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Results for “Microinjection”

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PubMed1998

Cryopreservation of in vitro-derived bovine blastocysts microinjected with foreign DNA at the pronuclear stage.

Days 6 and 7 bovine blastocysts derived from in vitro-fertilized and DNA-injected zygotes (day of IVF = Day 0) were cryopreserved either by conventional two-step freezing or by vitrification. Foreign DNA used for microinjection was the green fluorescent protein gene under the control of the immediate early promoter of human cytomegalovirus. All blastocysts were produced by an in vitro system and were harvested on Days 6 and 7. The proportion of DNA-injected zygotes developing into blastocysts on Days 6 and 7 (total 8%) was lower than that of nontreated zygotes (total 19%; P < 0.01). After cryopreservation in 1.5 M ethylene glycol, the survival rates of DNA-injected blastocysts assessed by re-expansion at 24 h of culture (Day 6: 59%, Day 7: 71%) were comparable with those of nontreated blastocysts (Day 6: 76%, Day 7: 71%). The post-thaw hatching rate within 72 h of culture of DNA-injected Day 7 blastocysts (38%) was not different from that of nontreated Day 7 blastocysts (40%), but the hatching rate of DNA-injected Day 6 blastocysts (23%) was lower than that of nontreated Day 6 blastocysts (47%; P < 0.05). After vitrification in 7.2 M ethylene glycol, 0.0026 M Ficoll-70 and 0.3 M sucrose, the survival and hatching rates of DNA-injected Day 7 blastocysts (61 and 28%, respectively) were similar to those of nontreated Day 6 (71 and 33%, respectively) and Day 7 (75 and 36%, respectively) blastocysts. However, the post-warming survival rate of DNA-injected Day 6 blastocysts was only 30%, and none of the blastocysts hatched (P < 0.01). The mean cell number of DNA-injected Day 6 blastocysts (100.3 +/- 36.4 cells) was lower than that of nontreated Day 6 blastocysts (130.5 +/- 37.1 cells; P < 0.01), while those of DNA-injected and nontreated Day 7 blastocysts were not different (111.2 +/- 42.8 and 119.6 +/- 31.4 cells, respectively). These results indicate that Day 7 IVMFC bovine blastocysts derived from DNA-injected zygotes can be successfully cryopreserved by conventional two-step freezing or vitrification.

Animals↗

PubMedDate not supplied

Microinjection of human placenta. I: Techniques.

An apparatus is described which has been used successfully to microinject the syncytiotrophoblast of chorionic villi, the mesenchymal core of chorionic villi, and cultured cells grown from first-trimester human trophoblast. It will accurately and repeatedly pulse picolitre quantities of injectate from the micropipette.

Female↗

PubMed1997

A pressure system for the microinjection of substances into the brain of awake monkeys.

We describe a method for placing pressure microinjections of drugs or anatomical tracers in physiologically defined sites in the brain of awake monkeys. This method provides a means to record neuronal activity from the tip of an injection cannula so that an injection can be made at a physiologically defined location. It uses pressure in a closed system to precisely control the amount of fluid injected and provides a visible means for monitoring injection volume. The injection cannula is easy to make with readily available components and can be used repeatedly for multiple recording sessions and injections.

Animals↗

PubMed2002

Pressor response to microinjection of orexin/hypocretin into rostral ventrolateral medulla of awake rats.

Orexin A (or hypocretin 1)-immunoreactive neurons in the rat lateral hypothalamus project to several areas of the medulla oblongata that are closely associated with cardiovascular regulation. The present study was undertaken to further strengthen the hypothesis that orexin A accelerates cardiovascular response by activating sympathoexcitatory neurons in the rat rostral ventrolateral medulla (RVLM). First, immunohistochemical studies revealed the presence of orexin A-immunoreactive fibers in the RVLM. Double labeling the sections with orexin A- and tyrosine hydroxylase (TH)-antisera further showed that orexin A-immunoreactive fibers are in close proximity with TH-immunoreactive neurons, some of which may be barosensitive, bulbospinal neurons in the RVLM. Second, microinjection of orexin A (6.35, 12.7 and 38.1 microM) into the RVLM, which was verified later by histological examination, caused a significant increase of mean arterial pressure (MAP) and a moderate increase of heart rate (HR) in awake rats. L-glutamate (33.3 mM) injected into the same sites, caused a larger increase in MAP, but a decrease in HR; whereas, saline injection was without significant effect. Results from this study suggest that orexin A, which may be released from the nerve fibers originating from the neurons in the lateral hypothalamus, acting on RVLM neurons in the medulla, increases sympathetic outflow targeted to the heart and blood vessels in awake animals.

Animals↗

PubMed2001

Place aversion induced by microinjections of C-fragment of substance P into the dorsal periaqueductal gray of rats is mediated by tachykinin NK1 receptors.

Neural circuits in the dorsal periaqueductal gray matter (dPAG) play an important role in the integration of defensive behavior. The neurokinin substance P causes conditioned place aversion when administered into this region. The present study examined whether these effects may be mimicked by its carboxy-terminal amino acid sequence and whether they are influenced by prior treatment with the tachykinin NK1 receptor antagonist WIN51,708. The behavioral testing apparatus is a circular open field consisting of 4 uniform quadrants that are equally preferred by the rats prior to drug treatments. For conditioning, rats received drug injections on three consecutive days and were placed into their assigned quadrant. The carboxy-terminal analog (17.5 pmol/0.2 microl) applied into the dPAG produced place aversion effects with reduced time spent in the drug-paired quadrant on the testing day. The effects of the carboxy-terminal analog was antagonized by pretreatment with WIN51,708 (20 mg/kg, i.p.). Microinjection of WIN51,708 (20 mg/kg, i.p.), by its own, did not produce significant effects. These findings suggest that previous reports showing conditioned place aversion effects of SP injected into the dPAG are encoded by its carboxy-terminal sequence and due to its action on tachykinin NK1 receptors.

Androstanes↗

PubMed2002

Clearance of manganese from the rat substantia nigra following intra-nigral microinjections.

Chronic exposure to manganese (Mn) positively correlates with the occurrence of Parkinsonism but little is known about mechanisms of its neurotoxicity. In the present study, we determined the clearance of Mn from rat substantia nigra after its nigral injection and correlated it with the establishment of apomorphine-induced rotational behaviour and loss of striatal tyrosine hydroxylase (TH) immunoreactivity. Our results suggest that Mn is slowly cleared from the substantia nigra, following a first-order kinetics with a t(1/2) of 3 days. Appearance of apomorphine-induced rotational behaviour and loss of TH immunoreactivity within the striatum follows metal clearance were both detected 24 hours after intra-nigral Mn microinjection and maximal 72 hours after injection. The present data suggest that the cellular mechanisms induced by Mn and leading to dopaminergic cell death, occurred shortly after its injection and that the metal concentration needs to reach a threshold value to induce neurotoxic effects. This would indicate that nigral damages are a direct consequence of Mn accumulation.

Animals↗

PubMed2003

Role of the dorsomedial hypothalamus in thermogenesis and tachycardia caused by microinjection of prostaglandin E2 into the preoptic area in anesthetized rats.

Prostaglandin E2 (PGE2) acts in the preoptic area (POA) of the mammalian hypothalamus to increase body temperature and heart rate. Chemical stimulation of the dorsomedial hypothalamus (DMH), a region richly innervated by neurons in the POA, evokes sympathetically-mediated increases in heart rate and body temperature. We tested the hypothesis that neurons in the DMH mediate hyperthermia and tachycardia resulting from the action of PGE2 in the POA. Microinjection of PGE2 150 pmol/15 nl into the POA in urethane-anesthetized rats caused increases in body temperature and heart rate that were sharply reversed after injection of muscimol 80 pmol/100 nl into the DMH but not after similar injection of saline vehicle. Therefore, thermogenic and tachycardic actions of PGE2 in the POA are at least in part a consequence of neuronal activity in the region of the DMH.

Anesthetics, Intravenous↗

PubMed2003

Effects of microinjection of melatonin into various brain regions of Japanese quail on locomotor activity and body temperature.

We previously reported that peripheral administration of melatonin resulted in simultaneous decreases in locomotor activity and body temperature in the Japanese quail. To examine the site of these melatonin-induced effects, we performed microinjection of melatonin into various brain regions of the Japanese quail. On the basis of the injection site the effects of melatonin were dissociated into four groups: decrease in only body temperature, decrease in only locomotor activity, decrease in both body temperature and locomotor activity, and a change in neither. The decrease in only body temperature was observed when melatonin was injected into areas of the diencephalon including the thalamus and hypothalamus, and the decrease in only locomotor activity was observed when melatonin was injected close to the nuclei septalis medialis and septalis lateralis. These results suggest that melatonin-induced decreases in body temperature and locomotor activity occur by melatonin acting at discrete sites in the brain.

Animals↗

PubMed1999

GABA may function tonically via GABA(A) receptors to inhibit hypotension and bradycardia by L-DOPA microinjected into depressor sites of the nucleus tractus solitarii in anesthetized rats.

We have proposed that DOPA is a transmitter of the primary baroreceptor afferents terminating in the rat nucleus tractus solitarii (NTS). GABA is a putative inhibitory neuromodulator for baroreflex inputs in the NTS. GABA may inhibit DOPAergic transmission. Drugs were microinjected into depressor sites of the NTS in anesthetized rats. DOPA (10-60 ng) elicited dose-dependent depressor responses. GABA (3-300 ng) elicited dose-dependent pressor responses. Nipecotic acid (100 ng) elicited pressor responses. Bicuculline (10 ng) elicited depressor responses. Responses to DOPA (30 ng) were inhibited by pretreatment with GABA and nipecotic acid, but potentiated by bicuculline, when vascular responses to pretreated drugs returned to basal levels. DOPA ME, a competitive DOPA antagonist, did not displace specific [3H]GABA binding. Prior DOPA ME (1 microg) inhibited by one-half pressor responses to 300 ng GABA. GABA seems to inhibit tonically via GABA(A) receptors depressor responses to DOPA and to elicit pressor responses partially by inhibition of tonic function of endogenous DOPA to activate depressor sites in the NTS. These findings further support the above proposal.

Anesthesia↗

PubMed1999

Inhibition of nicotine-induced hippocampal norepinephrine release in rats by alpha-conotoxins MII and AuIB microinjected into the locus coeruleus.

Hippocampal norepinephrine (NE) is secreted by neurons projecting from the locus coeruleus (LC) to the hippocampus; LC nicotinic receptors (NAchRs) are involved in the effects of systemic nicotine on this pathway. To clarify the NAchR subtypes, NAchR antagonists, termed alpha-conotoxins, were microinjected into the LC before nicotine; MII and AuIB were used to assess the potential involvement of alpha3beta2 and alpha3beta4 subunit-containing NAchRs, respectively. Nicotine dose-dependently stimulated hippocampal NE release (P < 0.01). MII (>0.25 pmol) reduced the NE response to nicotine (67% decrease; P < 0.05), as did AuIB (44% reduction by 25 pmol; P < 0.05). Administered together, however, MII and AuIB were no more effective than MII. Thus, MII and AuIB are capable of interacting with NAchR subtypes other than those previously defined as alpha3beta2 and alpha3beta4, respectively. NAchRs containing both beta2 and beta4-subunits may be involved.

Animals↗

PubMed1999

Progesterone microinjections into the pontine reticular formation modify sleep in male and female rats.

It has been reported that progesterone (P4) induces changes in sleep, but the brain regions involved in these actions are unknown. We studied the effects of P4 microinjections into the pontine reticular formation (PRF) upon rat sleep. Intact adult male and ovariectomized female rats were unilaterally injected with P4 into the PRF and the sleep-waking cycle was recorded for 6 h. P4 (1.0 and 5.0 microg/0.2 microl) did not modify sleep, but at a higher dose (7.5 microg/0.2 microl) it produced a marked decrease in rapid eye movement sleep (REM) latency in both male (55%) and female (63%) rats. A non-significant increase in the number of REM episodes was observed after P4 administration. These findings suggest that P4 should participate in the mechanisms related to REM initiation in the rat through its effects in the PRF.

Animals↗

PubMed2001

Reversible analgesia, atonia, and loss of consciousness on bilateral intracerebral microinjection of pentobarbital.

Concussion, asphyxia, and systemically administered general anesthetics all induce reversible depression of the organism's response to noxious stimuli as one of the elements of loss of consciousness. This is so even for barbiturate anesthetics, which have only modest analgesic efficacy at subanesthetic doses. Little is known about the neural circuits involved in this form of antinociception, although for anesthetic agents, at least, it is usually presumed that the drugs act in widely distributed regions of the nervous system. We now report the discovery of a focal zone in the brainstem mesopontine tegmentum in rats at which microinjection of minute quantities of pentobarbital induces a transient, reversible anesthetic-like state with non-responsiveness to noxious stimuli, flaccid atonia, and absence of the righting reflex. The behavioral suppression is accompanied by slow-wave EEG and, presumably, loss of consciousness. This zone, which we refer to as the mesopontine tegmental anesthesia locus (MPTA), apparently contains a barbiturate-sensitive 'switch' for both cortical and spinal activity. The very existence of the MPTA locus has implications for an understanding of the neural circuits that control motor functions and pain sensation, and for the cerebral representation of consciousness.

Adjuvants, Anesthesia↗

PubMed2000

Evidence for striatal dopaminergic overactivity in paroxysmal dystonia indicated by microinjections in a genetic rodent model.

Mutant dystonic hamsters (dt(sz)), a model of primary paroxysmal dystonia, display attacks of generalized dystonia in response to mild stress in an age-dependent manner. Recent studies in dystonic hamsters have revealed decreased densities of dopamine D(1) and D(2) in the dorsal striatum. This finding has been interpreted as a down-regulation in response to enhanced dopamine release because systemic treatments with neuroleptics reduced the severity of dystonia while levodopa exerted prodystonic effects. Therefore, in the present study we investigated the effects of amphetamine as well as of selective D(1) or D(2) receptor agonists and antagonists on the severity of dystonia after systemic administrations and after microinjections into the dorsal striatum. Amphetamine and the dopamine D(2) agonist quinpirole increased the severity of dystonia after systemic and striatal injections, while the dopamine D(1) agonist SKF 38393 exerted only moderate prodystonic effects after systemic administration of a high dose but not after striatal injections. These results suggest that a predominant overstimulation of D(2) receptors is pathogenetically involved in the dystonic syndrome. Combined systemic or striatal administrations of the D(1) and D(2) receptor agonists did not reveal synergistic prodystonic effects at the examined doses. The selective D(1) antagonist SCH 23390 as well as the D(2) antagonist raclopride tended to decrease the severity of dystonia after systemic administration but failed to exert significant effects after striatal injection. The coadministration of ineffective doses of the antagonists SCH 23390 and raclopride, however, exerted an enormous antidystonic efficacy after both systemic and striatal injections. Since striatal injections of compounds which enhance dopaminergic activity aggravated dystonia, while coinjections of dopamine D1 and D2 receptor antagonists reduced the severity of dystonia, the present findings clearly support the hypothesis that striatal dopaminergic overactivity plays a crucial role for the manifestation of dystonic attacks in the hamster model of paroxysmal dystonia.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed2000

Enhanced intake of high-fat food following striatal mu-opioid stimulation: microinjection mapping and fos expression.

Our previous studies have shown that stimulation of mu-opioid receptors within the nucleus accumbens preferentially enhances intake of palatable food containing sucrose and fat; thus, opioids in this brain area may mediate the rewarding characteristics of food by modulating taste and macronutrient preference. The present study was designed to further explore the nature of the involvement of striatal opioids in feeding behavior, such as the location of sensitive subregions of the ventral striatum and the brain neural circuits involved in opioid-mediated hyperphagia. In Experiment 1, we conducted a microinfusion mapping study of feeding behavior by microinfusion of the mu receptor agonist, D-Ala(2),NMe-Phe(4), Glyol(5)-enkephalin (0, 0.025 and 0.25 microg/0.5 microl per side; equivalent to 0, 0.04 and 0.40 nmol/0.5 microl per side), into several striatal subregions. In Experiment 2, detection of the expression of the immediate early gene, c-fos, was used to examine brain areas activated following intra-striatal microinfusion of D-Ala(2), NMe-Phe(4),Glyol(5)-enkephalin. The microinjection mapping study demonstrated a broad anatomical gradient within the striatum, with sensitivity highest in relatively more lateral and ventral regions of the striatum (ventrolateral striatum, lateral shell and core). The Fos mapping study demonstrated that circuitry including hypothalamic areas, the ventral tegmental area, the substantia nigra and the nucleus of the solitary tract was recruited by stimulation of mu receptors within the nucleus accumbens. A similar pattern was observed following stimulation of mu receptors in the dorsal striatum; however, the extent of activation was much smaller in magnitude. These results suggest that the role of mu receptors within the striatum in palatable feeding primarily involves ventral and lateral regions. Moreover, the pattern of activation in hypothalamic, midbrain and gustatory-visceral relay areas suggests that striatal mu receptors may participate in integrating motivational, metabolic and autonomic aspects of ingestive behavior.

Analgesics, Opioid↗

PubMed1997

Microinjections of muscimol into lateral superior colliculus disrupt orienting and oral movements in the formalin model of pain.

An important reaction in rodent models of persistent pain is for the animal to turn and bite/lick the source of discomfort (autotomy). Comparatively little is known about the supraspinal pathways which mediate this reaction. Since autotomy requires co-ordinated control of the head and mouth, it is possible that basal ganglia output via the superior colliculus may be involved; previously this projection has been implicated in the control of orienting and oral behaviour. The purpose of the present study was therefore, to test whether the striato-nigro-tectal projection plays a significant role in oral responses elicited by subcutaneous injections of formalin. Behavioural output from this system is normally associated with the release of collicular projection neurons from tonic inhibitory input from substantia nigra pars reticulata. Therefore, in the present study normal disinhibitory signals from the basal ganglia were blocked by injecting the GABA agonist muscimol into different regions of the rat superior colliculus. c-Fos immunohistochemistry was used routinely to provide regional estimates of the suppressive effects of muscimol on neuronal activity. Biting and licking directed to the site of a subcutaneous injection of formalin (50 microliters of 4%) into the hind-paw were suppressed in a dose-related manner by bilateral microinjections of muscimol into the lateral superior colliculus (10-50 ng; 0.5 microliter/side); injections into the medial superior colliculus had little effect. Bilateral injections of muscimol 20 ng into lateral colliculus caused formalin-treated animals to re-direct their attention and activity from lower to upper regions of space. Muscimol injected unilaterally into lateral superior colliculus elicited ipsilateral turning irrespective of which hind-paw was injected with formalin. Oral behaviour was blocked when the muscimol and formalin injections were contralaterally opposed; this was also true for formalin injections into the front foot. Interestingly, when formalin was injected into the perioral region, injections of muscimol into the lateral superior colliculus had no effect on the ability of animals to make appropriate contralaterally directed head and body movements to facilitate localization of the injected area with either front- or hind-paw. These findings suggest that basal ganglia output via the lateral superior colliculus is critical for responses to noxious stimuli which entail the mouth moving to and acting on the foot, but not when the foot is the active agent applied to the mouth. The data also suggest that pain produces a spatially non-specific facilitation of units throughout collicular maps, which can be converted into a spatially inappropriate signal by locally suppressing parts of the map with the muscimol.

Animals↗

PubMed2002

Microinjection of procaine into the pedunculopontine tegmental nucleus suppresses hippocampal theta rhythm in urethane-anesthetized rats.

It was found that the cholinergic component of the pedunculopontine tegmental nucleus (PPN) is involved in the generation of theta rhythm in the hippocampus. However, it is still not known how important PPN is in the brainstem theta-generating system, where the nucleus reticularis pontis oralis is regarded as a primary generator. In the present experiment, performed on urethane-anesthetized rats, we studied the effect on the tail pinch-elicited hippocampal theta of unilateral inactivation of PPN by means of direct procaine microinjection. Procaine induced ipsilateral suppression of theta rhythm, manifested as desynchronization of hippocampal EEG, a shift of the fast Fourier transformation (FFT) power peak toward lower frequencies, and a reduction of FFT peak magnitude at theta band. Hippocampal field activity returned to normal (both its FFT peak frequency and magnitude) within 30 min after the injection. The results obtained indicate that PPN is critical for hippocampal theta generation but it may not be involved in encoding theta frequency.

Anesthetics, Intravenous↗

PubMed1997

An easily constructed pipette for pressure microinjections into the brain.

A simple device for making pressure microinjections into the brain, its application for delivery of a tracer substance, biotin dextran amine, and an example of the resulting axonal transport are described. The device is based on the use of a Luer Up Hamilton microliter syringe mated directly to the plastic hub of an injection needle assembly in which the metal needle has been replaced by a glass pipette. In model experiments, the injection sites were measured in brain sections of rats, which were perfused with fixative immediately after administration of the tracer, and the relationship between the injected volume and the area of the injection site was evaluated. The results showed that the device provided accurate and reproducible delivery of the fluid.

Animals↗

PubMed1999

Morphological changes of the endolymphatic sac induced by microinjection of artificial endolymph into the cochlea.

Morphological changes of the endolymphatic sac were analyzed in guinea pigs following microinjection of artificial endolymph into the cochlea or withdrawal of a quantity of native endolymph. Injections were performed into the second turn of scala media with a micro-pump at a rate of 60-100 nl/min, lasting for a period of 4, 7. 5, 15 or 18 min. In withdrawal experiments, endolymph was aspirated from the second cochlear turn over a period of 8 min. For each procedure the contralateral (non-treated) ear served as a histological control. Following artificial endolymph injections of 7. 5 min or more there was an almost total absence of the normal intraluminal homogeneous substance (HS) on the injected side. Our observations suggest that the disappearance of the HS occurs by both enzymatic and macrophagic activity. After endolymphatic withdrawals the ES was found to contain increased amounts of HS. The results could suggest that the volume of fluid in the ES, and hence the volume of the entire membranous labyrinth, may be regulated by a dynamic relationship between active secretion and enzymatic degradation of a lumen-expanding substance that is intimately related to the intraluminal macrophages. The exact mechanism governing these regulatory systems, and their relationship to ion and water movements across the epithelium of the sac, remain to be elucidated.

Animals↗