An fMRI Study of the Brain Network Involved in Teeth Tapping in Elderly Adults.

Cortical activity during jaw movement has been analyzed using various non-invasive brain imaging methods, but the contribution of orofacial sensory input to voluntary jaw movements remains unclear. In this study, we used functional magnetic resonance imaging (fMRI) to observe brain activities during a simple teeth tapping task in adult dentulous (AD), older dentulous (OD), and older edentulous subjects who wore dentures (OEd) or did not wear dentures (OE) to analyze their functional network connections. (1) To assess the effect of age on natural activation patterns during teeth tapping, a comparison of groups with natural dentition-AD and OD-was undertaken. A general linear model analysis indicated that the major activated site in the AD group was the primary sensory cortex (SI) and motor cortex (MI) (p < 0.05, family wise error corrected). In the OD group, teeth tapping induced brain activity at various foci (p < 0.05, family wise error corrected), including the SI, MI, insula cortex, supplementary motor cortex (SMC)/premotor cortex (PMA), cerebellum, thalamus, and basal ganglia in each group. (2) Group comparisons between the OD and OEd subjects showed decreased activity in the SI, MI, Brodmann's area 6 (BA6), thalamus (ventral posteromedial nucleus, VPM), basal ganglia, and insular cortex (p ¡ 0.005, uncorrected). This suggested that the decreased S1/M1 activity in the OEd group was related to missing teeth, which led to reduced periodontal afferents. (3) A conjunction analysis in the OD and OEd/OE groups revealed that commonly activated areas were the MI, SI, cerebellum, BA6, thalamus (VPM), and basal ganglia (putamen; p < 0.05, FWE corrected). These areas have been associated with voluntary movements. (4) Psychophysiological interaction analysis (OEd vs OE) showed that subcortical and cortical structures, such as the MI, SI, DLPFC, SMC/PMA, insula cortex, basal ganglia, and cerebellum, likely function as hubs and form an integrated network that participates in the control of teeth tapping. These results suggest that oral sensory inputs are involved in the control of teeth tapping through feedforward control of intended movements, as well as feedback control of ongoing movements.

Developmental increase in hyperpolarization-activated current regulates intrinsic firing properties in rat vestibular ganglion cells.

The primary vestibular neurons convey afferent information from hair cells in the inner ear to the vestibular nuclei and the cerebellum. The intrinsic firing properties of vestibular ganglion cells (VGCs) are heterogeneous to sustained membrane depolarization, and undergo marked developmental changes from phasic to tonic types during the early postnatal period. Previous studies have shown that low-voltage-activated potassium channels, Kv1 and Kv7, play a critical role in determining the firing pattern of VGCs. In the present study, we explored the developmental changes in the properties of hyperpolarization-activated current (Ih) in rat VGCs and the role played by Ih in determining the firing properties of VGCs. Tonic firing VGCs showed a larger current density of Ih as compared to phasic firing VGCs, and tonic firing VGCs became phasic firing in the presence of ZD7288, an Ih channel blocker, indicating that Ih contributes to control the firing pattern of VGCs. The amplitude of Ih increased and the activation kinetics of Ih became faster during the developmental period. Analysis of developmental changes in the expression of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels revealed that expression of HCN1 protein and its mRNA increased during the developmental period, whereas expression of HCN2-4 protein and its mRNA did not change. Our results suggest that HCN1 channels as well as Kv1 channels are critical in determining the firing pattern of rat VGCs and that developmental up-regulation of HCN1 transforms VGCs from phasic to tonic firing phenotypes.

Nagoya university training system for neuroendovascular therapists.

SUMMARY: It is very important to train more neuroendovascular therapists (NETists) in response to Japan's growing social needs. Since the supply of qualified NETists is still insufficient to cover Japanese institutions, therefore some emergency cases with indications for endovascular therapy may be overlooked, untreated or treated by other methods resulting in unfavorable outcomes. Thus, neuroendovascular therapies are situated as one of the important tratment modalities for neurosurgical diseases (1,2). We studied our present states of the endovascular neurosurgery and introduce our training system.

Treatment Strategy for Cerebral Aneurysms Based on the Evidence of the Efficacy of GDC Embolization.

SUMMARY: The authors reviewed 531 patients with cerebral aneurysms treated with Guglielmi detachable coils (GDCs) over 5 years to clarify both the advantages and disadvantages of embolization based on the evidence of complications by aneurysm profile. There were 52 technical complications, 25 of which resulted in unfavorable patient outcomes. Intraoperative rupture, the most serious complication exacerbating the patient's condition, occurred in 19 patients, 4 of whom expired. All of these aneurysms were very small and were mostly located in the AcomA and PICA portions. Thirteen patients encountered thromboembolic complications, 6 of whom were elderly with acute ruptured aneurysms at MCA and the tip of BA. For large or giant aneurysms manifesting the mass effect, particularly those in the ICA-C2 portion compressing the optic nerve, the saccular packing did little to ameliorate the symptoms, and subsequent surgical or endovascular trapping was needed. Therefore, saccular embolization of endovascularly difficult, very small AcomA aneurysms and large C2 aneurysm with visual symptoms should be used sparingly based on a risk-benefit assessment.

Treatment strategy for aneurysms of the posterior cerebral artery.

SUMMARY: The authors carried out a retrospective review of the records of 12 patients with aneurysms of the posterior cerebral artery (PCA). Four were asymptomatic, 1 presented with a mass effect, and 7 with a subarachnoid haemorrhage (SAH). Of the 7 ruptured aneurysms, 3 were embolized and 2 were clipped. However, 2 patients died from rebleeding before any treatment. Of the 5 unruptured aneurysms, 1 was embolized with coils but the remaining 4 have been conservatively observed. No aneurysms have ruptured during the follow-up period, and 3 have thrombosed spontaneously. According to our results, the PCA aneurysms should be treated aggressively in the early phase. Although the preservation of the anatomical integrity of the PCA should naturally be one of the prime objectives, PCA occlusion may sometimes be inevitable when treating large or fusiform aneurysms. On the other hand, conservative therapy is one of the options for the treatment of incidentally encountered unruptured ones, because these have the possibility of spontaneous thrombosis.

Radiological and pathological changes in the sinus of an experimental arteriovenous fistula of the rat.

SUMMARY: The object of this study is to evaluate the radiological and pathological changes in the sinus of an experimental arteriovenous fistula of the rat. Twenty-five male Sprague-Dawley rats, including two control rats, were used for this study. A venous hypertension model in the transverse sinus was induced by means of anastomosis of a common carotid artery (CCA) to the ipsilateral external jugular vein (EJV). Rats were sacrificed 11 to 42 weeks after the procedure, then histopathological and immunohistopathological examinations were performed for the resected transverse sinus. Follow-up angiography was performed two to three weeks after the anastomosis in every case, and five months later in two rats. Patency of the anastomosed portion was confirmed in 12 of the 23 anastomosed rats. An ipsilateral carotid angiogram demonstrated a highflow arteriovenous (AV) shunt from the CCA to the sigmoid-to-transverse sinus and draining into the contralateral juglar vein. A contralateral angiogram displayed a steal phenomenon via the communicating artery. Histopathologically, the vein of the anastomosed portion and the transverse sinus were markedly dilated in with cases. There was a thickening the connecting tissue and a proliferation of fibroblast in four (50%) of the eight cases. Thrombus formation in the transverse sinus was found in one case. VEGF stained strongly in the endothelial hypertrophied area and in fibrous connective tissue around the transverse sinus compared to the control sinuses. Our results from this long-term observation of the radiological and pathological changes in the sinus exposed to hypertension resembled the clinical findings of a dural AV fistula.

Coil retrieval following embolization of cerebral aneurysms.

SUMMARY: Failed coil embolization of cerebral aneurysms may be occasionally followed by direct surgical treatment. We had 5 patients who underwent coil retrieval and surgical clipping after coil embolization because of periprocedural complications. The patients, ranging in age from 40 to 71, had wide-neck aneurysms located at the anterior communicating artery (AcomA) in 3 patients, the middle cerebral artery (MCA) in 1, and the internal carotidophthalmic artery (IC-Ophthalmic) in 1. They were embolized with Guglielmi detachable coils (GDCs), which had to be retrieved within 8 days because of coil protrusion and migration in 3 patients, aneurysm rupture in 1, and increased mass effect due to coil compaction in 1. Coils were successfully removed with aneurysmotomy or arteriotomy under temporary trapping, aneurysms were then clipped or trapped. Three patients had a good outcome, but one suffered permanent visual disturbance and the other had a motor deficit. Our study revealed that a small AcomA aneurysm had a high risk of complication in a case of complex anatomy of the AcomA-A1-A2 complex with its difficult access. In addition, insufficient packing of the inflow zone in a large and symptomatic aneurysm may cause coil compaction and regrow with increasing mass effect. The indication and treatment strategy for these aneurysms should be carefully determined.

Quantal components of the excitatory postsynaptic currents at a rat central auditory synapse.

1. Paired whole-cell recordings were made from a glutamatergic giant nerve terminal, the calyx of Held, and its postsynaptic target cell in the medial nucleus of the trapezoid body (MNTB) in the brainstem slice of juvenile rat. Excitatory postsynaptic currents (EPSCs) were evoked by presynaptic action potentials triggered by brief (1 ms) depolarizing pulses. 2. In normal artificial cerebrospinal fluid (ACSF), EPSCs of several nanoamperes in amplitude were evoked at a relatively constant latency with no failure, whereas in low [Ca(2+)](o)-high [Mg(2+)](o) solutions, EPSCs fluctuated both in amplitude and latency, and stochastic failures of transmitter release were observed in response to presynaptic action potentials. 3. After blocking action potentials with tetrodotoxin (TTX), direct depolarization of the calyceal preterminal elicited asynchronous release of miniature EPSCs (mEPSCs). When the magnitude of depolarization was increased, mEPSCs increased in frequency. Being consistent with their quantal nature, their mean amplitude remained constant over a wide range of frequencies. The amplitude distribution of mEPSCs was slightly skewed (skewness = 1.06), with a mean conductance of 0.45 nS and a coefficient of variation (c.v.) of 0.43. 4. Single-channel conductance underlying mEPSCs was estimated using non-stationary fluctuation analysis. The weighted mean single channel conductance was 20.4 pS, suggesting that a single quantum opens 22 postsynaptic glutamate receptor channels on average. 5. After washing out TTX, EPSCs evoked by presynaptic action potentials were tested for quantal analysis based upon the mean amplitude of mEPSCs and their variance. In low [Ca(2+)](o)-high [Mg(2+)](o) solutions, quantal contents estimated from the EPSC/mEPSC ratio, rate of failures or c.v. assuming Poisson's statistics, coincided with each other. Evoked EPSCs could be fitted by integer multiples of mEPSCs with an assumption of incremental variance more adequately than the constant variance assumption. 6. It is concluded that the rat central auditory synaptic transmission is made in a quantal manner as at the frog neuromuscular junction.

Cellular localization of metabotropic glutamate receptors mGluR1, 2/3, 5 and 7 in the main and accessory olfactory bulb of the rat.

The cellular localization of metabotropic glutamate receptors (mGluRs) (mGluR1alpha, 2/3, 5a and 7) in the main and accessory olfactory bulb (MOB and AOB) of adult rats was compared by using affinity purified polyclonal antibodies directed to their C-termini. mGluR1alpha and mGluR5a immunoreactivities were located in comparable structures of the MOB and AOB with different levels of intensity. mGluR5a reactivity was high in the AOB. mGluR2/3 showed a different pattern of expression in the MOB compared to that observed in the AOB; the periglomerular region of the MOB was strongly stained, but in the AOB it was the mitral/tufted cell layer that was intense. The mitral cell bodies in the MOB were strongly immunoreactive for mGluR7. These differences in the distribution of mGluRs in the MOB and AOB may reflect differences in synaptic transmission and sensitivity to neuromodulation in the two systems.

Microscopic kinetics and energetics distinguish GABA(A) receptor agonists from antagonists.

Although agonists and competitive antagonists presumably occupy overlapping binding sites on ligand-gated channels, these interactions cannot be identical because agonists cause channel opening whereas antagonists do not. One explanation is that only agonist binding performs enough work on the receptor to cause the conformational changes that lead to gating. This idea is supported by agonist binding rates at GABA(A) and nicotinic acetylcholine receptors that are slower than expected for a diffusion-limited process, suggesting that agonist binding involves an energy-requiring event. This hypothesis predicts that competitive antagonist binding should require less activation energy than agonist binding. To test this idea, we developed a novel deconvolution-based method to compare binding and unbinding kinetics of GABA(A) receptor agonists and antagonists in outside-out patches from rat hippocampal neurons. Agonist and antagonist unbinding rates were steeply correlated with affinity. Unlike the agonists, three of the four antagonists tested had binding rates that were fast, independent of affinity, and could be accounted for by diffusion- and dehydration-limited processes. In contrast, agonist binding involved additional energy-requiring steps, consistent with the idea that channel gating is initiated by agonist-triggered movements within the ligand binding site. Antagonist binding does not appear to produce such movements, and may in fact prevent them.

Recurrence of a neurenteric cyst with malignant transformation in the foramen magnum after total resection. Case report.

A 53-year-old man presented with recurrence of a neurenteric cyst with malignant transformation in the foramen magnum 3.5 years after total resection of the original tumor had been reported. For 2 years following the initial surgery, the patient had been in good condition, but then underwent ventriculoperitoneal shunt placement for intracranial hypertension. At the time there was no evidence of recurrence of the tumor on magnetic resonance (MR) images. One and one-half years later, he presented with headache and anorexia. A massive recurrent tumor was identified on MR images. The tumor was severely adhesive to the brainstem, cranial nerves, and vessels, allowing only partial resection. Histological examination of tumor specimens obtained during the first and second craniotomies indicated a malignant change from a typical neurenteric cyst with a one-layer epithelium in the first specimen to an adenocarcinoma with papillary proliferation in the second. The results of various immunohistochemical studies of the first specimen were typical of those of a neurenteric cyst. The second specimen displayed stronger staining of carbohydrate 19-9 and carcinoembryonic antigens than the initial specimen. The percentage of Ki-67 antigen (MIB-1)-positive cells increased from 0% in the first specimen to 6.7% in the second. To the authors' knowledge this is the first case in which malignant transformation has been demonstrated after total resection of a neurenteric cyst in the foramen magnum.

Hemodynamic changes in drainage systems following treatment of cerebral arteriovenous malformations.

SUMMARY: The authors studied 61 patients undergoing effective embolization for cerebral arteriovenous malformations (AVMs) and analyzed the hemodynamic changes in their drainage systems following embolization. The changes were classified into following 5 types: type A, disappearance of all the draining veins; type B, disappearance of a part of the cortical veins; type C, disappearance of a part of the deep-seated veins; type D, combined type Band C patterns; type E, disappearance of reflux into normal cortical veins. Each case was evaluated on the basis of these criteria from comparing pre- and post-embolization angiograms. The delay and reduction of shunt were observed in all cases. Forty-nine of them showed obvious hemodynamic changes in the draining systems including type A change in 9, type B in 19, C in 5, Din 3 and E in 13 cases, respectively. Two cases showed a spontaneous shift in the dominance of the main drainers. Thirteen of 15 cases showing successful results in subsequent radiosurgery exhibited various changes in draining pattern. Changes in drainage systems may be affected by the compartmentalization of the nidus, reduction in shunt flow, and spontaneous or progressive thromboses. These can be promoted by embolization and may be regarded as one of the indicators of successful pretreatment for radiosurgery.

Gene transfer for experimental saccular aneurysms.

SUMMARY: Reinforcing an aneurysmal wall is one possible way to prevent from aneurysm rupture. We preliminarily tried focal gene transfer against the wall of experimental aneurysms to aim the transgene remodeling of aneurysmal wall. Two experimental saccular aneurysms were created on canine common carotid artery with an artificial dissecting method, which resemble clinical aneurysms. Adenovirus vector (AxCALacZ, 10(8) pfu) was slowly injected into the aneurysm cavity for over 30 minutes under the condition of intraaneurysmal flow arrest using balloon-assisted neck-plasty technique. The arteries and aneurysms were evaluated 48 hours after the transduction with X-gal staining, and beta-galactosidase expression was detected mainly in the intima in both cases. No adverse effects on the normal carotid wall and no systemic complications were observed after the procedure. This experimental study suggests the possibility of gene therapy for cerebral aneurysms.

A new class of neurotoxin from wasp venom slows inactivation of sodium current.

The effects of alpha-pompilidotoxin (alpha-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that alpha-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action of alpha-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and alpha-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of alpha-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that alpha-PMTX slowed the Na+ channels inactivation process without changing the peak current-voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that alpha-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that alpha-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of alpha-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.

Defining affinity with the GABAA receptor.

At nicotinic and glutamatergic synapses, the duration of the postsynaptic response depends on the affinity of the receptor for transmitter (Colquhoun et al., 1977;Pan et al., 1993). Affinity is often thought to be determined by the ligand unbinding rate, whereas the binding rate is assumed to be diffusion-limited. In this view, the receptor selects for those ligands that form a stable complex on binding, but binding is uniformly fast and does not itself affect selectivity. We tested these assumptions for the GABAA receptor by dissecting the contributions of microscopic binding and unbinding kinetics for agonists of equal efficacy but of widely differing affinities. Agonist pulses applied to outside-out patches of cultured rat hippocampal neurons revealed that agonist unbinding rates could not account for affinity if diffusion-limited binding was assumed. However, direct measurement of the instantaneous competition between agonists and a competitive antagonist revealed that binding rates were orders of magnitude slower than expected for free diffusion, being more steeply correlated with affinity than were the unbinding rates. The deviation from diffusion-limited binding indicates that a ligand-specific energy barrier between the unbound and bound states determines GABAA receptor selectivity. This barrier and our kinetic observations can be quantitatively modeled by requiring the participation of movable elements within a flexible GABA binding site.

Dendrodendritic inhibition in the olfactory bulb is driven by NMDA receptors.

At many central excitatory synapses, AMPA receptors relay the electrical signal, whereas activation of NMDA receptors is conditional and serves a modulatory function. We show here quite a different role for NMDA receptors at dendrodendritic synapses between mitral and granule cells in the rat olfactory bulb. In whole-cell patch-clamp recordings in bulb slices, stimulation of mitral cells elicited slowly decaying, GABAA receptor-mediated reciprocal IPSCs that reflected prolonged GABA release from granule cells. Although granule cells had a normal complement of AMPA and NMDA receptors, the IPSC was completely blocked by the NMDA receptor antagonist D,L-AP-5, suggesting that NMDA receptor activation is an absolute requirement for dendrodendritic inhibition. The AMPA receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxobenzo[f]quinoxaline-7-sulfonamide (NBQX) had no effect on IPSCs in the absence of extracellular magnesium but modestly reduced IPSCs in 1 mM magnesium, indicating that the primary effect of the AMPA receptor-mediated depolarization was to facilitate the unblocking of NMDA receptors. Granule cell voltage recordings indicated that effective spike stimulation in granule cells depended on the slow NMDA receptor kinetics. Granule cells also showed a pronounced delay between synaptic stimulation and action potential generation, suggesting that their intrinsic membrane properties underlie the ineffectiveness of brief AMPA receptor-mediated EPSPs. NMDA receptors also seem to have a central role in dendrodendritic inhibition in vivo, because intraperitoneal dizocilpine maleate (MK-801) injection in young adult rats resulted in disinhibition of mitral cells as measured by the generation of c-fos mRNA. The unique dependence of dendrodendritic inhibition on slow EPSPs generated by NMDA receptors suggests that olfactory information processing depends on long-lasting reciprocal and lateral inhibition.

Activation kinetics of AMPA receptor channels reveal the number of functional agonist binding sites.

AMPA and NMDA receptor channels are closely related molecules, yet they respond to glutamate with distinct kinetics, attributable to differences in ligand binding and channel gating steps (for review, see Edmonds et al., 1995). We used two complementary approaches to investigate the number of functional binding sites on AMPA channels on outside-out patches from cultured hippocampal neurons. The activation kinetics of agonist binding were measured during rapid steps into low concentrations of selective AMPA receptor agonists and during steps from a competitive AMPA receptor antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione, into a saturating concentration of agonist. Both approaches revealed sigmoidal kinetics, which suggests that multiple agonist binding steps or antagonist unbinding steps are needed for channel activation. A kinetic model with two independent binding sites gave a better fit to the activation phase than models with one or three independent sites. A more refined analysis incorporating cooperative interaction between the two binding sites significantly improved the fits to the responses. The affinity of the first binding step was two to three times higher than the second step. These results demonstrate that binding of two agonist molecules are needed to activate AMPA receptors, but the two binding sites are not identical and independent. Because NMDA receptors require four ligand molecules for activation (two glycine and two glutamate; Benveniste and Mayer, 1991; Clements and Westbrook, 1991), it may be that some binding sites on AMPA receptors are functionally silent.

Glutamate receptor subunits GluR5 and KA-2 are coexpressed in rat trigeminal ganglion neurons.

To determine the subunit composition of high-affinity kainate receptors in native neurons is a challenging problem because of the expression of more than one GluR subunit. In the present study the question of whether GluR5 and/or GluR6 subunits combine with KA-1 or KA-2 subunits in vivo is addressed by performing detailed physiological, pharmacological, and molecular characterization of functional kainate receptor channels in acutely dissociated trigeminal ganglion (TG) neurons. The results show that (1) smaller diameter TG neurons (<30 microm) respond to L-glutamate and kainate, and the currents gated by kainate desensitize with prolonged agonist exposure; (2) all kainate receptor subunits are detected to some extent by reverse transcriptase-PCR, whereas glutamate receptor subunits GluR5 and KA-2 are expressed at high levels in the TG; (3) there is an obvious similarity between the features of native kainate receptor channels in TG neurons and of heteromeric recombinant GluR5(R)/KA-2 channels in pharmacological properties, desensitization, rectification, ion permeability, and mean channel conductance; and (4) the age-dependent increase in GluR5 and KA-2 RNA levels in the TG is correlated well with an increased number of kainate-sensitive cells during postnatal development. Our data suggest that the heteromeric GluR5/KA2 combination actually occurs in TG neurons and give a clue as to the subunit composition of native kainate receptor channels.

Hypoglossal premotor neurons in the rostral medullary parvocellular reticular formation participate in cortically-induced rhythmical tongue movements.

Premotor neurons projecting to the hypoglossal (XII) nucleus and participating in cortically-induced rhythmical tongue movements were defined by extracellular recording in the cat. Two thirds (37/57) of antidromically identified XII premotor neurons sampled in the rostral medullary parvocellular reticular formation showed changes in their firing pattern during cortically-induced rhythmical activity of XII motoneurons. Fifteen of the 37 neurons showed a firing in phase with rhythmical activity of either the medial or lateral branch of the XII nerve (phasic-type). The remaining 22 neurons showed an increase in discharge with no apparent correlation with cortically-induced rhythmical activity of the XII nerve (non-phasic-type). Among the phasic- and non-phasic-type neurons, 30 neurons received inputs from the cortical masticatory area, and 14 neurons received further excitatory inputs from the inferior alveolar nerve. By systematic mapping of the stimulation sites effective for antidromic activation, four phasic-type neurons were confirmed to project to either tongue-protruding or -retracting XII motoneuron pools in accordance with their burst firing, suggesting that the phasic-type premotor neurons contribute to excitation of XII motoneurons during cortically-induced rhythmical activity. It is concluded that there are the XII premotor neurons driving cortically-induced rhythmical activity of XII motoneurons in the rostral medullary parvocellular reticular formation.

Estradiol transcriptionally and posttranscriptionally up-regulates thyrotropin-releasing hormone receptor messenger ribonucleic acid in rat pituitary cells.

17 beta-Estradiol (E2) has been shown to up-regulate the binding activity of the TRH receptor in rat pituitary cells. In this report, we investigated whether and how E2 alters TRH receptor expression at the mRNA level using the oocyte mRNA expression system, Northern blot analysis, and nuclear run-on assay. In oocytes injected with mRNA from the anterior pituitaries or GH3 cells, a TRH-induced 45Ca2+ efflux appeared. This efflux was dependent on the amount of TRH and injected RNA, and was inhibited by simultaneous addition of chloridazepoxide, an antagonist of the TRH receptor. Treatment of GH3 cells with E2 increased TRH receptor mRNA activity, as assessed in the oocyte expression system; the E2 effect became apparent after 3 h of treatment and reached a maximum (3- to 4-fold) between 6-72 h after addition. Northern blot analysis with a 412-basepair cDNA fragment or 3.7-kilobasepair full-length cDNA of the TRH receptor as a probe showed that E2 maximally (5-fold) increased the TRH receptor mRNA level of GH3 cells, with a half-maximal concentration of 0.1 nM after 6 h of treatment. The elevated level of mRNA induced by E2 was augmented, rather than impeded, by cycloheximide, indicating that ongoing protein synthesis was not required for the induction. The rate of transcription of the TRH receptor gene in isolated nuclei taken from GH3 cells was increased 3-fold by 2 h of treatment with E2. Furthermore, the half-life of the TRH receptor mRNA in GH3 cells was prolonged by E2 (from 2.3 to 4.4 h). These results demonstrate that E2 up-regulates the TRH receptors of the pituitary cells at the mRNA level by increasing both the transcription rate and stability.