Stereotaxic Apparatus Research Articles

GnRH is expressed in GnRH-deficient hypogonadal (hpg) mice after GnRH gene transfer with a herpes simplex virus (HSV) amplicon vector

The challenge of gene therapy is to develop non-toxic vectors that can achieve stable transgene expression in target cells. The HSV amplicon is a unique vector that has essentially no toxicity, a large transgene capacity, and the potential to integrate the transgene into a specific site in the genome, thus making it a useful tool for gene therapy. Hpg mice are genetically deficient in GnRH production in the hypothalamus, resulting in many deficits in the reproductive system including infertility. Our goal was to determine neuronal expression of GnRH and the recovery of gonadal function after CNS delivery of an HSV amplicon vector containing the GnRH gene using hpg mice. An HSV amplicon containing the GnRH gene under its cognate promoter was prepared and packaged using a helper virus-free packaging system. This vector was injected into the preoptic area (POA) of the hypothalamus of hpg mice using a stereotaxic apparatus. Following injection, we determined GnRH neuronal expression by immunohistochemistry (IHC) and recovery of gonadal function using vaginal cytology and blood hormonal assays. Eight adult female hpg mice were studied. Five mice were injected with the HSV-GnRH vector in the POA, and 3 mice were injected with vehicle only as control. The HSV-GnRH vector (1× 108 transducing units/ml) was injected into the POA of hpg mice under anesthesia using a stereotaxic apparatus. Mice were sacrificed 10 days (n=4) or 50 days (n=1) after injection. Neuronal expression of GnRH was analyzed by IHC in brain tissue. Plasma FSH and LH levels were measured by radioimmunoassay. The weights of the ovary, uterus and oviduct were compared between the study and control groups. Vaginal cytology was also checked each day after HSV amplicon injection to evaluate estrous cyclicity. GnRH expression was present in neurons near the injection site, as determined by IHC 10 days and 50 days following the vector injections. However, there was no significant difference in plasma FSH (2.0 ± 0.7 ng/ml vs. 4.9 ± 0.9 ng/ml; study vs. control), LH (0.07 ± 0.01 ng/ml vs. 0.06 ± 0.02 ng/ml) levels, or in the weight of reproductive organs (24.2 ± 6.4 mg vs. 17.9 ± 2.7 mg) between the study and control groups. We observed only diestrus specific morphology in the vaginal epithelial cells. Expression of GnRH was observed in the hypothalamic neurons in the POA in GnRH-deficient hpg mice following GnRH gene transfer using an HSV amplicon vector. These data suggest that such HSV vectors may be useful in correcting the reproductive defect in these animals. Further studies are necessary to determine if normal hypothalamic-pituitary-gonadal axis function can be restored in hpg mice by gene therapy using this approach.

A novel stereotaxic approach to the hypothalamus for the use of push-pull perfusion cannula in Holstein calves

To determine secretory patterns of growth hormone-releasing hormone (GHRH) and somatostatin (SS) and their roles in the regulation of growth hormone (GH) secretion, a method for collecting hypothalamic perfusates, a push-pull perfusion method was developed in calves. With the use of the stereotaxic apparatus for cattle, a cannula was implanted into the hypothalamus of four male calves based upon cerebral ventriculography. Push-pull perfusates were collected at 10 min intervals for 6 h and GHRH and SS concentrations in perfusates and plasma GH concentration were determined by EIAs and RIA, respectively. A cannula was implanted into the hypothalamus based on the image of the third ventricle and maintained for 1 month. GHRH and SS showed pulsatile secretion and the pulses for GHRH and SS were irregular in conscious animals. Neither GHRH nor SS secretion had a clear relationship with GH secretion. In the present study, we thus (1) established a stereotaxic technique for approaching the hypothalamus using cerebral ventriculography for calves, and (2) demonstrated that GHRH and SS secretion were pulsatile but not closely related to GH profile in conscious calves. The technique is useful for the study of the functions of the hypothalamus in the control of pituitary hormones in cattle.

A technique for long-term implantation of a microcatheter into the third ventricle of post-pubertal Chinese Meishan pigs based on ventriculography

The objectives of this study were to implant a microcatheter into the third ventricle of post-pubertal Chinese Meishan pigs, and to maintain the microcatheter for a long time without causing stress. Fourteen pigs (45–60 kg BW) were used. Each pig was anesthetized and the head was orientated on the stereotaxic apparatus. A radiopaque dye was placed into the ventricle via a guide cannula inserted 3.5 mm forward of the bregma. A microcatheter was inserted into the third ventricle using ventriculography, and fixed with dental cement to a metal-mesh protector and screw anchors embedded into the skull. The opposite end of the microcatheter was externalized from the dorsal neck so that corticotropin-releasing hormone (CRH) could be injected easily. Simultaneously, a catheter was fitted in the jugular vein, and the free end of the catheter was externalized from the dorsal neck. Microcatheter-implanted pigs showed a normal progesterone concentration profile, and a constant cortisol level during at least two estrous cycles. Furthermore, intracerebroventricular injections of CRH (25 μg/500 μl) resulted in an increased plasma cortisol concentration ( P<0.05). Thus, the technique developed in this study enables us to approach the third ventricle in post-pubertal freely-moving pigs effectively over a long time, without causing stress.

Effects of basic fibroblastic growth factor on the growth of human medulloblastoma xenografts.

The purpose of this study was to evaluate the effect of basic fibroblastic growth factor (bFGF) on the growth of human UM-MB1 medulloblastoma xenografts injected intracranially in nude mice. Under general anesthesia, a homogenous suspension of UM-MB1 cells (10(5)/10 microl) were injected in the caudoputamen nuclei of the right cerebral hemisphere using a stereotaxic apparatus. The treatment group (n = 9) received 10 microl of a bFGF solution (20 microg/ml) at 3 and 6 days following the inoculation of the cells at the injection site using the same stereotaxic coordinates. The control group (n = 9) were injected intracranially with a phosphate-buffered saline vehicle using a similar protocol. Mice were sacrificed 3 weeks following the xenograft surgery and the brains were prepared for histological observations as well as tumor volume evaluations. The mean volume of bFGF-treated tumors (mean volume +/- SD = 50.0 +/- 32.9 +/- mm3) was significantly smaller than for the non-treated xenografts (mean volume +/- SD = 199.0 +/- 42.1 mm3) (t-test, p < 0.001). Compared to non-treated tumor cells, bFGF-treated medulloblastoma cells had a greater cytoplasm volume and their nuclei contained more euchromatin suggesting that bFGF may initiate differentiation. In conclusion, our results suggest that bFGF may offer a new chemotherapeutic modality for the treatment of medulloblastoma.

Inhibitory effect of iodophenpropit, a selective histamine H 3 antagonist, on amygdaloid kindled seizures

The effect of histamine H 3 antagonist, iodophenpropit on amygdaloid kindled seizures in rats was studied in comparison with those of other H 3 antagonists. Under pentobarbital anesthesia, the rats were fixed to a stereotaxic apparatus and bipolar electrodes were implanted into the amygdala. Electrodes were connected to a miniature receptacle, which was embedded in the skull with dental cement. To cause kindled seizures, electrical stimulation was applied to the amygdala bipolarly every day by a constant current stimulator, and electroencephalogram and convulsive behavior were observed. Drug effects were estimated in rats showing generalized kindled seizures. Intraperitoneal injection of H 3 antagonists, iodophenpropit, thioperamide, AQ0145 and clobenpropit, resulted in a dose-related inhibition of amygdaloid kindled seizures. The effect of iodophenpropit on amygdaloid kindled seizures was more potent than those of thioperamide, AQ0145 and clobenpropit. In conclusion, iodophenpropit may be useful for the treatment of partial epilepsy and/or secondary generalized seizures in humans.

Actions of noradrenaline on substantia gelatinosa neurones in the rat spinal cord revealed by in vivo patch recording.

To elucidate the mechanisms of antinociception mediated by the descending noradrenergic pathway in the spinal cord, the effects of noradrenaline (NA) on noxious synaptic responses of substantia gelatinosa (SG) neurones, and postsynaptic actions of NA were investigated in rats using an in vivo whole-cell patch-clamp technique. Under urethane anaesthesia, the rat was fixed in a stereotaxic apparatus after the lumbar spinal cord was exposed. In the current-clamp mode, pinch stimuli applied to the ipsilateral hindlimb elicited a barrage of EPSPs, some of which initiated an action potential. Perfusion with NA onto the surface of the spinal cord hyperpolarized the membrane (5.0-9.5 mV) and suppressed the action potentials. In the voltage-clamp mode (V(H), -70 mV), the application of NA produced an outward current that was blocked by Cs(+) and GDP-beta-S added to the pipette solution and reduced the amplitude of EPSCs evoked by noxious stimuli. Under the blockade of postsynaptic actions of NA, a reduction of the evoked and spontaneous EPSCs of SG neurones was still observed, thus suggesting both pre- and postsynaptic actions of NA. The NA-induced outward currents showed a clear dose dependency (EC(50), 20 microM), and the reversal potential was -88 mV. The outward current was mimicked by an alpha(2)-adrenoceptor agonist, clonidine, and suppressed by an alpha(2)-adrenoceptor antagonist, yohimbine, but not by alpha(1)- and beta-antagonists. These findings suggest that NA acts on presynaptic sites to reduce noxious stimuli-induced EPSCs, and on postsynaptic SG neurones to induce an outward current by G-protein-mediated activation of K(+) channels through alpha(2)-adrenoceptors, thereby producing an antinociceptive effect.

Intracerebroventricular administration of histamine H3 receptor antagonists decreases seizures in rat models of epilepsia

The effects of histamine H3 antagonists on amygdaloid kindled and maximal electroshock seizures in rats were studied to determine their potential as new antiepileptic drugs. Under pentobarbital anesthesia, rats were fixed to a stereotaxic apparatus and a stainless steel guide cannula for drug administration was implanted into the lateral ventricle. In amygdaloid kindled seizures, electrodes were implanted into the right amygdala and electroencephalogram was recorded bipolarly; stimulation was applied bipolarly every day by a constant current stimulator and continued until a generalized convulsion was obtained. In the maximal electroshock (MES) seizure test, electroconvulsion was induced by stimulating animals through ear-clip electrodes, and the durations of tonic and clonic seizures were measured. Thioperamide, clobenpropit, iodophenpropit, VUF5514, VUF5515 and VUF4929 caused a dose-dependent inhibition of both seizure stage and afterdischarge (AD) duration of amygdaloid kindled seizures. The duration of tonic seizure induced by MES was also inhibited by H3 antagonists, but the duration of clonic seizures were unchanged. Among the H3 antagonists tested, clobenpropit and iodophenpropit were somewhat more potent than the other drugs on amygdaloid kindled seizures and MES seizures, respectively. These results indicate that some H3 antagonists may be useful as antiepileptic drugs, especially for secondary generalized seizures and/or tonic-clonic seizures in humans.

The effect of acute hypoxemia and hypotension on adenosine-mediated depression of evoked hippocampal synaptic transmission

The present study was designed to investigate the relative contributions of arterial P O 2 , local cerebral blood flow, and oxygen delivery to the adenosine A 1 receptor-mediated depression of evoked synaptic transmission recorded in the rat hippocampus. Urethane-anesthetized rats were given a unilateral common carotid artery occlusion and then placed in a stereotaxic apparatus for stimulation and recording of bilateral hippocampal field excitatory postsynaptic potentials (fEPSPs). Arterial blood gases, mean arterial blood pressure (MAP), and bilateral hippocampal blood flow (HBF) were also measured. Arterial P O 2 , HBF, and oxygen delivery were manipulated using normoxic hypotension, hypoxic hypotension, and hypoxic normotension. Both hypoxic hypotension and normoxic hypotension resulted in decreased HBF, decreased oxygen delivery, and a depression of the evoked fEPSP limited to the hippocampus ipsilateral to the occlusion. The enhanced HBF and oxygen delivery associated with increased MAP resulted in a restoration and maintenance of hippocampal fEPSPs despite sustained hypoxemia. The adenosine A 1 receptor-mediated depression of the fEPSP was more strongly correlated with changes in HBF and oxygen delivery than with arterial P O 2 . We propose that adenosine plays an important role mediating the depression of neuronal activity associated with reduced oxygen delivery characteristically observed in ischemic brain tissue.

Differential roles of tumor necrosis factor-α and interleukin-1 β in lipopolysaccharide-induced brain injury in the neonatal rat

Increasing data provide support for the hypothesis that inflammatory cytokines mediate inflammation-induced injury to developing white matter. In the present study, roles of tumor necrosis factor-α (TNFα) and interleukin-1 β (IL-1β) in mediating lipopolysaccharide (LPS)-induced brain injury were investigated by co-administration of LPS with IL-1 receptor antagonist (IL-1ra) or TNFα antibody in the 5-day-old rat brain. Intracerebral injection of LPS and other agents was performed in a stereotaxic apparatus at the location of 1.0 mm posterior and 1.0 mm lateral to the bregma, and 2.0 mm deep to the skull surface at the left hemisphere. Brain injury was examined in brain sections 3 and 11 days after LPS injection. LPS-induced inflammatory responses were evidenced by great increases in TNFα and IL-1β concentrations in the neonatal rat brain 6 h after LPS injection. White matter rarefaction was observed in 71% (five out of seven) of the rat brains 3 days after LPS injection and bilateral ventricle dilation was found in 71% (five out of seven) of the P8 rat brains and in 100% of the P16 rat brains (four out of four). These alterations were not found in the control rat brains. No apparent histological changes in gray matter were observed in the LPS-injected rat brains. LPS injection also resulted in injuries to oligodendrocytes (OLs) and hypomyelination, as indicated by reduced immunostaining for O4 and myelin basic protein (MBP). Increased astrogliosis, as indicated by increased glial fibrillary acidic protein (GFAP) immunostaining, was also observed in the LPS-injected, but not the control rat brain. Co-administration of LPS with IL-1ra, but not with TNFα antibody, significantly attenuated LPS-induced white matter injury, as indicated by decreases in ventricle dilation, white matter rarefaction, GFAP positive staining and by improved O4 and MBP immunostaining. Co-administration of LPS with IL-1ra significantly reduced LPS-induced elevation of caspase-3 activity in the rat brain. While TNFα antibody had no effect on LPS-induced elevation of caspase-3 activity, co-administration of LPS with TNFα antibody partially, but significantly, decreased LPS-stimulated increase in IL-1β in the neonatal rat brain. These data suggest that IL-1β may play an important role in mediating LPS-induced brain injury and TNFα may have complicated, probably dual, effects in LPS-induced brain injury.

Stereotaxic assembly and procedures for simultaneous electrophysiological and MRI study of conscious rat.

A stereotaxic restraining assembly was designed and developed for simultaneous electrophysiological recordings and functional MRI (fMRI) data acquisition from a conscious rat. The design of the nonmagnetic stereotaxic apparatus facilitated the restraining of head and body of the unanesthetized conscious animal during MRI experiments. The apparatus was made of Teflon and Perspex materials with an appropriate size and shape for a 4.7 T / 40 cm animal MRI scanner. Electrodes made from nonmagnetic silver wire were implanted on the skull for recording the electroencephalogram (EEG), the electro-oculogram (EOG), and the electromyogram (EMG), while polycarbonate screws were used for anchoring the electrode assembly. There were no major distortions or artifacts observed in the electrophysiological tracings and MR images. Electrophysiological recordings during fMRI acquisitions are useful to study different neurophysiological mechanisms of sleep and pathophysiology of seizure activity. Integration of electrophysiological recordings (with their good temporal resolution) and MRI (with its superior spatial resolution) is helpful in characterizing the functional state of different brain regions.

Role of the subfornical organ in the chronic hypotensive response to losartan in normal rats.

Angiotensin II is known to act at a unique set of brain regions known as the circumventricular organs. These structures lack the normal blood-brain barrier and are therefore thought to participate in the central nervous system processing of neuroendocrine signals. We have reported that chronic treatment with the angiotensin type 1 (AT1) receptor antagonist, losartan, decreases arterial pressure in normotensive rats. Furthermore, this hypotension is attenuated in area postrema-lesioned rats, suggesting a role of endogenous angiotensin II at this circumventricular organ. Another circumventricular organ, the subfornical organ (SFO), has also been shown to mediate actions of angiotensin II. The present study tested the hypothesis that the SFO is a central site of action of endogenous angiotensin II at AT1 receptors. Adult male Sprague-Dawley rats were anesthetized and placed in a stereotaxic apparatus, and the SFO was sham or electrolytically lesioned. One week later, rats were instrumented with venous catheters and radiotelemetry pressure transducers for continuous infusion and monitoring of mean arterial pressure, respectively. After 3 days of control, losartan was administered intravenously (10 mg x kg(-1) x d(-1)) for 10 days in both SFO-lesioned and sham rats. By day 4 of losartan administration, mean arterial pressure had decreased to 75+/-2 mm Hg in sham rats (n=9) but had only fallen to 83+/-2 mm Hg in lesioned rats (n=10). This attenuated hypotensive response in SFO-lesioned rats continued through day 10 of losartan treatment. These results support the hypothesis that the SFO mediates part of the hypotensive effects of chronic AT1 receptor blockade in the normotensive rat.

Distinct patterns of gene transfer to gerbil hippocampus with recombinant adeno-associated virus type 2 and 5

Genetic modification of the gerbil hippocampal neuronal cells in vivo helps us understand the mechanisms of neuronal function under various circumstances such as ischemic insult. In this study, we examined the distinct distribution of the recombinant adeno-associated virus type 2 (rAAV2) and rAAV5 vectors for gene delivery to primary cultured cells and the gerbil hippocampus. Mixed cortical cultures containing both neurons and astrocytes from E17 rat embryos were infected with rAAVs containing the Cytomegalovirus virus (CMV) promoter. rAAV2 was preferably transduced to neurons, whereas rAAV5 was inclined to be transduced to astrocytes in vitro. rAAV2 and rAAV5 vectors, each with the CMV or Rous sarcoma virus (RSV) promoter, were injected into the gerbil hippocampus using a stereotaxic apparatus. Five days after injection, transgene expression was analyzed with X-gal staining. In the gerbil hippocampus, rAAV5 with the CMV promoter achieved a higher overall transgene expression than rAAV2 with the CMV promoter. The transgene expression of rAAV2 with the RSV promoter was found in the pyramidal and granular cells, while the transgene expression of rAAV5 with the RSV promoter was preferentially found in the granular cells. These findings would be valuable in optimizing rAAV-mediated gene transfer to the gerbil hippocampus.

A procedure for making precision stereotaxic surgery in the goldfish

A new device and a surgical procedure for placing electrodes in goldfish are described. They are used in stereotaxic surgery that is performed in lesion and electrophysiology studies. We modified DKI's stereotaxic apparatus in which the subject is fixed on a mouthpiece and two rubber plates in order to produce highly localized lesions in fish. A body holder and a new method of administering anesthesia were also developed. By stabilizing the water flow into the subject's gill using a three-pronged pump, we were able to maintain the anesthetic effect.

Ability of NMDA and non-NMDA receptor antagonists to inhibit cerebral ischemic damage in aged rats

Although stroke is a major cause of death and disability in the elderly, the inhibitory effects of neuroprotectants in acute stroke have been investigated using experimental cerebral ischemic models of young animals. Recent clinical trials have found that few neuroprotectants are effective. These observations indicate that effects in the clinical setting do not always reflect data from young animals. Thus, we compared the effects of the NMDA receptor antagonist MK-801 and of the AMPA receptor antagonist NBQX [2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo( F)quinixaline] on ischemic cerebral damage in the photothrombosis model of aged and young rats. MK-801 administered immediately after MCA occlusion significantly ( P<0.05) reduced the extent of cerebral damage in young, but not in aged, rats and the effects of NBQX were similar. In separate experiments, we evaluated brain damage after microinjecting NMDA or kainic acid into the cortex using a stereotaxic apparatus. We found no significant differences in focal cerebral damage caused by NMDA between young and aged rats. On the other hand, kainic acid caused all of the aged rats tested to die, but none of the young rats. Our observations indicate that NMDA and AMPA receptor antagonists are less effective in aged, than in young, rats and that cerebral damage by receptor agonists depends on the type of receptor, such as NMDA and AMPA.

Differential effects of dihydropyridine calcium channel blockers in kainic acid-induced experimental seizures in rats

The anticonvulsant effects of the dihydropyridine calcium channel blockers nifedipine, nicardipine and nimodipine were studied on experimental seizures induced by intra-hippocampal injection of kainic acid (KA) in chloralhydrate anesthetized Wistar rats. The rats were anesthetized and placed in a stereotaxic apparatus. After midline incision four screw electrodes were placed over the left and right frontal and parietal cortex and KA was injected into left dorsal hippocampus via 5-μl Hamilton microsyringe. The changes in electroencephalograph (EEG) activity and EEG power spectra were recorded in basal conditions and 5, 10, 15, 30 and 60 min following KA injection. KA-induced excitatory changes in the surface EEG activity were associated with the marked increase in EEG power spectra in the frequency range from 14.5–22 Hz. Pretreatment with nifedipine, nicardipine and nimodipine revealed that they exerted certain differences in their anticonvulsant properties. Nimodipine significantly delayed the onset of seizures and prevented the KA-induced changes in EEG and in EEG power spectra in all recorded channels and in a dose dependent manner. Nifedipine exerted significant anticonvulsant effect only in channel four, while nicardipine was ineffective. Our results suggest that anticonvulsant action of some dihydropyridine calcium channel blockers, especially nimodipine may be in part independent of its antagonism on L-type voltage-gated calcium (Ca 2+) channels.

Mechanism of the inhibitory effect of histamine on amygdaloid-kindled seizures in rats.

The mechanism of the inhibitory effect of histamine on amygdaloid-kindled seizures was investigated in rats. Under pentobarbital anesthesia, rats were fixed to a stereotaxic apparatus, and bipolar electrodes were implanted into the right amygdala. A guide cannula made of stainless steel tubing was implanted into the right lateral ventricle. Electrodes were connected to a miniature receptacle, which was embedded in the skull with dental cement. EEG was recorded with an electroencephalograph; stimulation of the amygdala was applied bipolarly every day by a constant-current stimulator and continued until a generalized convulsion was obtained. Intracerebroventricular (i.c.v.) injection of histamine at doses of 2-10 microg resulted in a dose-related inhibition of amygdaloid-kindled seizures. I.c.v. injection of calcium chloride at doses of 10-50 microg and A23187 at doses of 2-10 microg also caused dose-dependent inhibition of amygdaloid-kindled seizures. Calcium chloride at a dose of 10 microg, which showed no significant effect on amygdaloid-kindled seizures when used alone, significantly potentiated the effect of histamine. Similar findings were observed with A23187 at a dose of 2 microg. In addition, EGTA and EGTA/AM antagonized the inhibition of kindled seizures induced by histamine. Moreover, the inhibition of kindled seizures induced by histamine was antagonized by KN62. However, calphostin C did not antagonize the inhibitory effect of histamine. These results indicated that histamine-induced inhibition of amygdaloid-kindled seizures may be closely associated with a calcium calmodulin-dependent protein kinase II activation pathway.

In vivo microdialysis in the visual cortex of awake cat: I: Surgery, animal training and sampling

Sampling and monitoring release of excitatory and inhibitory amino acids in the striate cortex of mammals will provide important information for visual system research. A method allowing repeated microdialysis in the cortical layers of area 17 of the awake cat is described. Under visual control through a surgical microscope and using a stereotactic instrument, four probe guides are permanently implanted in area 17 of one hemisphere of the anesthetized animal and two fixation bars are mounted on the skull to allow fixation of the cat in a stereotactic frame. The implantation of four probe guides in the same hemisphere allows simultaneous sampling from different cortical regions subserving different parts of the visual field. A removable transparent cover protects the probe guides. After recovery from surgery the awake cats are trained to adapt to a fixation in a stereotaxic apparatus. Once adapted to that situation, the cats are used for 5 h in vivo microdialysis experiments without anesthesia.

Adenosine induces initial hypoxic-ischemic depression of synaptic transmission in the rat hippocampus in vivo.

The present study was designed to investigate the role of adenosine in the hypoxic depression of synaptic transmission in rat hippocampus. An in vivo model of hypoxic synaptic depression was developed in which the common carotid artery was occluded on one side in the urethane-anesthetized rat. Inspired oxygen levels were controlled through a tracheal cannula. Rats were placed in a stereotaxic apparatus for stimulation and recording of bilateral hippocampal field excitatory postsynaptic potentials. The percent inspired oxygen could be reduced to levels that produced a reversible and repeatable depression of evoked synaptic transmission restricted to the hippocampus ipsilateral to the occlusion. Further reduction in the level of inspired oxygen depressed synaptic transmission recorded from both hippocampi. The adenosine nonselective antagonist caffeine and the A(1) selective antagonist 8-cyclopentyltheophylline prevented the initial depression in synaptic transmission. We conclude that the initial depression of synaptic transmission observed in the rat hippocampus in vivo is due to endogenous adenosine acting at neuronal adenosine A(1) receptors.

Differential regulation of preovulatory luteinizing hormone and follicle-stimulating hormone release by opioids in the proestrous rat

We have investigated the role of m- and k-opioid receptors in the central control of preovulatory LH and FSH release in the proestrous rat. Animals were anesthetized with chloral hydrate at 14:00 h on proestrus day. Following femoral artery cannulation, they were mounted in a stereotaxic apparatus. Morphine and U-50488H (benzene-acetamide methane sulphonate) were infused intracerebroventricularly either alone or in combination with naloxone and MR1452, respectively. Controls received sterile saline alone. Blood samples were obtained at hourly intervals between 15:00 h and 17:00 h. Plasma LH and FSH levels were measured by radioimmunoassay. Morphine did not significantly change plasma LH levels at 15:00 h and 16:00 h sampling intervals. A significant increase was observed at 17:00 h compared to the controls (p&lt;0.05). U-50488H significantly increased LH levels at 16:00 h and 17:00 h (p&lt;0.05). The co-administration of naloxone and MR1452 with m- and k-agonist had no significant effect on LH levels at any sampling interval. In all groups, LH levels showed a linear rise over the sampling period between 15:00 h and 17:00 h. None of the treatments significantly altered plasma FSH levels which however, declined towards the end of the afternoon surge. In conclusion, we suggest that the secretion of LH and FSH is differentially regulated by m- and k-opioid receptors. It is thought that in all groups chloral hydrate interfered with the LH surge secretory systems.

Effect of NMDA-induced lesion of the subfornical organ on the angiotensin II binding sites density and acetylcholinesterase or NADPH-diphorase activities in the lamina terminalis of the rat brain.

1. Neural angiotensinergic circuitry located in the lamina terminalis has been proposed to be involved in blood pressure regulation and fluid homeostasis. 2. ANG II binding sites have been described to be localized throughout the lamina terminalis including the subfornical organ (SFO), the median preoptic nucleus (MnPO), and the organum vasculosum lamina terminalis (OVLT). 3. The present experiment was designed to investigate the ANG II binding sites localization in the lamina terminalis. For this purpose, we have compared the ANG II binding sites, acetylcholinesterase, and NADPH-diaphorase distributions throughout the lamina terminalis. Additionally, we have studied the effect of the preferential lesion of SFO neuronal cell bodies by local injection of NMDA on the ANG II binding sites density in different areas of the lamina terminalis. 4. Male Wistar rats were anesthetized, immobilized in a stereotaxic apparatus, and 500 nl of saline or 250 nmol NMDA was injected into the SFO. 5. Animals were sacrificed 1 week later, the brain was removed, frozen, and sagittal 16 microm slices were cut in a cryostat. Alternate brain slices were incubated with [125I]-Sar1-ANG II for receptor autoradiography or histochemically stained for visualization of acetylcholinesterase and NADPH-diaphorase activities. Binding capacity was determined by computerized quantitative densitometry of autoradiograms. The intensity of histochemical reactions was measured as relative units obtained by computerized densitometry processing of the brain slices stained for either activity. 6. Acetylcholinesterase staining was mainly located in the SFO, with faint staining reaction in other areas of the lamina terminalis. NADPH-diaphorase staining was homogeneously distributed throughout the lamina terminalis. A significant positive correlation was observed between acetylcholinesterase and NADPH-diaphorase stainings in the SFO of control and NMDA-lesioned rats. 7. ANG II binding sites were localized throughout the lamina terminalis. A significant positive correlation was observed between the density of ANG II binding sites and the intensity of acetylcholinesterase or NADPH-diaphorase staining in the SFO of control and NMDA-lesioned rats. 8. The distribution of the NADPH-diaphorase staining was found to closely match the distribution of the ANG II binding sites in the lamina terminalis. 9. Neuronal lesion of the SFO caused significant reductions in the density of ANG II biding sites in the SFO (-68%) and the MnPO (-48%). No changes were observed either in the OVLT or outside the lamina terminalis in the superior colliculus. 10. The present results indicate the following: first, the presence of high levels of acetylcholinesterase staining in the SFO and of NADPH-diaphorase throughout the lamina terminalis; second, that ANG II binding sites in the SFO and possibly in the MnPO are localized in neuronal cell bodies; third, that SFO lesion did not affect the expression of ANG II binding sites in the OVLT, thus suggesting that these binding sites correspond to different angiotensinergic system: and finally, the existence of a striking correlation between the distribution of the ANG II binding sites and NADPH-diaphorase throughout the lamina terminalis, thus suggesting a interrelation between angiotensinergic and nitrergic systems in the lamina terminalis.