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PubMed2003

Angiotensin II modulates the cardiovascular responses to microinjection of NPY Y1 and NPY Y2 receptor agonists into the nucleus tractus solitarii of the rat.

The aim of this work was to investigate the modulation of the cardiovascular effects of neuropeptide Y (NPY) in the nucleus tractus solitarii (NTS) by angiotensin II (Ang II) and to determine the NPY receptor subtype involved in this modulation. Anesthesized Sprague-Dawley rats received microinjections in the NTS of Ang II (threshold and ED(50) doses) with NPY Y(1) agonist Leu(31)Pro(34)NPY and NPY Y(2) agonist NPY(13-36) (threshold and ED(50) doses). The changes in mean arterial pressure (MAP) and heart rate (HR) recorded in the femoral artery were analyzed during 60 min after the microinjections. The injection of threshold doses of Ang II, Y(1) agonist or Y(2) agonist alone did not produce any change in cardiovascular parameters. However, the co-injections into the NTS of threshold doses of both Ang II and the Y(1) agonist elicited significant increases of MAP and HR of about 12 and 10%, respectively. The co-administration of threshold doses of Ang II with the Y(2) agonist also induced a significant vasopressor response. The vasodepressor and bradycardiac effect of an ED(50) dose of the Y(1) agonist was significantly counteracted (P<0.01) by a threshold dose of Ang II. The vasopressor effect elicited by an ED(50) dose of the Y(2) agonist was significantly enhanced by a threshold dose of Ang II (P<0.01). No significant change of cardiovascular responses elicited by an ED(50) dose of Ang II was observed in the presence of threshold doses of the Y(1) agonist or of the Y(2) agonist. The present study gives functional evidences for a differential modulatory activity of Ang II on the cardiovascular responses mediated by Y(1) and Y(2) receptor subtypes, which may be of relevance for central cardiovascular regulation in the NTS.

Angiotensin II↗

PubMed1997

Antinociception induced by opioid or 5-HT agonists microinjected into the anterior pretectal nucleus of the rat.

The changes in the latency for tail withdrawal in response to noxious heating of the skin induced by microinjection of opioid or serotonergic agonists into the anterior pretectal nucleus (APtN) was studied in rats. The mu-opioid agonist DAMGO (78 and 156 picomol), but not the delta-opioid agonist DADLE (70 and 140 pmol), the kappa-opioid agonist bremazocine (0.24 and 0.48 nanomol) or the sigma-opioid agonist N-allylnormetazocine (0.54 nanomol), produced a dose-dependent antinociceptive effect. The 5-HT1 agonist 5-carboxamidotryptamine (19 and 38 nanomol) and the 5-HT1B agonist, CGS 12066B (1.12 and 2.24 nanomol), but not the non-selective 5-HT agonist m-CPP (41 to 164 nanomol), 5-HT2 agonist alpha-methylserotonin (36 and 72 nanomol) and 5-HT3 agonist 2-methylserotonin (36 and 72 nanomol), produced a dose-dependent antinociceptive effect. These results indicate that the antinociceptive effects of opioid or serotonergic agonists microinjected into the APtN depend on drug interaction with local mu or 5-HT1B receptors, respectively.

Analgesics↗

PubMed1998

Microinjection of GABA-A agonist muscimol into the dorsal but not the ventral hippocampus impairs non-mnemonic measures of delayed non-matching-to-position performance in rats.

Anatomical studies of afferent and efferent connections suggest that the hippocampus may have more than one information processing role that varies along the septo-hippocampal axis. The present study was conducted to test whether a distinct functional specialization for the dorsal versus ventral extents of the hippocampus could be detected. The effects of a GABA-A receptor agonist, muscimol, microinjected into either the dorsal or ventral hippocampus on an operant, spatial delayed non-matching-to-position (DNMTP) task were measured. A decrease in the number of trials completed per session and disruption of several DNMTP discrimination parameters were produced by muscimol microinjection into the dorsal hippocampus but not into the ventral hippocampus. Muscimol injected into either site did not impair the measure of working memory, delayed choice accuracy. These results are consistent with the view that hippocampal function varies along the septo-temporal axis, and that the dorsal hippocampus is relatively more critical to visual discrimination performance than the ventral hippocampus.

Animals↗

PubMed1998

Morphine microinjected into the nucleus raphe magnus does not block the activity of spinal trigeminal nucleus oralis convergent neurons in the rat.

This study investigated the effects of morphine microinjection into the nucleus raphe magnus (RMg) on electrically evoked C-fiber activities of convergent neurons in the spinal trigeminal nucleus oralis (Sp5O), in halothane-anesthetized rats. Although the neurons could be depressed by systemic morphine (6 mg/kg, i.v.) in a naloxone-reversible fashion, morphine microinjected into the RMg (2. 5 microgram or 5 microgram) neither depressed their C-fiber-evoked responses, nor the diffuse noxious inhibitory controls acting on them. It is concluded that the RMg is not involved in reinforcing descending inhibitory controls that are tonic or triggered by noxious stimuli acting on Sp5O convergent neurons.

Animals↗

PubMed1999

Anatomical basis for cannabinoid-induced antinociception as revealed by intracerebral microinjections.

Cannabinoids suppress behavioral and neurophysiological responses to noxious stimuli in rodents when administered systemically. The purpose of this study was to extend previous studies of the site of cannabinoid analgesia. Rats were tested in the tail flick test before and after microinjections of the cannabinoid agonist WIN55, 212-2 (5 microg) into one of 17 different brain regions. WIN55,212-2 significantly elevated tail-flick latencies when injected into the amygdala, the lateral posterior and submedius regions of the thalamus, the superior colliculus and the noradrenergic A5 region. By contrast, pain behavior was unaffected by microinjections of the cannabinoid into the other 11 areas examined (prefrontal cortex, nucleus accumbens, lateral hypothalamus, substantia nigra, cuneiform nucleus, anterior pretectal, intralaminar, parafasicular, posterior, thalamic nuclei, as well as the ventral medial, ventral lateral nuclei in the posterior thalamus).

Amygdala↗

PubMed2000

U-69,593 microinjection in the infralimbic cortex reduces anxiety and enhances spontaneous alternation memory in mice.

The present report investigated the contributions of the ventromedial prefrontal cortex to the control of spontaneous alternation/working memory and anxiety-related behaviour. In Experiment 1, we examined the effects of microinjections of the selective kappa(1) receptor agonist, U-69,593, in the infralimbic cortex (IL) of CD-1 mice on several ethologically-derived anxiety indices in the elevated plus-maze (EPM) and defensive/withdrawal (D/W) anxiety in the open field, as well as on memory in the EPM transfer-latency (T-L) test and implicit spontaneous alternation memory (SAP) in the Y-maze. In week 1, pretreatment with one injection of vehicle, 1, 10 or 25 nmol/1.0 microliter U-69,593 in the IL dose-dependently prolonged T-L and produced a dose-dependent anxiolytic behavioural profile in the first EPM trial. Following a 24-h delay, the same mice were given a drug-free second trial in the EPM tests of T-L memory and anxiety. Whereas T-L memory was not disturbed, small but detectable carry-over effects were observed in trial-2 EPM behaviour relative to vehicle-treated animals. In week 2, the same groups of mice were again pretreated with one injection of the same doses of U-69,593 in the IL and given a D/W test in an open field, followed immediately by an 8-min SAP trial in the Y-maze. The smallest U-69,593 dose was anxiolytic in the D/W test, and SAP/working memory was dose-dependently enhanced in the Y-maze. In Experiment 2, we evaluated whether 0.5 microliter volume microinjections would produce comparable behavioural and carry-over effects in the IL of three new groups of CD-1 mice, in the event that the 1.0 microl volume injections used in Experiment 1 diffused beyond the IL and therefore may have confounded some effects. Experiment 2 procedures were carried out in the same manner as in Experiment 1, except the animals were tested in reverse order. Thus in week 1, SAP memory was tested in the Y-maze followed by D/W anxiety in the open field for half of the animals in each group, and the other half was tested in reverse order. In week 2, T/L memory and anxiety were tested in the EPM in 2 trials as described in Experiment 1. Pretreatment with one injection of vehicle, 10 or 25 nmol/0.5 microliter U-69,593 in the IL reduced D/W anxiety and enhanced SAP memory regardless of testing order in week 1. In week 2, the same groups of mice were again pretreated with one injection of the same doses of U-69,593 in 0.5 microliter volumes in the IL and tested in the EPM. In a similar fashion to Experiment 1, U-69,593 dose-dependently prolonged T/L and produced an anxiolytic behavioural profile in the first EPM trial. Following a 24-h delay, T/L recall memory was again not significantly influenced, but a robust anxiolytic behavioural profile was observed in the second drug-free anxiety trial in the EPM relative to vehicle-treated animals. Results are discussed relative to a) injection volumes and testing order, b) the possible influence kappa receptors may exert on neurochemical responsivity to anxiety-provoking environments in the IL area of the mPFC, c) the possibility that kappa-mediated anxiolysis from the IL in CD-1 mice results from interactions with neurochemical systems involved in the blunting of incoming anxiety-provoking information, d) evidence that SAP memory may be an implicit subtype of working memory, and e) the possibility that IL implicit working memory processes may modulate the induction and expression of anxiety-related behaviour.

Animals↗

PubMed1999

Bombesin microinjection into the basolateral amygdala influences feeding behavior in the rat.

It has been demonstrated that the basolateral amygdala (ABL) represents a satiety mechanism. Experimental data indicate that peripheral or central applications of neuropeptide bombesin (BN) and BN-like peptides inhibit feeding. Since the amygdala (AMY) is rich in BN-like immunoreactive elements, the present study was performed to determine whether 10 or 40 ng doses of BN microinjected bilaterally into the ABL could modify solid food intake. Twenty nanograms of BN (10 ng per injection site) in 24-h deprived rats caused transient inhibition of food intake and 80 ng resulted in a significant reduction of food consumption for 1 h. This inhibitory effect of BN on feeding was eliminated by prior BN antagonist treatment. Results of behavioral tests showed that BN microinjections into the ABL specifically reduced food intake without altering behavioral patterns or influencing the body temperature. Present results suggest that BN-like peptides may act as a complex satiety signal in the ABL.

Amygdala↗

PubMed1999

Modulatory effect of p-chlorophenylalanine microinjected into the dorsal and median raphe nuclei on cocaine-induced behaviour in the rat.

The present study examined whether a potentiation of cocaine-induced behaviour in rats following peripheral pretreatment with the 5-hydroxytryptamine (5-HT) biosynthesis inhibitor p-chlorophenylalanine may be due to depletion of 5-HT in the dorsal raphe nucleus and/or median raphe nucleus. Following peripheral pretreatment with p-chlorophenylalanine (100 mg/kg, i.p.) for 3 consecutive days, a potentiation of cocaine-induced locomotor activity and rears was observed. To investigate a possible involvement of serotonergic neurones arising in the midbrain raphe nuclei in the observed potentiation, p-chlorophenylalanine (0.5 microg) was microinjected in either the dorsal raphe nucleus or median raphe nucleus followed by behavioural testing 48 h later. Application of p-chlorophenylalanine in the dorsal raphe nucleus resulted in an enhancement of cocaine-induced locomotor activity and head bobs. In contrast, the stimulant effect of cocaine on behaviour was not altered by microinjection of p-chlorophenylalanine in the median raphe nucleus. Peripheral and central administration of p-chlorophenylalanine did not consistently alter the baseline behaviour of saline-treated animals. Biochemical results indicated only a moderate depletion of 5-HT in the midbrain raphe nuclei following peripheral p-chlorophenylalanine administration. Surprisingly, the central application of p-chlorophenylalanine in the dorsal raphe nucleus and median raphe nucleus did not alter the 5-HT levels in the midbrain raphe nucleus investigated. In addition, peripheral and central administration of p-chlorophenylalanine did not alter the 5-HT levels in the nucleus accumbens. In conclusion, the behavioural results suggest that the potentiation of cocaine-induced behaviour following peripheral p-chlorophenylalanine administration may be attributed to the dorsal raphe nucleus but not the median raphe nucleus suggesting that, serotonergic dorsal raphe nucleus neurones may normally mediate a tonic inhibitory effect on cocaine-induced behaviour. Furthermore, the biochemical data may indicate the existence of neurochemical resistance of the midbrain raphe nuclei to the 5-HT depleting effects of p-chlorophenylalanine.

Animals↗

PubMed2003

Gap junctional communication in microinjected human limbal and peripheral corneal epithelial cells cultured on intact amniotic membrane.

The aim of the study was to determine if human limbal epithelial cells (HLEC) do not form gap junctions (GJ) during ex vivo expansion on preserved and intact human amniotic membrane (AM). Thereby, we attempt to evaluate if characteristic features of the limbal epithelial progenitor cells are preserved on AM. Primary human limbal (HLEC) and peripheral corneal (HPCEC) epithelial cells from limbal and peripheral corneal explants were cultured with SHEM either on intact AM or plastic. After 3-4 weeks, cell cultures were terminated and processed for immunofluorescence. In all cell cultures, formations of GJs were analyzed with a mouse monoclonal antibody to connexin 43 (Cx43) and a rabbit affinity purified antibody against connexin 26 (Cx26). Sections of human limbus and cornea served as positive control. Lucifer yellow (LY) known to be a GJ permeant dye was used to analyse functionality of GJ. Microinjection of LY into single cells was performed with a pressure microinjection device under visual control and with the aid of phase contrast optics. Dye spread of LY into adjacent cells indicating intercellular communication was compared between HLEC and HPCEC cultured either on AM or plastic. In vivo, a punctate pattern of Cx43 was typically found in basal and suprabasal corneal epithelial cells and labeling for Cx26 was observed in all cell layers of the human corneal epithelium, however, subpopulations of limbal basal epithelial cells lacked detectable fluorescence signals for both connexins. In HLEC cultured on AM, a scanty immunolabeling for Cx43 (12.6%) was noted, but HPCEC cultured on AM as well as HLEC cultured on plastic showed a higher labeling index (LI) for Cx43 (42.7 and 52.3%, respectively). A significant lower immunostaining for Cx26 was observed in HLEC cultured on AM (LI: 35.16%) in comparison to HLEC cultured on plastic (68.4%), as well as, HPCEC cultured either on AM or plastic (61% and 79.3%, respectively; p<0.001). Gap junctional communication was evidenced more frequently in HLEC cultured on plastic (51%, p<0.05) in contrast to HLEC cultures on AM, which exhibited a limited dye spread in 29.7% of injected cells. A significant difference in dye coupling was also evidenced between HPCEC on AM (52.9%; p<0.05) and HLEC on AM. Subpopulations of HLEC cultured on AM remain Cx43 and Cx26 negative and without functional GPs indicating that characteristic features of limbal epithelial progenitor cells might be preserved during ex-vivo expansion on AM. These data provide support to the use of the ex-vivo expansion of HLEC as an alternative therapeutic strategy for corneal surface reconstruction in distinct ocular surface diseases.

Adult↗

PubMed2002

Brugia malayi: transient transfection by microinjection and particle bombardment.

To develop a method for the introduction of DNA into filarial parasites, several methods that have proven successful in other organisms were evaluated for their ability to transform Brugia malayi. Luciferase activity was detectable in embryos bombarded with gold particles coated with a construct consisting of a luciferase reporter gene under the control of the 5S rRNA intergenic spacer (SL promoter). Similar results were seen in adult parasites and infective larvae bombarded with this construct, or in adult female parasites microinjected with the plasmid. In similar experiments employing the SL promoter driving a green fluorescent protein (GFP) reporter, expression of the reporter was detectable in the intrauterine embryos of the microinjected adult parasites, and in the sub-cuticular tissues of biolistically transfected adult female parasites. A similar pattern of GFP expression to that seen in the SL promoter construct transfected parasites was noted in parasites transfected with constructs consisting of the upstream domain derived from an aspartyl aminoacyl tRNA synthetase gene of B. malayi. The ability to transfect B. malayi embryos may provide a foundation for studies of the regulation of gene expression and function in these organisms.

Animals↗

PubMed2001

Rescue of mouse embryos from 2-cell blocks by microinjection of maturation promoting factor.

OBJECTIVE: To examine the rescue of mouse embryos from 2-cell blocks by the microinjection of maturation promoting factor (MPF) extracted from matured Xenopus eggs into one of the blastomeres of 2-cell stage mouse embryos. DESIGN: Controlled laboratory study. SETTING: First Department of Obstetrics and Gynecology, Toho University School of Medicine, Tokyo, Japan. ANIMAL(S): Eight- to 10-week-old female Crj:CD-1(ICR) mice. INTERVENTION(S): One of the blastomeres of the mouse 2-cell embryos was injected with MPF (MI group) or mHTF medium (MED group) at 28--32 hours after insemination. MAIN OUTCOME MEASURE(S): The developmental rate to blastocyst. RESULT(S): The developmental rate to blastocyst in the MI group (48.0%) was significantly higher than that in the MED group (0%). CONCLUSION(S): The 2-cell block was specifically rescued by the microinjection of MPF and not by the insertion of pipettes.

Animals↗

PubMed1999

Microinjection of mitochondria into zygotes creates a model for studying the inheritance of mitochondrial DNA during preimplantation development.

OBJECTIVE: To determine the effect of mutant mitochondria on preimplantation embryo development and of preimplantation embryo development on the survival of mutant mitochondrial DNA. DESIGN: Laboratory research. SETTING: Academic research laboratory. PATIENT(S): None. INTERVENTION(S): Mutant and wild-type mitochondria, fractionated from tissue obtained from a patient with MELAS syndrome, a mitochondrial disease, were microinjected into mouse zygotes. Control zygotes received either no injection or sham injection. MAIN OUTCOME MEASURE(S): Preimplantation embryo development and survival of mutant mitochondrial DNA as determined by polymerase chain reaction analysis. RESULT(S): After microinjection into zygotes, the MELAS mutation could be identified by polymerase chain reaction until the hatched blastocyst stage of embryo development. The survival of MELAS-injected zygotes, observed for 4 days after injection, did not differ from the survival of zygotes injected with wild-type mitochondria or from the survival of uninjected or sham-injected controls. CONCLUSION(S): It appears that preimplantation embryo development does not screen out mitochondrial DNA mutations introduced into fertilized oocytes, and low levels of mutant mitochondrial DNA do not disrupt early embryo development.

Animals↗

PubMed2002

Melatonin microinjection into the medial preoptic area increases sleep in the rat.

A wide variety of hypnotic compounds including triazolam, pentobarbital, ethanol and adenosine have been reported to enhance sleep when microinjected into the medial preoptic area (MPA) of the anterior hypothalamus of the rat. It is uncertain whether the pineal hormone melatonin, which may alter sleep/wake physiology in mammals, acts at this site. A previous report has indicated that a more widespread injection of melatonin into the hypothalamus of the cat induces sleep. In the present study we have examined the possibility that the MPA may mediate this effect. Nine adult rats were microinjected with melatonin 1 and 50 ug and vehicle into the MPA during the daytime in a repeated measures design study. It was found that melatonin increased total sleep time in a dose-dependent manner, primarily by increasing NREM sleep, and that wake time after sleep onset was significantly reduced. These data add melatonin to the growing list of compounds that increase total sleep after administration into the MPA, and suggest that the MPA may be a common site of action for such agents from a variety of pharmacologic classes. Based on previous studies, the possibility is raised that this sleep enhancement results from an alteration in function of the GABA(A)-benzodiazepine receptor complex.

Animals↗

PubMed1996

Sleep induction by microinjection of pentobarbital into the medial preoptic area in rats.

In a previous study we have shown that microinjection of the benzodiazepine hypnotic triazolam into the medial preoptic area increases sleep in rats. In order to determine whether this effect is specific to benzodiazepines, or whether it occurs with hypnotic medications from other pharmacologic classes, we have microinjected pentobarbital (1 and 100 micrograms) and vehicle in random sequence into rats and performed two hour sleep studies in the daytime with the lights on. Both doses significantly decreased sleep latency and increased nonREM and total sleep. The amount of REM sleep, REM latency, and intermittent waking time were not significantly altered. These data are consistent with the hypothesis that the medial preoptic area may be involved in sleep induction by both benzodiazepine and barbiturate hypnotic medications.

Animals↗

PubMed1998

Cardiorespiratory responses to glutamate microinjected into the medullary raphé.

The involvement of the medullary raphé in modulating cardiorespiratory activity was examined by microinjecting L-glutamate (L-Glu) into the raphé of rats. Animals were vagotomized, paralyzed, artificially ventilated, maintained at 37 degrees C, and instrumented to record arterial blood pressure (BP) and phrenic nerve activity (PNA). Mock cerebrospinal fluid (mCSF, 10 nl, pH 7.4; control) and L-Glu dissolved in mCSF (10, 100, 1000 mM; 10 nl; pH 7.4; experimental) were microinjected into the raphé. L-Glu affected both BP and PNA in a dose dependent manner. Blood pressure was reduced by 6.30 +/- 0.97 and 12.98 +/- 1.29% by 100 and 1000 mM L-Glu, respectively, without affecting heart rate. PNA increased by 23 and 38% with 100 and 1000 mM L-Glu, respectively. Mock CSF and 10 mM L-Glu had no effect. It is concluded that there are sites in the medullary raphé that affect blood pressure only and other sites which can affect both blood pressure and respiration.

Animals↗

PubMed2005

Microinjection as a tool to explore small GTPase function.

Microinjection overcomes the plasma membrane barrier to the introduction of charged or large nonlipid soluble molecules into cells by the direct insertion of a hollow capillary micropipette into the cell. With the application of pressure, aqueous solution is then directly transferred into either the cytosol or the nucleus. I give specific examples of the application of this approach to the functional study of small GTPases of the Sar1, ARF, and rab family in membrane trafficking between the Golgi apparatus and endoplasmic reticulum (ER). The principles illustrated by these examples should be generally applicable to other small GTPases. Detailed protocols for capillary microinjection using semiautomated equipment are given.

Animals↗

PubMed1997

Kainate microinjection into the dorsal raphe nucleus induces 5-HT release in the amygdala and periaqueductal gray.

Earlier results obtained in one of our laboratories showed that microinjection into the dorsal raphe nucleus (DRN) of the excitatory amino acid kainic acid, the benzodiazepine (BZD) inverse agonist FG 7142, and the 5-HT1A receptor agonist 8-OHDPAT changed the behavior of rats in the elevated T-maze, an animal model of anxiety. The present study investigates biochemical correlates of these results in awake rats by measuring 5-HT release with in vivo microdialysis in two brain structures innervated by the DRN-the amygdala (Am) and the dorsal periaqueductal gray matter (DPAG)-that have been implicated in anxiety. Microinjection of kainic acid (60 pmol) into the DRN significantly increased 5-HT release in both the Am and the DPAG. In the DPAG, the increase was 14-fold higher with respect to the baseline and occurred only at the first sample, which was collected 30 min after the injection. In the Am, the increase was less pronounced (nearly fourfold) but persistent, lasting until the fourth sample, which was collected 120 min from the injection. FG 7142 (40 pmol) and 8-OH-DPAT (8 nmol) were ineffective. Because only intra-DRN kainate both increased inhibitory avoidance and decreased one-way escape in the elevated T-maze, the present behavioral results support the suggestion that 5-HT facilitates conditioned fear in the Am and inhibits unconditioned fear in the DPAG.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1999

In vivo development of microinjected embryos from superovulated prepuberal slaughter lambs.

This study was performed to investigate the developmental potential of microinjected embryos recovered from superovulated prepuberal lambs. Fifty-nine mixed-bred lambs (about 3 mo old) were superovulated either with 18 mg FSH-P with (Group FSH/+S) or without (Group FSH/-S) progestagen treatment, or with 10 mL Ovagene following progestagen treatment (Group OVA/+S). All animals received hCG to induce ovulation. Ovulation rates and the number of ova recovered per animal for the different groups were 8.7 and 4.7 (55%, FSH/+S); 7.3 and 3.2 (42%, FSH/-S); and 6.4 and 4.0 (65%, OVA/+S), respectively. No significant differences were seen in the ovulation and the recovery rates, but animals without progestagen treatment showed a significantly lower fertilization rate (44%) when compared with progestagen-treated groups (87%; P<0.001). Foreign DNA was microinjected into the pronuclei of fertilized ova (n = 155), which were transferred (n = 98) into synchronized recipient ewes (n = 21). Two animals were detected pregnant and both gave birth to a single lamb. Results of superovulation and embryo recovery from prepuberal lambs were promising, but the low rate of development to term indicates that ova recovered from prepuberal lambs have reduced developmental competence in vivo. Although 2 lambs were born, it seems that this is not a successful method for use in future gene transfer programs.

Animals↗