Dissertations / Theses on the topic 'Rat cholinergic neurones'

Many basal forebrain and mesopontine tegmental cholinergic projection systems tend to overlap in their origins. This raises the possibility that these projection systems are collateralized to innervate divergent areas. In experiment one, the degree to which basal forebrain and mesopontine tegmental neurons that innervate the reticular thalamic nucleus have axons that collateralize to innervate the cortex as well was examined with a retrograde fluorescence labeling method combined with immunohistochemistry. A significant portion of the labeled neurons in the region of the nucleus basalis magnocellularis and pedunculopontine tegmental nucleus projecting to the reticular thalamic nucleus were observed to be also labeled (double-labeled) following intracortical tracer injections. Many of these double-labeled neurons displayed choline acetyltransferase choline acetyltransferase immunoreactivity. It was also shown that numerous basal forebrain neurons that innervated the reticular thalamic nucleus contained the calcium-binding protein, parvalbumin. These neurons tended to be located more rostrally than the ChAT immunoreactive neurons; primarily in the region of the ventral pallidum. There was some indication that parvalbumin-containing neurons in the basal forebrain that innervate the reticular thalamic nucleus also have axons that branch to innervate the cortex. Finally, none of the basal forebrain neurons innervating the reticular thalamic nucleus was found to contain somatostatin. In experiment two, the degree to which basal forebrain neurons have axons that collateralize to innervate the interpeduncular nucleus and hippocampus was examined with retrograde fluorescence labeling methods. Labeled neurons projecting to both of these limbic structures were observed only occasionally. Comparison of the distribution of single labeled neurons innervating each of these structures revealed that within the region of origin, in the horizontal limb of the diagonal band, neurons innervating the interpeduncular nucleus tended to be located dorsally to those innervating the hippocampus. The results of these experiments are discussed in relation to their anatomical and functional implications toward a greater understanding of the basal forebrain and mesopontine cholinergic and non-cholinergic projection systems.<br>Medicine, Faculty of<br>Graduate

The two neuron types that initially degenerate with Alzheimer's Disease are the cholinergic projections from the septum to the hippocampus and from the substantia innominata to the cortex, and the somatostatinergic neurons in the hippocampus and cortex. The functional relationship between these two types of neurons was investigated using folic acid, a neuro-excitant, and cysteamine, a somatostatin depleter. Folic acid causes a neuron to fire at a much higher rate than normal (Spector, 1971). Folic acid was injected into either the septum or the substantia innominata, and the long-term effect of the resulting acute hyperactivity of the cholinergic neurons on somatostatin neurons was measured as somatostatin-like immunoreactivity in the hippocampus and cortex. Glutamic acid decarboxylase activity, a marker for gamma-amino butyric acid (GABA) neurons, was also measured because it has been shown to decrease in the cortex after injection of folic acid into the substantia innominata. The administration of folic acid to the cholinergic neurons did not have a significant long-term effect on somatostatin-like immunoreactivity nor glutamic acid decarboxylase activity; therefore, a hyperactivity of the cholinergic neurons did not result in degeneration of GABAergic nor somatostatinergic neurons. Cysteamine causes a short-term depletion of somatostatin. Cysteamine was injected subcutaneously and the effect of an acute decrease of brain somatostatin on the cholinergic neurons was studied by measuring high affinity choline uptake, an indicator of cholinergic activity. Administration of cysteamine had no measured effect on high affinity choline uptake in the hippocampus or frontal cortex; therefore, a depletion of somatostatin did not effect cholinergic activity. The assay for high affinity choline uptake was tested by injection of pentobarbital, a drug known to decrease high affinity choline uptake. We detected a decrease in high affinity choline uptake after pentobarbital administration, indicating that if cysteamine were decreasing high affinity choline uptake, the assay would have detected it.<br>Master of Science

Etude in vivo chez le rat adulte et âgé à l'aide d'approches multidisciplinaires des caractéristiques des neurons cholinergiques des voies septo-hippocampique et basalo-corticale. Les caractéristiques physiologiques ont été décrites par enregistrements extra et intracellulaires, les propriétés pharmacologiques par applications iontophorétiques, les modifications de ces propriétés ont été étudiées au cours du vieillissement. Enfin une étude anatomique par marquage histologique à la peroxydase a été menée pour définir la voie basalo-corticole.

Les systèmes cholinergique et dopaminergique jouent un rôle prépondérant dans les fonctions cognitives. Ce rôle est exercé principalement grâce à leur action modulatrice de l’activité des neurones pyramidaux du cortex préfrontal. L’interaction pharmacologique entre ces systèmes est bien documentée mais les études de leurs interactions neuroanatomiques sont rares, étant donné qu’ils sont impliqués dans une transmission diffuse plutôt que synaptique. Ce travail de thèse visait à développer une expertise pour analyser ce type de transmission diffuse en microscopie confocale. Nous avons étudié les relations de microproximité entre ces différents systèmes dans le cortex préfrontal médian (mPFC) de rats et souris. En particulier, la densité des varicosités axonales en passant a été quantifiée dans les segments des fibres cholinergiques et dopaminergiques à une distance mutuelle de moins de 3 µm ou à moins de 3 µm des somas de cellules pyramidales. Cette microproximité était considérée comme une zone d’interaction probable entre les éléments neuronaux. La quantification était effectuée après triple-marquage par immunofluorescence et acquisition des images de 1 µm par microscopie confocale. Afin d’étudier la plasticité de ces relations de microproximité, cette analyse a été effectuée dans des conditions témoins, après une activation du mPFC et dans un modèle de schizophrénie par déplétion des neurones cholinergiques du noyau accumbens. Les résultats démontrent que 1. Les fibres cholinergiques interagissent avec des fibres dopaminergiques et ce sur les mêmes neurones pyramidaux de la couche V du mPFC. Ce résultat suggère différents apports des systèmes cholinergique et dopaminergique dans l’intégration effectuée par une même cellule pyramidale. 2. La densité des varicosités en passant cholinergiques et dopaminergiques sur des segments de fibre en microproximité réciproque est plus élevée comparé aux segments plus distants les uns des autres. Ce résultat suggère un enrichissement du nombre de varicosités axonales dans les zones d’interaction. 3. La densité des varicosités en passant sur des segments de fibre cholinergique en microproximité de cellules pyramidales, immunoúactives pour c-Fos après une stimulation visuelle et une stimulation électrique des noyaux cholinergiques projetant au mPFC est plus élevée que la densité des varicosités de segments en microproximité de cellules pyramidales non-activées. Ce résultat suggère un enrichissement des varicosités axonales dépendant de l’activité neuronale locale au niveau de la zone d'interaction avec d'autres éléments neuronaux. 4. La densité des varicosités en passant des fibres dopaminergiques a été significativement diminuée dans le mPFC de rats ayant subi une déplétion cholinergique dans le noyau accumbens, comparée aux témoins. Ces résultats supportent des interrelations entre la plasticité structurelle des varicosités dopaminergiques et le fonctionnement cortical. L’ensemble des donneès démontre une plasticité de la densité locale des varicosités axonales en fonction de l’activité neuronale locale. Cet enrichissement activité-dépendant contribue vraisemblablement au maintien d’une interaction neurochimique entre deux éléments neuronaux.<br>The cognitive functions of the rat medial prefrontal cortex (mPFC) are modulated by ascending modulatory systems such as the cholinergic and dopaminergic afferent systems. However, despite the well-documented pharmacological interactions between the cholinergic and dopaminergic afferents and pyramidal cells in the PFC, there is only scarce neuroanatomical data on the reciprocal interrelationships between these neuronal elements in the mPFC. This might be due to the diffuse rather than synaptic transmission mode of intercellular communication of the cholinergic system in the mPFC. For these reasons, the neuroanatomical relationships between the cholinergic and dopaminergic systems and pyramidal cells in the mPFC are examined, with an emphasis on the local density of the cholinergic and dopaminergic axon varicosities. To analyze the plasticity of these interrelationships, the two systems were examined in condition of increased neuronal activity in the mPFC, or of decrease dopaminergic activity in a model of schizophrenia. The microproximity relationships between cholinergic and dopaminergic fibers as well as with pyramidal cells were studied in the mPFC of rats and mice. In particular, the number of axon varicosities in cholinergic and dopaminergic fiber segments within 3 µm from each other or from pyramidal cells were quantified. This microproximity was considered as a possible interaction zone between two neuronal elements. Quantification was performed using triple immunofluorescence labeling and acquisition of 1 µm optic sections using confocal microscopy. To assess the plasticity of these relationships, the analysis has been performed in control condition as well as after a cortical activation or a decreased dopaminergic input in a schizophrenia model. Our results demonstrate a neuroanatomical convergence of cholinergic and dopaminergic fibers on the same pyramidal cell from layer V (output) of mPFC, suggestinggests the integration of different types of inputs by the same pyramidal cell, which may be transmitted to subcortical areas to execute prefrontal cognitive control. Close apposition between cholinergic and dopaminergic fibers could also be seen in the mPFC. There was an increase of the density of cholinergic and dopaminergic en passant varicosities on those fiber segments within microproximity of each other, compared to those outside the reciprocal microproximity, supporting functional importance of the close apposition between those two ascending neuromodulatory systems into the mPFC. There was enrichment of cholinergic en passant varicosities on the fiber segments within microproximity of c-Fos activated pyramidal cells in the mPFC of visually and HDB electrically stimulated rats, indicating association between axonal varicosity density and the local neuronal activity. There was decrease of dopaminergic en passant varicosities in the mPFC of rats with ChAT depletion in the N.Acc., compared to controls. This evidence supports the association between dopaminergic axonal varicosities and relevant neuronal activity in a complex neuronal network. This thesis shows that the density of cholinergic and dopaminergic axonal varicosity density in the mPFC is influenced by and contributes to the relevant local neuronal activity from the interactions of different transmitter systems. Such interactions of different systems in a complex and intricate prefrontal neuronal network endeavour to maintain the delicate balance for cognitive processes.

碩士<br>長庚大學<br>基礎醫學研究所<br>93<br>The suprachiasmatic nucleus (SCN) is the master pacemaker in mammals, with two anatomically and functionally distinct divisions of dorsal (dSCN) and ventral SCN (vSCN). In the SCN, both muscarinc and nictonic cholinergic receptors have been shown to be present and acetylcholine acts directly on SCN neurons. In this study, I used the cell-attached recording technique to investigate the effects of cholinergic agents on the SCN neurons, focusing on the time-dependent responses of both dSCN and vSCN neurons. I found that cholinergic agents altered the spontaneous firing rate (SFR) and the effects exhibited a circadian rhythm. Comparing the response profiles of muscarine (Musc) and nicotine (Nict) to that of carbachol (CCh) indicated that the carbachol responses were most likely mediated by the mAChR such as M1-mAChR on the dSCN neurons. The cholinergic responses of dSCN and vSCN neurons were in a similar way. However, the muscarinic responses of dSCN and vSCN neurons differed during the early night and during the low and high concentration. These data suggested that cholinergic effects on the neurons of the SCN might play an important role.

碩士<br>臺灣大學<br>動物學研究所<br>98<br>Acetylcholine (ACh) is one of principal neurotransmitters involved in pain modulation. Many behavioral studies have shown that central or peripheral ACh administrations can evoke analgesia, and have proved that cholinergic agonists can serve as a synergistic role of α2 adrenergic receptors-mediated antinociception in the spinal cord. Moreover, recent behavioral researches also indicate that there might be supraspinal interactions between muscarinic cholinergic system and noradrenergic (NAergic) pain descending pathway. Nevertheless, there is currently no direct evidence to support this argument. In this study, we investigated the effect of carbachol (CCh), a cholinergic agonist, on NAergic neurons of A7 catecholamine cell group, which projects NAergic fibers to the dorsal horn of the spinal cord to modulate nociceptive signaling. Whole-cell recordings were made from A7 neurons in voltage-clamp mode with membrane voltage clamped at -70 mV in brainstem slices taken from rat pups. Bath application of 25 μM CCh evoked inward currents, which were blocked by 1.5 μM atropine, a muscarinic acetylcholine receptor (mAChR) antagonist, suggesting that carbachol-induced currents (ICCh) were mediated through mAChR. Furthermore, ICCh were significantly attenuated with the existence of high concentration of himbacine, a dose-selective antagonist of mAChRs, showing that mAChRs on NAergic A7 neurons activated by CCh were M1-like mAChRs. Surprisingly, the ICCh were not blocked with internal administration of GDP-β-S, a non-catalytic analogue of GDP, suggesting that the ICCh were G-protein-independent. Bath application of U73122, a phospholipase C inhibitor, slightly but significantly blocked the ICCh, showing that phospholipase C was not the major participant in ICCh. The ICCh were reversed at about -12.6 mV and blocked by extracellular application of NMDG substituted for Na+, showing that ICCh were caused through opening a nonselective cation channel, presumably by transient receptor potential (TRP) channels. Indeed, ICCh were significantly attenuated by several antagonists of TRP channels, including 2APB, SKF96365 and ruthenium red. Besides, high frequency stimulation at pedunculopontine tegmental nucleus (PPTg) evoked an inward current partially blocked by atropine, suggesting PPTg projected their axons to NAergic A7 neurons. There was an auto-inhibition in PPTg-A7 synaptic transmission. These results indicate that mAChR modulate the NAergic A7 neurons via activating TRP channels without the requirement of G-protein and phospholipase C, and there is endogenous ACh released from PPTg onto NAergic A7 neurons. The above results provide an evidence of supraspinal interaction between muscarinic cholinergic system and NAergic descending pain pathway.

Body size in multicellular organisms is determined by the integartion of two factors: the rate of growth and the duration of growth. In most animals, the rate of growth is controlled cell autonomously by the insulin-stimulated Pi3 kinase (Pi3K) pathway. However, the duration of growth is controlled in a more complex manner that involves endocrine factors that act cell non-autonomously. For example, in insects such as Drosophila, the duration of each larval phase is regulated by the timing of release of the molting hormone ecdysone from the prothoracic gland (PG). The molecular mechanisms by which the rate of growth and the duration of growth are integrated remain poorly understood. To help shed light in this area, I have investigated the intracellular signaling events that regulate ecdysone release in the Drosophila PG. I have found that expressing activated Ras, or the targets of Ras signaling Raf or Pi3K, in the PG reduces fly size and accelerates larval development via precocious synthesis and release of ecdysone. In contrast, expression of dominant-negative (dn) Ras, Raf, or Pi3K increases fly size and prolongs larval development via delayed synthesis and release of ecdysone. These results indicate that Ras-Raf and Pi3K signaling act in the PG to regulate the duration of growth by altering the timing of ecdysone synthesis and release. Conversely, I have found that expressing dn-Ras or dn-Raf, but not dn-Pi3K, in cholinergic neurons increases fly size and prolongs larval development, whereas, expression of activated Ras or Raf, but not Pi3K, in cholinergic nerurons decreases fly size, but delays larval development. Inhibition of insulin signaling in flies, via chromosomal loss-of-function mutations, also decreases fly size and delays development, raising the possibility that Ras-Raf signaling in cholinergic neurons may affect fly size by controlling the rate of growth via systemic insulin signaling.

碩士<br>國立臺灣大學<br>動物學研究所<br>100<br>Acetylcholine (ACh) is one of principal neurotransmitters involved in pain modulation. By behavioral researches, it has been found that there might be some supraspinal interactions between muscarinic cholinergic system and noradrenergic (NAergic) pain descending pathway. Moreover, morphological and electrophysiological data indicates that the cholinergic neuron group CH5, which distributes most of its neurons in pedunculopontine tegmental nucleus (PPTg), projects its fibers to downstream NAergic neurons of A7 catecholamine cell group, which further project NAergic fibers to the dorsal horn of the spinal cord to modulate nociceptive signaling. By electrically stimulating PPTg, glutamatergic projection to NAergic A7 neurons can be recorded and further inhibited by DNQX. In this study, excitatory postsynaptic currents (EPSCs) on NAergic A7 neurons evoked by electrical stimulation at PPTg were recorded under whole-cell patch clamp with membrane voltage at -70 mV. By application of solution with high extracellular calcium concentration, the proportion of asynchronous neurotransmitter release, which is believed to allows for the modulation of postsynaptic excitability and the alteration of action potential firing patterns increased. This result suggests that the change of extracellular calcium concentration, which in turn influences the calcium influx and neurotransmitter release, may provide a way for neural activity regulation. Under current-clamp mode, delayed stimulation-evoked action potentials was blocked by EGTA-AM, indicates that asynchronous neurotransmitter release could not only modulates neural activity but communicate with downstream neurons. Previous studies showed that PPTg might regulate downstream NAergic A7 neurons through M1-like mAChRs. However, application of atropine, a non-selective mAChR antagonist, increased stimulation-evoked asynchronous neurotransmitter release, suggesting that there might be other types of mAChR between PPTg and NAergic A7 neurons, and these mAChRs might modulate downstream NAergic A7 neurons by blocking the glutamatergic neurons projected from PPTg. By applying genistein with the concentration to be a tyrosine kinase inhibitor, the inward current induced by carbachol, a cholinergic agonist did not changed, indicating that the mAChRs might not transmit signals by activating the Src family of tyrosine kinases (SFKs).