Biomedical subjects

O K Ronnekleiv

Publications and source records attributed to O K Ronnekleiv.

At least 19 recordsLinked to original sources

PubMed2001

Estrogen biphasically modifies hypothalamic GABAergic function concomitantly with negative and positive control of luteinizing hormone release.

The principal role of estrogen is its control of the female ovulatory cycle via negative and positive feedback on gonadotropin secretion. However, a detailed, cohesive picture of how the steroid specifically regulates the excitability of hypothalamic neurons involved in the central control of gonadotropin secretion is still emerging. Here, we used an ovariectomized female guinea pig model to test the hypothesis that estrogen acts on GABAergic neurons in the preoptic area (POA) to elicit a biphasic profile of luteinizing hormone (LH) secretion. Intracellular electrophysiological recordings revealed that estradiol benzoate (EB; 25 microgram, s.c.) decreased the hyperpolarizing response of GABAergic neurons to the GABA(B) receptor agonist baclofen 24 hr after treatment. This effect of GABA(B) receptor stimulation in unidentified POA neurons was still depressed 42 hr after EB administration. By the use of a ribonuclease protection assay, however, EB reduced glutamic acid decarboxylase mRNA expression 42 hr but not 24 hr after its administration. Thus, estrogen attenuated the autoinhibition of GABAergic POA neurons during the initial LH suppressive (i.e., negative feedback) phase and subsequently reduced GABAergic function during the LH surge (i.e., positive feedback). These studies demonstrate that the effects of estrogen on hypothalamic GABAergic neurons coincide with the inhibitory and stimulatory actions, respectively, of the steroid on LH secretion. Furthermore, the data provide novel insights into the mechanism by which estrogen regulates hypothalamic GABAergic neurons, which are critical for the biphasic modulation of LH release observed over the course of the female ovulatory cycle.

Action Potentials↗

PubMed2000

The role of intrinsic and agonist-activated conductances in determining the firing patterns of preoptic area neurons in the guinea pig.

Whole-cell and intracellular recordings were made in coronal hypothalamic slices prepared from ovariectomized female guinea pigs. 62% of preoptic area (POA) neurons fired action potentials in a bursting manner, and exhibited a significantly greater afterhyperpolarization (AHP) than did non-bursting POA neurons. The majority (70%) of POA neurons (n=76) displayed a time-dependent inward rectification (I(h)) that was blocked by CsCl (3 mM) or by ZD 7288 (30 microM). In addition, 51% of the cells expressed a low-threshold spike (LTS) associated with a transient inward current (I(T)) that was blocked by NiCl(2) (200 microM). A smaller percentage of POA neurons (29%) expressed a transient outward, A-type K(+) current that was antagonized by a high concentration of 4-aminopyridine (3 mM). Moreover, POA neurons responded to bath application of the mu-opioid receptor agonist DAMGO (93%) or the GABA(B) receptor agonist baclofen (83%) with a membrane hyperpolarization or an outward current. These responses were accompanied by a decrease in input resistance or an increase in conductance, respectively, and were attenuated by BaCl(2) (100 microM). In addition, the reversal potential for these responses closely approximated the Nernst equilibrium potential for K(+). These results suggest that POA neurons endogenously express to varying degrees an AHP, an I(h), an I(T) and an A-type K(+) current. The vast majority of these neurons also are inhibited upon mu-opioid or GABA(B) receptor stimulation via the activation of an inwardly-rectifying K(+) conductance. Such intrinsic and transmitter-activated conductances likely serve as important determinants of the firing patterns of POA neurons.

4-Aminopyridine↗

PubMed2000

Effect of the mu-opioid agonist DAMGO on medial basal hypothalamic neurons in beta-endorphin knockout mice.

The endogenous opioid neurotransmitter beta-endorphin (beta-END), a product of the proopiomelanocortin (POMC) gene, is strongly implicated in the control of the female reproductive cycle, stress responses, and antinociception. Using selective gene targeting, we have generated a strain of mice that do not express any beta-END. These mice exhibit both normal reproduction and normal basal and stress-induced hypothalamic-pituitary-axis activity, but exhibit a significantly attenuated opioid-mediated stress-induced analgesia. To further understand the cellular bases of these responses, we have studied mediobasal hypothalamic (MBH) neurons, including POMC neurons, using whole-cell patch recording in an in vitro slice preparation. Twenty-seven MBH cells were recorded in wild-type and 25 MBH cells were recorded in beta-END knockout mice. Neurons from both genotypes showed a significant positive correlation between DAMGO concentration (from 30 nM to 10 microM) and the induced outward K(+) current. The genotypes did not differ, however, in either the DAMGO-induced maximum outward current response or EC(50), or for the maximal response to the GABA(B) agonist baclofen. Furthermore, quantitative receptor autoradiography utilizing (3)H-DAMGO did not reveal any differences in total mu-opioid receptor binding between genotypes. Therefore, we conclude that the complete absence of beta-END throughout development did not alter either the expression of mu-opioid receptors or their coupling to K(+) channels in MBH neurons.

Animals↗

PubMed1999

Cocaine upregulates the dopamine transporter in fetal rhesus monkey brain.

Cocaine is a highly addictive drug that binds to the dopamine transporter (DAT), inhibits the reuptake of dopamine, and initiates multiple actions within midbrain dopaminergic systems. Using the rhesus monkey, we have investigated the consequences of in utero cocaine exposure on the expression of DAT in the fetal brain. By using the selective DAT ligand [125I]RTI-121 and tyrosine hydroxylase (TH) immunocytochemistry, we found that DAT binding sites are highly developed by day 70 of gestation and show a distribution pattern similar to TH. The rank order of specific 3beta-(4-[125I]iodophenyl)tropane-2beta-carboxylic acid isopropyl ester ([125I]RTI-121) binding densities was substantia nigra-ventral tegmental area > putamen > caudate > lateral hypothalamus > accumbens > linear/interfascicular nuclei >/= globus pallidus > prefrontal cortex. Furthermore, we observed that DAT mRNA was differentially expressed within fetal midbrain dopamine neurons with the highest levels detected in the ventral tier of the substantia nigra pars compacta, and the lowest levels in the ventral tegmental area and the linear/interfascicular nuclei. In utero cocaine exposure between days 22 and 70 significantly increased DAT mRNA expression, and the density of [125I]RTI-121 binding sites within midbrain dopamine neurons in the 70-d-old fetus. This increased DAT expression is accompanied by other presynaptic and postsynaptic neuronal changes, which collectively suggest that midbrain dopamine neurons are hypoactive after prolonged cocaine exposure, a state that may be a contributing factor in the development of attention deficit disorders observed in subjects exposed prenatally to cocaine.

Animals↗

PubMed1997

Modulation of G protein-coupled receptors by an estrogen receptor that activates protein kinase A.

17beta-Estradiol (E2) rapidly (<20 min) attenuates the ability of mu-opioids to hyperpolarize guinea pig hypothalamic (beta-endorphin) neurons. In the current study, we used intracellular recordings from guinea pig hypothalamic slices to characterize the receptor and intracellular effector system mediating the rapid effects of E2. E2 acted stereospecifically with physiologically relevant concentration dependence (EC50 = 8 nM) to cause a 4-fold reduction in the potency of a mu-opioid agonist to activate an inwardly rectifying K+ conductance. Using Schild analysis to estimate the affinity of the mu-opioid receptor for an antagonist (naloxone), we found that estrogen did not compete for the mu-opioid receptor or alter the affinity of the mu receptor. Both the nonsteroidal estrogen diethylstilbestrol and the "pure" antiestrogen ICI 164,384 blocked the actions of E2, the latter with a subnanomolar affinity. The protein synthesis inhibitor cycloheximide did not block the estrogenic uncoupling of the mu-opioid receptor from its K+ channel, implying a nongenomic mechanism of action by E2. The actions of E2 were mimicked by the protein kinase A (PKA) activators forskolin and cAMP, Sp-isomer triethylammonium salt. Furthermore, the selective PKA antagonists cAMP, Rp-isomer triethylammonium salt and KT5720, which have different chemical structures and modes of action, both blocked the effects of E2. Thus, estrogen binds to a specific receptor that activates PKA to rapidly uncouple the mu-opioid receptor from its K+ channel. Because we have previously shown that gamma-aminobutyric acidB receptors are also uncoupled by estrogen, this mechanism of action has the potential to alter synaptic transmission via G protein-coupled receptors throughout the brain.

Analgesics↗

PubMed1995

Distribution of dopamine D1, D2, and D5 receptor mRNAs in the monkey brain: ribonuclease protection assay analysis.

The distribution of the mRNAs encoding the dopamine D1, D2 and D5 receptors was determined in brain tissues obtained from intact female rhesus monkeys, using a ribonuclease protection assay. Tissue blocks from the frontal cortex, striatum, thalamus, hippocampus and substantia nigra were dissected and total RNA was extracted. Dopamine D2 and D5 receptor DNA fragments were generated from rhesus monkey genomic DNA using polymerase chain reaction. To generate dopamine receptor subtype-specific cRNA probes, DNA fragments corresponding to the carboxy terminus of the rhesus monkey D1 and D2 receptor genes and to the putative transmembrane domain regions (IV-VI) of the D5 receptor gene, were subcloned into the pGEM3Z/4Z vectors. Expression of D1 receptor mRNA exhibited significant regional differences: striatum > > > cerebral cortex > or = hippocampus > or = lateral thalamus. D1 receptor mRNA was found in low quantities in the medial thalamus, but was not consistently expressed in the substantia nigra area. In contrast, D2 receptor mRNA was detected in all regions that were studied: striatum > > > substantia nigra > > hippocampus > or = cerebral cortex > or = medial thalamus > or = lateral thalamus. D5 receptor mRNA was also expressed in all regions, with highest levels in the cerebral cortex, striatum and lateral thalamus, and moderate levels in the substantia nigra, medial thalamus and the hippocampus. The D5 receptor mRNA appears to be widely distributed in the monkey brain. Most interesting is the expression of D5 receptor mRNA in tissues of the substantia nigra area.

Animals↗

PubMed1995

Characterization and distribution of a cloned rat mu-opioid receptor.

We have cloned and expressed a rat brain cDNA, TS11, that encodes a mu-opioid receptor based on pharmacological, physiological, and anatomical criteria. Membranes were prepared from COS-7 cells transiently expressing TS11 bound [3H]diprenorphine with high affinity (KD = 0.23 +/- 0.04 nM). The rank order potency of drugs competing with [3H]diprenorphine was as follows: levorphanol (Ki = 0.6 +/- 0.2 nM) approximately beta-endorphin (Ki = 0.7 +/- 0.05 nM) approximately morphine (Ki = 0.8 +/- 0.5 nM) approximately [D-Ala2, N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO; Ki = 1.6 +/- 0.5 nM) uch much greater than U50,488 (Ki = 910 +/- 0.78 nM) > [D-Pen2,5]- enkephalin (Ki = 3,170 +/- 98 nM) > dextrorphan (Ki = 4,100 +/- 68 nM). The rank order potencies of these ligands, the stereospecificity of levorphanol, and morphine's subnanomolar Ki are consistent with a mu-opioid binding site. Two additional experiments provided evidence that this opioid-binding site is functionally coupled to G proteins: (a) in COS-7 cells 50 microM 5'-guanylylimidodiphosphate shifted a fraction of receptors with high affinity for DAMGO (IC50 = 3.4 +/- 0.5 nM) to a lower-affinity state (IC50 = 89.0 +/- 19.0 nM), and (b) exposure of Chinese hamster ovary cells stably expressing the cloned mu-opioid receptor to DAMGO resulted in a dose-dependent, naloxone-sensitive inhibition of forskolin-stimulated cyclic AMP production. The distribution of mRNA corresponding to the mu-opioid receptor encoded by TS11 was determined by in situ hybridization to brain sections prepared from adult female rats. The highest levels of mu-receptor mRNA were detected in the thalamus, medial habenula, and the caudate putamen; however, significant hybridization was also observed in many other brain regions, including the hypothalamus.

Amino Acid Sequence↗

PubMed1995

Chronic cocaine exposure in the fetal rhesus monkey: consequences for early development of dopamine neurons.

The effects of cocaine on the development of neurons containing tyrosine hydroxylase (TH) were investigated in fetal rhesus macaques. Pregnant monkeys were treated with cocaine 3 mg/kg or saline i.m., four times a day from day 18 of pregnancy until days 40 or 60. Cocaine concentration in plasma from females thus treated was approximately 800 ng/ml 10-20 min following injection. At the time of C-section, plasma levels of cocaine in fetal blood were 231 +/- 70 ng/ml. The brains from 40 and 60 d old fetuses were examined using immunocytochemistry and in situ hybridization. The appearance of neurons containing TH by day 40 of gestation was not different between control and cocaine-treated fetal monkeys. In both groups (N = 3 each) TH-IR neurons and TH mRNA were located in the mesencephalon and dorsal hypothalamus by day 40, and fiber projections extended to the developing striatum. Also in the day 60 fetuses, the TH-IR neurons were distributed similarly in both groups (N = 5 each), but the TH mRNA content, measured by quantitative in situ hybridization, was reduced in the substantia nigra (SN) and ventral tegmental area (VTA) after cocaine treatment. These data suggest that exposure to cocaine in fetal life does not affect the development of TH or the expression of its mRNA on day 40 of gestation. By day 60, however, the expression of TH mRNA was significantly reduced. This latter effect can be explained by reduced dopamine synthesis in the cocaine-treated fetuses.

Animals↗

PubMed1994

The potency of mu-opioid hyperpolarization of hypothalamic arcuate neurons is rapidly attenuated by 17 beta-estradiol.

The mu-opioid agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) hyperpolarizes the majority of arcuate hypothalamic (ARC) neurons by opening an inwardly rectifying potassium conductance. The EC50 for the DAMGO-induced hyperpolarization was 60 +/- 3 nM in ARC neurons from ovariectomized guinea pigs. Superfusion of 17 beta-estradiol (E2; 100 nM) for 20 min in vitro resulted in a significant decrease in DAMGO potency (EC50 = 212 +/- 16 nM) in 40% of the neurons that were tested. This rapid effect of E2 on the mu-opioid response was not mimicked by the biologically inactive isomer 17 alpha-estradiol. Multiple concentrations of E2 were used to generate an E2 concentration-response curve, with an EC50 of 9 nM and a maximal increase in the DAMGO EX50 of 411% of controls. The membrane properties and firing rate of E2-sensitive and E2-insensitive neurons were not different. Streptavidin-FITC labeling did not reveal any significant morphological differences between the groups, but a higher number of E2-sensitive cells was found in the lateral ARC and cell-poor zone. Moreover, immunocytochemical staining of the recorded cells revealed that beta-endorphin neurons were among those sensitive to E2. Therefore, E2 could increase beta-endorphin release by decreasing the potency of beta-endorphinergic autoinhibition, thus increasing the tonic opioid inhibition of E2-insensitive cells. Furthermore, the diffuse projections of hypothalamic beta-endorphin neurons would allow E2 to alter processes throughout the brain, as well as having local effects in the hypothalamus.

Animals↗

PubMed1993

Electrophysiology of guinea-pig supraoptic neurones: role of a hyperpolarization-activated cation current in phasic firing.

1. Immunocytochemically identified magnocellular neurosecretory cells (MNCs) in the guinea-pig supraoptic nucleus (SON) were studied using the in vitro intracellular recording technique. Cells were identified as containing arginine vasopressin (AVP) or oxytocin (OT) following recordings made with biocytin-filled electrodes. Both AVP and OT MNCs demonstrated a fusiform or pyramidal shape (15-20 microns by 26-39 microns), with two to three processes. There were no significant differences in the proportion of AVP and OT cells in the retrochiasmatic (caudal) versus the rostral slices. 2. No significant differences in passive membrane properties were observed between AVP and OT cells, except that AVP cells exhibited a significantly broader action potential width (1.51 +/- 0.1 ms, n = 11) than did OT cells (1.01 +/- 0.08 ms, n = 7). 3. Firing patterns were recorded for 100 MNCs, 41% of which fired in a phasic manner (repeated clustering of action potentials into bursts). Of the seventy-seven cells which were immunocytochemically identified, only AVP-containing MNCs displayed phasic firing. Phasic firing occurred only in MNCs demonstrating a depolarizing potential which followed hyperpolarizing after-potentials (HAPs). The presence of the depolarizing potential was not always associated with phasic firing, however, as both OT cells and non-phasic AVP cells sometimes exhibited a depolarizing potential. 4. In 160 MNCs examined for the presence of the time-dependent inward rectification (TDR in current clamp, or Ih in voltage clamp), a significant difference in the proportion of cells expressing the Ih was observed in the two cell types. The Ih was expressed in forty-five of fifty-four AVP MNCs (83%) and in six of fifteen OT MNCs (40%). No significant association was found with firing pattern. 5. The Ih exhibited properties similar to those found in other CNS and peripheral tissues. It appeared on steps to potentials more hyperpolarized than -65 mV. It was augmented by raising the extracellular potassium concentration, blocked by 2 mM CsCl, and insensitive to 100-500 microM BaCl2. Activation followed a single exponential, and the time constant of activation was voltage dependent. 6. The adenylate cyclase activator forskolin increased the Ih and shifted its activation curve to more depolarized levels. In cells recorded for several hours, the Ih varied in amplitude, suggesting intrinsic modulation, possibly by intracellular second messenger systems. The Ih in guinea-pig SON MNCs appears to serve an excitatory role, bringing cells closer to firing threshold.

Action Potentials↗

PubMed1993

Role of a T-type calcium current in supporting a depolarizing potential, damped oscillations, and phasic firing in vasopressinergic guinea pig supraoptic neurons.

Guinea pig magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) were studied using the in vitro slice preparation. Intracellular recordings were made with biocytin-filled electrodes, permitting immunocytochemical identification of the recorded cells as arginine vasopressin- (AVP) versus oxytocin- (OT) containing. Only AVP cells displaying a depolarizing potential (DP) fired phasically. The DP was associated with a transient inward current measured in voltage clamp, which exhibited a number of properties of the T-type calcium current: activation threshold of -64 mV, time course of up to 250 ms, blockade by nickel and augmentation by barium chloride. This current has not been reported previously in SON neurons. The T-type current (IT) was always associated with a damped oscillation of the membrane following the offset from hyperpolarizing steps. In all cells tested, an apamin-sensitive afterhyperpolarization (AHP) was observed, similar to the calcium-dependent potassium current (IK, Ca) described in rat SON and other CNS regions. Therefore, as with other CNS regions displaying damped oscillations, guinea pig SON cells possess both an IT and an IK, Ca. We have previously described an Ih activating at hyperpolarized potentials in these cells, which depolarizes the membrane to a range in which the IT and IK, Ca can interactively support oscillations. In summary, the IT and associated depolarizing potential appears to be a requisite feature for phasic firing in AVP cells of guinea pig SON.

Animals↗

PubMed1992

Estrogen suppresses mu-opioid- and GABAB-mediated hyperpolarization of hypothalamic arcuate neurons.

The effects of estrogen on the response of hypothalamic arcuate neurons to mu-opioid and GABAB agonists were investigated. Intracellular recordings were made from arcuate neurons in slices prepared from ovariectomized guinea pigs that were pretreated with estrogen or vehicle. Estrogen shifted the dose-response curve to the mu-opioid agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol) by 3.4-fold; the EC50 for DAMGO was 240 +/- 25 nM in estrogen-treated females versus 70 +/- 12 nM in the controls. The maximal hyperpolarization induced by DAMGO was equivalent in neurons from both groups. The Ke for the naloxone antagonism of the DAMGO response was similar in both groups, which would indicate that the affinity of the mu-receptor was unchanged. To explore where in the receptor/G-protein/K+ channel cascade estrogen may be acting to attenuate the mu-opioid-mediated hyperpolarization, the response to the GABAB agonist baclofen was also tested. Estrogen treatment also shifted the dose-response curve for the baclofen-induced hyperpolarization by 3.3-fold without altering the maximum hyperpolarization; the EC50 shifted from 11.0 +/- 4.0 microM to 36.0 +/- 5.0 microM. All of the neurons were identified after linking the intracellular biocytin with streptavidin-FITC, and a subpopulation of cells in both groups were immunoreactive for beta-endorphin. We conclude that estrogen decreases the functional coupling of the mu-opioid and GABAB receptors to the inwardly rectifying K+ channel possibly through an action on the G-protein.

Animals↗

PubMed1991

Neurons in the rat arcuate nucleus are hyperpolarized by GABAB and mu-opioid receptor agonists: evidence for convergence at a ligand-gated potassium conductance.

Both gamma-aminobutyric acid (GABA) and the endogenous opioid peptides have pervasive effects on neuroendocrine function. This study examined the effects of selective activation of GABAB and/or mu-opioid receptors on neurons of the arcuate nucelus (ARC) of the rat hypothalamus using intracellular recording of cells in a hypothalamic slice. Some recorded neurons were filled with biocytin allowing subsequent identification and immunocytochemical evaluation for the presence of beta-endorphin. ARC neurons exhibited a broad array of active and passive conductances. Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGOL), a mu-opioid receptor agonist, inhibited spontaneous firing, hyperpolarized 68% of ARC cells in a dose-dependent manner and increased cell conductance. Baclofen, a GABAB receptor agonist, hyperpolarized all cells tested. The reversal potentials for both the DAGOL- and baclofen-induced currents were near that of a potassium conductance. Maximal activation by either of the agonists blocked the effects of the other agonist. Identified beta-endorphin cells were inhibited by both DAGOL and baclofen. The results of these in vitro studies suggest that GABAB and mu-opioid receptors are coupled to the same set of potassium channels and that these channels directly and powerfully inhibit most ARC cells, including beta-endorphin neurons. We propose that convergence of inhibitory influences at the ligand-gated potassium conductance described here may be an important site of interaction for opioidergic, GABAergic and other putative neurotransmitter systems in the control of neuroendocrine circuits by the ARC.

Animals↗

PubMed1990

Acute administration of estrogen suppresses LH secretion without altering GnRH release in ovariectomized rhesus macaques.

The pattern of hypothalamic gonadotropin-releasing hormone (GnRH) release was examined during estrogen (E)-induced suppression of plasma luteinizing hormone (LH) in ovariectomized (OVX) rhesus macaques. In Expt. 1, 4 OVX macaques were fitted with a jugular catheter and a push-pull cannula (PPC) directed into the median eminence (ME). Push-pull perfusion (PPP) was initiated 10 h before and continued for 10 h after subcutaneous estradiol benzoate (EB) injection (42 micrograms/kg b.wt.). In Expt. 2, 4 additional monkeys were subjected to local intrahypothalamic perfusion with estradiol-17 beta (E2, 3 microM) for the last 10 h of a 20-h PPP. In Expt. 2, OVX animals were challenged with 5 micrograms exogenous GnRH 3 h before and 8 h after EB injection to test for changes in altered LH release. Integrated 10-min ME perfusate and intermittent 10- or 60-min peripheral plasma samples were assayed for GnRH and LH by radioimmunoassay and bioassay, respectively. In addition, 2 other OVX macaques that received similar ME-PPC placement were sacrificed 2 days after the completion of a PPP for immunocytochemical labeling of GnRH neurons at the perfusion site. The results show that after EB, hypothalamic GnRH (MBH-GnRH) release remained unaltered while LH levels declined rapidly (Expt. 1). Similarly, intrahypothalamic perfusion of E2 failed to change the pattern of MBH-GnRH release in any of 4 monkeys (Expt. 2). Conversely, plasma LH release in response to exogenous GnRH was greatly reduced after EB (Expt. 3).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Opioids hyperpolarize beta-endorphin neurons via mu-receptor activation of a potassium conductance.

Intracellular recordings were made from hypothalamic arcuate (ARC) neurons with biocytin-filled electrodes under current- and voltage-clamp in slices prepared from ovariectomized guinea pigs which were pretreated with estradiol. Forty-three neurons were identified after linking the intracellular biocytin with streptavidin-FITC and subsequently were examined for beta-endorphin immunoreactivity. Ten of these neurons were immunoreactive for beta-endorphin. beta-Endorphin neurons displayed the following passive membrane properties: RMP:-56 +/- 2 mV; Rin: 439 +/- 66 M omega; tau: 17.5 +/- 2.4 ms; and often fired spontaneously (5.9 +/- 2.2 Hz). These membrane characteristics were not different from identified neurons in the ARC that were not immunoreactive for beta-endorphin. beta-Endorphin neurons exhibited instantaneous inward rectification and time-dependent rectification. The mu-opioid agonist Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO) decreased spontaneous firing, induced membrane hyperpolarization (12 +/- 2 mV; range 6-22 mV) and decreased the Rin (38 +/- 4%) of the beta-endorphin neurons. These effects of DAGO were blocked by the opioid antagonist naloxone (1 microM) and were not blocked by 1 microM TTX. DAGO-responsive cells were unaffected by either kappa- or delta-receptor opioid agonists. These results indicate that mu-receptors may be autoreceptors on ARC beta-endorphin neurons and that activation of opioid mu-receptors hyperpolarizes beta-endorphin neurons via an increase in K+ conductance. Therefore, opioid peptides may modulate opioid tone through an 'ultra-short loop' feedback control mechanism.

Action Potentials↗

PubMed1990

Ontogeny of gonadotropin-releasing hormone-containing neurons in early fetal development of rhesus macaques.

We studied the ontogeny of GnRH neurons in fetal rhesus macaques from days 36-135 of gestation. The nasal region, pituitary, and brain were dissected, fixed in 4% paraformaldehyde, sectioned on a cryostat at 10 microns, and mounted on slides. Immunocytochemistry and in situ hybridization were performed for GnRH, pro-GnRH, and pro-GnRH mRNA on nasal and brain tissues. Immunoreactive LH, FSH, and PRL were determined in developing pituitary glands. At 36 days, clusters of GnRH cells were found in the nasal region only. GnRH fibers extended into the brain, and large bundles projected laterally toward the basal hypothalamus. By day 38 GnRH cells were also localized in the olfactory region of the brain. With increasing fetal age a gradual caudal extension of GnRH cells occurred. These cells were first observed in the basal hypothalamus at 47 days. Cells containing PRL and gonadotropins (LH and FSH) were detected in the pituitary at 47 and 50 days, respectively. Low levels of pro-GnRH mRNA were present in the nasal epithelium and septum by day 38 of gestation. The levels of pro-GnRH mRNA in the nasal area and the brain remained low in the day 50 fetus, but were increased significantly by day 135. These data suggest that GnRH neurons in the primate brain originate in the nasal region. Furthermore, GnRH neurons exhibit low levels of synthetic activity at the early fetal stages, but higher synthetic activity close to term.

Animals↗

PubMed1990

Testosterone regulates progonadotropin-releasing hormone levels in the preoptic area and basal hypothalamus of the male rat.

GnRH is synthesized as part of a larger 92-amino acid prohormone (pro-GnRH). In the present study, we examined the effect of gonadectomy and hormone replacement on the processing of pro-GnRH to GnRH in adult male rats. Immunoreactive levels of pro-GnRH and GnRH in acid extracts of preoptic area (POA) and basal hypothalamus (BH) from intact, castrate, and testosterone (T)-treated castrate males were quantified by RIA. In addition, we used immunocytochemistry to detect pro-GnRH-and GnRH-positive neurons and determine the effects of hormone treatment. Three weeks after castration, the pro-GnRH content of the POA was 2-fold greater than the amount found in intact males. Treatment with T for 1 week lowered the prohormone content to intact levels. Although the pro-GnRH content in the BH was about 50% lower than that in the POA, the BH responded to castration and steroid replacement in a manner identical to the POA. The GnRH content of the BH, but not that of the POA, was significantly reduced after castration and increased by T treatment. On the other hand, the total number of pro-GnRH and GnRH cell bodies visualized by immunocytochemistry was not significantly altered by either treatment. These results show that changes in pro-GnRH content vary in inverse relation to changes in GnRH content and suggest that gonadectomy inhibits the enzymatic processing of precursor, while T treatment promotes it.

Animals↗

PubMed1990

Membrane properties and response to opioids of identified dopamine neurons in the guinea pig hypothalamus.

The electrophysiological properties and opioid responsiveness of the dopamine-containing neurons in the arcuate nucleus of the guinea pig hypothalamus were examined. Dopamine-containing neurons, identified immunocytochemically by the presence of tyrosine hydroxylase, had a mean length-to-width profile of 14.9 +/- 4.4 x 11.5 +/- 3.1 microns (N = 14). The Na+ action potential of these neurons was of short duration, and induction of repetitive firing (20-50 Hz) caused an afterhyperpolarization of 6-9 mV in amplitude, with a decay half-time of approximately 1.5 sec. Dopamine-containing cells exhibited a low threshold spike, which induced 1-4 Na+ action potentials. This potential had a threshold close to -65 mV, could not be induced without prior hyperpolarization and was not sensitive to TTX. Dopamine-containing neurons also exhibited a time- and voltage-dependent inward current at potentials negative to -70 mV, and Cs+ blocked this conductance. The mu-opioid agonist Tyr-D-Ala-Gly-mePhe-Gly-ol hyperpolarized (14 +/- 3 mV) dopamine neurons via induction of an outward current (93 +/- 44 pA near the resting membrane potential) which had a reversal potential similar to that expected for a selective potassium conductance. TTX (1 microM) did not block the opioid effects. These results show that dopamine neurons of the arcuate nucleus differ in their intrinsic conductances and their responsiveness to opioids from other CNS dopaminergic neurons. Furthermore, opioid activation of a potassium conductance resulted in a direct hyperpolarization of dopamine neurons of the arcuate nucleus, and we suggest that this mechanism may underlie the effects of opioids on dopamine-mediated prolactin release.

Action Potentials↗