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PubMed1992

Heterologous expression of the human potassium channel Kv2.1 in clonal mammalian cells by direct cytoplasmic microinjection of cRNA.

The cloned human delayed rectifying K+ channel Kv2.1 (drk1) was expressed in clonal mouse fibroblasts (L-cells) and rat basophilic leukemia cells (RBL-1) by direct cytoplasmic microinjection of complementary RNA (cRNA). Within six hours, cells microinjected with Kv2.1 cRNA expressed a large sustained outward current as determined from whole-cell patch-clamp recordings. Nearly 100% of cells injected with cRNA expressed outward current. Current density was 30-70 pA/pF when measured at a potential of +50 mV. Steady-state activation and inactivation parameters for Kv2.1 were similar when expressed in either L-cells or RBL-1 cells. These results are the first to demonstrate that functional ion channel proteins can be expressed in mammalian clonal cell lines by direct cytoplasmic microinjection of cRNA.

Animals↗

PubMed1976

Sites of action of morphine involved in the development of physical dependence in rats. II. Morphine withdrawal precipitated by application of morphine antagonists into restricted parts of the ventricular system and by microinjection into various brain areas.

Morphine withdrawal was precipitated by injection of various morphine antagonists into restricted parts of the ventricular system or by microinjection of levallorphan into specific brain areas of rats made dependent on morphine by repeated pellet implantation. When the antagonists could spread only within the lateral ventricles and the 3rd ventricle, a weak withdrawal syndrome was induced; by antagonist administration into the restricted 4th ventricle, however, strong withdrawal signs like jumping were elicited even at small dosages. In microinjection experiments, structures in the midbrain and the lower brain stem proved to be the most sensitive to antagonist action. Although microinjections into thalamic nuclei also had some effect, it could not be excluded that the effects were due to uncontrolled spreading of the drug. This became especially clear from experiments with tritium-labeled levallorphan. It is concluded that brain structures located in the anterior parts of the floor of the 4th ventricle and/or caudal parts of the periaqueductal gray matter are important sites of action for the development of physical dependence on morphine.

Animals↗

PubMed1991

A case of human pregnancy after microinjection of capacitated sperm into the perivitelline space.

Microinjection of capacitated sperm into the perivitelline space of oocyte was offered to one couple with persistent infertility of mixed origin. The husband's semen was subnormal, whereas his wife had definitive tubal occlusion and polycystic ovaries. Four previous in vitro fertilization (IVF) attempts were performed but no fertilization was obtained. After superovulation, 13 oocytes were collected. Ten were submitted to microinjection and two were damaged during the procedure. One of the remaining eight had two pronuclei 18 hr after microinjection and progressed to a four-cell embryo after 48 hr. After reimplantation, a normal pregnancy was initiated, caryotype (46XX) was checked at 17 weeks. A normal and healthy girl has been delivered at term.

Adult↗

PubMed1995

Transgene detection during early murine embryonic development after pronuclear microinjection.

The polymerase chain reaction (PCR) technique was used to detect a whey acidic protein (WAP) gene and transgene presence in mouse ova cultured to various stages of development after pronuclear microinjection at the one-cell stage. The PCR technique detected an endogenous 442 bp WAP DNA sequence in 78% of one-cell, 88% of two-cell and 94% of four-cell ova, and in 95% of morulae and 97% of blastocysts. The heterologous WAP-human protein C transgene was detected in 88% of one-cell, 88% of two-cell and 44% of four-cell ova, and in 40% of morulae and 29% of blastocysts. For comparison, the integration frequency for transgenic mouse production using the same DNA construct was 22%. After five days of in vitro culture, embryos that were either developmentally arrested or fragmented were tested for the presence of the transgene. The injected construct was detected in 83% of arrested one-cell, 85% of arrested two-cell, and 85% of fragmented ova. In culture, only 28% of zygotes microinjected with DNA developed to the blastocysts stage compared to 74% of noninjected zygotes, while 63% of zygotes developed to the blastocyst stage after injection of buffer alone. Pronuclear injection of the transgene at concentrations of 1.5, 15 and 50 micrograms ml-1 resulted in 28, 11 and 9% development to blastocysts and 29, 86 and 88% transgene detection, respectively. Transgene detection was 85, 96 and 97% in degenerate embryos at the respective doses of DNA. These data show that pronuclear microinjection of the transgene is detrimental to subsequent embryonic development.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Effects of accumbens DALA microinjections on brain stimulation reward and behavioral activation in intact and 6-OHDA treated rats.

The effects of bilateral nucleus accumbens microinjections of d-ala-met-enkephalinamide (DALA) were assessed in behavioral activation and lateral hypothalamic self-stimulation (LHSS) rate-frequency curve-shift paradigms in normal and accumbens 6-OHDA (4.0 micrograms) treated rats. Microinjections of DALA (2.5 micrograms/microliters) in the behavioral activation paradigm had little effect on normal activity; however, DALA administered to 6-OHDA treated rats produced a significant overall increase in locomotion. The 6-OHDA DALA-induced locomotion effect peaked at 2 weeks after 6-OHDA treatment and then returned to baseline levels by week 5 post-treatment. Using LHSS, DALA tested over a range of doses (2.5, 5, 10, 20 micrograms/microliters) displayed a weak biphasic reward effect only at the highest dose, which was characterized by an initial suppression followed by an elevation. DALA significantly depressed initial operant motor/performance in LHSS in a dose dependent fashion. Microinjections of the normally ineffective low dose of DALA (2.5 micrograms/microliters) following accumbens 6-OHDA treatment produced a significant LHSS reward decrease 2 weeks post-treatment, while LHSS motor/performance was relatively unaffected. Results are discussed in terms of opiate-dopamine and limbic-motor interactions.

Animals↗

PubMed1994

Anxiolytic effect of glycine antagonists microinjected into the dorsal periaqueductal grey.

To investigate if blockade of the modulatory glycine site of NMDA receptors in the dorsal periaqueductal grey (DPAG) would produce anxiolytic effects, groups of 9-14 rats received microinjections into this structure of 7-chloro-kynurenic acid (7-Cl-KY, 4 and 8 nmol) or 3-amino-1-hydroxypyrrolid-2-one (HA-966, 30 or 100 nmol), two selective antagonists at the strychnine-insensitive glycine modulatory site, and were submitted to the elevated plus-maze, an ethologically based animal model of anxiety. Both drugs increased the percentage of entries and of time spent in open arms as compared to rats receiving isotonic saline. Injections of the active compounds outside the DPAG were not effective. In another experiment microinjections of 7-Cl-KY (8 nmol) and HA-966 (100 nmol) into the DPAG raised the threshold of aversive electrical stimulation of the rat DPAG. These results indicate that microinjections of 7-Cl-KY and HA-966 into the DPAG cause anxiolytic effects in two different models of anxiety and support the proposal that NMDA-mediated neurotransmission in the DPAG may be related to anxiety and panic.

Animals↗

PubMed1994

Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos.

The effect of DNA microinjection at various times after in vitro insemination on DNA detection and survival rates of bovine embryos was investigated. Oocytes were inseminated 24 h after maturation with frozen/thawed semen prepared with a Percoll separation procedure. At 11, 15 and 19 h after insemination, embryos were centrifuged to visualize pronuclei and microinjected with a murine whey acidic protein-human protein C genomic DNA construct. After culture for 7 days on Buffalo Rat Liver cells, embryos were assessed for stage of development and assayed for the presence of the transgene by polymerase chain reaction. Of zygotes in the 11 h after insemination treatment, 16% (25/152) of non-injected and 7% (11/161) of injected embryos developed to the morula or blastocyst stage. Comparable development of non-injected and injected embryos treated at 15 h after insemination was 15% (23/158) and 4% (6/159) and treated at 19 h after insemination was 14% (23/162) and 1% (1/165), respectively. Development of injected embryos was greater (p < 0.05) when injection was performed at 11 h after insemination compared to 19 h after insemination. Development of non-injected embryos was greater (p < 0.01) than that of injected embryos. There was no difference in transgene detection frequency in embryos of all developmental states between treatments (53% at 11; 50% at 15; 48% at 19 h after insemination). Injected embryos testing positive for the presence of the transgene exhibited increased development over negative embryos (p < 0.01). Greater development efficiencies can be obtained in microinjected bovine embryos when injection is performed early in pronuclear formation.

Animals↗

PubMedDate not supplied

Effects of microinjection of scopolamine into the neostriatum of rats on performance of a food conditioned reflex at different levels of fixation.

Chronic experiments performed on 32 Sprague-Dawley rats using a movement-feeding operant reflex (Skinner box) model showed that microinjection of scopolamine into the neostriatum had effects on this reflex which depended on the stage of learning. In animals with weakly fixed reflexes (prior to reaching the stage of memory consolidation), bilateral microinjection of 0.3 microgram of scopolamine into the caudate nucleus completely inhibited the reflex for a prolonged period of time. When the operant habit was well fixed, bilateral microinjection of the same doses of scopolamine into the neostriatum had no effect on the reflex. These results suggest that the neostriatum cholinergic system is critically involved in forming the motor engram. The cholinergic system of the caudate nucleus either takes no part in realizing the well-fixed conditioned reflex movement response and/or other forebrain structures are involved in the reflex, compensating for the disturbance in neostriatal cholinergic function.

Animals↗

PubMed2002

Effects of serotonin (5-HT)(1B) receptor ligands, microinjected into accumbens subregions, on cocaine discrimination in rats.

Recent data indicate a significant input of serotonin (5-HT) on mesoaccumbens dopamine-dependent behavioral effects of cocaine in rats. The present study investigated the role of 5-HT(1B) receptors in nucleus accumbens subregions (the shell and the core) and the effect of stimulation of those receptors in the discriminative stimulus effects of cocaine in rats. Male Wistar rats were trained to discriminate cocaine (10 mg/kg, i.p.) from saline (i.p.) in a two-choice, water-reinforced fixed-ratio 20 procedure. After reaching the cocaine-saline discrimination criterion, rats were stereotaxically implanted with bilateral cannulae in the accumbens shell or core, and then were microinjected with selective 5-HT(1B) receptor ligands. In substitution studies, microinjections of the 5-HT(1B) receptor antagonist GR 55562 (0.1-10 microg/side) or the 5-HT(1B) receptor agonist CP 93129 (0.1-10 microg/side) into accumbens subregions did not evoke cocaine-lever responding. Pretreatment with the 5-HT(1B) receptor antagonist GR 55562 (0.1-10 microg/side) in the accumbens shell or core failed to modulate the discriminative stimulus effects of cocaine (5 mg/kg). Combination tests using a fixed dose of CP 93129 (1-10 microg/side) into the accumbens shell prior to cocaine administration (0.6-5.0 mg/kg) did not affect cocaine discrimination. CP 93129 (1 microg/side, but not 0.1 microg/side) microinjected in the accumbens core, and low doses of systemic cocaine (0.6-2.5 mg/kg) produced a leftward shift in the cocaine dose-response curve and a decrease in its ED(50) value. GR 55562 (1 microg/side) significantly attenuated the enhancement of cocaine discrimination evoked by a combination of CP 93129 (1 microg/side) and cocaine (1.25 mg/kg or 2.5 mg/kg). These results seem to exclude a major role for the accumbens shell and core 5-HT(1B) receptors in controlling the discriminative stimulus effects of cocaine. However, they do suggest that the stimulation of 5-HT(1B) receptors in the accumbens core, but not in the shell, enhances cocaine discrimination in rats.

Animals↗

PubMed1998

Microinjection of sigma ligands into cranial nerve nuclei produces vacuous chewing in rats.

Many typical neuroleptics carry a high risk for producing motor side effects in humans, and have significant affinities for sigma (sigma) receptors. Sigma receptors are densely concentrated in cranial nerve nuclei that comprise the final common pathways for lingual, facial and masticatory movements; thus, they may serve as important substrates for some of the unwanted movements that can accompany neuroleptic treatment. Therefore, the purpose of this study was to evaluate whether microinjection of sigma ligands into the facial nucleus or spinal trigeminal nucleus, oralis would cause orofacial dyskinesias, and whether these effects could be attenuated with sigma receptor antagonists. Microinjection of the high affinity sigma ligands, di-o-tolylguanidine or haloperidol (0-10 nmol/0.5 microl), produced a marked increase in vacuous chewing and facial tremors in rats, while coadministration of the functional sigma antagonists, BD1047 or BD1063 (5 nmol), greatly attenuated these drug-induced movements. Sulpiride and clozapine (10 nmol/0.5 microl), sigma inactive/dopamine active atypical antipsychotic drugs with a much reduced risk for producing motor side effects in humans, were unable to elicit orofacial dyskinesias when microinjected into the facial or spinal trigeminal nucleus, oralis. These studies indicate that sigma receptors may contribute to some forms of motor side effects resulting from antipsychotic drug treatment.

Animals↗

PubMed2000

Cochlear microinjection and its effects upon auditory function in the guinea pig.

Microinjection through the round window membrane has been found to represent a method for vector delivery in intracochlear gene transfer in animal models but breaches the round window membrane, making it necessary to evaluate animals for possible postinjection hearing loss. In the present study healthy guinea pigs were evaluated for their baseline click auditory brainstem response (ABR) thresholds. Each animal was then injected with saline via the round window membrane. After 1 week auditory function was evaluated by click ABR. Animals with increased ABR thresholds were retested at 4 weeks. Animals with 1-week postoperative ABRs similar to baseline were not retested. Results showed that postoperative ABR thresholds in five animals (71%) remained unchanged from baseline, while two animals had increases of 20-25 dB in ABRs after 1 week but recovered baseline ABRs after 4 weeks. The mean baseline ABR threshold was 25.7 dB and was 27.9 dB after 1 week after injection. The difference between preoperative and 1-week postoperative averages was not significant (P = 0.707). In this preliminary study saline microinjection through the round window membrane did not cause permanent hearing loss in the guinea pigs tested, and any damage caused by microinjection appeared to be reversible.

Animals↗

PubMed2005

The effects of microinjection of the selective blocker of muscarinic M1 receptors pirenzepine into the neostriatum on the motor behavior of rats.

A discrimination conditioned active avoidance reflex (CAAR) model in a T maze was used in 18 rats to study the effects of bilateral microinjections of the selective muscarinic M1 receptor blocker pirenzepine into the neostriatum on the acquisition of the CAAR and behavior in an open field test. There was sharp degradation of learning of the CAAR and a significant improvement in motor activity both in the open field test and in the maze itself in rats given bilateral microinjections (pirenzepine, 0.004 mg) into the neostriatum as compared with intact controls. This suggests that changes in motor behavior (a sharp increase in locomotor activity) may be among the reasons for difficulty in learning the CAAR in rats after pirenzepine microinjections. Another reason for difficulty in learning the CAAR in these animals may be impairment of the perception of the conditioned signals (a flashing light) and poor differentiation. This is particularly indicated by the delay in the start chamber (double that seen in intact animals) on presentation of conditioned signals despite the high level of motor activity. These results and published data provide evidence for the complex nature of changes induced by blockade of muscarinic M1 receptors in the neostriatum.

Animals↗

PubMed1981

Baroreflex actions of substance P microinjected into the nucleus tractus solitarii in rat: a consequence of local distortion.

Biologically active substance P (SP) (1000 ng in 0.1 microliter saline) microinjected into the nucleus tractus solitarii (NTS) of 25 rats did not affect arterial pressure, heart rate, or the baroreceptor reflex. However, microinjection of saline alone in volumes greater than 0.3 microliter consistently elicited hypotension and bradycardia followed occasionally by transient hypertension. These data suggest that previously reported cardiovascular effects of SP microinjected into the NTS resulted from local distortion.

Afferent Pathways↗

PubMed1986

Increased food intake after opioid microinjections into nucleus accumbens and ventral tegmental area of rat.

The nucleus accumbens (ACC) and ventral tegmental area (VTA), two areas believed to subserve reinforcement and increases in locomotor activity produced by opioid microinjections, were examined for their involvement in opioid-produced changes in ingestive behavior. Opioids were infused bilaterally, and food and water intakes were measured for 1 h thereafter. Different morphine doses were administered and, with placements in globus pallidus and lateral ventricles as controls for diffusion, it was found that only ACC and VTA microinjections (0.1-10 nmol) produced dose-related increases in food intake. In both the ACC and VTA low doses of morphine also produced increases in water intake while in ACC high doses produced a decrease. Administration of morphine and an enkephalin analogue (Tyr-D-Met-Gly-Phe(4-NO2)-Pro-NH2) at different depths in the ACC indicated that the increase in food intake occurred at a site separate from that of the decrease in water intake. Using levorphanol, dextrorphan and morphine mixed with naloxone, it was shown that the effects were due to activation of opioid receptors. Additional experiments demonstrated that food intake is increased by ACC morphine under different levels of deprivation, with different times of testing and with availabilities of various goal objects in addition to food. The effect also did not appear to undergo development of tolerance or sensitization. It was concluded that there are sites in the ACC and VTA where increased activity of endogenous opioid peptide systems reliably increase food intake and it was hypothesized that these sites may contribute to changes in ingestive behavior after systemic morphine administration. Also, together with other effects produced by opioid microinjections into the ACC and VTA, the present findings suggest that increased opioid activity in these areas produce a pattern of behaviors similar to that produced in normal animals by food conditioned stimuli.

Animals↗

PubMed1986

Effect of chronic haloperidol on dopamine release following microinjection of GABA into the substantia nigra zona reticulata in the rat.

To investigate the influence of the striatonigral gamma-aminobutyric acid (GABA) system on the nigrostriatal dopamine (DA) system, the release of DA and/or 3,4-dihydroxyphenylacetic acid in the striatum ipsilateral to the injection side was examined by in vivo voltammetry following microinjection of GABA into the substantia nigra zona reticulata (SNR). The microinjection of GABA (100-300 micrograms/2 microliters) into the SNR produced a dose-dependent increase in the electrochemical signals recorded from the caudate nucleus ipsilateral to the injection side. Following chronic treatment with haloperidol, microinjection of GABA into the SNR produced only a slight (non-significant) increase in the electrochemical signals recorded from the caudate nucleus ipsilateral to the injection side. These results provide additional evidence to support the concept that DA cells in the substantia nigra zona compacta are regulated by the SNR non-DA neurons in an inhibitory manner. It appears, furthermore, that chronic treatment with haloperidol reduces the release of DA in the striatum ipsilateral to the injection side and that this effect may be due to a gradual development of depolarization block of DA cells by chronic administration of haloperidol.

Animals↗

PubMed1986

Elicitation of intraspecific defensive behaviors in the rat by microinjection of picrotoxin, a gamma-aminobutyric acid antagonist, into the midbrain periaqueductal gray matter.

Behavioral reactions induced in the rat by microinjections of a gamma-aminobutyric acid (GABA) antagonist (picrotoxin; 25 and 50 ng in 0.25 microliter) into the midbrain periaqueductal gray matter were measured in an open-field test and when the animal was confronted by a conspecific introduced into its cage (i.e. resident-intruder paradigm). In the open-field, microinjections of picrotoxin significantly increased backward locomotion while decreasing self-grooming. In the resident-intruder paradigm, microinjections of picrotoxin selectively increased defensive reactions (defensive uprights, defensive sideways, retreat) while offensive behaviors were rather reduced. In addition, the actual nature of the effects was found to depend upon the intruder's relative position. Defensive reactions were significantly increased when the partner was on the side contralateral to the injection site, whereas social approach behaviors (fur investigation, anogenital investigation) were decreased when the partner was located on the ipsilateral side. These data suggest the involvement of GABAergic synapses within the midbrain periaqueductal gray matter in the control of intraspecific defensive behaviors in the rat.

Aggression↗

PubMed1987

Microinjection of a benzodiazepine into substantia nigra elevates kindled seizure threshold.

The purpose of these experiments was to initiate investigations of the brain site(s) at which the benzodiazepines exert their anticonvulsant effect. We examined the effects of microinjections of clonazepam into substantia nigra (SN) on seizure threshold in the kindling model. We also examined the distribution of microinjected [3H]methylclonazepam with autoradiographic methods. Microinjection of clonazepam bilaterally into substantia nigra pars reticulata (SNR), but not nearby, produced a 75% elevation of generalized seizure threshold. Quantitative analysis of autoradiographic studies indicated that the vast majority of [3H]methylclonazepam was distributed within 400 micron of the injection cannula tip; even optimally placed injections did not result in drug access throughout the entire SN. The data demonstrate that local application of an anticonvulsant benzodiazepine to the substantia nigra alone is sufficient to suppress seizures. We suggest that the substantia nigra is one site at which systemically administered benzodiazepines act to suppress seizures.

Animals↗

PubMed1988

Pressor responses evoked by microinjections of L-glutamate into the caudal ventrolateral medulla of the rat.

The caudal medulla of the rat was mapped for cardiovascular sensitive regions by recording changes in mean arterial pressure (MAP) and heart rate evoked by microinjections of L-glutamate (1 nmol/50 nl). Using this technique to selectively activate cell soma in the brainstem, a new pressor area in the caudal ventrolateral medulla has been identified. Several sites located approximately 1-1.5 mm posterior to the caudal medullary depressor zone were found where L-glutamate evoked pressor responses of 10-45 mm Hg. The most responsive area was located just dorsal to the lateral aspect of the lateral reticular nucleus at the level of the pyramidal decussation and the caudalmost pole of the inferior olives. Pressor responses at this site averaged 37 +/- 2 mm Hg. Changes in heart rate were inconsistent and both tachycardia and bradycardia were observed. Increases in arterial pressure elicited from the caudal pressor area (CPA) were abolished by ganglionic blockade. Pressor responses evoked from the CPA were also eliminated after functional inactivation of vasopressor neurons in the rostral ventrolateral medulla (RVM) was produced by microinjections of muscimol. Inhibition of CPA neurons by microinjections of GABA had no effect on MAP while GABA markedly reduced MAP when injected into the RVM. These studies demonstrate that a circumscribed region of the caudal ventrolateral medulla contains a population of 'vasopressor' neurons distinct from those located in the rostral medulla. No evidence was obtained to suggest that neural activity in the CPA contributes to the maintenance of arterial pressure. The precise functional role of the CPA in central cardiovascular regulation remains to be determined.

Animals↗