A pressure system for the microinjection of substances into the brain of awake monkeys
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Lateral hypothalamus
Methiodide
GABA receptor antagonist
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The present study evaluated the role of ventrolateral periaqueductal gray (vlPAG)-located orphanin-FQ (OFQ) in the opioid tolerance induced by repeated microinjections of morphine (MOR) into vlPAG. Microinjection of MOR (5 µg/0.5 µl) into vlPAG caused antinociception as quantified with the tail flick and the hot plate tests. When MOR microinjection was repeated twice daily, the antinociceptive effect disappeared within 2 days (tolerance). However, if MOR microinjection was preceded by the OFQ receptor antagonist nocistatin (NST; 1 ng/0.5 µl), the microinjections of MOR did not induce tolerance. If NST microinjections were suspended, subsequent MOR microinjections induced tolerance. In MOR-tolerant rats, a single NST microinjection into vlPAG was enough to restore the antinociceptive effect of MOR. Furthermore, if OFQ (1 ng/0.5 µl) was microinjected into vlPAG, then a MOR microinjection administered 15 min later into vlPAG did not elicit antinociception. Finally, opioid tolerance induced by repeated systemic MOR injections (5 mg/kg, i.p.) was reversed by a single microinjection of NST into vlPAG. This emphasizes the central importance of vlPAG-located OFQ in the MOR tolerance.
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SUMMARY 1. Activation of peripheral chemoreceptors with KCN in the working heart–brainstem preparation from young male Wistar rats (70–90 g) increases phrenic (PNA; +105 ± 18%) and thoracic (tSNA; +44 ± 6%) sympathetic nerve activity compared with baseline and reduces heart rate (HR; from 377 ± 27 to 83 ± 6 b.p.m.). 2. Microinjections of increasing doses of ATP (1, 5, 25, 100 and 500 mmol/L; n = 7) into the intermediate nucleus tractus solitarius (NTS) produced a dose‐dependent reduction in PNA (from −6 ± 3 to −82 ± 1%) and in HR (from −12 ± 4 to −179 ± 47 b.p.m.). Microinjections of ATP into the intermediate NTS also produced a reduction in tSNA (from −3 ± 3 to −26 ± 5%), which was not dose dependent. 3. Microinjections of ATP into the caudal NTS ( n = 5) produced a dose‐dependent increase in PNA (from 0.2 ± 3 to 115 ± 27%) and minor changes in HR and tSNA, which were not dose dependent. 4. The data show that microinjection of ATP into distinct subregions of the NTS produces different respiratory and autonomic responses and suggest that ATP in the caudal NTS is involved in the respiratory but not in the sympathoexcitatory component of the chemoreflex.
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The ventrolateral periaqueductal gray (PAG) is a key structure for the development of opioid tolerance. An increased activity of 'anti-opioids' like cholecystokinin (CCK) has been proposed as a possible mechanism for opioid tolerance. The present study evaluates the role of PAG-located CCK in the opioid tolerance induced by repeated microinjections of morphine (MOR) into PAG. Male rats were implanted with chronic guide cannulae aimed at the PAG. Microinjection of MOR (0.5 microg in 0.5 microl) into PAG caused antinociception as quantified with the tail flick and the hot plate tests. When MOR microinjection was repeated twice daily, the antinociceptive effect disappeared within 2 days (tolerance). However, if each MOR microinjection was preceded (within 15 min) by a microinjection of the non-selective CCK receptor antagonist proglumide (PRO), (0.4 microg in 0.5 microl) into the same PAG site, the microinjections of MOR always produced antinociception and did not induce tolerance. If PRO microinjections were suspended, subsequent MOR microinjections induced tolerance. In MOR-tolerant rats, a single PRO microinjection into the same PAG site was enough to restore the antinociceptive effect of MOR. On the other hand, if CCK (1 ng in 0.5 microl) was microinjected into PAG, then MOR microinjection administered 15 min later into the same PAG site did not elicit antinociception. These results show that CCK has anti-opioid activity in PAG and that tolerance to MOR in PAG can be prevented or reversed if CCK receptors are blocked with PRO. Finally, opioid tolerance induced by repeated systemic MOR injections (5mg/kg intraperitoneal ) was reversed by a single microinjection of PRO into PAG. This emphasizes the central importance of PAG in the MOR/CCK interactions that lead to opioid tolerance.
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Proglumide
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Drug tolerance
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Dorsal raphe nucleus
Raphe nuclei
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Local microinjection of met-enkephalin into both the nucleus reticularis paragigantocellularis (Pgc) and into the midbrain periaqueductal gray (PAG) altered the spontaneous discharge of many nociresponsive units in the nucleus raphe magnus (NRM) of the urethane-anesthetized rat. The activity of the majority of units (11 of 14) was suppressed following microinjections into both the PAG and Pgc. Two units showed an increase in spontaneous activity after PAG microinjections and a decrease after Pgc microinjections. One cell showed a decrease in activity after microinjection into Pgc and was unaffected by the PAG microinjection. These findings demonstrate that many nociresponsive units in NRM are affected by microinjections of met-enkephalin into both Pgc and the PAG, indicating a convergent influence of these two regions on single NRM neurons.
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Nucleus raphe magnus
Periaqueductal gray
Raphe nuclei
Dorsal raphe nucleus
Raphe
Met-enkephalin
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INTRODUCTIONThe quantitative microinjection of drugs, antibodies, toxins, and manipulated RNAs and proteins into Drosophila embryos--the "pharmacological approach"--provides a unique opportunity to analyze cellular functions in the developing embryo, and provides spatial and temporal resolution that is not readily available through genetic studies. These studies require that the observed effects reflect a dose-response relationship so that the data can be accurately interpreted. Quantitative microinjections can be readily achieved with the addition of a fluorescent tracer to the solution to be injected. Analysis of the resulting integrated fluorescent intensity following injection can then be used to determine the volume and hence the concentration of the solution injected. This protocol outlines the procedures for the microinjection and quantification of aqueous solutions during high-resolution observation of early development in the Drosophila embryo.
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Rostral ventrolateral medulla
Medulla
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