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At least 19 recordsLinked to original sources
PubMed2004

Microinjection of living adherent cells by using a semi-automatic microinjection system.

Testing in vitro is an alternative to animal experimentation. The capillary pressure microinjection technique is a supporting technology for efficient in vitro testing. The main benefit of the technique is the possibility of injecting large molecules into a single living cell. The ultimate goal of the research discussed in this paper is to increase the cell survival rate in capillary pressure microinjection. A method to reliably evaluate cell survival rate is therefore needed. A three-phase evaluation process is presented in this paper. The first phase determines the success rate of the injection capillary to penetrate the cell membrane. The second phase studies the success rate of delivering the injection substance inside the cell, while the third phase studies cell survival after the microinjection. In addition to the three-phase evaluation process, this paper describes the initial results of penetration and injection tests performed by using a semi-automatic capillary pressure microinjection system developed by the research group. Three adherent cell lines, namely, retinal pigment epithelial cells, MCF-7 human breast cancer cells and SH-SY5Y neuroblastoma cells, were used in the experiments. The results of the penetration tests show that the average success rate of penetrating the cell membrane using the micromanipulator was 87%. The goal of the injection tests was to demonstrate the successful microinjection of living cells and to study the injection success rate. Fluorescein dextran was injected into MCF-7 cells, and preliminary results showed an injection success rate of 49%. In the survival tests, the neuronal cells were microinjected with KCl. During long-term observation after the microinjection, the microinjected cells first decreased their adhesion to the plate, but later adhered to the bottom of the plate and even grew some dendrites. In the next phase of the study, more tests will be performed in order to obtain a statistically reliable value for the survival rate.

Animal Testing Alternatives↗
PubMed1988

Factors related to successful sperm microinjection of hamster eggs: The effect of sperm species, technical experience, needle dimensions, and incubation medium on egg viability and sperm decondensation following microinjection.

A series of experiments was designed to identify factors in a sperm microinjection system that could influence egg viability and decondensation of sperm nuclei after microinjection. Egg viability and sperm decondensation rates were not different among eggs microinjected with rodent sperm. The microinjection of ram sperm required a larger diameter needle for injection, which resulted in low egg viability and sperm decondensation in the first 3 mo of the study but improved greatly after 9 mo of technical experience. The degree of technical experience (3 vs 9 mo) also improved (P<0.05) egg viability after microinjection with rodent sperm; however, the rate of sperm decondensation remained unaffected. Altering the dimensions of the injection needle from a tapered needle barrel to a more uniform needle barrel increased egg viability from 61 to 96% and sperm decondensation from 3 to 27%. The use of medium 199 for incubating microinjected eggs further increased (P<0.05) the percentage of eggs containing decondensed sperm nuclei (52%) compared to eggs incubated in Holmes defined medium (28%). By altering the dimensions of the injection needle, by selecting an appropriate incubation medium, and by gaining technical experience in microinjection, the efficiency of a sperm microinjection system was improved for both rodents and domestic animals.

Journal Article↗
PubMed1988

Cytoplasmic Ca2+ release induced by microinjection of Ca2+ and effects of microinjected divalent cations on Ca2+ sequestration and exocytosis of cortical alveoli in the medaka egg.

Intracellular release of Ca2+ by microinjection of Ca2+ was analyzed by measuring the luminescence of aequorin loaded in eggs of the medaka (Oryzias latipes). Microinjection of Ca2+ into the cortical cytoplasm induced propagative waves of cytoplasmic Ca2+ release and exocytosis of cortical alveoli initiated at the injection point. The Ca2+ wave was initiated with a time lag after some was sequestered at the region of the microinjection. Microinjection of Mg2+ or Mn2+ failed to trigger Ca2+ release and exocytosis. When the aequorin-loaded eggs were inseminated after microinjection of Mg2+, Mn2+, or Co2+ into a restricted region of the vegetal hemisphere, the wave of Ca release was propagated through the injected region toward the vegetal pole, but neither Ca sequestration (fall in Ca-aequorin luminescence) nor exocytosis occurred at the area of cortex where the eggs were injected with these divalent cations. These results suggest that a significant period is required to induce Ca2+ release from cytoplasmic stores by the increased Ca2+ concentration and that both the phenomena of Ca2+ release and Ca sequestration are involved in the process of exocytosis.

Animals↗
PubMed1982

Microinjection of macromolecules into normal murine lymphocytes by means of cell fusion. II. Enhancement and suppression of mitogenic responses by microinjection of monoclonal anti-cyclic AMP into B lymphocytes.

Reproducible methods are now available for introducing protein molecules such as antibodies into normal murine lymphocytes by fusion with protein molecule-containing erythrocyte ghosts. Monoclonal antibodies against cyclic AMP were raised by hybridoma technique and packed into erythrocyte ghosts. Then, monoclonal anti-cyclic AMP containing ghosts were fused with splenic B lymphocytes by polyethylene glycol-mediated fusion at various intervals after LPS stimulation. This method made it possible for us to quantitatively microinject antibodies into B lymphocytes. Microinjection of anti-cyclic AMP antibody molecules into lymphocytes at a very early stage of LPS stimulation resulted in a marked enhancement of DNA synthetic responses as well as increased numbers of plaque-forming cells. Intracellular cyclic AMP levels were found to be markedly decreased after microinjection of monoclonal anti-cyclic AMP, suggesting that lowering the intracellular cyclic-AMP level in the B lymphocytes at an early stage of stimulation might have induced the enhanced proliferative as well as differentiative responses to LPS. Similar enhancing effects on cell proliferation were obtained when antibodies were injected 18 hr after stimulation. Microinjection of anti-cyclic AMP at 12 hr after culture, however, inhibited the DNA synthetic responses, and induction of plaque-forming cells was suppressed when anti-cyclic AMP was injected 6 hr after LPS stimulation. The present data suggest the biphasic regulatory roles of cyclic AMP at the early stage of B lymphocyte activation. This approach may be useful in identifying regulatory molecules in B lymphocyte induced by mitogenic or antigenic stimulation.

Animals↗
PubMed1982

Microinjection of macromolecules into normal murine lymphocytes by cell fusion technique. I. Quantitative microinjection of antibodies into normal splenic lymphocytes.

Human erythrocyte ghosts loaded with various kinds of protein molecules were fused with mouse splenic lymphocytes by means of polyethylene glycol supplemented with poly-L-arginine and dimethylsulfoxide. This fusion method made quantitative microinjection of IgG and other proteins into intact lymphocytes possible. The injection itself did not alter cell viability, and lymphocytes given protein molecules retained intact response activity when they were stimulated with mitogens. Rabbit anticyclic AMP was purified by affinity chromatography and injected into lymphocytes. Antibody activity in the cell lysates was measured by using 125I-labeled cyclic AMP as an antigen, and it was shown that antibody molecules were quantitatively injected and immunologically active in the cells. Antigen binding activity of anti-cyclic AMP antibodies in the nonstimulated lymphocytes was stable and intact even 24 hr after microinjection, whereas the activity rapidly decreased in mitogen-stimulated lymphocytes, indicating that some immunologic or enzymatic mechanisms for inactivating antibodies were induced in mitogen-stimulated cells. Furthermore, microinjection of anti-cyclic AMP markedly enhanced the proliferative responses of lymphocytes to mitogens such as Con A or LPS and reversed the effect of a known elevator of intracellular cyclic AMP. These observations have implications for the role of cyclic AMP in early lymphocyte activation events.

Animals↗
PubMed1991

Dopamine and conditioned reinforcement. II. Contrasting effects of amphetamine microinjection into the nucleus accumbens with peptide microinjection into the ventral tegmental area.

It has been shown that infusion of certain neuropeptides into the ventral tegmental area (VTA) results in increased motor activity and enhanced dopamine turnover in the nucleus accumbens. In the present experiments, substance P (SP), neurotensin (NT), d-ala-metenkephalin (DALA) and morphine sulfate (MS) were injected bilaterally into the VTA and their effects on conditioned reinforcement were assessed. These effects were compared with infusion of amphetamine into the nucleus accumbens, which has previously been shown to strongly enhance responding for conditioned reinforcers. For these experiments, hungry rats were trained to associate a compound stimulus (light and click) with the presentation of food. In the test phase, responding on one lever (CR lever) resulted in the presentation of the stimulus but no food. Responding on the other (NCR lever) had no consequences. Different groups of animals received microinjections (0.5 microliter, bilaterally) of SP (0, 0.03, 0.3, 3.0 micrograms), NT (0, 0.025, 0.25, 0.5 microgram), DALA (0, 0.01, 0.1, 1.0 microgram) or morphine (0, 0.025, 0.25, 2.5 micrograms) into the VTA. SP infusion into the VTA resulted in a small increase in responding which was not selective for the CR lever. NT, DALA and morphine had no effect on responding for conditioned reward. In contrast, amphetamine (0, 0.2, 2.0, 20 micrograms) injected into the nucleus accumbens markedly enhanced responding for conditioned reward. These findings suggest that stimulation of the mesolimbic system at the level of the DA cell bodies, which induces a small increase in DA turnover, is not sufficient to potentiate responding for conditioned reward. On the other hand, an important requirement for potentiation may be excessive release of dopamine in the nucleus accumbens.

Animals↗
PubMed1992

Neuroethological evaluation of audiogenic seizures and audiogenic-like seizures induced by microinjection of bicuculline into the inferior colliculus. II. Effects of nigral clobazam microinjections.

Male Wistar rats were classified as susceptible (S) and resistant (R) to audiogenic seizures (AS) by evaluation of their response to high-intensity sound stimulation (110.3 dB). R rats injected with bicuculline into the inferior colliculus (IC) preferentially displayed audiogenic-like seizures with gyri, jumping and atonic falling, without important tonic-clonic components but with postictal contralateral asymmetry and hyperreactivity. These audiogenic-like seizures were blocked by clobazam microinjection into the substantia nigra (SN) and partially modified by SN vehicle injection. Injection of vehicle or clobazam into the SN of susceptible rats (S) did not modify the occurrence of AS. This may suggest the participation of GABAergic regulation in the development of audiogenic-like seizures in R rats and a defect in GABAergic neurotransmission in S rats.

Acoustic Stimulation↗
PubMed1983

Red cell ghost-mediated microinjection of RNA into HeLa cells. I. A comparison of two techniques for the entrapment and microinjection of tRNA and mRNA.

We have compared two techniques for introducing RNA into red blood cell ghosts. In the pre-swell technique, RNA is introduced into red cells without prior removal of endogenous contents. In the multiple lysis technique, the red cells are subjected to two or three cycles of of reversible lysis, prior to introducing the RNA, in order to first remove the normal red cell constituents. The pre-swell technique offers much greater entrapment of both tRNA and protamine messenger RNA (mRNA), but the RNA appears to be degraded during the procedure. This may be due either to nucleolytic degradation or oxidation by the high concentration of endogenous hemoglobin. The multiple lysis technique offers much lower entrapment but also results in diminished degradation of the entrapped RNA. Although some degradation is apparent, a significant portion of the biological activity of the entrapped protamine mRNA is retained. We have also fused red cells loaded with protamine mRNA by the multiple lysis technique to HeLa cells using polyethylene glycol 6000. The recipient HeLa cells are capable of translating this heterologous message into protamine, a trout testis chromosomal protein.

Cell Fusion↗
PubMed1975

Protein migration into nuclei. II. Frog oocyte nuclei accumulate a class of microinjected oocyte nuclear proteins and exclude a class of microinjected oocyte cytoplasmic proteins.

Nuclear contents or cytoplasm from Xenopus oocytes labeled with (35-S)methionine or (3-H)proline (donor oocytes) were reinjected into unlabeled oocytes (recipient oocytes). The radioactivity injected as nuclear contents was found to enter and accumulate in the recipient oocyte nucleus. In contrast, the radioactivity injected as cytoplasm was found to enter but not to accumulate in the recipient oocyte nucleus. Sodium dodecyl sulfate (SDS) gel electrophoresis of the nucleus and cytoplasm of donor oocytes revealed the existence of three classes of labeled proteins in these oocytes: those proteins found predominantly in the nucleus (N proteins), those found predominantly in the cytoplasm (C proteins), and those found in both the nucleus and cytoplasm at similar concentrations (B proteins). SDS gel electrophoresis of the nucleus and cytoplasm of recipient oocytes showed that N proteins entered and accumulated in the nucleus but that B proteins partitioned about equally between the nucleus and cytoplasm. A similar analysis of oocytes injected with labeled cytoplasm showed that C proteins did not enter the nucleus but again B proteins partitioned about equally between the nucleus and cytoplasm.

Animals↗
PubMed2003

Involvement of local cholecystokinin in the tolerance induced by morphine microinjections into the periaqueductal gray of rats.

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.

Analgesics, Opioid↗
PubMed1993

Neuronal and cardiovascular responses to adenosine microinjection into the nucleus tractus solitarius.

This study investigated neuronal, blood pressure, and heart rate responses to adenosine microinjection into caudal and rostral NTS of anesthetized rats. The site of recording and microinjection was verified chemically by observing the responses to a test dose of l-glutamate (5 nmol) and histologically at the conclusion of the experiment. Neuronal firing rate increased (+29.4 +/- 5.3%) and decreased (-48 +/- 9.4%) in response to l-glutamate microinjection into the rostral and caudal NTS, respectively. These opposite neuronal responses were followed by depressor (-32.4 +/- 8.3 vs. -36 +/- 5.5 mmHg) and bradycardic (-25.2 +/- 7.7 vs. -25.8 +/- 3.4 beats/min) responses to l-glutamate microinjection into the two subareas of the NTS. Microinjection of a submaximal dose (1 nmol) of adenosine into the NTS produced site-dependent cardiovascular responses which were preceded by similar inhibition of neuronal firing (-60 +/- 4 vs. -55.9 +/- 1.7%). Whereas adenosine microinjection into the rostral NTS elicited modest pressor (+10.1 +/- 2.1 mmHg) and tachycardic (+9 +/- 3.9 beats/min) responses, its microinjection into the caudal NTS produced depressor (-29.2 +/- 5.3 mmHg) and bradycardic (-14.6 +/- 1.7 beats/min) responses. These findings suggest that compared to l-glutamate, adenosine produces opposite (rostral) and similar (caudal) neuronal and cardiovascular effects in the two subareas of the NTS. In the caudal NTS, adenosine (0.1, 1, and 10 nmol) elicited dose-related inhibitory neuronal and cardiovascular responses that were attenuated by systemic theophylline but not 8-(p-sulfophenyl) theophylline (8-SPT) administration. The neuronal and cardiovascular responses to adenosine microinjection into the caudal NTS were also attenuated by microinjection of 8-SPT into the same area. Finally, single-unit activity inhibited by adenosine or l-glutamate microinjection into the caudal NTS was also inhibited by baroreceptor loading and excited by baroreceptor unloading. These findings suggest a) l-glutamate elicits opposite neuronal responses in the rostral and caudal NTS; b) the distinct hemodynamic responses elicited by adenosine in the two subareas may be related, at least in part, to their differing responses to l-glutamate; and c) the similarity between the neuronal responses to adenosine and l-glutamate microinjection into the caudal NTS and the response of the same neurons to baroreceptor activation support the hypothesis that adenosine plays a neuromodulatory role in the processing of baroreceptor information.

Action Potentials↗
PubMed1984

Analysis of BHK cell growth kinetics after microinjection of catalytic subunit of cyclic AMP-dependent protein kinase.

The effect of catalytic subunit (C) of cyclic AMP-dependent protein kinase on cell growth kinetics of BHK cells was assessed by microinjection with chicken erythrocyte ghosts as vehicles for introduction of the protein into the cytosol of large populations of cells. The advantage in using chicken erythrocytes for microinjection is that the inactive erythrocyte nuclei serve as a probe for identifying and analyzing microinjection events. By utilizing this procedure, BHK cells were microinjected with an amount of C that was 5- to 10-fold greater than their endogenous levels. Growth kinetics were analyzed by [3H]thymidine incorporation and autoradiography. Cells were stained after autoradiography to more clearly reveal the chicken nuclei, and at each time point, cells were categorized into four groups: (i) not microinjected, not in S phase, (ii) not microinjected, in S phase, (iii) microinjected, not in S phase, (iv) microinjected, in S phase. Those cells not microinjected served as internal controls. Two experimental protocols were used to test the notion that C is involved in blocking cell progression through G1 phase of the cell cycle. First, cells were arrested in G0 phase by serum deprivation, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum or purified growth factors. Second, cells were collected in mitosis, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum. The results of these studies indicate that a 5- to 10-fold increase in the intracellular concentration of C is not a sufficient signal to arrest cell growth in G1 phase. Thus, growth-inhibitory effects of cyclic AMP on BHK cells are unlikely to be the result of activation of cyclic AMP-dependent protein kinase.

Animals↗
PubMed2004

Cardiovascular responses to microinjection of ATP into the nucleus tractus solitarii of awake rats.

Microinjection of increasing doses of ATP (0.31, 0.62, 1.25, and 2.5 nmol/50 nl) into the nucleus tractus solitarii (NTS) produced a dose-dependent pressor response. Prazosin abolished the pressor response and produced no change in the bradycardic response to ATP. Microinjection of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (0.25 nmol/50 nl), a nonselective P2 receptor antagonist into the NTS, reduced the bradycardic response but had no effect on the pressor response to microinjection of ATP (1.25 nmol/50 nl) into the NTS. Microinjection of suramin (2 nmol/50 nl), another nonselective P2 receptor antagonist, had no effect on the pressor and bradycardic responses to microinjection of ATP (1.25 nmol/50 nl) into the NTS. Antagonism of A1 receptors of adenosine with 1,3-dipropyl-8-cyclopentylxanthine also produced no changes in the cardiovascular responses to microinjection of ATP into the NTS. The involvement of excitatory amino acid (EAA) receptors in the pressor and bradycardic responses to microinjection of ATP into the NTS was also evaluated. Microinjection of kynurenic acid, a nonselective EAA receptor antagonist (10 nmol/50 nl), into the NTS reduced the bradycardic response and had no effect on the pressor response to microinjection of ATP into the NTS. The data show that 1) microinjection of ATP into the NTS of awake rats produced pressor and bradycardic responses by independent mechanisms, 2) the activation of parasympathetic component may involve an interaction of P2 and EAA receptors in the NTS, and 3) the sympathoexcitatory response to microinjection of ATP into the NTS was not affected by the blockade of P2, A1, or EAA receptors.

Adenosine A1 Receptor Antagonists↗
PubMed1990

Improved developmental potential of rabbit oocytes fertilized by sperm microinjection into the perivitelline space enlarged by hypertonic media.

The objectives of the present study were: 1) to develop a simple and more efficient technique for sperm microinjection than is currently available, using the rabbit as a model, and 2) to evaluate the development of rabbit oocytes fertilized by single or multiple sperm microinjection. Hyperosmotic sucrose in phosphate-buffered saline (SPBS) was employed to dehydrate oocytes to increase the perivitelline space for sperm microinjection and prevent possible injury to the vitellus. In the first experiment, 58% (n = 29) oocytes treated with 0.5 M SPBS developed to morulae following multiple sperm microinjection compared, respectively, to 47% (n = 34) and 60% (n = 15) for control IVF with or without sucrose exposure (P greater than 0.05). Blastocyst development from microinjected oocytes, however, was much lower (P less than 0.05) than that of controls (14% vs. 42% and 40%, respectively). Sham operation by puncturing the zona pellucida of the sucrose-treated oocytes with the microinjection pipette did not increase parthenogenesis (P greater than 0.05). In Experiment 2 a smaller-size injection pipette and shorter sucrose exposure time after sperm microinjection resulted in 41% (n = 42) of the oocytes developing into blastocysts for the microinjection group, whereas only 21% (n = 24) developed to blastocysts in the control IVF group (P less than 0.05). When relatively older oocytes (17 hr post ovulation injection) were used to test if microinjection could reduce the time to fertilization and cleavage (Expt. 3), an average of 27% (n = 63) blastocysts resulted from microinjection vs. 0% (n = 28) for the control IVF group.

Animals↗
PubMed1992

Additive effects of dopamine and 8-OH-DPAT microinjected into the nucleus ambiguus in eliciting vagal bradycardia in rats.

The effects of combined microinjection into the nucleus ambiguus (NA) of dopamine (DA) and the 5-hydroxytryptamine-1A (5HT-1A) receptor agonist 8-hydroxy-2-[di-n-propylamino]tetralin (8-OH-DPAT) on arterial pressure (AP) and heart rate (HR) were studied in 24 urethane-anaesthetized, artificially ventilated spinal (C1) rats. Sites from which bradycardia was elicited by microinjection of L-glutamate (GLU) were selected for microinjection of DA and 8-OH-DPAT into the NA. Microinjections of 8-OH-DPAT (60-300 pmol in 10 nl) elicited a dose-dependent bradycardia; the HR responses elicited by microinjection of 300 pmol of 8-OH-DPAT were significantly larger (-34.0 +/- 3.0) than responses elicited by the threshold dose of 60 pmol (-3.1 +/- 0.1 bpm). The onset latency of the HR responses elicited by the 300 pmol of 8-OH-DPAT was 6.5 +/- 0.5 s and the peak was reached in 40.0 +/- 8.0 s. The duration of these responses was 615 +/- 3.5 s. Microinjection of DA (1 nmol in 10 nl) within 3-5 s from the time of 8-OH-DPAT microinjection (60 pmol in 2 nl) into the NA at sites previously shown to elicit decreases in HR following microinjection of GLU, produced significant additive effects in eliciting bradycardia (20.4 +/- 2.9 bpm) when compared with decreases in HR elicited by microinjection of DA (11.1 +/- 1.8 bpm) or of 8-OH-DPAT (3.1 +/- 0.1 bpm) alone. There were no changes in AP after microinjections of DA, 8-OH-DPAT or of these two substances combined.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed2000

Bradycardic and hypotensive responses to microinjection of L-glutamate into the lateral aspect of the commissural NTS are blocked by an NMDA receptor antagonist.

Baroreflex activation by phenylephrine infusion produces a bradycardic response while microinjection of L-glutamate into the most lateral aspect of the commissural nucleus tractus solitarius (NTS, 0.8 mm lateral to the midline) produces bradycardic and hypotensive responses. In the present study we investigated the role of NMDA receptors in the lateral aspect of the commissural NTS (0.8 mm lateral to the midline) in the bradycardic and hypotensive responses to microinjection of L-glutamate as well as in the processing of the bradycardic response to the baroreflex activation. The hypotensive and bradycardic responses to L-glutamate microinjection into the NTS were blocked by methyl-atropine (intravenous, i.v.), indicating that the hypotensive response was secondary to the bradycardia. Microinjection of L-glutamate (1 nmol/50 nl) into the NTS was performed before and after microinjection of increasing doses of phosphonovaleric acid (AP-5, a selective NMDA antagonist) at the same site. The microinjection of AP-5 [0.5 (n = 9), 2.0 (n = 8) and 10.0 nmol/50 nl (n = 7)] into the NTS (0.8 mm lateral to the midline) produced a dose-dependent blockade of the bradycardic and hypotensive responses to L-glutamate. In a specific group of rats the microinjection of 10 nmol/50 nl of AP-5 produced a significant reduction in baroreflex sensitivity 2 min after microinjection into the lateral NTS [gain = -1.48 +/- 0.12 vs. -0.5 +/- 0.2 beats/mmHg, (n = 5)], which was reversible. The data show that the bradycardic responses produced by microinjection of L-glutamate into the most lateral aspect of the commissural NTS or by activation of the baroreflex were blocked by microinjection of AP-5, indicating that the neurotransmission of the parasympathetic component of the baroreflex in the neurons of the lateral aspect of the commissural NTS involves NMDA receptors.

2-Amino-5-phosphonovalerate↗
PubMed2005

Mechanism of cardiovascular effects of nociceptin microinjected into the nucleus tractus solitarius of the rat.

Microinjections (100 nl) of 0.15, 0.31, 0.62, and 1.25 mmol/l of nociceptin into the medial nucleus tractus solitarius (mNTS) elicited decreases in mean arterial pressure (11 +/- 1.8, 20 +/- 2.1, 21.5 +/- 3.1, and 15.5 +/- 1.9 mmHg, respectively) and heart rate (14 +/- 2.7, 29 +/- 5.5, 39 +/- 5.2, and 17.5 +/- 3.1 beats/min, respectively). Because maximal responses were elicited by microinjections of 0.62 mmol/l nociceptin, this concentration was used for other experiments. Repeated microinjections of nociceptin (0.62 mmol/l) into the mNTS, at 20-min intervals, did not elicit tachyphylaxis. Bradycardia induced by microinjections of nociceptin into the mNTS was abolished by bilateral vagotomy. The decreases in mean arterial pressure and heart rate elicited by nociceptin into the mNTS were blocked by prior microinjections of the specific ORL1-receptor antagonist [N-Phe(1)]-nociceptin-(1-13)-NH(2) (9 mmol/l). Microinjections of the ORL1-receptor antagonist alone did not elicit a response. Prior combined microinjections of GABA(A) and GABA(B) receptor antagonists (2 mmol/l gabazine and 100 mmol/l 2-hydroxysaclofen, respectively) into the mNTS blocked the responses to microinjections of nociceptin at the same site. Prior microinjections of ionotropic glutamate receptor antagonists (2 mmol/l NBQX and 5 mmol/l d-AP7) also blocked responses to nociceptin microinjections into the mNTS. These results were confirmed by direct neuronal recordings. It was concluded that 1) nociceptin inhibits GABAergic neurons in the mNTS, 2) GABAergic neurons may normally inhibit the release of glutamate from the terminals of peripheral afferents in the mNTS, and 3) inhibition of GABAergic neurons by nociceptin results in an increase in the release of glutamate in the mNTS, which in turn elicits depressor and bradycardic responses via activation of ionotropic glutamate receptors on secondary mNTS neurons.

Animals↗
PubMed1996

Microinjection strategies for the study of mitogenic signaling in mammalian cells.

First used in the analysis of dynamic changes in cell structure, microneedle microinjection allows in situ study of individual living cells as opposed to large scale metabolic analysis of heterogeneous cell culture. In addition, microinjection also offers the possibility to examine in vivo regulated processes by modulating the intracellular levels and activity of key regulatory proteins and genes in both a specific and controlled manner. A number of different strategies have been developed over the past 5 years to examine the pathways and effectors that are involved in mitogenic signaling as well as in the regulation of gene expression during the proliferative response to growth factors by normal fibroblasts. These strategies include: 1. Direct in vivo competition for various trans-activating DNA binding activities by microinjection of double-stranded oligonucleotides, microinjection of monospecific antibodies against transcription factors and microinjection of dominant negative mutants of transcription factors based upon their DNA binding domain. 2. Microinjection of purified enzymes (kinases and phosphatases) or peptides and antibodies that specifically inhibit these activities. 3. Microinjection of expression plasmids which encode various normal and epitope-tagged regulatory molecules. In many of the experiments described below, c-fos gene expression was monitored as an early marker of mitogenic response. The c-fos gene belongs to a family of genes whose transcription is activated very early after addition of growth factor (1-4). For in vivo studies, the c-fos promoter offers several unique advantages. Primarily, it is easy to manipulate. In practical terms, when mammalian fibroblasts are made quiescent (by replacing the normal growth media, with growth factors-depleted media) and subsequently activated by re-adding mitogen (growth factors, serum), c-fos RNA expression is restored within 15 minutes and the protein is specifically detected in the nuclei of cells after 90 minutes, but is no longer detectable after 3 hours. Secondly, results obtained with the c-fos promoter are directly applicable to cell growth since expression of c-fos is itself a prerequisite for proliferation as demonstrated by microinjection of anti-fos antibodies which prevented proliferation in mammalian cells (5). Thirdly, the c-fos promoter is exquisitely sensitive to agents which cause cell stress. In this respect, heat-shock, poor microinjection or microinjection in the presence of heavy metals or chelating agents in the culture media all rapidly stimulate c-fos expression. However, when compared to c-fos expression in the proliferative response, stress mediated c-fos expression is induced both more rapidly and strongly, reverses more slowly (the protein is still detectable after 5-6 hours) and does not result in cell proliferation (unpublished observation). As such, it provides an excellent internal control for identifying poor treatment and manipulation of cells . Finally, the c-fos promoter is subject to several levels of auto-regulation enabling the analysis of not only components involved in transcriptional activation , but also various aspects of transcriptional down regulation and shut-off.

Animals↗