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PubMed1995

Effect of microinjection time during postfertilization S-phase on bovine embryonic development.

Microinjection into bovine zygotes was performed to evaluate the effects of the timing of injection during the phase of DNA replication on the subsequent in vitro development of embryos and expression of injected chicken beta-actin promoter-lac Z gene construct. The period of DNA replication of bovine zygotes, determined by 3H-thymidine incorporation, begins between 12 hr and 13 hr postinsemination (hpi) of in vitro matured oocytes, reaches a maximum from 17 hpi to 19 hpi, and is complete by 21-22 hpi. Aphidicolin, an inhibitor of DNA polymerase alpha, was used to synchronize the pronuclei and the zygote population. Treatment with aphidicolin at 9-18 hpi arrested DNA replication without affecting formation of the pronuclei or embryo development. Cycloheximide, an inhibitor of protein synthesis, was used for nucleocytoplasmic resynchronization of the aphidicolin-treated zygotes. Microinjection was performed at 15 (early), 18 (mid), and 21 (late S phase) hpi. Embryonic development was affected following each of the three microinjection times. The development of zygotes injected at 18 hpi was significantly higher (P < 0.01) after 5 days of culture than those injected at 15 hpi or 21 hpi. Expression of the marker gene was observed in the higher stage of development (> 16 cells) only in the zygotes injected at 18 hpi. At the earlier stages of development, the proportions of embryos showing expression of the foreign gene were the same for all microinjection times. In aphidicolin- and cycloheximide-treated zygotes, expression of the marker gene followed the same curve as development, i.e., expression was low when injected early or late and higher (P < 0.005) when injected in the middle of zygotic S phase. The ability of the embryos to survive microinjection and to express the marker gene as a function of hpi seems to be influenced mostly in the cytoplasm processing stage rather than the pronuclei processing stage.

Animals↗

PubMedDate not supplied

Dynamic analysis of drug action on in vitro reconstituted thyroid follicle by microinjection of tracer molecules and videomicroscopy.

Thyroid cells isolated from the gland by trypsinization are capable in culture of reconstituting histiotypic structures, the thyroid follicles. This morphological differentiation requires the presence of the main thyroid regulator; thyrotropin. We have analyzed some structural and functional aspects of in vitro reconstituted thyroid follicles (RTF) using microinjection of fluorescent probes and videomicroscopy. This experimental approach allowed to visualize biological processes and actions of drugs, signalling factors, etc. in living cells. We describe here some examples of what can be studied with this powerful still-undervalued method. Microinjection of a cell-impermeant fluorescent probe of either high or low molecular mass into the lumen of RTF allowed to check the tightness of this compartment and therefore to analyze the control of tight junctions assembly. A small cell-impermeant probe like Lucifer Yellow microinjected into a cell was used to demonstrate and then to study the regulation of cell to cell communication via gap junctions. The presence of calcium in the lumen of RTF was detected by microinjection of a properly designed probe: Calcium Green which becomes fluorescent in the presence of the ligand. The lumen to cell transport or endocytosis of thyroglobulin, the thyroid prohormone, which is stored into the lumen of the follicles, is currently studied by microinjection of TRITC-labeled thyroglobulin. Coupled to image processing and videorecorder systems, kinetic analysis and quantitative measurements can be performed.

Animals↗

PubMed1996

Microinjection technique for pharmacological evaluation of microvascular permeability in human skin.

A new technique which combines skin microinjection of minute amounts of drugs (0.5 microliter) and measurement of capillary permeability by intravital fluorescence videomicroscopy and densitometry is introduced. Glass micropipettes with a tip diameter of 7-9 microns are inserted by a micromanipulator into the stratum papillare containing the capillary loops and used for microinjection by microsyringe and special dispenser. Transcapillary diffusion of sodium fluorescein applied by intravenous bolus injection is visualised by fluorescence videomicroscopy and stored on videotape. Perivascular fluorescent light intensity (FLI) is measured in arbitrary units (AU) by videodensitometry around the site of microinjection during playback of the videotapes. The method was tested by microinjection of 0.5 microliter histamine (1% solution) at the distal tibial plateau. Mean FLI values representing microvascular permeability were 2186 AU 10 min after microinjection of histamine, 420 AU after physiologic saline and 1228 AU after histamine combined with oral intake of 20 mg cetirizine. Cetirizine significantly reduced (P < 0.01) the increased permeability induced by histamine. However, the mean values after injection of the H1-blocker were still significantly enhanced (P < 0.01) when compared to the mean values observed after injection of physiologic saline solution, which provoked only minor microtrauma. The technique offers new scope for pharmacological testing in man.

Adult↗

PubMed1987

Erythrocyte-mediated microinjection of horseradish peroxidase into BHK cells. Subcellular localization by light and electron microscopy.

In order to establish the distribution with time of proteins microinjected into mammalian cells, horseradish peroxidase (HRP) was microinjected into baby hamster kidney (BHK) cells using chicken erythrocyte ghosts. At time intervals following initiation of fusion between ghosts and target cells, samples were fixed with aldehydes and the peroxidase visualized by reaction with diaminobenzidine and viewing by light and electron microscopy. At 10 min, the reaction product was observed within the cytoplasm of 60% of the microinjected cells, but was excluded from the nucleus and membranous organelles. In the other 40% of microinjected cells, the reaction product was also observed within the nucleus. At 30 min, the reaction product was observed to be evenly distributed throughout the cell, including the nucleus but excluded from organelles. By 6 h, the reaction product was present almost exclusively within the nucleus of 63% of microinjected cells. At all time points, 20-30% of the erythrocytes ghosts appear to have been taken up by cells by phagocytosis rather than fusion, as evidenced by the presence of peroxidase reaction product within intact and fragmented erythrocyte ghosts in the cytoplasm of target cells. Cells incubated with a lanthanum solution following fusion excluded this electron dense tracer, indicating that the cytoplasmic compartment is not opened during exposure to polyethylene glycol.

Animals↗

PubMed1990

Swallowing responses induced by microinjection of glutamate and glutamate agonists into the nucleus tractus solitarius of ketamine-anesthetized rats.

Swallowing is a patterned motor activity generated by neurons located within the nucleus tractus solitarius (NTS). An excitatory amino acid (EAA) neurotransmitter, such as glutamate (GLU), is suspected of being involved in the initiation of swallowing by NTS neuronal components. However, swallowing can still be elicited in animals anesthetized with ketamine, an antagonist of the N-methyl-D-aspartate (NMDA) subclass of EAA receptors. The present experiments were therefore designed to investigate the influence of EAA administration within the NTS on the swallowing motor activity of rats anesthetized with ketamine. Pressure microinjections of GLU in doses ranging from 25 to 500 pmol elicited swallowing. This effect was dose-dependent and was not reproduced when control injections of the vehicle solution were performed. Microinjections of the GLU agonists, quisqualate (QUIS) and NMDA, in doses ranging between 2.5 and 50 pmol, also induced swallowing motor activities. QUIS, like GLU, elicited a short series of swallows at a brief latency while NMDA generated long-lasting rhythmic swallowing with a longer latency. Swallowing induced by GLU microinjections (100 pmol) was suppressed almost completely by local pretreatment with either the broad spectrum EAA receptor antagonist, gamma-D-glutamylglycine (250 pmol), or the more selective non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (50-100 pmol), but not by pretreatment with the selective NMDA antagonist, DL-2-amino-5-phosponovalerate (250 pmol). On the other hand, pretreatment with DL-2-amino-5-phosphonovalerate (50 pmol) suppressed the deglutitions induced by NMDA microinjections (10 pmol) but not those elicited by QUIS microinjections (10 pmol).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Cardiovascular responses elicited by microinjection of monoamines into mesencephalic nucleus dorsalis raphe in cats.

The effect of monoaminergic agonists and antagonists microinjected into mesencephalic nucleus dorsalis raphe has been studied on blood pressure and heart rate to elucidate the nature and role of these monoaminergic receptors in cardiovascular regulation. Microinjection of monoamines, noradrenaline, phenylephrine and 5-hydroxytryptamine (5-HT) into nucleus dorsalis raphe elicited hypertension and tachycardia which could be blocked by local pretreatment with piperoxan (an alpha-adrenoceptor blocker) and methysergide (a 5-HT receptor blocker) respectively. However, isoprenaline microinjections failed to evoke any response. Bilateral vagotomy did not prevent these cardiovascular responses evoked by monoamines microinjection, while cervical spinal cord (C1) transection with bilateral vagotomy prevented these responses. These monoaminergic receptors seem to be localized in nucleus dorsalis raphe since microinjection of monoamines into neural structures adjoining nucleus dorsalis raphe, failed to induce any cardiovascular response. Monoaminergic receptors are present in nucleus dorsalis raphe which modulate cardiovascular activity by influencing sympathetic preganglionic neurons in the intermediolateral columns of the spinal cord.

Adrenergic alpha-Agonists↗

PubMed1988

Microinjected ribonuclease A as a probe for lysosomal pathways of intracellular protein degradation.

There are multiple pathways of intracellular protein degradation, and molecular determinants within proteins appear to target them for particular pathways of breakdown. We use red cell-mediated microinjection to introduce radiolabeled proteins into cultured human fibroblasts in order to follow their catabolism. A well-characterized protein, bovine pancreatic ribonuclease A (RNase A), is localized initially in the cytosol of cells after microinjection, but it is subsequently taken up and degraded by lysosomes. This lysosomal pathway of proteolysis is subject to regulation in that RNase A is taken up and degraded by lysosomes at twice the rate when serum is omitted from the culture medium. Subtilisin cleaves RNase A between residues 20 and 21, and the separated fragments are termed RNase S-peptide (residues 1-20) and RNase S-protein (residues 21-124). Microinjected RNase S-protein is degraded in a serum-independent manner, while RNase S-peptide microinjected alone shows a twofold increase in degradation in response to serum withdrawal. Furthermore, covalent linkage of S-peptide to other proteins prior to microinjection causes degradation of the conjugate to become serum responsive. These results show that recognition of RNase A and certain other proteins for enhanced lysosomal degradation during serum withdrawal is based on some feature of the amino-terminal 20 amino acids. The entire S-peptide is not required for enhanced lysosomal degradation during serum withdrawal because degradation of certain fragments is also responsive to serum. We have identified the essential region to be within residues 7-11 of RNase S-peptide (Lys-Phe-Glu-Arg-Gln; KFERQ). To determine whether related peptides exist in cellular proteins, we raised antibodies to the pentapeptide. Affinity-purified antibodies to KFERQ specifically precipitate 25-35% of cellular proteins, and these proteins are preferentially degraded in response to serum withdrawal. Computer analyses of known protein sequences indicate that proteins degraded by lysosomes at an enhanced rate in response to serum withdrawal contain peptide regions related, but not identical, to KFERQ. We suggest two possible peptide motifs related to KFERQ and speculate about possible mechanisms of selective delivery of proteins to lysosomes based on such peptide regions.

Amino Acid Sequence↗

PubMed1994

Behavioral and frontal cortical metabolic effects of apomorphine and muscimol microinjections into the mediodorsal thalamic nucleus.

To study sensorimotor correlates of dopamine (DA) and gamma-amino butyric acid (GABA) neurotransmission in the thalamus, we microinjected the DA agonist apomorphine (APO), the GABA agonist muscimol and vehicle into the mediodorsal thalamic nucleus (MdT) of rats and monitored catalepsy, sensorimotor asymmetries and the acoustic startle response. Unilateral MdT muscimol microinjections (50 ng) produced a lateralization of the removal of adhesive disks placed simultaneously on both forelegs in a tactile extinction task, but did not measurably influence any aspects of startle behavior. The sensorimotor asymmetry consisted of perferential orientation to the adhesive disk on the side ipsilateral to the microinjection. Vehicle and APO microinjections produced no significant behavioral results. In a follow-up study, unilateral MdT muscimol microinjections significantly depressed medial prefrontal cortical metabolism (measured by 2-fluorodeoxyglucose uptake) by 24%, but did not affect nucleus accumbens metabolic activity. Together, these findings are consistent with the concept that GABA-mediated inhibition of thalamocortical neurons in the MdT influences tactile extinction behavior, most likely by selectively suppressing excitatory input to the frontal cortex. The sensorimotor asymmetry observed in the present study resembles attentional and spatial memory deficits associated with frontal cortical lesions, and in conjunction with the 2-fluorodeoxyglucose results, suggests that elevated GABA neurotransmission in the thalamus may be involved in attentional and functional metabolic deficits in humans.

Animals↗

PubMed1995

Reward shifts and motor responses following microinjections of opiate-specific agonists into either the core or shell of the nucleus accumbens.

Differences in pharmacology, anatomical connections, and receptor densities between the "core" and "shell" of the nucleus accumbens suggest that behavioral activity normally modulated by the accumbens, such as reward and motor functions, may be differentially regulated across the mediolateral axis. This study investigated the effects of opiate receptor-specific agonists on reward and motor functions in either the accumbens core or shell, using the intracranial self-stimulation (ICSS) rate-frequency curve-shift method. Microinjections of the mu opiate receptor-specific agonist, DAMGO (vehicle, 0.03 nmol, and 0.3 nmol), or the delta opiate receptor-specific agonist DPDPE (vehicle, 0.3 nmol, 3.0 nmol), were administered bilaterally in a random dose order with a minimum of 3 days between injections. Rats were tested over three consecutive 20-min rate-frequency curves immediately following a microinjection to investigate the time course of drug effects. Both opiate agonists decreased the ICSS frequency necessary to maintain half-maximal response rates when injected into the medial and ventral shell region of the accumbens. However, DAMGO microinjections into the lateral accumbens core or the control site of the caudate increased the frequency necessary to elicit half-maximal response rates, while DPDPE microinjections into these regions had no effect. Evaluation of motor effects show that administration of DAMGO resulted in a suppression of activity in all locations. In contrast, DPDPE microinjections resulted in little or no effect on lever pressing activity at any location.

Analgesics↗

PubMed1985

Red blood cell-mediated microinjection: methodological considerations.

Red blood cell-mediated microinjection is a powerful approach to introducing proteins into the cytoplasm of cultured cells. In the course of our microinjection studies of intracellular protein degradation, we have encountered several potential problems with certain proteins. The microinjection procedure may be accompanied by denaturation of protein by radiolabeling procedures, binding of protein to red cell ghosts during loading, degradation of protein by the red cell ghost prior to microinjection, and adsorption of protein that leaks from red cell ghosts in the presence of fusogen to the fibroblast monolayer. We conclude with a list of points that must be considered prior to use of red cell-mediated microinjection to study a particular protein.

Cells, Cultured↗

PubMed1986

Cardiorespiratory effects produced by microinjecting L-glutamic acid into medullary nuclei associated with the ventral surface of the feline medulla.

The purpose of our study was to use microinjections of L-glutamic acid to better localize the cell bodies in the intermediate area of the ventral medullary surface that exert control over cardiorespiratory activity. L-glutamic acid (200 nl of a 1-M solution) was microinjected into the nucleus paragigantocellularis lateralis, lateral reticular nucleus and into an area which is part of the 'glycine-sensitive area', which lies in the center of the intermediate area. Normally, when L-glutamic acid is applied to the surface of the intermediate area, increases in arterial pressure and tidal volume are observed. Increases in tidal volume were never observed upon microinjection into the 3 sites associated with the intermediate area, suggesting that the tidal volume change elicited from surface application occurs because of L-glutamic acid interacting with cell bodies either on the surface or extremely close to the surface. Pressor responses were elicited with microinjection of L-glutamic acid into the lateral reticular nucleus and the 'glycine-sensitive area', but not the nucleus paragigantocellularis lateralis; indeed, microinjection of L-glutamic acid into the nucleus paragigantocellularis lateralis caused hypotension. Hence, cell bodies responsible for raising arterial pressure may reside in either the lateral reticular nucleus or the 'glycine-sensitive area'.

Animals↗

PubMed1986

Facilitation of avoidance behavior by vasopressin fragments microinjected into limbic-midbrain structures.

Effects of arginine vasopressin (AVP1-9) and its behaviorally active fragments [Cyt6]AVP5-9 and [Cyt6]AVP5-8 were studied on the retention of one-trial learning passive avoidance behavior in rats. Peptides were microinjected into various limbic and midbrain structures (ventral or dorsal hippocampus or the dorsal raphe nucleus) and were administered either immediately after the learning trial (post-learning treatment) or shortly before the 24 h retention session (pre-retention treatment). Doses for intracerebral microinjections were selected after preliminary experiments with subcutaneous and intracerebroventricular peptide administration. AVP1-9 facilitated passive avoidance behavior when the peptide was microinjected into either brain structure, however, the ventral hippocampus appeared to be the most sensitive. In this limbic region, AVP1-9 facilitated passive avoidance behavior in an amount of 8 pg (bilaterally), both when given as post-learning or pre-retention treatment. [Cyt6]AVP5-9 and [Cyt6]AVP5-8 were more effective than the parent nonapeptide in terms that a lower amount of these peptide fragments facilitated passive avoidance behavior in all brain regions investigated. The ventral hippocampus appeared to be the most sensitive brain site for the behaviorally active vasopressin fragments as well. Following microinjections into the ventral hippocampus, [Cyt6]AVP5-8 was more effective in a post-learning than in a pre-retention treatment schedule. [Cyt6]AVP5-9 on the other hand was more effective when injected shortly before the retention trial. The data indicate that limbic-midbrain structures are sensitive to AVP1-9 and behaviorally active putative metabolites of this neuropeptide. The active fragments selectively influence different phases of information processing upon limbic microinjections.

Animals↗

PubMed1986

Cardiovascular effects of microinjections of angiotensin II into the nucleus tractus solitarii.

The role of the angiotensin II system within the nucleus tractus solitarii (NTS) in central cardiovascular control was investigated by local microinjections of angiotensin II and the angiotensin II receptor antagonist saralasin. Microinjections of 1 ng angiotensin II into the NTS resulted in a monophasic depressor response (-7.3 +/- 1.7 mm Hg), while higher doses were characterized by a biphasic response, with an initial decrease followed by a subsequent increase in blood pressure (10 ng: -4.7 +/- 1.3/+7.9 +/- 1.1 and 100 ng: -2.4 +/- 1.2/+7.5 +/- 1.2 mm Hg). Heart rate decreased significantly following microinjections of 1 and 10 ng angiotensin II (-27 +/- 5.0 and -15 +/- 5.9 bpm), while with 100 ng angiotensin II there was no significant effect on heart rate. Prior i.v. administration of atropine (1 mg/kg) abolished the bradycardia, but did not significantly affect the blood pressure response. Microinjections of saralasin into the NTS elicited a dose-dependent pressor response (10 ng: 6.0 +/- 1.5 mm Hg; 100 ng: 16.8 +/- 3.4 mm Hg) and tachycardia (10 ng: 5 +/- 3.2 bpm; 100 ng: 17 +/- 4.4 bpm). Our data support the hypothesis that angiotensin II acts on specific receptors within the NTS to modulate peripheral cardiovascular responses. The cardiovascular effects elicited by microinjections of the peptide exhibit complicated dose-response relationships. The effects on heart rate, but not on blood pressure, appear to be mediated by parasympathetic activation.

Angiotensin II↗

PubMed1988

Microinjection of GABA antagonists into the posterior hypothalamus elicits locomotor activity and a cardiorespiratory activation.

It is well known that electrical stimulation of an area (subthalamic locomotor region, STLR) of the posterior hypothalamus evokes locomotion as well as increases in cardiorespiratory activity. Uncertainty exists over whether these responses are due to stimulation of STLR neurons or to activation of fibers of passage originating outside this area. The purpose of the present study was to determine if stimulation of STLR neurons alone would elicit the cardiorespiratory and locomotor responses. Neurons were stimulated by microinjections of gamma-aminobutyric acid (GABA) antagonists (picrotoxin and bicuculline) into the posterior hypothalamus of anesthetized cats. Both picrotoxin and bicuculline produced increases in arterial pressure, heart rate and minute ventilation which were accompanied by locomotor movements of the limbs. Increases in arterial pressure, heart rate and phrenic nerve activity were also caused by picrotoxin microinjections in paralyzed, ventilated cats. Microinjections of GABA or a GABA agonist (muscimol) reversed all of these responses. In contrast, microinjection of GABA or muscimol into the STLR without a prior antagonist injection had only small, transient effects upon cardiorespiratory activity. However, microinjection of muscimol prevented the responses to a subsequent injection of bicuculline. These results indicate that: (1) stimulation of cell bodies alone in the subthalamic locomotor region of the hypothalamus produces all the cardiorespiratory and locomotor responses evoked by electrical stimulation; (2) the responses evoked by picrotoxin and bicuculline are due to an interaction with GABA receptors and (3) a GABAergic mechanism exerts a tonic depressive influence over the cardiorespiratory and locomotor systems by an action in the posterior hypothalamus.

Animals↗

PubMed1991

CRF microinjected into the dorsal vagal complex inhibits TRH analog- and kainic acid-stimulated gastric contractility in rats.

The effect of CRF microinjected into the dorsal vagal complex (DVC) on centrally-stimulated gastric contractility was investigated in fasted, urethane-anesthetized rats. Miniature strain gauge force transducers were acutely implanted on the corpus of the stomach and contractility was analyzed by computer. Microinjection of the stable thyrotropin-releasing hormone (TRH) analog, RX 77368, (26 pmol) into the DVC induced a 12.2-fold stimulation of gastric contractility within 30 min. Corticotropin-releasing factor (CRF) (63-210 pmol) microinjected into the DVC concomitantly with RX 77368 (26 pmol) induced a dose-related inhibition of stimulated gastric contractility. Neither CRF alone (210 pmol) nor vehicle modified basal gastric contractility. Microinjection of kainic acid (141 pmol) into the raphe pallidus nucleus induced a 3.6-fold stimulation of gastric contractility after 45 min. This stimulation was suppressed by bilateral microinjection of CRF (105 pmol/site) into the DVC. These results demonstrate that CRF acts in the DVC to inhibit centrally-stimulated gastric contractility and suggest that TRH and CRF may interact in the DVC to regulate gastric motor function.

Animals↗

PubMed1993

Antinociception produced by microinjection of L-glutamate into the ventromedial medulla of the rat: mediation by spinal GABAA receptors.

This study examined whether the antinociception produced by glutamatergic stimulation of neurons in the nucleus raphe magnus (NRM) or nucleus reticularis gigantocellularis pars alpha (NGCp alpha) is mediated by activation of GABAA receptors in the spinal cord. Two approaches were used. The first approach determined the ability of intrathecally (i.t.) administered bicuculline, a competitive GABAA receptor antagonist, to attenuate the antinociception produced by microinjection of L-glutamate in the NRM or NGCp alpha. Bicuculline was selected on the basis of an initial study that determined that 0.3 micrograms i.t. bicuculline caused a 3.4-fold rightward shift in the dose-effect relationship of the i.t.-administered GABAA agonist, isoguvacine, without producing allodynia or alterations in nociceptive threshold. The GABAA antagonist SR 95531 was judged unsuitable because it caused only a modest 1.7-fold rightward shift in the dose-effect relationship of isoguvacine at doses that did not produce allodynia. The second approach determined the ability of i.t. administered diazepam, a benzodiazepine receptor agonist, to enhance the antinociception produced by microinjection of L-glutamate in these nuclei. Diazepam was selected because of its ability to enhance the actions of GABA at the GABAA receptor. Rats were pretreated with i.t. administration of 0.3 micrograms bicuculline, 40 micrograms diazepam, or vehicle, after which 30 nmol L-glutamate was microinjected into the NRM or NGCp alpha. Microinjection of L-glutamate into the NRM or NGCp alpha in vehicle-pretreated rats significantly increased tail flick latency. The antinociception produced by microinjection of L-glutamate in the NGCp alpha was antagonized by bicuculline and enhanced by diazepam. In contrast, the antinociception evoked from sites in the NRM was only partially attenuated by bicuculline and was not enhanced by diazepam. In an ancillary experiment, i.t. administration of 0.3 micrograms bicuculline or 40 micrograms diazepam did not alter tail skin temperature or nociceptive threshold, suggesting that their effects on glutamate-induced antinociception were not secondary to alterations in tail skin temperature. Taken together, these results support the hypothesis that the antinociception produced by activation of neurons in the NGCp alpha, but not the NRM, is mediated in part by an action of GABA at GABAA receptors in the spinal cord.

Animals↗

PubMed1996

Microinjection of thyrotropin-releasing hormone analogue into the central nucleus of the amygdala stimulates gastric contractility in rats.

The effect on gastric contractility following bilateral microinjection of thyrotropin-releasing hormone (TRH) analog, RX 77368, into the central nucleus of the amygdala was examined in fasted, urethane-anesthetized rats. Extraluminal force transducers were used to measure gastric corpus contractility. Bilateral microinjection of RX 77368 (0.5 microgram, 1.0 microgram, n = 6 each) stimulated gastric contractility for up to 120 min post-injection, P < 0.05. Gastric contractility was not significantly stimulated by microinjection of 0.1 microgram RX 77368, 0.1% bovine serum albumin (BSA) into the central nucleus or RX 77368 (0.5 microgram, 1.0 microgram) into sites adjacent to the central nucleus. Peak responses (1.0 microgram) occurred 40 min post-injection and represented a 16-26-fold increase over basal values. The frequency of gastric contraction waves was attenuated for 0-90 min in rats receiving central amygdaloid microinjection of RX 77368 (0.1, 0.5 or 1.0 microgram) versus rats microinjected with the vehicle or RX 77368 into sites adjacent to the central nuclei. The stimulatory effect of RX 77368 (1.0 microgram) on gastric contractility was abolished by subdiaphragmatic vagotomy. These results indicate that the TRH analog, RX 77368, acts within the central amygdala to vagally stimulate gastric contractility.

Amygdala↗

PubMed1988

Persistence and replication of plasmid DNA microinjected into early embryos of Xenopus laevis.

The persistence and replication of defined circular and linear plasmid DNA molecules microinjected into fertilized eggs of Xenopus laevis were analyzed. For all plasmids tested, a small fraction of microinjected circular molecules was replicated; however, the overall copy numbers of either free form I or form II molecules usually did not increase through blastulation. In contrast, extensive amplification of input DNA sequences was seen whenever the microinjected DNA was assembled into high molecular weight concatemers. Moreover, the appearance and subsequent replication of injected sequences in high molecular weight DNA were enhanced when linear (form III), rather than circular, molecules were microinjected. The injected form III DNA was rapidly converted into long linear concatemers. All possible orientations of monomeric molecules within the concatemers were observed although, on occasion, head-to-tail orientations were favored. Long linear concatemers were replicated very efficiently, irrespective of the sequence of the input DNA. Form I and form II DNA molecules were also formed in the embryo from microinjected form III DNA. A small fraction of these circular forms was replicated, although overall copy numbers did not increase significantly. Form III molecules that remained monomeric were not observed to be replicated at all within our limits of detection. In some batches of embryos, form I and form II DNA molecules were replicated to the extent that overall copy number increased. Even in these cases, however, the amplification of long linear concatemers of the input DNA sequences was more efficient.

Animals↗