors (non-M1) are presynaptic on cholinergic interneurons (hence, autoreceptors), and a subpopulation of dopamine D2 receptors are postsynaptic on cholinergic interneurons. Furthermore, dopamine D1, muscarinic M1 and 3Hforskolin-labeled adenylate cyclase are not localized to striatal cholinergic interneurons.
Poly (ADP-ribose) polymerase (PARP-1) is a nuclear enzyme that facilitates DNA repair and may be important in neuronal cell death in a variety of diseases including cardiac arrest/cardiopulmonary ...resuscitation (CPR) and stroke. This chapter briefly reviews the characteristics of PARP, its activation, and its role in both focal (stroke) and global (cardiac arrest/CPR) cerebral ischemia. We demonstrate PARP deficiency to be effective in reducing neuronal reperfusion injury after stroke and cardiac arrest/CPR in mouse models. These data suggest a selective role of PARP-1 in glutamate excitotoxicity and that strategies of inhibiting PARP-1 may offer substantial neuroprotection in focal and global cerebral ischemia.
Cortical choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), tryptophan hydroxylase (TPH), muscarinic receptors and sodium-dependent, high-affinity, choline uptake (SDHACU) sites were ...examined in the rat brain following unilateral stereotaxic injection of the cholinotoxin, AF64A, into the nucleus basalis magnocellularis (NBM). Injection of AF64A resulted in a significant loss of presynaptic cholinergic markers in the cortex without alteration in TH and TPH activity. The binding to SDHACU sites was reduced to background values in the NBM and increased in the central amygdala (Ce) and cortex. The increase in cortical 3HQNB binding was the result of a change in muscarinic receptor number (BMAX) and not a change in receptor affinity (KD). Examination of muscarinic receptor subtypes demonstrated a reduction of M1 receptor binding in the cortex and NBM without any alteration in the Ce. Non-M1 binding was significantly increased in all the laminae of the cortex and in the Ce, but decreased in the NBM. These data suggest that there exists a population of M1 receptors on NBM projections to the cortex and that NBM projections influence a population of postsynaptic receptors in the cortex and Ce which are not of the M1 subtype.
The autoradiographic distribution and density of muscarinic receptors was studied in the neostriatum of rats with long-term unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal ...dopaminergic pathway and in lesioned rats who had additionally received embryonic substantia nigra grafts in the dopamine denervated striatum. Muscarinic receptors were labeled with 3Hquinuclidinyl benzilate (QNB), M1 receptors were directly labeled with 3Hpirenzepine (PZ) and non-M1 receptors were labeled by the competition of 100 nM PZ with 3HQNB. The density and distribution of muscarinic receptors were directly compared to the sodium-dependent, high-affinity, choline uptake sites as labeled with 3Hhemicholinium-3 (HC-3). In the 6-OHDA-lesioned animals, there was a 25% reduction in muscarinic receptors labeled with 3HQNB. Subtype analysis showed that there was a reduction of both M1 (-26%) and non-M1 (-33%) receptors. A normal density of both muscarinic receptor populations was found in animals with successful transplants. Saturation analysis demonstrated that the changes, in muscarinic receptor density, were due to a change in receptor number (Bmax) and not affinity (Kd). There was no significant change in 3HHC-3 binding in the 6-OHDA-lesioned or transplanted animals, indicating that alterations in muscarinic receptors were not due to transynaptic degeneration of striatal cholinergic interneurons. The findings of downregulation of muscarinic receptors following long-term dopamine denervation and the subsequent normalization of muscarinic receptor density after fetal mesencephalic transplantation suggests that transplanted substantia nigra cells are able to restore inhibitory control on striatal cholinergic interneurons.
Excitotoxicity is a common pathological process in many neurodegenerative disorders; and this process involves over-stimulation of glutamate receptors and an excessive influx of calcium into cells. ...Cell death in excitotoxicity is unique in that it does not involve caspase dependent pathways. Overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) is an early pathological event in excitotoxicity that leads to a unique form of cell death called parthanatos. Biochemical events in parthanatos include early accumulation of poly (ADP-ribose) (PAR) and nuclear translocation of apoptosis inducing factor (AIF) from the mitochondria.
Stereotaxic injection of AF64A, into the medial septum of the rat, resulted in significant loss of presynaptic cholinergic markers in this structure. No significant change was observed for the ...presynaptic neuronal markers for dopamine- and serotonin-containing neurons in either the medial septum or hippocampus. The AF64A lesion also resulted in a significant reduction of muscarinic receptors as demonstrated by a loss of 3HQNB binding in the medial septum. Subtype analysis showed the decrease of receptor binding in the medial septum to be due to a loss of M1 receptors as well as other muscarinic receptor subtypes. In the hippocampal formation, 3Hhemicholinium-3 binding was significantly reduced in the molecular layer of the dentate gyrus, and in the stratum oriens and stratum radiatum of the hippocampus. AF64A lesion resulted in a significant increase (Bmax) in non-M1 muscarinic receptors in hippocampal stratum oriens, in areas CA2, CA3, and CA4. AF64A lesion of the medial septum did not result in muscarinic receptor alterations in any other region of the hippocampal formation examined. These results indicate that postsynaptic muscarinic receptors in the stratum oriens of the CA2 to CA4 region of the hippocampus mediate primarily the function of the cholinergic cell bodies of the medial septum. These receptors are not of the M1 subtype.
Unilateral fimbria-fornix lesions were made by aspiration in female Sprague-Dawley rats. In a group of these rats, fetal septal tissue was transplanted into the lesion cavity. Lesion of the ...fimbria-fornix resulted in a reduction of cholinergic input to the hippocampal formation as indicated by the loss of acetylcholinesterase (AChE)-positive staining in all ipsilateral hippocampal laminae and a loss of 3Hhemicholinium-3 binding to cholinergic terminals in the strata oriens (82% reduction) and radiatum (77% reduction) of areas CA2 and CA3 and in the molecular layer of the dentate gyrus (83% reduction). In contrast, the density of muscarinic receptor binding (3HQNB) increased in the strata oriens (80% increase) and radiatum (70% increase) in areas CA2-CA4. This was shown to be due to an actual increase in receptor number (Bmax) and not to a change in affinity (KD). Analysis of muscarinic receptor subtypes indicated that the increase in receptor binding in the stratum radiatum was of the M-1 subtype (3H-pirenzepine) and in the stratum oriens was of the M-2 subtype (3HQNB + 100 nM pirenzepine). In the host hippocampus after fetal septal graft, the staining for AChE, the binding of 3Hhemicholinium-3, and the binding of muscarinic receptors (both the M-1 and M-2 receptor subtypes) were all comparable to nonlesioned control values. These data indicate that the fetal septal grafts have reinnervated the host hippocampus and have made synaptic contact with host cells in a manner capable of regulating postsynaptic muscarinic receptors.
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