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PubMed1996

Effects of electrical stimulation and morphine microinjection into periaqueductal gray on 5-hydroxyindole oxidation current in spinal cord of cats.

1. The effects of electrical stimulation and microinjection of morphine into the periaqueductal gray (PAG) on the 5-hydroxyindole oxidation current (280-300 mV) in laminae I-VI (800-2500 microns) of the spinal cord were examined using in vivo voltammetry in anesthetized cats. 2. Electrical stimulation of the PAG (PAG-S) both enhanced and attenuated the current. PAG-S increased the signal by 13.3 +/- 3.7% (laminae I-II: 800-1200 microns) or 19.0 +/- 3.6% (laminae V-VI: 1700-2500 microns) in comparison to control values. Attenuation by PAG-S decreased the signal by 15.3 +/- 2.6% (laminae I-II) or 13.3 +/- 2.0% (laminae V-VI) of control values. Naloxone antagonized signal enhancement by PAG-S. 3. Morphine (10 micrograms/microliter) microinjected into the PAG significantly increased the height of the signal (laminae I-II: 15.0 +/- 3.4%, laminae V-VI: 12.2 +/- 1.5%). Enhancement by microinjected morphine was antagonized by naloxone. In contrast, microinjected morphine also significantly decreased the signal by 10.4 +/- 2.7% (laminae I-II) and by 10.3 +/- 1.3% (laminae V-VI) of control values. 4. Microinjection of morphine and electrical stimulation of the PAG was observed both to enhance and attenuate the oxidation current of 5-hydroxyindole in the superficial and deeper dorsal horn. PAG may function to regulate the RVM-spinal serotonergic pathway, which modulates the transmission of nociceptive messages at the spinal cord.

Animals↗

PubMed2000

Focal microinjection of carbachol into the periaqueductal gray induces seizures in the forebrain of the rat.

Previous studies have reported that the repetition of running-bouncing and tonic-clonic seizures mediated by brainstem structures eventually elicits seizure activity in the forebrain. The purpose of the present study was to determine if the periaqueductal gray (PAG) region is a component of the neural network through which brainstem seizures elicit forebrain seizures. Bilateral microinjection of 40 nmol carbachol into the PAG region of rats induced arrested, staring behavior accompanied by epileptiform electrocorticogram (ECoG) afterdischarge recorded from the parietal cortex. In two animals limbic seizure activity similar to kindled amygdala seizures was also induced. The carbachol effect was dose-related as the 40 nmol dose induced a significantly greater duration of ECoG afterdischarge than a 20 nmol dose. The carbachol effect was mediated by muscarinic receptors as bilateral 50 nmol atropine microinjection 1 min prior to 40 nmol carbachol microinjection inhibited all seizure activity. Immunohistochemical detection of the proto-oncogene c-fos was used to verify that seizure activity was induced in forebrain regions. Rats with seizures induced by PAG carbachol microinjections exhibited dense c-fos-like immunoreactivity in the dentate gyrus but not the CA(1) or CA(3) regions, amygdala, piriform cortex, perirhinal cortex or hypothalamus. In addition, PAG microinjection of 10 nmol N-methyl-D-aspartic acid (NMDA) induced wild-running convulsions while 400 pmol bicuculline induced clonic spasms, myoclonic activity or limbic seizures. These results indicate that stimulation of the PAG, a brainstem structure, is sufficient to induce forebrain seizures. Since the forebrain seizures were induced by a single carbachol administration, it is proposed that the PAG serves as a pathway for caudal-rostral seizure generalization.

Animals↗

PubMed2003

Cardiovascular responses to microinjection of AMPA into the rostral commissural nucleus tractus solitarius of awake rats.

Previous studies have shown that microinjection of NMDA or non-NMDA receptor agonists into the nucleus tractus solitarius of anesthetized rats produces bradycardia and hypotension. In the present study, we evaluated the autonomic components of the cardiovascular responses to the microinjection of alpha-amine-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) into the nucleus tractus solitarius of awake rats. AMPA (0.05 nmol 50 nl(-1)) was microinjected into the nucleus tractus solitarius before and at 2, 10, 30 and 180 min after intravenous injection of methylatropine (2 mg kg(-1)). The control response to the microinjection of AMPA into the lateral aspect of the commissural nucleus tractus solitarius consisted of bradycardia and hypotension, (-190+/-20 bpm and -49+/-7 mm Hg), which were effectively blocked at 2 min (-5+/-2 bpm and -4+/-5 mm Hg), 10 min (-3+/-2 bpm and -6+/-3 mm Hg) and 30 min (-9+/-4 bpm and -5+/-5 mm Hg) after methylatropine. Although the bradycardic and hypotensive responses remained significantly reduced 180 min after injection of methylatropine, the responses tended to normalize. The data show that microinjection of AMPA into the rostral commissural nucleus tractus solitarius of awake rats produced intense bradycardia essentially mediated by parasympathetic excitation, which was the cause of the concomitant hypotensive response.

Animals↗

PubMed2002

Exogenous DNA expression in eukaryotic cells following microinjection.

Microinjection of nucleic acids, DNA, RNA, proteins, and any soluble material into living eukaryotic cells makes it possible to design experiments focused on single cells. In contrast facilitated transfer protocols requires hundreds of thousands of cells from which the expressed gene or intracellular effect must be detected within the culture. In addition to the immediate observable nature of the expressed product and intracellular reaction, microinjection bypasses the uptake toxicity associated with facilitated transfer of foreign material into cultured cells. The direct injection of material into the nucleus or cytoplasm allows the number of treated cells to be monitored and expression efficiencies to be observed directly. Microinjection of a hundred cells grown on small glass coverslips and subsequently counted for expression of the foreign material determines expression efficiency as a percentage of cells injected. The efficiency is based on detection of the foreign inserted gene product and does not control for relative promoter efficiency between constructs. The purpose is not to compare two constructs to each other but to monitor dual expression. The creation of marker fluorescent proteins, such as the green fluorescent protein (GFP) in the same expression plasmid with a test gene allows the immediate observation of the GFP injected cells and within the same cells the positive or negative expression of the test gene. Expression of a foreign gene, such as SV40 T antigen cloned into an expression vector can be detected four hours after microinjection of the DNA. Fusing GFP into the same expression region of the T coding sequence labels T-GFP as a fusion protein with characteristic T immunological staining nuclear patterns but allows the cells to be studied without fixation through sequential periods of observation. The direct nature of microinjection allows comparison of gene expression in a variety of cells and the determination of the number of cells expressing the exogenous material in relationship to the number of cells injected.

Animals↗

PubMed1983

Erythrocyte-mediated microinjection, a technique to study protein degradation in muscle cells.

The technique of erythrocyte-mediated microinjection has been successfully adapted for use with cultured muscle cells. Erythrocytes were fused with primary chick myotube cultures with poly(ethylene glycol), and fluorescent antibodies to haemoglobin demonstrated that this protein was injected into the sarcoplasm of myotubes. The microinjection treatment did not significantly alter protein metabolism in the muscle cells as monitored by rates of synthesis and degradation of muscle proteins. 125I-labelled ribonuclease A and bovine serum albumin were degraded with the expected exponential decay kinetics after microinjection into muscle cells, and the half-life of ribonuclease A (40 h) was approximately twice that of bovine serum albumin (17 h). The degradation of ribonuclease A in the muscle cells was enhanced 1.6-fold in the absence of horse serum and chick-embryo extract, whereas the degradation of bovine serum albumin was not altered during deprivation. These results are characteristic of the breakdown of microinjected ribonuclease A and bovine serum albumin in other cell types. Therefore, our experiments indicate the erythrocyte-mediated microinjection is a valid technique to study protein degradation in primary chick muscle cultures.

Animals↗

PubMed1988

Degradation of native and modified forms of fructose-bisphosphate aldolase microinjected into HeLa cells.

The uptake and degradation of radiolabelled rabbit muscle fructose-bisphosphate aldolase (EC 4.1.2.13) was studied in HeLa cells microinjected by the erythrocyte ghost fusion system. Labelled aldolase was progressively modified by treatment with GSSG or N-ethylmaleimide (NEM) before microinjection to determine whether these agents, which inactivate and destabilize the enzyme in vitro, affect the half-life of the enzyme in vivo. Increasing exposure of aldolase to GSSG or NEM before microinjection increased the extent of aldolase transfer into the HeLa cells and decreased the proportion of the protein that could be extracted from the cells after water lysis. Some degradation of the GSSG- and NEM-inactivated aldolases was observed in the ghosts before microinjection; thus a family of radiolabelled proteins was microinjected in these experiments. In spite of the above differences, the 40 kDa subunit of each aldolase form was degraded with a half-life of 30 h in the HeLa cells. In contrast, the progressively modified forms of aldolase were increasingly susceptible to proteolytic action in vitro by chymotrypsin or by cathepsin B and in ghosts. These studies indicate that the rate of aldolase degradation in cells is not determined by attack by cellular proteinases that recognize vulnerable protein substrates; the results are most easily explained by a random autophagic process involving the lysosomal system.

Cathepsin B↗

PubMed2000

Effects of glutamate agonist versus procaine microinjections into the basal forebrain cholinergic cell area upon gamma and theta EEG activity and sleep-wake state.

Serving as the ventral, extra-thalamic relay from the brainstem reticular activating system to the cerebral cortex, basal forebrain neurons, including importantly the cholinergic cells therein, are believed to play a significant role in eliciting and maintaining cortical activation during the states of waking and paradoxical sleep. The present study was undertaken in rats to examine the effects upon electroencephalogram (EEG) activity and sleep-wake state of inactivating basal forebrain neurons with microinjections of procaine versus activating them with microinjections of agonists of glutamate, which is the primary neurotransmitter of the brainstem reticular activating system. Microinjections into the basal forebrain were performed using a remotely controlled device in freely moving, naturally sleeping/waking rats during the day when they are asleep the majority of the time. Procaine produced a decrease in gamma (30-60 Hz) and theta (4-8 Hz) EEG activities, and an increase in delta (1-4 Hz) associated with a loss of paradoxical sleep, despite the persistence of slow wave sleep. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) produced an increase in gamma and a decrease in delta, while eliciting waking. In addition, NMDA, which has been shown in vitro to induce rhythmic bursting in the cholinergic cells, significantly increased theta activity. Following the microinjections of NMDA, c-Fos protein, which has been shown to reflect neural activity, was found in numerous cholinergic, and also GABAergic (gamma-aminobutyric acid) and other non-cholinergic neurons, in the substantia innominata and magnocellular preoptic nucleus near the microinjection cannulae. These results substantiate the role of cholinergic, possibly together with other, basal forebrain neurons in cortical activation, including elicitation of gamma and theta activities that underlie cortical arousal during waking and paradoxical sleep.

Acetylcholine↗

PubMed2000

Opioid tolerance induced by metamizol (dipyrone) microinjections into the periaqueductal grey of rats.

Non-opioid analgesics have been shown to elicit antinociception by an action upon central nervous system structures, in addition to their well known action upon peripheral tissues. Microinjection of metamizol (dipyrone), a widely used nonopioid analgesic, into the periaqueductal grey matter (PAG) of rats activates pain-modulating systems in the nucleus raphe magnus and inhibits spinal nociceptive neurons and the tail-flick reflex. Since these effects involve an activation of endogenous opioidergic systems, the possibility that metamizol induces opioid tolerance was investigated. Microinjection of metamizol into the ventrolateral PAG in awake rats induced antinociception, as demonstrated in the heat-elicited tail flick and hot plate tests. When microinjected into the ventrolateral PAG twice daily for 2 days, metamizol induced tolerance, i.e. a progressive loss of its antinociceptive effect. In contrast to rats repeatedly microinjected with saline, metamizol-tolerant rats were also tolerant to morphine microinjection into the same PAG site, and displayed signs of opioid withdrawal upon systemic administration of naloxone. These and other results suggest that metamizol activates endogenous opioid systems and that nonopioid analgesics may, by an action upon the central nervous system, lead to opioid tolerance and the risk of opioid withdrawal.

Animals↗

PubMed1984

Microinjected pBR322 stimulates cellular DNA synthesis in Swiss 3T3 cells.

When pBR322 is manually microinjected into the nuclei of quiescent Swiss 3T3 cells it stimulates the incorporation of [3H]thymidine into DNA. The evidence clearly shows that this increased incorporation that is detected by in situ autoradiography in microinjected cells represents cellular DNA synthesis and not DNA repair or plasmid replication. The effect is due to pBR322 and not due to impurities, mechanical perturbances due to the microinjection technique, or aspecific effects. This stimulation is striking in Swiss 3T3 cells. Some NIH 3T3 cells show a slight stimulation, but hamster cells, derived from baby hamster kidney (BHK) cells, are not stimulated when microinjected with pBR322. The preliminary evidence seems to indicate that the integrity of the pBR322 genome is important for the stimulation of cellular DNA synthesis in quiescent Swiss 3T3 cells. These results, although of a preliminary nature, are of interest because they indicate that a prokaryotic genome may alter the cell cycle of mammalian cells. From a practical point of view the stimulatory effect of microinjected pBR322 on cellular DNA synthesis has a more immediate interest, because pBR322 is the vector most commonly used for molecular cloning and 3T3 cells are very frequently used for gene transfer experiments.

Animals↗

PubMed1984

Artifacts caused by cell microinjection.

The effects of microinjection on Rana pipiens oocytes were determined using cryomicrodissection to measure Na, K, water, and injected radiolabeled sucrose (in gelatin) in the nucleus, animal, and vegetal ooplasm and injected bolus (reference phase, RP). The results point to potential problems in the interpretation of microinjection experiments. When oocytes were injected and incubated in Ringer's solution, nucleus, ooplasm, and RP lost K and sucrose and gained Na. Patterns of loss and gain were complex but were consistent with continuous solute leakage at the injection site causing artifactual intracellular diffusion gradients. In spite of leakage, oocytes completed scheduled meiotic maturation when exposed to progesterone. When oocytes were microinjected and incubated in paraffin oil (a medium in which polar solutes cannot exchange), nuclear and ooplasmic Na, K, and water concentrations remained identical to those in uninjected cells. Neither microinjection per se nor the injected bolus affected intraoocytic solute distributions. These findings imply that, after microinjection in aqueous media, metabolites are lost from and redistribute in cells, and that these artifactual changes are inadequately reflected in the ability of the cell to carry out a complex process. They also show that injection artifacts can be avoided by injecting and incubating cells under paraffin oil.

Animals↗

PubMed1997

Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-xL has activity independent of cytochrome c release.

Bcl-xL, an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types. Recent reports have demonstrated that cytochrome c is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation of proapoptotic caspases and subsequent cell death. Furthermore, it has been demonstrated that Bcl-2 can prevent the release of cytochrome c from mitochondria in cells triggered to undergo apoptosis. This has led to the hypothesis that the antiapoptotic effects of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation. In the present report, we use microinjection techniques to investigate the relationship between cytochrome c release, induction of apoptosis, and Bcl-xL activity in intact cells. We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis. In contrast, MCF7 breast carcinoma cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32), are totally resistant to microinjection of cytochrome c. However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores cytochrome c sensitivity. Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase-dependent manner in response to either tumor necrosis factor or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-xL expression. Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-xL. Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochrome c release is not sufficient to induce death in all cells. Importantly, the ability of Bcl-xL to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria.

Apoptosis↗

PubMed1990

Microinjected centromere [corrected] kinetochore antibodies interfere with chromosome movement in meiotic and mitotic mouse oocytes.

Kinetochores may perform several functions at mitosis and meiosis including: (a) directing anaphase chromosome separation, (b) regulating prometaphase alignment of the chromosomes at the spindle equator (congression), and/or (c) capturing and stabilizing microtubules. To explore these functions in vivo, autoimmune sera against the centromere/kinetochore complex are microinjected into mouse oocytes during specific phases of first or second meiosis, or first mitosis. Serum E.K. crossreacts with an 80-kD protein in mouse cells and detects the centromere/kinetochore complex in permeabilized cells or when microinjected into living oocytes. Chromosome separation at anaphase is not blocked when these antibodies are microinjected into unfertilized oocytes naturally arrested at second meiotic metaphase, into eggs at first mitotic metaphase, or into immature oocytes at first meiotic metaphase. Microtubule capture and spindle reformation occur normally in microinjected unfertilized oocytes recovering from cold or microtubule disrupting drugs; the chromosomes segregate correctly after parthenogenetic activation. Prometaphase congression is dramatically influenced when antikinetochore/centromere antibodies are introduced during interphase or in prometaphase-stage meiotic or mitotic eggs. At metaphase, these oocytes have unaligned chromosomes scattered throughout the spindle with several remaining at the poles; anaphase is aberrant and, after division, karyomeres are found in the polar body and oocyte or daughter blastomeres. Neither nonimmune sera, diffuse scleroderma sera, nor sham microinjections affect either meiosis or mitosis. These results suggest that antikinetochore/centromere antibodies produced by CREST patients interfere with chromosome congression at prometaphase in vivo.

Anaphase↗

PubMed1992

Pathway of incorporation of microinjected lamin A into the nuclear envelope.

When microinjected into the cytoplasm of 3T3 cells, biotinylated human lamin A rapidly enters the nucleus and gradually becomes incorporated into the nuclear lamina region as determined by immunofluorescence. The incorporation of the microinjected material takes several hours and progresses through a series of morphologically identifiable stages. Within minutes after microinjection, lamin A is found in spots distributed throughout the nucleus, except in nucleolar regions. Over a time course of up to 6 h, these spots appear to decrease in size and number as the biotinylated lamin A becomes associated with the endogenous nuclear lamina. Eventually, the typical nuclear rim staining pattern normally revealed by immunofluorescence with nuclear lamin antibodies is seen with antibiotin. This latter rim staining property is passed on to daughter cells following mitosis. These results indicate that the microinjected biotinylated nuclear lamin A retains those properties required for its integration into the lamina, as well as those necessary for the disassembly and subsequent reassembly of the nuclear lamina during cell division. The initial rapid accumulation into foci and the subsequent slower incorporation into the nuclear lamina appear to be analogous to the stages of incorporation following the microinjection of cytoskeletal intermediate filament proteins such as vimentin and keratin (Vikstrom, K., G. G. Borisy, and R. D. Goldman. 1989. Proc. Natl. Acad. Sci. USA. 86:549-553; Miller, R. K., K. Vikstrom, and R. D. Goldman. 1991. J. Cell Biol. 113:843-855). Foci are also observed in some uninjected cells using nuclear lamin antibodies, indicating that these features are a genuine component of nuclear substructure. Evidence is presented that shows the appearance of these nuclear structures is cell cycle dependent.

3T3 Cells↗

PubMed1984

Immunoprecipitation of nonerythrocyte spectrin within live cells following microinjection of specific antibodies: relation to cytoskeletal structures.

The intracellular precipitation of nonerythrocyte spectrin has been achieved by the microinjection into cells of either a monoclonal antibody (IgM) directed against the alpha chain of nonerythrocyte spectrin or an affinity-purified polyclonal antibody raised against bovine brain spectrin (fodrin). This antibody-induced precipitation of spectrin was observed in fibroblastic and epithelial cell types, including embryonic bovine tracheal fibroblasts, a bovine kidney epithelial cell line (MDBK), Hela cells, gerbil fibroma cells, and fibroblast lines of human and mouse origins. The precipitation of the spectrin was specific and two proteins with a similar distribution to the nonerythrocyte spectrin were not induced to co-precipitate in the spectrin aggregates. Comparing the two types of antibody microinjected, the affinity-purified polyclonal antibody resulted in more compact aggregates of spectrin and these were frequently aligned with microfilament bundles. The rate at which the spectrin aggregates were cleared into presumptive lysosomes varied with different cell types: in some such as the bovine kidney epithelial cells, this appeared complete within 3 h after microinjection, whereas in some of the fibroblasts the spectrin aggregates were prominent in the cytoplasm at 24 and even 48 h after microinjection. Microfilament bundles appeared unaffected by the aggregation of spectrin. We conclude that the integrity of the actin microfilament bundles does not require nonerythrocyte spectrin and that most probably these structures are linked at their termini to the membrane through proteins other than nonerythrocyte spectrin. No effect of the intracellular spectrin precipitation was observed on cell shape, or on the distribution of coated vesicles or microtubules. The aggregation of the nonerythrocyte spectrin, however, did affect the distribution of the vimentin type of intermediate filaments in most of the cell types studied. These filaments became more distorted and condensed, but generally did not collapse around the nucleus as occurs following microtubule disruption induced by colchicine treatment. The clumped intermediate filaments were frequently seen to coincide with regions of aggregated spectrin. This aggregation of intermediate filaments was not induced by microinjection of irrelevant antibodies, nor was it induced by the monoclonal antibody against spectrin in cells with which it did not cross-react.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Chromosomal analysis of human oocytes fertilized by microinjection of spermatozoa into the perivitelline space.

In-vitro fertilization by sperm microinjection techniques have been shown to be effective (the birth of one child has resulted), but concerns about the chromosomal normality of embryos derived from this process have been raised. The British Interim Licensing Authority has set, as a pre-condition to the clinical application of sperm microinjection, the chromosomal evaluation of pre-embryos derived by this technique. In this study, we demonstrate that four of 18 (22%) eggs fertilized by sperm microinjection into the perivitelline space are chromosomally abnormal as compared with nine of 30 (30%) fertilized by conventional IVF procedures. These results are not significantly different. In addition, we demonstrate that nine out of 20 couples who did not previously have fertilization in multiple cycles of IVF had between one and three oocytes fertilized by sperm microinjection. These findings demonstrate that sperm microinjection does not increase the incidence of chromosomally abnormal eggs and provide reassurance and support for the clinical implementation of a technique that appears to be effective for the treatment of certain forms of male infertility.

Aneuploidy↗

PubMed1984

Antinociception from a stereospecific action of morphine microinjected into the brainstem: a local or distant site of action?

Morphine (1 microgram) was microinjected into rats in the midline medullary nucleus raphe magnus (NRM); 1 mm lateral into nucleus reticularis paragigantocellularis (NRPG); 2 mm lateral into the VIIth nerve nucleus and 3 mm lateral into the Vth nerve nucleus. The time course of changes in the sensitivity to noxious heat was followed by the tail flick test. Significant and prolonged antinociception was seen following microinjection into NRPG. At sites 1 mm from NRPG very weak effects were seen and at 2 mm from NRPG no antinociception occurred. It is concluded that 1 microgram of morphine microinjected into the brainstem is unlikely to cause antinociception by entering the circulation and having effects at remote sites. The distance diffused by morphine to cause significant antinociception after microinjection of 1 microgram is less than 1 mm. Levorphanol (1 microgram) had very similar effects to morphine but dextrorphan and saline were ineffective. It is concluded that although the concentrations achieved following microinjections may be high, they are not excessive as the effects show stereospecificity. The concentrations of endogenous substances released into the synaptic cleft may also be high.

Analgesics↗

PubMed2004

Differential conditioned place preference responses to endomorphin-1 and endomorphin-2 microinjected into the posterior nucleus accumbens shell and ventral tegmental area in the rat.

An unbiased conditioned place preference (CPP) paradigm was used to evaluate the reward effects of endogenous mu-opioid receptor ligands endomorphin-1 (EM-1) and endomorphin-2 (EM-2) from the mesolimbic posterior nucleus accumbens (Acb) shell and the ventral tegmental area (VTA) in CD rats. EM-1 (1.6-8.1 nmol) microinjected into posterior Acb shell produced CPP, whereas EM-2 (8.7-17.5 nmol) given into the same Acb shell produced conditioned place aversion (CPA). EM-1 (1.6-16.3 nmol) microinjected into the VTA produced CPP, whereas EM-2 (8.7 and 17.5 nmol) given into the same VTA site did not produce any effect, but at a high dose (35 nmol) produced CPP. EM-1 (3.3 nmol) or EM-2 (17.5 nmol) microinjected into the nigrostriatal substantia nigra was not significantly different from vehicle-injected groups. D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) at 94.13 pmol or 3-methoxynaltrexone at 0.64 pmol microinjected into the posterior Acb shell blocked EM-1-induced CPP and EM-2-induced CPA. At a higher dose, CTOP (941.3 pmol) and 3-methoxynaltrexone (6.4 pmol) produced CPA and CPP, respectively. Coadministration with antiserum against dynorphin A(1-17) (Dyn) (10 microg) microinjected into the posterior Acb shell blocked EM-2-induced CPA. However, it did not affect EM-1-induced CPP. It is concluded that EM-1 and EM-2 produce site-dependent CPP and CPA, respectively, by stimulation of different subtypes of mu-opioid-receptors; stimulation of one subtype of mu-opioid-receptor at the posterior Acb shell and VTA by EM-1 induces CPP, whereas stimulation of another subtype of mu-opioid receptor at the posterior Acb shell, but not the VTA, by EM-2 induces the release of Dyn to produce CPA.

Animals↗

PubMed1992

TRH analogue microinjected into specific medullary nuclei stimulates gastric serotonin secretion in rats.

Brain nuclei involved in vagal release of gastric serotonin (5-HT) were investigated in urethan-anesthetized rats acutely implanted with gastric cannula. Gastric secretions were collected every 10-15 min for 2 h, and measurement of serotonin 5-HT was performed by high-performance liquid chromatography (HPLC) with electrochemical detection and that of acid by titration. The stable thyrotropin-releasing hormone (TRH) analogue, RX 77368, microinjected into the dorsal vagal complex (DVC) (1.5-77 pmol/50 nl) induced a dose-related and long-lasting increase in gastric 5-HT and acid output. TRH (276 pmol) microinjected into the DVC also stimulated gastric 5-HT secretion. TRH action was abolished by vagotomy. RX 77368 (77 pmol) increased 5-HT release after microinjection into the raphe obscurus, raphe pallidus, and nucleus ambiguus, although the responses were significantly lower compared with that induced by DVC microinjection. No changes in gastric 5-HT release were observed when the peptide was microinjected into the lateral hypothalamus, hypoglossal, paramedian reticular, inferior olive, lateral paragigantocellular or spinal trigeminal nucleus. The potent gastric 5-HT release induced by TRH or TRH analogue into the DVC added to the presence of TRH receptors and TRH-LI nerve terminals in the DVC suggest a role of medullary TRH in the vagal regulation of gastric 5-HT release.

Animals↗