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PubMed1985

A technique for the microinjection of macromolecules into viable chromaffin cells in culture.

A technique is described by which macromolecules can be microinjected into chromaffin cells in monolayer culture. This technique employs erythrocyte ghosts as the vehicles for microinjection, phytohemagglutinin, a plant lectin, as an attachment agent, and polyethylene glycol as the fusagen. High erythrocyte ghost-chromaffin cell fusion indices have been obtained, with an average of 46.2 +/- 1.1% (n = 14) of the total chromaffin cell population efficiently injected. Cell viability is well maintained during and after fusion with an average cell loss of 12 +/- 0.4% (n = 14) of the total cell population. The functional parameters which characterize the chromaffin cells in culture are unaltered after fusion-induced microinjection. The endogenous catecholamine content, the uptake of exogenous catecholamines via the high-affinity uptake mechanism for catecholamines, as well as the cell's response to various secretagogues remain unchanged. This procedure which allows a large number of cultured cells to be injected rapidly without significant loss of cell viability will aid in the study of the molecular and cell biology in this system.

Adrenal Medulla↗

PubMed1994

Effects of microinjection of kainic acid into the nucleus tractus solitarius on fluid and NaCl absorption across the jejunum.

Effects of bilateral chemical inactivation of the nucleus tractus solitarius (NTS) by microinjection of kainic acid (KA) on fluid and NaCl absorption across the jejunum were examined in anesthetized Sprague-Dawley rats. Jejunal fluid and NaCl absorption was measured in a jejunal loop before and after the microinjection of artificial cerebrospinal fluid (aCSF) or KA into the NTS. Net fluid and NaCl absorption was not altered by a microinjection of aCSF. However, net fluid (from 1.12 +/- 0.07 to 1.66 +/- 0.06 ml/30 min) and NaCl (Na+ from 164.5 +/- 10.1 to 243.3 +/- 7.1 muEq/30 min; and Cl- from 175.9 +/- 7.5 to 260.8 +/- 6.5 muEq/30 min) absorption was significantly increased by the chemical inactivation of the NTS. To examine the efferent mechanism of the increased net absorption induced by the NTS inactivation, mesenteric nerve activity (MNA) was measured before and after the inactivation of the NTS. MNA was significantly increased by 165.9 +/- 74.2% after the bilateral inactivation of the NTS. Furthermore, absorption experiments were conducted in rats with pretreatment of atropine (acetylcholine-antagonist) or yohimbine (specific alpha 2-antagonist). In atropine treated rats, net jejunal absorption was significantly increased by the inactivation of the NTS. However, the increase in net absorption induced by the inactivation of the NTS was completely abolished by pretreatment with yohimbine. These results suggest that the NTS has a tonic suppression on jejunal absorption through alpha 2-adrenergic mechanism.

Animals↗

PubMed1996

Glycine produced pressor responses when microinjected in the pressor areas of pons and medulla in cats.

In 24 cats under chloralose/urethane anesthesia changes of systemic arterial pressure (SAP) and sympathetic vertebral nerve activities (VNA) were induced by microinjection of glycine (Gly, 1.0 M, 50 nl) into the pressor areas of the rostral pons, i.e., locus coeruleus-parabrachial nucleus (LC-PBN), nucleus of gigantocellular tegmental field-lateral tegmental field (FTG-FTL), and dorsomedial (DM) and ventrolateral (VLM) medulla. The effects were compared with those induced by microinjection of sodium glutamate (Glu, 0.25 M, 50 nl) into the same sites. In about 60% of the injections Gly produced increases in SAP and VNA similar to that of Glu. The increase in SAP was greater in VLM, while the increase in VNA was more marked in DM. In the rest of microinjections Gly and Glu produced changes of SAP and VNA in different combinations. The latency of Gly-induced increases in SAP and VNA was 1 to 3 s longer than that induced by Glu. Our findings show that although Gly is classified as an inhibitory transmitter, it often produced excitation of the pressor neurons in the pons and medulla similar to that of Glu. Whether Gly acts through the same cardiovascular neurons that respond to Glu or through activation of different kinds of neurons remains to be elucidated.

Animals↗

PubMed1985

Changes in body temperature and metabolic rate following microinjection of Met-enkephalinamide in the preoptic/anterior hypothalamus of rats.

The effects of Met-enkephalinamide (MET-ENKamide) on brain temperature (Tb) and metabolic rate (MR) were assessed following direct administration into the preoptic/anterior hypothalamus (PO/AH) of freely moving rats. Bilateral microinjections of saline or MET-ENKamide (1-25 micrograms/microliter) were delivered through cannula guide tubes previously implanted in nine animals. Thiorphan, an enkephalinase inhibitor, was microinjected into the PO/AH of two of the animals. All injections were made remotely at an ambient temperature of 22 +/- 1 degree C in a volume of 1 microliter. Measurements of Tb (via a brain-dwelling thermistor) and MR were recorded continuously. The ability of naloxone to antagonize the effects of MET-ENKamide was investigated by fashioning a double-barreled injection cannula to fit within each guide tube; 1 microliter of saline or naloxone (1-10 micrograms) was delivered bilaterally into the PO/AH followed by 1 microliter of MET-ENKamide (25 micrograms) 5-10 min later. PO/AH administration of MET-ENKamide (1-25 micrograms) produced dose-dependent increases in Tb preceded by dose-dependent increases in MR, with a characteristic time course of approximately 30 min. Naloxone antagonized the rise in Tb and MR, either partially or completely, depending on dose. When administered alone, naloxone had no effect on Tb or MR. Microinjection of thiorphan (10 micrograms) into the PO/AH evoked increases in Tb and MR that were similar to those responses induced by MET-ENKamide. These results support a role for endogenous Met-enkephalin in the regulation of Tb in the rat.

Animals↗

PubMedDate not supplied

Peptides and self-stimulation of the medial prefrontal cortex in the rat: effects of intracerebral microinjections of substance P and cholecystokinin.

The effects of intracerebral microinjections of substance P and cholecystokinin on self-stimulation of the medial prefrontal cortex of the rat were studied. Intracerebroventricular administration of substance P at doses of 2.5, 5, 10 and 20 micrograms produced a dose-related decrease in self-stimulation of the medial prefrontal cortex; spontaneous motor activity, measured as a control, was not affected. Unilateral microinjections into the medial prefrontal cortex of substance P at doses of 10 and 20 micrograms produced a decrease of self-stimulation of the ipsilateral side, but self-stimulation of the contralateral cortex, used as a control, was not affected. On the contrary, cholecystokinin in both intracerebroventricular administration at doses of 100, 200 and 400 ng, or intracortical microinjections into the medial prefrontal cortex at doses of 200, 400 and 800 ng, had no effect on self-stimulation of this cortical area. These results suggest that substance P, but not cholecystokinin, could be part of the neurochemical substrate underlying self-stimulation of the medial prefrontal cortex in the rat.

Animals↗

PubMedDate not supplied

Proteolytic conversion of oxytocin in vivo after microinjection in the rat hippocampus.

Since previous studies in vivo have shown that oxytocin is metabolized by rat synaptic membrane-bound aminopeptidase- and endopeptidase-like enzymes, the proteolytic conversion of oxytocin was studied in vivo after microinjection in the rat hippocampus, a brain area that contains oxytocinergic nerve endings and receptors. Isolation of the formed peptide fragments from the injected brain area after homogenization and adsorption on a Sep-Pak cartridge by high performance liquid chromatography, and their characterization by amino acid analysis, revealed that, when oxytocin (50 nmol in 0.5 microliter) was microinjected in the CA1 field of the rat hippocampus, only the N-terminal fragment oxytocin(1-8) was formed in such amount that could be characterized. The microinjection of [3H-Tyr2]oxytocin (10 pmol) revealed that in addition to oxytocin(1-8), free [3H]tyrosine was formed. Taken together with previous findings showing that C-terminal oxytocin fragments as well oxytocin(1-8) are formed by membrane-bound aminopeptidases and endopeptidases in vitro, respectively, the results suggest that, in addition to aminopeptidases, endopeptidase-like enzymes are involved in the proteolysis of endogenous brain oxytocin.

Amino Acids↗

PubMed1994

Neuropeptide Y microinjected into the suprachiasmatic region phase shifts circadian rhythms in constant darkness.

The geniculohypothalamic tract (GHT) is a projection from the intergeniculate leaflet to the suprachiasmatic nucleus (SCN). The GHT exhibits neuropeptide Y (NPY) immunoreactivity and appears to communicate photic information to the SCN. Microinjection of NPY into the SCN has been found to phase shift circadian rhythms of hamsters housed in constant light in a manner similar to the phase shifts produced by pulses of darkness or triazolam injections. In the present study, NPY was injected into the SCN of Syrian hamsters housed in constant darkness and was found to produce phase shifts similar to those seen in hamsters housed in constant light. Microinjections were not followed by wheel running during the subjective day (the time when NPY microinjections are followed by significant phase advances). These data suggest that NPY produces phase shifts by some mechanism other than by inducing wheel running or by inhibiting the response of SCN neurons to light and supports a role for NPY in nonphotic shifting of the circadian clock.

Animals↗

PubMed1994

Microinjection of Cdc25 protein phosphatase into Xenopus prophase oocyte activates MPF and arrests meiosis at metaphase I.

Microinjection of bacterially expressed human cdc25A protein into Xenopus prophase oocytes provokes the activation of p34cdc2 kinase and the tyrosine dephosphorylation of p34cdc2 in the presence or absence of protein synthesis. The level of p34cdc2 kinase activity then drops in parallel with the degradation of cyclin B2 and finally increases again to stabilize at a high level. Cdc25 microinjection induces the assembly of a metaphase I spindle which is abnormally located in the deep cytoplasm. Moreover, oocytes arrest at the metaphase I stage and do not reach metaphase II even 10 h after cdc25 microinjection. The extended metaphase I period observed in cdc25-injected oocytes results from an equilibrium between degradation of cyclins and synthesis of new cyclins. This is in contrast with progesterone-stimulated oocytes where cyclin degradation is turned off when oocytes enter metaphase II. During metaphase I, the reactivation of MPF activity can be disrupted in two different ways: 1) cycloheximide, an inhibitor of protein synthesis, by preventing the synthesis of new cyclins, provokes the disappearance of MPF kinase activity and the reformation of a nucleus; 2) when the cAMP level is increased during the metaphase I period in cdc25-injected oocytes, MPF kinase activity drops following a rephosphorylation of tyrosine 15 of p34cdc2, while the cyclin turn-over remains unaffected. Moreover, increasing the cAMP level in prophase oocytes totally prevents the action of cdc25. Our results indicate that in Xenopus oocytes, the PKA pathway negatively regulates the activation of MPF and the activity of p34cdc2/cyclin B complex through tyrosine phosphorylation of p34cdc2 during metaphase I.

Animals↗

PubMed1985

Inhibition of flank-marking behavior in golden hamsters by microinjection of a vasopressin antagonist into the hypothalamus.

Microinjection of arginine-vasopressin (AVP) into the medial preoptic area of the hypothalamus of the hamster stimulates flank marking, a complex stereotypic motor behavior involved in olfactory communication. Microinjection of an antagonist of AVP, [1-deaminopenicillamine-2-(O-methyl)-tyrosine]arginine-vasopressin, into the same site blocks both the effect of microinjected AVP as well as the natural flank-marking behavior normally elicited by placing a hamster into the recently vacated home cage of another hamster. This finding supports the notion that AVP is a transmitter in the expression of flank marking.

Animal Communication↗

PubMed1987

The use of microinjected colloidal gold and immunocytochemistry to localise pressor sites in the rostral medulla oblongata of the rat.

A new technique for localising the centre of sites of microinjection of drugs in the brain using the silver intensification of colloidal gold is described. The technique is compatible with immunocytochemistry and in this study was used in Wistar-Kyoto rats to investigate the relationship between the location of phenylethanolamine-N-methyltransferase-like immunoreactive (PNMT-LI) cell bodies in the medulla and the precise sites where microinjection of L-glutamate elicited pressor responses. Colloidal gold provided a simple technique for localising the centre of sites of microinjection with a high degree of spatial resolution and allowed pressor sites to be precisely localised in the region of ventrolateral medulla with the highest density of PNMT-LI cells.

Animals↗

PubMed1988

Microinjected morphine suppresses the activity of locus coeruleus noradrenergic neurons in freely moving cats.

Microinjection of morphine (1.0 microgram/0.1 microliter) produced a significant suppression (approximately 60%) of the single unit activity of locus coeruleus noradrenergic neurons in freely moving cats. This effect was reversible by systemic administration of the opioid receptor antagonist, naloxone (1.0 mg/kg i.v.). The microinjection of naloxone (1.0 microgram/0.1 microliter), however, was without effect on the spontaneous activity of noradrenergic neurons in the locus coeruleus. Non-noradrenergic neurons recorded in the same vicinity showed no consistent response to the microinjection of morphine. These results suggest that the direct effect of opioids in the locus coeruleus is an inhibition of noradrenergic neuronal activity. Furthermore, it appears that opioid influences upon these neurons are not tonically active.

Action Potentials↗

PubMed1988

Microinjections of glutamate or morphine at coincident midbrain sites have different effects on nociceptive dorsal horn neurons in the rat.

Responses of single lumbar spinal neurons to noxious skin heating (50 degrees C, 10 s) were electrophysiologically recorded in barbiturate-anesthetized rats. Responses of all neurons were suppressed by electrical stimulation in the midbrain periaqueductal gray (PAG) or lateral reticular formation (LRF). Microinjection of glutamate (GLU, 0.1-0.3 microliter, 0.5 M) into the PAG rapidly (within 15 s) suppressed (to 13-55% of control) the responses of 6/16 neurons with recovery within 8 min. The remainder were affected less at even higher doses (0.5-1 microliter). Responses of 4/10 neurons were suppressed following GLU microinjected into the LRF. We also tested effects of microinjection of morphine (MOR, 5 micrograms/0.5 microliter) into GLU-sensitive and insensitive PAG sites. Responses of 4 neurons were unaffected, 4 were enhanced (to 130-155%), and 2 suppressed (to 43 and 57%) following MOR in PAG, with enhancement or suppression beginning within 12-20 min and lasting 40 to over 70 min. The differing effects of GLU and MOR may reflect different mechanisms for the descending modulation of spinal nociceptive transmission.

Animals↗

PubMed1995

Inhibition of spinal nociceptive neurons by microinjections of somatostatin into the nucleus raphe magnus and the midbrain periaqueductal gray of the anesthetized cat.

The effects of somatostatin (SOM) after intravenous application and intracerebral microinjection into the medullary nucleus raphe magnus (NRM) or into the periaqueductal gray (PAG) on the spinal nociceptive transmission was quantitatively studied in the anesthetized cat. Noxious heat-evoked responses of multireceptive lumbar spinal dorsal horn neurons were reversibly depressed to 56.6 +/- 9.7% of the control after systemically applied SOM (7 micrograms/kg i.v.; 7 micrograms/kg per h infusion rate). At 11 of 14 brainstem microinjection sites in the NRM and PAG, SOM (2.5 micrograms/microliter) attenuated the heat-evoked responses to 58.9 +/- 6.2% (n = 5) (NRM) and 64.4 +/- 6.3% (n = 6) (PAG) of the control. After microinjection, maximal inhibition was reached within 8-14 min (NRM) or 23-29 min (PAG), respectively. Inhibition was reversible within 60 min after the injection. Thus, SOM has an antinociceptive potency by activating descending inhibition of nociceptive dorsal horn neurons from the NRM and PAG.

Animals↗

PubMed1994

Dissociation of the alpha 2-adrenergic antinociception from sedation following microinjection of medetomidine into the locus coeruleus in rats.

It is well established that alpha 2-adrenoceptor agonists have sedative and antinociceptive properties. In the current behavioral study we tried to find out if the alpha 2-adrenergic sedative and antinociceptive effects can be dissociated. We tested the hypothesis that alpha 2-adrenergic sedation is mediated by the locus coeruleus (LC) and antinociception by spinal alpha 2-adrenoceptors. Also, we addressed the possibility that intracerebral injection of an alpha 2-agonist might produce its antinociceptive effect by an action directly at the spinal cord. Medetomidine, an alpha 2-adrenergic agonist, or atipamezole, an alpha 2-adrenergic antagonist, were microinjected bilaterally into the LC through chronic cannulae in unanesthetized Han-Wistar rats. The effect on locomotor activity (/vigilance), tail-flick and hot-plate response, and on formalin-induced pain behavior was determined. Medetomidine microinjected into the LC (1-10 micrograms/cannula) produced dose-dependently hypolocomotion (/sedation), increase of response latencies in the hot-plate and the tail-flick tests, and a decrease in the formalin-induced pain behavior. Hypolocomotion (/sedation) was obtained at a lower medetomidine dose (1 microgram/cannula) than antinociception (3-10 micrograms/cannula). The lowest medetomidine dose used (1 microgram/cannula), which induced significant hypolocomotion (/sedation), produced either no antinociception (hot-plate and tail-flick tests) or even a slight hyperalgesia (formalin test). The hypolocomotion (/sedation) but not antinociception (tail-flick test) induced by systemic administration of medetomidine (100 micrograms/kg s.c.) could be reversed by atipamezole (10 micrograms/cannula) microinjected into the LC. Only a high systemic dose of atipamezole (1 mg/kg s.c.) reversed the antinociceptive effects of medetomidine.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Antagonists↗

PubMed1984

Depression of neuronal activity in the hypothalamic ventromedial nucleus of the rat following microinjection of morphine in the amygdala or the periaqueductal gray.

The effects of morphine administered intravenously and intracerebrally in the cortical amygdala and periaqueductal gray were examined on spontaneous neuronal activity in the hypothalamic ventromedial nucleus in both intact and castrated adult male rats. Spontaneous extracellular single unit activity in the ventromedial nucleus was significantly faster in chronically castrated (19-23 days) rats compared with intact control rats (6.0 +/- 1.1 and 2.5 +/- 0.3 spikes/s, respectively). Neurons in the ventromedial nucleus exhibited a significant attenuation of neuronal activity following the intravenous administration of morphine; the maximal per cent decrease (to 25% of the control unit activity) was the same in both castrated and intact rats, but cell firing in castrated rats was affected more at lower doses of morphine. When microinjected bilaterally in the cortical amygdala or periaqueductal gray (5 micrograms/site), morphine produced the same maximal depression of unit activity in the ventromedial nucleus as when administered intravenously. The time required to significantly attenuate unit activity in the ventromedial nucleus was the same (3 min) whether morphine was administered intravenously or bilaterally in the periaqueductal gray. When microinjected bilaterally in the cortical amygdala, the depressant effect of morphine on cell firing in the ventromedial nucleus was significant first at 10 min. These data correlate well with the time courses for depression of serum levels of luteinizing hormone produced by morphine microinjected in the same doses in the same extrahypothalamic sites in the rat. In view of the relatively high levels of specific opiate receptor binding in both the periaqueductal gray and amygdala, this report further supports a role for modulation by extrahypothalamic areas of opioid-induced changes in the functional activity of the hypothalamic-pituitary axis, perhaps via the ventromedial nucleus.

Action Potentials↗

PubMed1992

Antinociception induced by microinjection of substance P into the A7 catecholamine cell group in the rat.

Stimulation of neurons in the ventromedial medulla produces antinociception that is mediated in part by indirect activation of pontospinal noradrenergic neurons. Substance P-containing neurons located in the ventromedial medulla project to the A7 catecholamine cell group and may serve as an excitatory link between these two cell groups. Thus, the antinociception induced by stimulation of the neurons in ventromedial medulla may be mediated by substance P released from these projections which activates spinally projecting noradrenergic neurons in the A7 cell group. This hypothesis was tested by determining whether microinjection of various doses of substance P into the A7 cell group of the rat could induce antinociception. The results indicated that substance P induced dose-dependent antinociception that was more pronounced in the hindlimb ipsilateral to the microinjections. This observation is consistent with anatomical observations that noradrenergic A7 neurons project predominantly to the ipsilateral spinal cord dorsal horn. Moreover, the antinociceptive effects of substance P microinjection appear to be mediated at least in part by activation of spinally projecting noradrenergic neurons in the A7 cell group, because intrathecal injections of the alpha-2 noradrenergic antagonists yohimbine and idazoxan blocked these antinociceptive effects. The results of these experiments support the hypothesis that the antinociception induced by stimulation of neurons in the ventromedial medulla is mediated in part by activation of substance P-containing neurons that project to, and activate, spinally projecting noradrenergic neurons located in the A7 catecholamine cell group.

Afferent Pathways↗

PubMedDate not supplied

14C-dopamine microinjected into the brain-stem of the rat: dispersion kinetics, site content and functional dose.

A morphological analysis was undertaken of both the dispersion characteristics and tissue content of dopamine (DA) microinjected acutely into the brain-stem of the anesthetized rat. 14C-DA, with a specific activity of 56-62 mCi/mMol, was infused unilaterally into the pars compacta of the substantia nigra in one of four test volumes: 0.5, 1.0, 4.0 or 8.0 microliters. The concentration of the 14C-DA solution was 1.0 microCi/microliter, equivalent to 3.01 micrograms/microliters, which was delivered at an injection rate of 1.0 microliter per 45 sec. At an interval of either one min or 15 min following the microinjection, the rat's brain was removed rapidly from its calvarium, flash frozen and then cut in the coronal plane on a freezing microtome in 500 micron slabs. After each of the respective serial slabs was mounted on glass, the Eik Nes-Brizzee trochar technique for the discrete removal of tissue samples was used to obtain 0.5 mm dia. cylindrical plugs of meso-diencephalic tissue at distances from the site of injection ranging from 0.5 to 2.5 mm, center to center. Each sample plug was subsequently solubilized and 14C-DA activity quantitated by liquid scintillation spectrometry. The results show that regardless of volume, the spatial patterning of the microinjected solution assumes a tear-drop or pear shape, not a sphere. Further, as the volume of the injection is increased from 0.5 to 8.0 microliters, the magnitude of the dispersion of 14C-DA is enhanced throughout the surrounding parenchyma, but not in a linear fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2005

Homeostatic alterations after intrapallidal microinjection of interleukin-1beta in the rat.

To elucidate the homeostatic consequences of direct pallidal administration of interleukin-1beta (IL-1beta), short- (2 h) and long-term (12 h) food intakes, water intake and body temperature were measured after bilateral microinjection of IL-1beta (with or without paracetamol/PAR/pretreatment) into the ventro-medial part of the globus pallidus (GP). The effects were compared with those found in vehicle- or vehicle+PAR-treated control animals and intraperitoneally (i.p.) injected IL-1beta, IL-1beta+PAR or control rats. Direct GP microinjection of IL-1beta, similar to the peripheral (i.p.) administrations, reduced remarkably short-term food intake in food deprived animals. However, there were no significant differences among the groups in long-term food intakes and in water intakes as well. IL-1beta microinjection into the GP caused a significant increase in body temperature. This IL-1beta induced hyperthermia was attenuated by PAR pretreatment, whereas food intake was not affected. Results of the electrophysiological experiments revealed substantial changes in GP extracellular single neuron activity in response to the microelectrophoretic administration of IL-1beta. The present findings, along with previous data, are discussed in terms of the glucose-monitoring neural circuitry through which cytokines exert their direct modulatory effect on central homeostatic regulation.

Animals↗