Ozhan Eyigor

Expression of ionotropic glutamate receptor subunit mRNAs in the hypothalamic paraventricular nucleus of the rat.

The hypopthalamic paraventricular nucleus (PVN) coordinates multiple aspects of homeostatic regulation, including pituitary‐adrenocortical function, cardiovascular tone, metabolic balance, fluid/electrolyte status, parturition and lactation. In all cases, a substantial component of this function is controlled by glutamate neurotransmission. In this study, the authors performed a high‐resolution in situ hybridization analysis of ionotropic glutamate receptor subunit expression in the PVN and its immediate surround. N‐methyl‐D‐aspartate (NMDA) receptor 1 (NMDAR1), NMDAR2A, and NMDAR2B mRNAs were expressed highly throughout the PVN and its perinuclear region as well as in the subparaventricular zone. NMDAR2C/2D expression was limited to subsets of neurons in magnocellular and hypophysiotrophic regions. In contrast with NMDA subunit localization, AMPA (α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate)‐preferring and kainate (KA)‐preferring receptor subunit mRNAs were expressed heterogeneously in the PVN and surround. Glutamate receptor 1 (GluR1) mRNA labeling was most intense in preautonomic subregions, whereas GluR2, GluR4, GluR5, and KA2 were expressed in hypophysiotrophic cell groups. It is noteworthy that GluR5 mRNA expression was particularly robust in the dorsolateral region of the medial parvocellular PVN, suggesting localization in corticotropin‐releasing hormone neurons. All four AMPA subunits and GluR6 and GluR7 mRNAs were expressed highly in the perinuclear PVN region and the subparaventricular zone. These data suggest the capacity for multifaceted regulation of PVN function by glutamate, with magnocellular neurons preferentially expressing NMDA subunits, preautonomic neurons preferentially expressing AMPA subunits, and hypophysiotrophic neurons preferentially expressing KA subunits. Localization of all species in the perinuclear PVN suggests that glutamate input to the immediate region of the PVN may modulate its function, perhaps by communication with local γ‐aminobutyric acid neurons. J. Comp. Neurol. 422:352–362, 2000.

Distribution of ionotropic glutamate receptor subunit mRNAs in the rat hypothalamus.

The excitatory amino acid neurotransmitter glutamate participates in the control of most (and possibly all) neuroendocrine systems in the hypothalamus. This control is exerted by binding to two classes of membrane receptors, the ionotropic and metabotropic receptor families, which differ in their structure and mechanisms of signal transduction. To gain a better understanding about the precise sites of action of glutamate and the subunit compositions of the receptors involved in the glutamatergic neurotransmission in the hypothalamus and septum, in situ hybridization was used with 35S‐labeled cRNA probes for the different ionotropic receptor subunits, including glutamate receptor subunits 1–4 (GluR1–GluR4), kainate‐2, GluR5–GluR7, N‐methyl‐D‐aspartate (NMDA) receptor 1 (NMDAR1), and NMDAR2A–NMDAR2D. The results showed that subunits of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionate‐preferring, kainate‐preferring, and NMDA‐preferring receptor subunits are distributed widely but heterogeneously and that the GluR1, GluR2, kainate‐2, NMDAR1, NMDAR2A, and NMDAR2B subunits are the most abundant in the hypothalamus. Thus, GluR1 subunit mRNA was prominent in the lateral septum, preoptic area, mediobasal hypothalamus, and tuberomammillary nucleus, whereas kainate‐2 subunit mRNA was abundant in the medial septum‐diagonal band, median and anteroventral preoptic nuclei, and supraoptic nuclei as well as the magnocellular portion of the posterior paraventricular nucleus. Regions that contained the highest levels of NMDAR1 subunit mRNA included the septum, the median preoptic nucleus, the anteroventral periventricular nucleus, and the supraoptic and suprachiasmatic nuclei as well as the arcuate nucleus. Together, the extensive distribution of the different GluR subunit mRNAs strengthen the view that glutamate is a major excitatory neurotransmitter in the hypothalamus. The overlap in the distribution of the various subunit mRNAs suggests that many neurons can express GluR channels that belong to different families, which would allow a differential regulation of the target neurons by glutamate. J. Comp. Neurol. 434:101–124, 2001.

Expression of Glutamate Receptor Subunit mRNAs in Gonadotropin-Releasing Hormone Neurons during the Sexual Maturation of the Female Rat

Excitatory amino acids, particularly glutamate, are thought to be important for the maturation of the brain-pituitary-gonadal axis and the induction of puberty in the rat. We have previously shown that, in the female rat, GnRH neurons preferentially express the KA2 and NMDAR2A receptor subunit mRNAs, but not AMPA or NMDAR1 mRNA. The aim of the present study was to determine whether the onset or rate of KA2 and NMDAR2A receptor expression in GnRH neurons is correlated with the onset of puberty. Dual in situ hybridization using digoxigenin-labeled GnRH cRNA probes and 35S-labeled glutamate receptor subunit probes, followed by autoradiography and image analysis were used to measure the KA2 or NMDAR2A mRNA content in GnRH neurons in 20- to 50-day-old female rats which were sacrificed at 08.00 or 17.00 h. The results show that: (a) the KA2 mRNA content of GnRH neurons and the number of GnRH neurons expressing KA2 mRNA increase progressively in the morning hours between postnatal days 20 and 40; (b) the diurnal pattern of KA2 mRNA levels in GnRH neurons changes between days 40 and 50 from high KA2 levels in the morning hours before day 40 to high KA2 mRNA levels in the afternoon in 45- and 50-day-old animals; (c) while the high levels of KA2 mRNA in GnRH neurons in the morning hours of 20- to 40-day-old animals are paralleled by an overall increase in KA2 expression in the preoptic area, the rise in KA2 mRNA in GnRH neurons in the afternoon of 45- and 50-day-old animals appears to be specific for the GnRH neurons, and (d) no significant differences were detected for the NMDAR2A mRNA content in GnRH neurons among the different age groups and the morning and afternoon values. Since the gradual increase in the KA2 mRNA content in GnRH neurons of animals reaching puberty as well as the reversal of diurnal rhythmicity in KA2 receptor mRNA content of GnRH neurons coincide with the times of vaginal opening and first ovulation, it is suggested that glutamate, acting through KA2 receptors directly on GnRH neurons is, at least in part, an important factor in the excitatory regulation of the postnatal sexual development of the female rat. In contrast, expression of the NMDA-preferring receptor, NMDAR2A, in GnRH neurons appeared to be unchanged during this development.

Identification of glutamate receptor subtype mRNAs in gonadotropin-releasing hormone neurons in rat brain

AbstractThe aims of the present study were to determine:1.If glutamate neurites can provide input to gonadotropin-releasing hormone (GnRH) neurons;2.Which glutamate receptor subtype mRNAs are expressed in GnRH neurons; and3.If GnRH neurons synthesize kainate 2 receptor (KA2) protein. Immunohistochemical double stainings for GnRH and glutamate or for GnRH and KA2-receptor protein were applied to rat brain sections containing the medial septum-diagonal band and preoptic area or the median eminence; in addition, dualin situ hybridization studies were carried out with digoxygenin-labeled cRNA probes encoding GnRH in combination with35S-labeled cRNA probes encoding the glutamate receptor subtypes GluR1–4, KA2, NMDA R1, or NMDA R2A-D. The results show that GnRH neurons are surrounded by glutaminergic neurites, which form puncta-like close appositions with the GnRH perikarya, and that an extensive overlap exists in the distribution of GnRH-positive axon terminals and glutaminergic neurites in the median eminence. Similarly, KA2-receptor immunoreactivity is present in the perikarya of many GnRH neurons and in their axon terminals in the median eminence. Dualin situ hybridization experiments show that about 32% of all digoxygenin-labeled GnRH neurons also contain KA2-receptor mRNA, 17% contain NMDA R2A mRNA, 8% contain NMDR R1, whereas <5% of the GnRH neurons express measurable amounts of GluR1–4 or NMDAR2B-D mRNA. The results suggest that glutaminergic neurons innervate the GnRH neuronal system directly through activation of KA2 receptors on GnRH neurons, whereas the effects of AMPA and NMDA on GnRH release are likely to be exerted indirectly through interneurons.

Kainate Receptor Subunit-Positive Gonadotropin-Releasing Hormone Neurons Express c-Fos during the Steroid-Induced Luteinizing Hormone Surge in the Female Rat1

During the preovulatory and estradiol-progesterone-induced GnRH-LH surge, a subpopulation of GnRH neurons transiently expresses the transcription factor c-fos, which is a useful marker of cell activation. To further characterize this subpopulation of GnRH neurons, multiple immunohistochemical procedures were applied to visualize GnRH, c-Fos, KA2, GluR5, GluR6, and GluR7 receptor subunits during different phases of the estrogen-progesterone-induced LH surge. The results show that the LH surge begins at 1400 h and peaks at 1600 h before returning to baseline late in the evening. At 1400 h, about 50% of the GnRH neurons contained c-Fos, and this percentage remained high at 65% at 1600 and 2000 h. During the surge, 50% of the c-Fos-positive GnRH neurons contained KA2 receptor subunit protein at 1400 h, 65% of the c-Fos-positive GnRH neurons expressed the KA2 subunit at 1600 h, and 50% of the c-Fos-positive GnRH neurons expressed the KA2 subunit at 2000 h. As KA2 subunits require other kainate-preferring subun...

Identification of neurones in the female rat hypothalamus that express oestrogen receptor-alpha and vesicular glutamate transporter-2.

Oestrogen exerts its effects in the brain by binding to and activating two members of the nuclear receptor family, oestrogen receptor (ER)‐α and ER‐β. Evidence suggests that oestrogen‐receptive neurones participate in the generation of reproductive behaviours and that they convey the oestrogen message to gonadotropin‐releasing hormone (GnRH) neurones. The aim of the present study was to identify the neurochemical phenotype of a subset of oestrogen receptor‐expressing neurones. To this aim, we focused on the glutamate neuronal system, which is one of the most important stimulators of GnRH synthesis and release. We used the presence of vesicular glutamate transporter‐2 (VGLUT2) mRNA as a specific marker to identify glutamate neurones and employed dual in situ hybridization to localize ERα mRNA‐(35S‐labelling) and VGLUT2 mRNA‐(digoxigenin‐labelling) expressing neurones within the hypothalamus. The results show that the overall distribution of VGLUT2 mRNA and ERα mRNA are consistent with previous data in the literature. Dual‐labelled neurones were localized in the ventrolateral part of the ventromedial nucleus where 81.3 ± 3.4% of the ERα mRNA containing neurones expressed VGLUT2 mRNA, in the anteroventral periventricular nucleus (30% colocalization) and in the medial preoptic nucleus (19% colocalization). Only 4.4% of the ERα expressing neurones in the arcuate nucleus contained VGLUT2 mRNA. These findings reveal that certain subpopulations of oestrogen‐receptive neurones are glutamatergic in select hypothalamic areas that are known to regulate reproductive behaviour and GnRH neurones in the female rat. Thus, the oestrogen signal could be propagated through glutamate neurones to distant sites and influence the activity of the postsynaptic neurones.

Identification of kainate-preferring glutamate receptor subunit GluR7 mRNA and protein in the rat median eminence.

In situ hybridization and immunohistochemistry were used to determine the presence of kainate-preferring glutamate receptor subunits GluR6 and GluR7 mRNA and protein in the median eminence of the rat. The results show that most tanycytes lining the ventral third ventricle and many astrocytes within the median eminence contain the GluR7 receptor subunit mRNA but not the GluR5 and GluR6 receptor subunit mRNA. Immunohistochemical stainings show that the GluR6/7 receptor protein was localized to tanycytic cell bodies, their basal processes and to many other astrocytes in different layers of the median eminence. The results suggest that glutamate can act directly on the glial cells in the median eminence by binding to the GluR7 subunit which may be important for the control of the secretion of releasing and inhibiting hormones from axon terminals in the external layer. In order to determine if these receptor subunits are functional, kainic acid was injected and c-fos expression monitored. Results show that kainic acid induced c-fos synthesis in most of these glial cells.

Clinical importance of the basal cavernous sinuses and cavernous carotid arteries relative to the pituitary gland and macroadenomas: quantitative analysis of the complete anatomy

BACKGROUND It is unusual to encounter hemorrhagic complications caused by arterial or venous damage during TSS. Problems with these structures can lead to permanent disability or death. Our aim was to quantitatively analyze anatomical and radiologic relationships among the BCS, the CCA, and the pituitary gland, as these structures are accessed during TSS. METHODS Forty-nine formaldehyde-fixed, sellar-parasellar tissue blocks from adult cadavers were used to simulate accessing the BCSs via TSS. In each specimen, size of the pituitary gland and specific characteristics of each BCS and the horizontal segment of each CCA were recorded. Nine other specimens were used for histologic investigation and microanatomical measurements. To attest correlation between clinical data and cadaveric measurements, coronal MRI scans of 22 healthy adults as well as of 28 patients with macroadenomas were analyzed. RESULTS In cadaveric specimens, distances between both CCAs in the BCS were 17.1 +/- 4.0 mm anteriorly, 20.3 +/- 4.2 mm medially, and 18.8 +/- 4.6 mm posteriorly. In this study, the anterior medial space of the BCS was dominant in 12 specimens on the right side and in 5 specimens on the left; the posterior medial space of the BCS was dominant in 23 specimens on the right side and in 9 specimens on the left side. The right medial BCS was dominant in 35 specimens. On histologic coronal sections, some part of the carotid arterys (CAs) diameter was located below the line passing from the basal dural layer ranging from 5.3% to 65.4%. In normal-sella images, distances between both CCAs were 15.4 +/- 1.8 mm anteriorly, 16.0 +/- 2.8 mm medially, 16.2 +/- 3.4 mm posteriorly. On coronal normal-sella images, some part of the CAs diameter was located below the line passing from the basal dural layer ranging from 16.4% to 66.7%. In macroadenomas, distances between both CCAs were 22.0 +/- 3.6 mm anteriorly, 21.5 +/- 3.8 mm medially, and 20.7.2 +/- 3.7 mm posteriorly . On coronal images, in only 6 of 28 macroadenomas, some part of the CAs diameter was located below the line passing from the basal dural layer ranging from 12.5% to 100%. CONCLUSIONS Our results indicate that a working area of 15.0 +/- 2.6 x 10.3 +/- 2.1 mm is safe during TSS. The position of the CCA posterior segment was notably more caudal than the anterior segment with respect to the basal dura, which should be taken into account during extended exposure. Also, preoperative recognition of the anatomical variations is beneficial for detection of the boundaries of dissection, which is particularly important in the BCS, where variable course of CCAs may transform the anatomical configuration. Slowly growing pituitary adenomas stretch out both CCAs considerably from medial to lateral directions, and they cause widening of intercarotid distances in all segments. Processing of fixation, decalcification, and paraffin embedding for the cadaveric tissue in contrast to physiologically hydrated tissues may change the accuracy of measurements. These measurements are significantly different than those in the radiologic images when arterial blood under pressure is in the CCA as well as when venous blood fills the cavernous sinus as is the case in vivo. In clinical practice, these facts must be taken into consideration in the cadaveric measurements.

Glutamate and Orexin Neurons

Orexin neurons are localized in the lateral hypothalamus and regulate many functions including sleep-wake states. Substantial number of neurotransmitters and neuromodulators has been proposed to influence orexinergic system. Glutamate, as the major excitatory amino acid neurotransmitter in the hypothalamus, was shown to mediate orexin neurons in the regulation of wakefulness and feeding. Glutamate is readily present in the lateral hypothalamus, and glutamate receptors are expressed by the neurons of this region. Glutamate agonists initiate excitatory postsynaptic currents in orexin neurons, and this can be blocked by specific antagonists of the glutamate receptors. It is reported that both NMDA and non-NMDA receptors contribute the glutamatergic neurotransmission which affects orexinergic functions. Glutamatergic axon terminals are demonstrated to make contacts with the orexin neurons, as revealed by the presence of vesicular glutamate transporter proteins in the terminals, and these contacts were ultrastructurally confirmed to establish synapses on orexin neurons. This chapter reviews the literature on the glutamatergic regulation of orexin neurons including the data from our laboratory.

KAINIC ACID ACTIVATES OXYTOCINERGIC NEURONS THROUGH NON-NMDA GLUTAMATE RECEPTORS

The present study assessed if kainic acid activates oxytocinergic neurons and this activation is blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Dual immunohistochemistry for oxytocin and c-Fos showed that oxytocin neurons in SON and PVN express c-Fos following kainic acid administration, a significant increase when compared to the control group. Administration of CNQX prior to kainic acid caused a significant reduction. The results suggested the participation of non-NMDA receptors in the regulation of oxytocin neurons because the administration of kainic acid activates these neurons and this activation is blocked by CNQX administration.